Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities

CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL2 and HDL3 remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu2+-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.


Introduction
High-density lipoproteins (HDLs) have antioxidant and anti-inflammatory activity to suppress the oxidation of low-density lipoproteins (LDLs) and the production of pro-inflammatory cytokines in the acute-phase response in the blood and lungs [1,2].
Apolipoprotein A-I (apoA-I), a major protein component in HDL of around 70%, is mainly responsible for the antioxidant, anti-inflammatory, and anti-tumorigenic activities with cholesterol efflux ability [3,4].ApoA-I can reduce systemic and lung inflammation in the acute phase by modulating innate and adaptive immunity [5].In the hyperinflammatory state, such as critical phase of COVID-19 with the acute elevation of tumor necrosis factor (TNF)-α and interleukin (IL)-6, serum concentrations of HDL-C and apoA-I significantly decreased and were negatively correlated with COVID-19 severity [6,7].Furthermore, the decreased apoA-I concentration was positively associated with the severity of COVID-19.It was negatively correlated with the production of IL-6 and high-sensitive C-reactive protein (CRP) [7], suggesting that apoA-I itself could exert anti-inflammatory and antiviral activities [8].
On the other hand, HDL can become dysfunctional with a decrease in cholesterol content and an increase in oxidation and glycation, particularly in the acute-phase response [9,10].In particular, the loss of HDL functionality is directly associated with the alteration of apoA-I by glycation, nitration, and myeloperoxidase-mediated oxidation [11].The higher modification extent of apoA-I was directly correlated with the larger loss of cholesterol efflux ability, which is linked with damage to HDL particle formation.Nonenzymatic glycation impaired the structural stability and functionality of apoA-I, such as decreased HDL particle size, cholesterol efflux ability, and paraoxonase activity [12,13].Interestingly, the apoA-I half-life was longer with lower glycated hemoglobin (HbA 1c ) levels.Specifically, the half-life of glycated apoA-I was three times shorter than that of native apoA-I [13].Among the advanced glycation end products (AGE), carboxymethyllysine (CML), a glycoxidation product of glycated lysine residues, caused an increase in atrial stiffness and pulmonary fibrosis [14,15].In the same context, previous reports showed that a CML treatment led to significant increase in the extent of glycation of HDL and apoA-I, accompanied by severe proteolytic degradation [16,17].High levels of CML in serum were also identified in patients with type 2 diabetes mellitus (T2DM) and cardiovascular diseases, who had extremely high inflammatory cytokines [18,19].
These studies helped develop a new pharmaceutical agent to treat the cytokine storm and hyperinflammation in acute and chronic inflammatory disease by protecting HDL and apoA-I from glycoxidation attack, maximizing its anti-infection and anti-inflammation activity.CIGB-258 (Jusvinza ® ), an altered peptide ligand composed of 27 amino acids with a molecular weight of 2987, is derived from heat shock protein HSP60.It has demonstrated protective effects on HDL and apoA-I against proteolytic degradation caused by glycation and oxidation, specifically, CIGB-258 dose-dependent protection for HDL and apoA-I from CML-induced glycation by stabilizing their protein structure and enhancing their antioxidant capacity.Moreover, CIGB-258 could bind strongly with apoA-I and transthyretin (TTR) in human serum from affinity chromatography [20], but the precise mechanism and purpose are unclear.
A previous study reported that CIGB-258 can effectively bind phospholipids and cholesterol, stabilizing apoA-I within the reconstituted HDL (rHDL) structure and leading to the formation of larger rHDL particles.The rHDL containing CIGB-258 exhibited improved in vitro antioxidant ability against LDL oxidation, enhanced anti-glycation activity to protect HDL, and showed in vivo anti-inflammatory effects against CML toxicity in embryos and adult zebrafish.The incorporation of apoA-I and CIGB-258 in the lipid-bound state (rHDL) resulted in synergistic interactions that enhanced the structural integrity and functional performance of rHDL in a dose-dependent manner.On the other hand, there are no reports of an interaction between apoA-I and CIGB-258 in the lipid-free state, either synergistically or in an uncooperative manner, depending on the increase in CIGB-258 in the mixture.The study aims to compare the putative collaboration effect between the apoA-I and CIGB-258 at molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1 to exert in vitro antioxidant activity and anti-glycation activity by stabilizing the lipid-free apoA-I structure.The apoA-I and CIGB-258 mixture were administrated to zebrafish embryos and adults to evaluate in vivo anti-inflammatory activities in the presence of CML, which can cause acute embryotoxicity, developmental defects, and acute paralysis and inflammatory death.

Antioxidant Activity of Lipid-Free apoA-I and CIGB-258
Native LDL (lane N) exhibited a distinct band intensity and the slowest electromobility (as indicated by the black arrow), while the band intensity of oxidized LDL (oxLDL, lane O) in the presence of Cu 2+ (final 1 µM) almost disappeared with the fastest electromobility (as indicated by the red arrow) (Figure 1A).Interestingly, the co-treatment of apoA-I and CIGB-258 inhibited the LDL oxidation with a thicker band intensity than oxLDL (lanes 1-4), suggesting that LDL was protected from the cupric ion-mediated oxidation.Quantification of the oxidized species in each LDL showed that native LDL had the lowest level of malondialdehyde (MDA), while oxLDL had the highest level of MDA, 11-fold higher MDA than native LDL (Figure 1B).Interestingly, a co-treatment with apoA-I and CIGB-258 at 1:0, 1:0.1, 1:0.5, and 1:1 molar ratio lowered the MDA level by 27% (p < 0.05), 44% (p < 0.01), 57% (p < 0.001), and 71% (p < 0.001) lower than that of oxLDL, respectively.These results suggest that a co-treatment of apoA-I and CIGB-258 up to a 1:1 molar ratio inhibited oxidation to protect the LDL band and lower the oxidized species via the putative stabilization of the apoA-I structure.
the apoA-I and CIGB-258 at molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1 to exert in vitro antioxidant activity and anti-glycation activity by stabilizing the lipid-free apoA-I structure.The apoA-I and CIGB-258 mixture were administrated to zebrafish embryos and adults to evaluate in vivo anti-inflammatory activities in the presence of CML, which can cause acute embryotoxicity, developmental defects, and acute paralysis and inflammatory death.

Antioxidant Activity of Lipid-Free apoA-I and CIGB-258
Native LDL (lane N) exhibited a distinct band intensity and the slowest electromobility (as indicated by the black arrow), while the band intensity of oxidized LDL (oxLDL, lane O) in the presence of Cu 2+ (final 1 μM) almost disappeared with the fastest electromobility (as indicated by the red arrow) (Figure 1A).Interestingly, the co-treatment of apoA-I and CIGB-258 inhibited the LDL oxidation with a thicker band intensity than ox-LDL (lanes 1-4), suggesting that LDL was protected from the cupric ion-mediated oxidation.Quantification of the oxidized species in each LDL showed that native LDL had the lowest level of malondialdehyde (MDA), while oxLDL had the highest level of MDA, 11fold higher MDA than native LDL (Figure 1B).Interestingly, a co-treatment with apoA-I and CIGB-258 at 1:0, 1:0.1, 1:0.5, and 1:1 molar ratio lowered the MDA level by 27% (p < 0.05), 44% (p < 0.01), 57% (p < 0.001), and 71% (p < 0.001) lower than that of oxLDL, respectively.These results suggest that a co-treatment of apoA-I and CIGB-258 up to a 1:1 molar ratio inhibited oxidation to protect the LDL band and lower the oxidized species via the putative stabilization of the apoA-I structure.

Enforcement of Antioxidant Ability of HDL by Co-Presence of CIGB-258
Native LDL (lane N) showed the highest distinct band intensity with the slowest electromobility (Figure 2A), while the band intensity of oxidized LDL (lane O) nearly vanished alongside the aggregated band at the loading position, as highlighted by the red arrowhead.The HDL 2 alone (lane 1) and HDL 3 alone (lane 5) treatments inhibited the oxidation and degradation of LDL, suggesting that HDL possesses adequate inhibition activity against LDL oxidation.Conversely, the co-presence of CIGB-258 led to stronger antioxidant ability of HDL 2 (lanes 2-4) and HDL 3 (lanes 6-8), in a dose-dependent manner, of CIGB-258.The higher CIGB-258 dosage caused the slower electromobility of LDL with more distinct band intensity under the same amount of HDL, suggesting that the co-presence of CIGB-258 can elicit the antioxidant ability of HDL.

Enforcement of Antioxidant Ability of HDL by Co-Presence of CIGB-258
Native LDL (lane N) showed the highest distinct band intensity with the slowest electromobility (Figure 2A), while the band intensity of oxidized LDL (lane O) nearly vanished alongside the aggregated band at the loading position, as highlighted by the red arrowhead.The HDL2 alone (lane 1) and HDL3 alone (lane 5) treatments inhibited the oxidation and degradation of LDL, suggesting that HDL possesses adequate inhibition activity against LDL oxidation.Conversely, the co-presence of CIGB-258 led to stronger antioxidant ability of HDL2 (lanes 2-4) and HDL3 (lanes 6-8), in a dose-dependent manner, of CIGB-258.The higher CIGB-258 dosage caused the slower electromobility of LDL with more distinct band intensity under the same amount of HDL, suggesting that the copresence of CIGB-258 can elicit the antioxidant ability of HDL.Oxidized LDL showed a 20-fold higher MDA level than native LDL (Figure 2B), while HDL2 or HDL3 alone-treated LDL showed 37% and 30% lower MDA levels.These results suggest that HDL2 and HDL3 alone could exert adequate inhibition activity against LDL oxidation via HDL-associated paraoxonase activity.On the other hand, the co-presence of CIGB-258 at an apoA-I:CIGB-258 molar ratio of 1:1 reduced the MDA level (up to 57% (in HDL2) and 46% (in HDL3) lower than oxLDL) in a dose-responsive manner.The findings imply that HDL and CIGB-258 exhibited synergistic antioxidant activity against LDL oxidation to protect LDL particles from proteolytic degradation and aggregation (Figure 2A).A further increase in the CIGB-258 ratio in HDL resulted in the detection of a lower MDA level in LDL (Figure 2B), suggesting that CIGB-258 could promote the antioxidant ability of HDL.Oxidized LDL showed a 20-fold higher MDA level than native LDL (Figure 2B), while HDL 2 or HDL 3 alone-treated LDL showed 37% and 30% lower MDA levels.These results suggest that HDL 2 and HDL 3 alone could exert adequate inhibition activity against LDL oxidation via HDL-associated paraoxonase activity.On the other hand, the co-presence of CIGB-258 at an apoA-I:CIGB-258 molar ratio of 1:1 reduced the MDA level (up to 57% (in HDL 2 ) and 46% (in HDL 3 ) lower than oxLDL) in a dose-responsive manner.The findings imply that HDL and CIGB-258 exhibited synergistic antioxidant activity against LDL oxidation to protect LDL particles from proteolytic degradation and aggregation (Figure 2A).A further increase in the CIGB-258 ratio in HDL resulted in the detection of a lower MDA level in LDL (Figure 2B), suggesting that CIGB-258 could promote the antioxidant ability of HDL.

HDL-Associated Paraoxonase Activity with CIGB-258
As depicted in Figure 3A, during 90 min incubation, the native HDL 3 showed 2.5-times higher paraoxonase (PON) activity than HDL 2 , suggesting that HDL 3 is the principal source of PON activity in total HDL.The addition of an apoA-I:CIGB-258 mixture to HDL 2 and HDL 3 elevated the PON activity as the CIGB-258 content was increased.The addition of a 1:1 mixture (apoA-I:CIGB-258) into HDL 2 and HDL 3 resulted in the highest PON activity, approximately 13% and 28% higher than that of native HDL 2 or HDL 3 alone, respectively.
In contrast, the 1:0.5 mixture (apoA-I:CIGB-258) resulted in the second-highest PON activity of HDL 2 and HDL 3 , approximately 14% and 18% higher than HDL alone.Interestingly, the addition of apoA-I alone or CIGB-258 alone showed a similar increase in PON activity, approximately 5-7% and 3-5% higher than HDL 2 and HDL 3 alone.These results suggest that a combination of apoA-I and CIGB-258 had the highest PON activity in HDL 2 and HDL 3 , with synergistic activity dependent on the CIGB-258 content.
As depicted in Figure 3A, during 90 min incubation, the native HDL3 showed 2.5times higher paraoxonase (PON) activity than HDL2, suggesting that HDL3 is the principal source of PON activity in total HDL.The addition of an apoA-I:CIGB-258 mixture to HDL2 and HDL3 elevated the PON activity as the CIGB-258 content was increased.The addition of a 1:1 mixture (apoA-I:CIGB-258) into HDL2 and HDL3 resulted in the highest PON activity, approximately 13% and 28% higher than that of native HDL2 or HDL3 alone, respectively.In contrast, the 1:0.5 mixture (apoA-I:CIGB-258) resulted in the second-highest PON activity of HDL2 and HDL3, approximately 14% and 18% higher than HDL alone.Interestingly, the addition of apoA-I alone or CIGB-258 alone showed a similar increase in PON activity, approximately 5-7% and 3-5% higher than HDL2 and HDL3 alone.These results suggest that a combination of apoA-I and CIGB-258 had the highest PON activity in HDL2 and HDL3, with synergistic activity dependent on the CIGB-258 content.Statistical significance is denoted by * and ** at p < 0.05, and p < 0.01, for PON activity observed in HDL3, while ## and ### denote the statistical significance at p < 0.01, and p < 0.001, for PON activity observed in HDL2 compared to the PBS group.
In the same context, native HDL2 displayed the restarted electromobility, characterized by a distinct sharp band intensity (Figure 3B), highlighted by the black arrow (lane N).Conversely, HDL2 subjected to cupric ion treatment exhibited rapid electromobility, migrating to the gel bottom with the diminished and diffused band intensity (lane O), as denoted by the red arrow, attributed to proteolytic degradation, and improved negative charge.On the other hand, adding the apoA-I and CIGB-258 mixture induced slower electromobility with a stronger band intensity, depending on the increase in CIGB-258 content (lanes 1-4).In particular, the co-addition of a 1:1 mixture (apoA-I:CIGB-258) into HDL2 resulted in the slowest electromobility among the oxidized lipoproteins (lane 4), suggesting that a greater elevation in CIGB-258 content induced more resistance to HDL2 oxidation.

Stabilization of apoA-I Structure by Co-Presence of CIGB-258
In the absence of urea, lipid-free apoA-I alone displayed 336.5 nm of WMF, while the addition of CIGB-258 caused a blue shift of apoA-I toward 335.9, 334.9, and 334.7 nm for In the same context, native HDL 2 displayed the restarted electromobility, characterized by a distinct sharp band intensity (Figure 3B), highlighted by the black arrow (lane N).Conversely, HDL 2 subjected to cupric ion treatment exhibited rapid electromobility, migrating to the gel bottom with the diminished and diffused band intensity (lane O), as denoted by the red arrow, attributed to proteolytic degradation, and improved negative charge.On the other hand, adding the apoA-I and CIGB-258 mixture induced slower electromobility with a stronger band intensity, depending on the increase in CIGB-258 content (lanes 1-4).In particular, the co-addition of a 1:1 mixture (apoA-I:CIGB-258) into HDL 2 resulted in the slowest electromobility among the oxidized lipoproteins (lane 4), suggesting that a greater elevation in CIGB-258 content induced more resistance to HDL 2 oxidation.

Stabilization of apoA-I Structure by Co-Presence of CIGB-258
In the absence of urea, lipid-free apoA-I alone displayed 336.5 nm of WMF, while the addition of CIGB-258 caused a blue shift of apoA-I toward 335.9, 334.9, and 334.7 nm for 1:0.1, 1:0.5, and 1:1, respectively, as depicted in Figure 4A.These findings imply that the copresence of CIGB-258 prompted the migration of intrinsic Trp, particularly Trp 108, within apoA-I towards the hydrophobic environment, possibly through a presumed interaction between the amphipathic helix regions of apoA-I and CIGB-258.
1:0.1, 1:0.5, and 1:1, respectively, as depicted in Figure 4A.These findings imply that the co-presence of CIGB-258 prompted the migration of intrinsic Trp, particularly Trp 108, within apoA-I towards the hydrophobic environment, possibly through a presumed interaction between the amphipathic helix regions of apoA-I and CIGB-258.Interestingly, SDS-PAGE displayed the fact that the intensity of the apoA-I band was increased by the co-presence of CIGB-258 in a dose-responsive manner up to a 1:1 molar ratio (Figure 4B).Compared to a 1:0 molar ratio of apoA-I:CIGB-258, the 1:0.5 and 1:1 apoA-I:CIGB-258 mixtures showed an up to 2.1-and 2.2-fold band intensity, respectively, due to the addition of CIGB-258.In lanes 3 and 4, the CIGB-258 band was detected at the base of the gel, as highlighted by the red arrowhead.In particular, a 1:1 blend of apoA-I and CIGB-258 showed the strongest band intensity (lane 4, Figure 4B).The results imply that the co-presence of CIGB-258 induced more stabilization of apoA-I structure to protect against the denaturation and proteolysis of apoA-I.
The isothermal denaturation of lipid-free apoA-I alone (1:0) by a urea treatment caused a 20.4 nm increase in WMF from 336.5 nm (without urea) to 356.9 nm (7 M urea), suggesting exposure of intrinsic Trp toward the hydrophilic phase by unfolding of the secondary structure.The sigmoidal curve of WMF showed a typical α-helix-enriched protein of apoA-I.Until 2 M urea treatment, 9 nm of WMF was increased from the baseline (0 M urea), indicating apoA-I was resistant to denaturation at 1-2 M urea (Figure 4A) with q mid-point of denaturation (D1/2) of 2.33 M of urea (Table 1).Conversely, the addition of CIGB-258 into apoA-I displayed a slower enhancement in the WMF upon the same urea exposure, a 6-7 nm rise in WMF with the 2 M urea exposure, indicating less exposure of intrinsic Trp via more resistance to denaturation.At 3 M urea treatment, the WMF of apoA-I was red-shifted to 351.9 nm, 351.4 nm, 347.3 nm, and 347.8 nm for apoA-I:CIGB-258 molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1, respectively.These results suggest that the copresence of CIGB-258 confers resistance of apoA-I against chaotropic agent-induced denaturation by stabilizing the α-helical domains and tertiary structure.Interestingly, SDS-PAGE displayed the fact that the intensity of the apoA-I band was increased by the co-presence of CIGB-258 in a dose-responsive manner up to a 1:1 molar ratio (Figure 4B).Compared to a 1:0 molar ratio of apoA-I:CIGB-258, the 1:0.5 and 1:1 apoA-I:CIGB-258 mixtures showed an up to 2.1-and 2.2-fold band intensity, respectively, due to the addition of CIGB-258.In lanes 3 and 4, the CIGB-258 band was detected at the base of the gel, as highlighted by the red arrowhead.In particular, a 1:1 blend of apoA-I and CIGB-258 showed the strongest band intensity (lane 4, Figure 4B).The results imply that the co-presence of CIGB-258 induced more stabilization of apoA-I structure to protect against the denaturation and proteolysis of apoA-I.
The isothermal denaturation of lipid-free apoA-I alone (1:0) by a urea treatment caused a 20.4 nm increase in WMF from 336.5 nm (without urea) to 356.9 nm (7 M urea), suggesting exposure of intrinsic Trp toward the hydrophilic phase by unfolding of the secondary structure.The sigmoidal curve of WMF showed a typical α-helix-enriched protein of apoA-I.Until 2 M urea treatment, 9 nm of WMF was increased from the baseline (0 M urea), indicating apoA-I was resistant to denaturation at 1-2 M urea (Figure 4A) with q mid-point of denaturation (D 1/2 ) of 2.33 M of urea (Table 1).Conversely, the addition of CIGB-258 into apoA-I displayed a slower enhancement in the WMF upon the same urea exposure, a 6-7 nm rise in WMF with the 2 M urea exposure, indicating less exposure of intrinsic Trp via more resistance to denaturation.At 3 M urea treatment, the WMF of apoA-I was red-shifted to 351.9 nm, 351.4 nm, 347.3 nm, and 347.8 nm for apoA-I:CIGB-258 molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1, respectively.These results suggest that the co-presence of CIGB-258 confers resistance of apoA-I against chaotropic agent-induced denaturation by stabilizing the α-helical domains and tertiary structure.
Regression analysis showed that the mid-points of denaturation were increased up to 2.35 M, 2.57 M, and 2.56 M of urea for apoA-I:CIGB-258 ratios of 1:0.1, 1:0.5, and 1:1, respectively, suggesting less exposure of intrinsic Trp via more stabilization of the α-helix domains of apoA-I.These results indicate that the secondary and tertiary configuration of apoA-I could be improved by the co-presence of CIGB-258 via the putative helix-helix interactions.The stabilization of apoA-I (Figure 4A) was linked with more protection of the apoA-I band intensity, with a concomitant increase in CIGB-258 (Figure 4B).

Anti-Glycation Activity of CIGB-258 against CML-Induced apoA-I Glycation
The CML exposure of lipid-free apoA-I caused the largest increase in yellowish fluorescence intensity (FI), 8.1-fold higher FI than apoA-I alone during 72 h, as shown in Figure 5A.Conversely, the co-treatment of CIGB-258 caused a smaller increase in FI in a dose-dependent manner: 3%, 17%, and 25% reduction for apoA-I:CIGB-258 molar ratios of 1:0.1, 1:0.5, and 1:1, respectively.With the largest increase in glycation extent by the CML treatment (final 200 mM), the WMF of the glycated apoA-I (349.5 nm) showed a 5.5 nm red shift at 72 h incubation compared to the baseline WMF at 0 h (344.0 nm), as shown in Figure 5B.Through glycation, the intrinsic Trp of apoA-I was more exposed to the hydrophilic phase, due to unfolding of the α-helix domain and an unstable tertiary structure formed.Nevertheless, the co-presence of CIGB-258 attenuated the increase in WMF, in a dose-dependent manner, after 72 h incubation: 346.9 nm, 345.8 nm, and 343.5 nm for apoA-I:CIGB-258 molar ratios of 1:0.1, 1:0.5, and 1:1, respectively.The results imply that the co-presence of CIGB-258 inhibited CML-mediated glycation to stabilize the α-helix domain and tertiary structure with the maintenance of Trp toward the hydrophobic phase.Regression analysis showed that the mid-points of denaturation were increased up to 2.35 M, 2.57 M, and 2.56 M of urea for apoA-I:CIGB-258 ratios of 1:0.1, 1:0.5, and 1:1, respectively, suggesting less exposure of intrinsic Trp via more stabilization of the α-helix domains of apoA-I.These results indicate that the secondary and tertiary configuration of apoA-I could be improved by the co-presence of CIGB-258 via the putative helix-helix interactions.The stabilization of apoA-I (Figure 4A) was linked with more protection of the apoA-I band intensity, with a concomitant increase in CIGB-258 (Figure 4B).

Anti-Glycation Activity of CIGB-258 against CML-Induced apoA-I Glycation
The CML exposure of lipid-free apoA-I caused the largest increase in yellowish fluorescence intensity (FI), 8.1-fold higher FI than apoA-I alone during 72 h, as shown in Fig- ure 5A.Conversely, the co-treatment of CIGB-258 caused a smaller increase in FI in a dosedependent manner: 3%, 17%, and 25% reduction for apoA-I:CIGB-258 molar ratios of 1:0.1, 1:0.5, and 1:1, respectively.With the largest increase in glycation extent by the CML treatment (final 200 mM), the WMF of the glycated apoA-I (349.5 nm) showed a 5.5 nm red shift at 72 h incubation compared to the baseline WMF at 0 h (344.0 nm), as shown in Figure 5B.Through glycation, the intrinsic Trp of apoA-I was more exposed to the hydrophilic phase, due to unfolding of the α-helix domain and an unstable tertiary structure formed.Nevertheless, the co-presence of CIGB-258 attenuated the increase in WMF, in a dose-dependent manner, after 72 h incubation: 346.9 nm, 345.8 nm, and 343.5 nm for apoA-I:CIGB-258 molar ratios of 1:0.1, 1:0.5, and 1:1, respectively.The results imply that the co-presence of CIGB-258 inhibited CML-mediated glycation to stabilize the α-helix domain and tertiary structure with the maintenance of Trp toward the hydrophobic phase.
(Em) of 370 nm and 440 nm, respectively.The measurement was conducted over a 72 h incubation period in the presence of 200 μM CML.(B) The degree of tryptophan (Trp) exposure in apoA-I was evaluated over the 72 h glycation process.Changes in the wavelength of maximum fluorescence (WMF) in apoA-I were observed across the molar ratios of apoA-I:CIGB-258 during CML-mediated glycation.As the extent of glycation and incubation time increased, alterations in Trp exposure were compared using WMF (Ex = 295 nm, Em range = 305-400 nm).Statistical significance is denoted by *, ** and *** at p < 0.05, p < 0.01 and p < 0.001, compared to the apoA-I + CML group.

Zebrafish Embryo Protection against CML-Toxicity
A microinjection of CML (500 ng) into zebrafish embryos led to acute death and the lowest survivability (21 ± 2% survivability) at 24 h post-injection (Figure 6).In contrast, the PBS-injected embryo showed the utmost survivability of 89 ± 3% (Figure 6).In the presence of CML, however, a co-injection of apoA-I (1.4 ng) alone or CIGB-258 alone (143 pg) resulted in a higher survivability of around 37 ± 5% and 33 ± 1%, respectively, suggesting that either apoA-I or CIGB-258 possessed adequate anti-inflammatory activity to neutralize the CML toxicity.Furthermore, in the presence of CML, co-injection of an apoA-I:CIGB-258 mixture yielded significantly higher embryo survivability, in a dose-dependent manner, of CIGB-258: 47 ± 3% (p < 0.01), 78 ± 2% (p < 0.001), and 82 ± 2% (p < 0.001) for apoA-I:CIGB-258 molar ratios of 1:0.1, 1:0.5, and 1:1, respectively.These findings indicate that the presence of CIGB-258 significantly improved the anti-inflammatory capabilities of apoA-I, effectively reducing acute embryo mortality through a potential synergistic effect of structural stabilization.The stereo image of the embryos showed that the PBS-alone group revealed a normal developmental speed and morphology at 5 h, 24 h, and 48 h (Figure 7A, photograph a).All the embryos in the PBS-alone group displayed the primordium-6 stage with the darkest eye pigmentation and tail elongation, along with the highest hatching (~78%) and somite counts (~34.6 ± 0.3) at 48 h post-treatment (Figure 7A,B).Conversely, the CML+PBSinjected embryo showed the most severe embryonic defects, with the least embryo hatching (~4%) (photograph b).The co-injection of apoA-I alone improved the CML-altered survivability (photograph c) and substantially improved the hatching (~18%) and somite The stereo image of the embryos showed that the PBS-alone group revealed a normal developmental speed and morphology at 5 h, 24 h, and 48 h (Figure 7A, photograph a).All the embryos in the PBS-alone group displayed the primordium-6 stage with the darkest eye pigmentation and tail elongation, along with the highest hatching (~78%) and somite counts (~34.6 ± 0.3) at 48 h post-treatment (Figure 7A,B).Conversely, the CML+PBS-injected embryo showed the most severe embryonic defects, with the least embryo hatching (~4%) (photograph b).The co-injection of apoA-I alone improved the CML-altered survivability (photograph c) and substantially improved the hatching (~18%) and somite counts (~22 ± 0.3) (Figure 7A,B).Likewise, similar results were observed for the only CGB-258-alone (0:1) injected group (photograph g).Interestingly, the 1:0.1 and 1:0.5 mixture of the apoA-I and CIGB-258 groups showed a much faster developmental speed (photograph d and e) than the apoA-I (1:0) and CIGB-258-alone (0:1) groups, with substantial high hatching and somite counts (Figure 7A,B).On the other hand, co-injection of a 1:1 mixture of (apoA-I and CIGB-258) resulted in the most improved developmental speed and morphology (photograph f) altered by CML, and all embryos showed the primordium-6 stage with the darkest eye pigmentation and tail elongation.As compared to the CML-injected group, the 1:1 mixture of (apoA-I and CIGB-258) resulted in 18-fold higher embryo hatching (~61%) and 32-fold higher somite counts (~32 ± 0.3) (Figure 7A,B).These results suggest that the co-presence of CIGB-258 helped to protect the embryos from CML-mediated embryotoxicity, in a dose-dependent manner, of CIGB-258.
counts (~22 ± 0.3) (Figure 7A,B).Likewise, similar results were observed for the only CGB-258-alone (0:1) injected group (photograph g).Interestingly, the 1:0.1 and 1:0.5 mixture of the apoA-I and CIGB-258 groups showed a much faster developmental speed (photograph d and e) than the apoA-I (1:0) and CIGB-258-alone (0:1) groups, with substantial high hatching and somite counts (Figure 7A,B).On the other hand, co-injection of a 1:1 mixture of (apoA-I and CIGB-258) resulted in the most improved developmental speed and morphology (photograph f) altered by CML, and all embryos showed the primordium-6 stage with the darkest eye pigmentation and tail elongation.As compared to the CML-injected group, the 1:1 mixture of (apoA-I and CIGB-258) resulted in 18-fold higher embryo hatching (~61%) and 32-fold higher somite counts (~32 ± 0.3) (Figure 7A,B).These results suggest that the co-presence of CIGB-258 helped to protect the embryos from CMLmediated embryotoxicity, in a dose-dependent manner, of CIGB-258.AO staining to detect cellular apoptosis showed that the CML-alone group had a 5.3-fold larger increase in apoptosis than the PBS group, suggesting that the CML injection induced acute cell death (Figure 7B).On the other hand, the co-injection of a 1:1 mixture resulted in the least apoptosis (~79% less apoptosis than the CML-alone group), while the apoA-I-alone (1:0) group showed no significant reduction: ~15% lower than the CML+PBS group.Interestingly, the (1:0.1)-and(1:0.5)-mixturegroups showed a more significant decrease in apoptosis (~23% and 73% reduction, respectively) than the CML+PBS group.Hence, the cytoprotective effect of apoA-I was enhanced by the co-presence of CIGB-258 in a dose-dependent manner.
DHE staining to detect ROS showed that the CML+PBS injection caused a 3.3-fold increase in ROS production compared to PBS alone (Figure 7B).A co-injection of apoA-I alone (1:0) resulted in the weakest activity for reducing ROS generation (~12% lower than the CML+PBS group).A co-injection of the 1:1 mixture had the strongest activity in lowering ROS generation (~66% reduction of ROS).The co-injection of 1:0.1 or 1:0.5 mixtures also reduced ROS production (~31% and ~63% lower than the CML+PBS group, respectively).Overall, all mixtures induced adequate protective activity against the CML toxicity, in a dose-dependent manner, of CIGB-258.The 1:1 mixture exerted the most potent activity in recovering the highest survivability and fastest development.
Although the PBS-alone group (photograph a) exhibited an active swimming pattern (Supplementary Video S1), all zebrafish in the CML+PBS group (photograph b) could not swim and were lying down on the bottom of the tank with occasional quivering (Figure 8C) despite being still alive at 30 min post-injection.At 1 h post-injection, 25% of the fish could swim again, with 58% survivability in the CML+PBS group, but the swimming pattern involved wobbling, seizure, and uncontrollable vertical movements (Supplementary Video S2).In contrast, the CML+apoA-I group showed an enhanced recovery of swimming ability of ~45 ± 3% and survivability of ~70% at 1 h post-injection, displaying a more improved swimming pattern, albeit with wobbling and seizure still detected (Supplementary Video S3).Similarly, the CML+CIGB-258 group showed an improved recovery of their swimming ability (~46 ± 10%) at 1 h post-injection with 80 ± 4% survivability (Supplementary Video S4).On the other hand, the CML+apoA-I+CIGB-258 group showed the fastest recovery of swimming ability of ~70 ± 6% and highest survivability of ~92 ± 3% at 1 h post-injection with the most active and natural swimming pattern (Supplementary Video S5).At 3 h post-injection, the CML+apoA-I+CIGB-258 group showed the highest survivability of ~85 ± 3%, while the CML+apoA-I and CML+CIGB-258 groups showed 65 ± 6% and 70 ± 5% survivability, respectively.These results suggest that a combination of apoA-I and CIGB-258 had a synergistic effect on improving swimming ability and survivability against the acute toxicity of CML.The CML+PBS group showed the lowest survivability (~58% and ~47% at 1 h and 3 h post-injection, respectively), indicating severe lethal toxicity of CML (Figure 8B).On the other hand, the CML+apoA-I and CML+CIGB-258 groups showed comparable survivability (~70-80% and 65-70% at 1 h and 3 h post-injection, respectively), suggesting that apoA-I and CIGB-258 had adequate anti-inflammatory activity, to a similar extent.On the other

Histologic Examination of Hepatic Tissue
The H&E analysis of the liver section as documented in Figure 9, displayed the hepatological changes in the different groups.A massive neutrophil infiltration was observed in the PBS+CML group, which was, significantly, 15.2-fold (p < 0.001) higher than the neutrophil counts observed in the PBS-injected control group (Figure 9B).The CML-induced neutrophil infiltration was effectively countered by the injection of apoA-I, CIGB-258 and apoA-I+CIGB-258, evident in a significant 2.8-fold, 2.7-fold and 8.8-fold reduced neutrophil count in the respective groups, as compared to the CML-injected group.However, the most promising effect was exerted by apoA-I+CIGB-258, as evident in significantly ~3-fold (p < 0.05) reduced neutrophil counts in the hepatic section of the apoA-I+CIGB-258-treated group compared to the apoA-I-and CIGB-258-treated groups, testifying enhanced protective activity against CML-induced hepatotoxicity when using the combination of apoA-I+CIGB-258.

Extent of ROS Production and Apoptosis in Hepatic Tissue
AO staining revealed the PBS-alone group to have the weakest green fluorescence (photo a1, Figure 10A), ~8 ± 2%, while the CML+PBS group had the highest green fluorescence area (photo a2), of ~41 ± 2% (Figure 10B), showing the greatest extent of cellular

Extent of ROS Production and Apoptosis in Hepatic Tissue
AO staining revealed the PBS-alone group to have the weakest green fluorescence (photo a1, Figure 10A), ~8 ± 2%, while the CML+PBS group had the highest green fluorescence area (photo a2), of ~41 ± 2% (Figure 10B), showing the greatest extent of cellular apoptosis, indicating that an IP injection of CML causes acute apoptosis.In contrast, the CML+apoA-I group showed a 37%-lower AO-stained area (p < 0.001) than the CML+PBS group, while the CML+CIGB-258 group exhibited a 53%-lower AO-stained area (p < 0.001).Interestingly, the CML+apoA-I+CIGB-258 group showed the smallest AO-stained area (~13 ± 1%), which was 67% lower than the CML+PBS group (Figure 10A,B).These results suggest that a co-injection of both apoA-I and CIGB-258 was more effective in preventing cellular apoptosis, even though injection of either apoA-I or CIGB-258 was also effective in preventing apoptosis in the presence of CML.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 14 of 24 apoptosis, indicating that an IP injection of CML causes acute apoptosis.In contrast, the CML+apoA-I group showed a 37%-lower AO-stained area (p < 0.001) than the CML+PBS group, while the CML+CIGB-258 group exhibited a 53%-lower AO-stained area (p < 0.001).Interestingly, the CML+apoA-I+CIGB-258 group showed the smallest AO-stained area (~13 ± 1%), which was 67% lower than the CML+PBS group (Figure 10A,B).These results suggest that a co-injection of both apoA-I and CIGB-258 was more effective in preventing cellular apoptosis, even though injection of either apoA-I or CIGB-258 was also effective in preventing apoptosis in the presence of CML.DHE staining also revealed the PBS-alone group to have the weakest red fluorescence of ~9 ± 1% (photo a2), while the CML+PBS group had the highest red fluorescence intensity of ~40 ± 2% (photo b2), representing the highest ROS production (Figure 10).By contrast, the CML+apoA-I group (photo c2) showed a 37%-lower DHE-stained area (p < 0.001) than the CML+PBS group, while the CML+CIGB-258 group (photo d2) exhibited a 47%-lower AO stained area (p < 0.001).Interestingly, the CML+apoA-I+CIGB-258 group (photo e2) showed the smallest AO-stained area (~12 ± 1%), which was 70% lower than the CML+PBS group.These results suggest that a co-injection of apoA-I and CIGB-258 was more effective in preventing ROS production, even though an injection of either apoA-I or CIGB-258 was DHE staining also revealed the PBS-alone group to have the weakest red fluorescence of ~9 ± 1% (photo a2), while the CML+PBS group had the highest red fluorescence intensity of ~40 ± 2% (photo b2), representing the highest ROS production (Figure 10).By contrast, the CML+apoA-I group (photo c2) showed a 37%-lower DHE-stained area (p < 0.001) than the CML+PBS group, while the CML+CIGB-258 group (photo d2) exhibited a 47%-lower AO stained area (p < 0.001).Interestingly, the CML+apoA-I+CIGB-258 group (photo e2) showed the smallest AO-stained area (~12 ± 1%), which was 70% lower than the CML+PBS group.These results suggest that a co-injection of apoA-I and CIGB-258 was more effective in preventing ROS production, even though an injection of either apoA-I or CIGB-258 was also effective in preventing oxidative stress in the presence of CML.Overall, a combination of apoA-I and CIGB-258 induced remarkable protection of hepatic tissue from cellular apoptosis and ROS production.

Immunohistochemistry for IL-6 Detection in Hepatic Tissue
The immunohistochemical detection of interleukin (IL)-6 in the hepatic tissue revealed the PBS-alone group to have the smallest stained area (photo a1) and red conversion area (photo a2) of ~3.7% (Figure 11A), while the CML+PBS group exhibited the largest stained area (photo b1) and red conversion area (photo b2) of ~22.9% (Figure 11B).On the other hand, the CML+apoA-I group showed a 15.2% IL-6-stained area, which was a ~33% further reduction than the CML+PBS group (Figure 11B), suggesting that co-injection of apoA-I (8.5 µg/zebrafish, final 1 µM) could alleviate the inflammatory response to lower the IL-6 level.Interestingly, the CML+CIGB-258 group showed a more remarkable decrease in IL-6 stained area (photo d1 and d2), a ~7.6% stained area, which was 67% smaller than the CML+PBS group, suggesting that an injection of CIGB-258 (0.9 µg, final 1 µM) was two times more effective in reducing the hepatic IL-6 level of CML-mediated inflammation than apoA-I (final 1 µM).Moreover, the CML+apoA-I+CIGB-258 group showed the smallest IL-6-stained area (photo e1 and e2), ~6.3%, which was 73% lower (p < 0.001) than that of the CML+PBS group, indicating the strongest synergistic anti-inflammatory activity through the co-presence of and CIGB-258.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 15 of 24 also effective in preventing oxidative in the presence of CML.Overall, a combination of apoA-I and CIGB-258 induced remarkable protection of hepatic tissue from cellular apoptosis and ROS production.

Immunohistochemistry for IL-6 Detection in Hepatic Tissue
The immunohistochemical detection of interleukin (IL)-6 in the hepatic tissue revealed the PBS-alone group to have the smallest stained area (photo a1) and red conversion area (photo a2) of ~3.7% (Figure 11A), while the CML+PBS group exhibited the largest stained area (photo b1) and red conversion area (photo b2) of ~22.9% (Figure 11B).On the other hand, the CML+apoA-I group showed a 15.2% IL-6-stained area, which was a ~33% further reduction than the CML+PBS group (Figure 11B), suggesting that co-injection of apoA-I (8.5 μg/zebrafish, final 1 μM) could alleviate the inflammatory response to lower the IL-6 level.Interestingly, the CML+CIGB-258 group showed a more remarkable decrease in IL-6 stained area (photo d1 and d2), a ~7.6% stained area, which was 67% smaller than the CML+PBS group, suggesting that an injection of CIGB-258 (0.9 μg, final 1 μM) was two times more effective in reducing the hepatic IL-6 level of CML-mediated inflammation than apoA-I (final 1 μM).Moreover, the CML+apoA-I+CIGB-258 group showed the smallest IL-6-stained area (photo e1 and e2), ~6.3%, which was 73% lower (p < 0.001) than that of the CML+PBS group, indicating the strongest synergistic anti-inflammatory activity through the co-presence of apoA-I and CIGB-258.

Change in the Serum Lipid Profile
After collecting plasma from each zebrafish group, plasma lipid quantification showed that the CML+ PBS group had the highest total cholesterol (TC) and triglyceride (TG) plasma levels, as shown in Figure 12A,B.In particular, the serum TC and TG levels in the CML+PBS group were approximately 1.5-fold and 2.5-fold higher than those of the PBS-alone group, respectively, suggesting an abrupt elevation in TC and TG by the blood infusion of CML.On the other hand, a co-injection of apoA-I or CIGB-258 resulted in a decrease in TC and TG levels to a similar extent: a 37-39% and 60-62% reduction in TC and TG, respectively.These results suggest that apoA-I alone or CIGB-258 alone could lower the plasma TC and TG with the concomitant suppression of the inflammatory cascade.Interestingly, a co-injection of apoA-I and CIGB-258 resulted in the lowest levels of TC and TG: 45% and 65% lower than those of the CML+PBS group, respectively, suggesting that a combination of apoA-I and CIGB-258 had higher lipid-lowering activity.

Change in the Serum Lipid Profile
After collecting plasma from each zebrafish group, plasma lipid quantification showed that the CML+ PBS group had the highest total cholesterol (TC) and triglyceride (TG) plasma levels, as shown in Figure 12A,B.In particular, the serum TC and TG levels in the CML+PBS group were approximately 1.5-fold and 2.5-fold higher than those of the PBS-alone group, respectively, suggesting an abrupt elevation in TC and TG by the blood infusion of CML.On the other hand, a co-injection of apoA-I or CIGB-258 resulted in a decrease in TC and TG levels to a similar extent: a 37-39% and 60-62% reduction in TC and TG, respectively.These results suggest that apoA-I alone or CIGB-258 alone could lower the plasma TC and TG with the concomitant suppression of the inflammatory cascade.Interestingly, a co-injection of apoA-I and CIGB-258 resulted in the lowest levels of TC and TG: 45% and 65% lower than those of the CML+PBS group, respectively, suggesting that a combination of apoA-I and CIGB-258 had higher lipid-lowering activity.Quantification of HDL-C in plasma showed that the combined apoA-I and CIGB-258 group showed the highest level of HDL-C (~123 mg/dL), while the apoA-I-alone group and CIGB-258-alone group exhibited a lower level (~69-72 mg/dL).On the other hand, the percentages of HDL-C in TC (HDL-C/TC (%)) were higher in the apoA-I-alone and CIGB-258-alone group, with 27-29% of HDL-C/TC (%), which were higher than that of the CML+PBS group.Surprisingly, a co-injection of the apoA-I and CIGB-258 group showed the highest HDL-C/TC (%), ~54%, suggesting a synergistic effect in increasing the HDL-C (mg/dL) and HDL-C/TC (%).

Change in the Serum AST and ALT
The CML+PBS group showed the highest AST and ALT levels (515 IU/L and 252 IU/L, respectively), which were 4.0-fold and 2.0-fold higher than those of the PBS-alone group, respectively (Figure 13).On the other hand, the CML+apoA-I group showed lower AST and ALT levels (442 IU/L and 197 IU/L, respectively), which were 15% and 22% lower than CML+PBS group, respectively.On the other hand, the CML+CIGB-258 group showed AST and ALT levels of 353 and 175 IU/L, respectively, which were 32% and 31% lower than those of the CML+PBS group, respectively.Interestingly, the CML+apoA-I+CIGB-258 group showed the lowest AST and ALT levels (253 IU/L and 147 IU/L, respectively), which were 51% and 42% lower than those of the CML+PBS group, respectively.Hence, a co-injection of apoA-I and CIGB-258 synergistically ameliorated the hepatic damage, particularly lowering the AST and ALT levels caused by CML toxicity.

Change in the Serum AST and ALT
The CML+PBS group showed the highest AST and ALT levels (515 IU/L and 252 IU/L, respectively), which were 4.0-fold and 2.0-fold higher than those of the PBS-alone group, respectively (Figure 13).On the other hand, the CML+apoA-I group showed lower AST and ALT levels (442 IU/L and 197 IU/L, respectively), which were 15% and 22% lower than CML+PBS group, respectively.On the other hand, the CML+CIGB-258 group showed AST and ALT levels of 353 and 175 IU/L, respectively, which were 32% and 31% lower than those of the CML+PBS group, respectively.Interestingly, the CML+apoA-I+CIGB-258 group showed the lowest AST and ALT levels (253 IU/L and 147 IU/L, respectively), which were 51% and 42% lower than those of the CML+PBS group, respectively.Hence, a coinjection of apoA-I and CIGB-258 synergistically ameliorated the hepatic damage, particularly lowering the AST and ALT levels caused by CML toxicity.

Discussion
CIGB-258 exhibits anti-inflammatory activity against acute toxicity of CML within a normal diet [16] and a high-cholesterol diet (HCD) [17].In normolipidemic zebrafish, the CIGB-258 group showed higher recovery of swimming ability and survivability than the Infliximab (Remsima ® ) and Tocilizumab (Actemra ® ) groups, with the least hepatic inflammation [16].In hyperlipidemic zebrafish, a co-injection of CIGB-258 resulted in a 2.2-fold faster recovery of swimming ability than the CML alone with the lowest IL-6 level in hepatic tissue compared to the Infliximab, Etanercept (Enbrel ® ), and Tocilizumab groups [17].These results suggest that CIGB-258 has similar efficacy to the IL-6 inhibitor rather than the TNF-α inhibitor regarding the lowest IL-6 level, with improvements in survivability and lipid profiles.These results also show good agreement with a previous report that HDL and apoA-I suppress IL-6 production via Toll-like receptor-4 signaling [21,22].

Discussion
CIGB-258 exhibits anti-inflammatory activity against acute toxicity of CML within a normal diet [16] and a high-cholesterol diet (HCD) [17].In normolipidemic zebrafish, the CIGB-258 group showed higher recovery of swimming ability and survivability than the Infliximab (Remsima ® ) and Tocilizumab (Actemra ® ) groups, with the least hepatic inflammation [16].In hyperlipidemic zebrafish, a co-injection of CIGB-258 resulted in a 2.2-fold faster recovery of swimming ability than the CML alone with the lowest IL-6 level in hepatic tissue compared to the Infliximab, Etanercept (Enbrel ® ), and Tocilizumab groups [17].These results suggest that CIGB-258 has similar efficacy to the IL-6 inhibitor rather than the TNF-α inhibitor regarding the lowest IL-6 level, with improvements in survivability and lipid profiles.These results also show good agreement with a previous report that HDL and apoA-I suppress IL-6 production via Toll-like receptor-4 signaling [21,22].In addition, in the presence of CML in zebrafish embryos, the co-addition of lipid-free apoA-I and CIGB-258 resulted in the highest hatching ratio and somite numbers with increasing CIGB-258 content (Figure 7B).
In addition to HDL, beyond cholesterol trafficking in blood, lipid-free apoA-I is involved in the multi-functional innate immune response and regulation of antiviral activity [23] and anti-inflammatory activity, and has a tumor-suppressive role [14,24].ApoA-I exerts antiviral activity to inhibit herpes simplex virus-induced cell fusion and prevent viral penetration [23].Furthermore, apoA-I also displays potent bactericidal activity [25], facilitation of complement-mediating bacterial killing, and protection against the invasion of protozoal parasites, such as trypanosome brucei [26].The maintenance of higher antioxidant ability and apoA-I content in HDL was reported to be critical for maximizing and preserving the broad spectrum of anti-infection activity [27].
In the current study, CIGB-258 helped apoA-I and HDL induce more antioxidant ability against LDL oxidation in a dose-dependent manner (Figures 1-3).During isothermal denaturation by adding urea, the co-presence of CIGB-258 helped stabilize the tertiary structure of apoA-I against proteolytic degradation and Trp exposure (Figure 4 and Table 1).CIGB-258 inhibited the CML-mediated glycation of apoA-I to enhance the structural stabilization via the blue shift in intrinsic Trp (Figure 5).In the presence of CML to induce the acute death of zebrafish embryos, a co-injection of an apoA-I:CIGB-258 mixture helped induce higher survivability and faster developmental speed with lower apoptosis and ROS production according to the CIGB-258 content (Figures 6 and 7).Regarding the acute inflammatory death of adult zebrafish by CML toxicity, a co-injection of apoA-I+CIGB-258 induced the highest survivability, lowest hepatic hyperinflammation, and lowest IL-6 levels (Figures 8-11).With the lowest ROS production and apoptosis extent in the liver, the co-injection of apoA-I+CIGB-258 resulted in the most desirable lipid profile with the highest HDL-C and least hepatic damage (Figures 12 and 13).These results suggest that the advantageous roles of apoA-I could be protected and enhanced by the co-presence of CIGB-258 via a putative interaction.
These synergistic interactions of apoA-I and CIGB-258 might help strengthen the antiviral activity.During infection and inflammation, a decrease in HDL-C and an increase in serum amyloid A, which can displace apoA-I from HDL, are major components of the acute-phase response.Although lipid-free apoA-I could bind with the dengue virus during attachment, apoA-I can neutralize nonstructural protein (NS)-1-induced cell activation and prevent NS-1-mediated dengue virus infection [28].Indeed, native HDL and apoA-I displayed potent virus-killing activity against SARS-CoV-2, while glycated HDL lost its antiviral activity [29].In addition, the paraoxonase activity was significantly impaired in the glycated HDL via the modification and loss of apoA-I, suggesting that the antioxidant activity was linked with the loss of antiviral activity.In the same context, the association of elevated apoA-I glycation and reduced HDL-associated PON activity in patients with T2DM has been reported [30].Interestingly, the PON-1 activity was positively correlated with the increase in HDL-C and apoA-I concentrations in the healthy control group but not in T2DM patients [30,31].These results showed that the inhibition of apoA-I glycation by CML might be a suitable pharmaceutical target to maximize the antioxidant and antiinfection activity by stabilizing the tertiary structure and functionality.Indeed, the PON-1 activities in human HDL 2 and HDL 3 were elevated by the co-addition of apoA-I:CIGB-258, in a dose-dependent manner, of CIGB-258 (Figure 3A), suggesting that the co-presence of CIGB-258 could enforce the antiviral activity of HDL, as reported elsewhere [32].
On the other hand, CIGB-258 was found to bind with apoA-I and transthyretin (TTR) in the serum, as identified by affinity chromatography and mass spectrometry [20,33].Interestingly, apoA-I is synthesized in the liver and intestine, whereas TTR, a transport protein for thyroxine (T4) and retinol, is synthesized in the liver and brain [34].Although the two proteins appear to have no relation with each other, TTR has several connections to the apoA-I metabolism: (1) serum TTR circulates in HDL through binding to apoA-Il; (2) TTR can cleave the C-terminus of apoA-I; and (3) the cleaved apoA-I by TTR impaired cholesterol efflux and promoted amyloidogenesis [35].Therefore, the co-presence of CIGB-258 with the apoA-I and TTR in the serum might suggest putative binding for protecting apoA-I, by CIGB-258, from cleavage by a TTR attack.Indeed, the amphipathic helix domain of CIGB-258, 81.4% of the α-helix content in 22mer amino acid within the 5-26 residue, was similar to an amphipathic helix domain of apoA-I, 74.9% of the α-helix in the entire sequence.In more detail, eight helix domains consisted of 22mer amino acids in apoA-I, which are highly homologous with the α-helix of CIGB-258.Future research should be carried out to determine which helix domain of apoA-I could bind to CIGB-258 to understand the physiological meaning of the co-presence of CIGB-258 between apoA-I and TTR.Furthermore, the next in vivo studies should be conducted separately on male and female zebrafish to determine the sex-based response of apoA-I and CIGB-258.

Materials
Jusvinza ® (CIGB-258) is a lyophilized powder formulation containing the recombinant peptide derived from HSP60, consisting of 27 amino acids (Lot# 1125J1/0; 1.25 mg/vial).The peptide was obtained from the Center for Genetic Engineering and Biotechnology (CIGB) in Havana, Cuba, for exclusive research use.Unless otherwise noted, all other chemicals and reagents were of analytical grade and used as supplied.

Isolation of Lipoprotrins from the Blood
The density gradient ultracentrifugation technique was used to isolate lipoprotein (LDL and HDL) from human blood [36].First, the serum was collected from the blood and subsequently processed for density gradient centrifugation in a density gradient mixture of NaCl (1.019 < d < 1.063) from LDL and (1.063 < d < 1.225) for HDL.The isolated LDL and HDL were processed for overnight dialysis using Tris-buffered saline (pH 8.0) and stored at −21 • C for further use.A detailed procedure is outlined in Supplementary Methods S1.

Purification of Human apoA-I
Apolipoprotein A-I (apoA-I) was extracted from the HDL using a previously described method [37], utilizing fast protein liquid chromatography with an AKTA purifier system (GE Healthcare, Uppsala, Sweden).The SDS-PAGE was performed to confirm the purity of the separated apoA-I.A detailed procedure is outlined in Supplementary Methods S1.

Effect of CIGB-258 on the Oxidation of LDL
The ability of apoA-I and the CIGB-258 mixture to prevent LDL oxidation was assessed using a thiobarbituric acid reactive substance (TBARS) assay [38] with malondialdehyde (MDA) standard and performing 0.5% agarose gel electrophoresis [39].In brief LDL (1 mg/mL) was treated with only CuSO 4 (10 µM) for 4 h or with the apoA-I:CIGB-258 mixture with molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1.In addition the effect of different concentrations of CIGB-258 in presence of 2 mg/mL of HDL 2 and HDL 3 was evaluated on the CuSO 4 (10 µM) mediated oxidation.A detailed procedure is outlined in Supplementary Methods S1.

Measurement of Trp Fluorescence of apoA-I during Isothermal Denaturation
The WMF of tryptophan (Trp) in apoA-I in the co-presence of CIGB-258 was determined using a previously described method [16,40].Briefly, to minimize tyrosine fluorescence interference, samples were excited at 295 nm, and emission spectra were registered from 305 to 400 nm.Isothermal denaturation experiments were performed to assess the impact of varying urea concentration (0 M to 7 M) on the secondary structures of apoA-I and CIGB-258, employing molar ratios of 1:0, 1:0.1, 1:0.5, and 1:1 in the lipid-free state.The exposure extent of Trp in apoA-I during denaturation in the presence of CIGB-258 was measured independently by fluorescence spectroscopy using the earlier described method [41].

Paraoxonase Assay
The HLD-associated paraoxonase (PON)-1 activity was determined by the hydrolysis of paraoxon into p-nitrophenol and diethylphosphate catalyzed by the ezyme, following the earlier described method [42].A detailed methodology is outlined in Supplementary Methods S1.

Figure 4 .
Figure 4. Isothermal denaturation of apoA-I and CIGB-258 in the lipid-free state by the urea treatment for 16 h incubation.(A) Change in the wavelength of maximum fluorescence (WMF) in apoA-I with different molar ratios of apoA-I:CIGB-258 during the urea treatment.The increase in urea concentration was used to assess the change in Trp exposure (excitation at 295 nm, emission spectra range 305-400 nm), presented as WMF.(B) Electrophoresis pattern of the apoA-I and CIGB-258 mixture at 0 M urea.Electrophoretic profiles of the apoA-I:CIGB-258 mixture were visualized by staining the protein bands with 0.125% Coomassie brilliant blue.The yellow numbers indicate the band intensity of each band.The red arrowhead indicates the band of CIGB-258 at the bottom of the gel.Lanes 1, 2, 3 and 4 represent apoA-I + CIGB-258 at the molar ratios of 1:0, 1:0.1, 1:0.5 and 1:1, respectively.Statistical significance *** denotes p < 0.001 between 1:0 and 1:1 of apoA-I:CIGB-258.

Figure 4 .
Figure 4. Isothermal denaturation of apoA-I and CIGB-258 in the lipid-free state by the urea treatment for 16 h incubation.(A) Change in the wavelength of maximum fluorescence (WMF) in apoA-I with different molar ratios of apoA-I:CIGB-258 during the urea treatment.The increase in urea concentration was used to assess the change in Trp exposure (excitation at 295 nm, emission spectra range 305-400 nm), presented as WMF.(B) Electrophoresis pattern of the apoA-I and CIGB-258 mixture at 0 M urea.Electrophoretic profiles of the apoA-I:CIGB-258 mixture were visualized by staining the protein bands with 0.125% Coomassie brilliant blue.The yellow numbers indicate the band intensity of each band.The red arrowhead indicates the band of CIGB-258 at the bottom of the gel.Lanes 1, 2, 3 and 4 represent apoA-I + CIGB-258 at the molar ratios of 1:0, 1:0.1, 1:0.5 and 1:1, respectively.Statistical significance *** denotes p < 0.001 between 1:0 and 1:1 of apoA-I:CIGB-258.

Figure 5 .
Figure 5.Effect of CIGB-258 in countering CML-mediated glycation of apolipoprotein (apoA-I).(A) The fluorescent intensity was assessed under excitation wavelength (Ex) and emission wavelength

Figure 5 .
Figure 5.Effect of CIGB-258 in countering CML-mediated glycation of apolipoprotein (apoA-I).(A) The fluorescent intensity was assessed under excitation wavelength (Ex) and emission wavelength (Em) of 370 nm and 440 nm, respectively.The measurement was conducted over a 72 h incubation period in the presence of 200 µM CML.(B) The degree of tryptophan (Trp) exposure in apoA-I was evaluated over the 72 h glycation process.Changes in the wavelength of maximum fluorescence (WMF) in apoA-I were observed across the molar ratios of apoA-I:CIGB-258 during CML-mediated glycation.As the extent of glycation and incubation time increased, alterations in Trp exposure were compared using WMF (Ex = 295 nm, Em range = 305-400 nm).Statistical significance is denoted by *, ** and *** at p < 0.05, p < 0.01 and p < 0.001, compared to the apoA-I + CML group.

Figure 11 .
Figure 11.Interleukin (IL)-6 production in the liver section of zebrafish injected with carboxymethyllysine (CML) and subsequently treated with different combinations of apolipoprotein A-I (apoA-I) and CIGB-258.(A) Images of immunohistochemistry (a1-e1).Red conversion images (a2-e2) are IHC-stained areas (brown color) interchanged with red color [at a threshold value of (20-100)] using Image J software to enhance visualization.(B) Quantification of IL-6-stained area employing Image

Figure 12 .
Figure 12.Comparison of plasma lipid profiles.(A) total cholesterol (TC), (B) triglyceride (TG), (C) high-density lipoprotein cholesterol (HDL-C), and (D) HDL-C/TC (%) in the serum of zebrafish injected with carboxymethyllysine (CML) alone or in conjunction with apoA-I, CIGB-258 and apoA-I+CIGB-258.Statistical significance is indicated by *, ** and *** at p < 0.05, p < 0.01 and p < 0.001, compared to the CML+PBS group; ns is a non-significant difference between the groups.Quantification of HDL-C in plasma showed that the combined apoA-I and CIGB-258 group showed the highest level of HDL-C (~123 mg/dL), while the apoA-I-alone group

Figure 13 .
Figure 13.Measurement of the zebrafish plasma hepatic enzyme levels (A) aspartate transaminase (AST) and (B) alanine transaminase (ALT) detected at 180 min post-injection of CML alone or in combination with apoA-I, CIGB-258 and apoA-I+CIGB-258.Statistical significance is denoted by *,** and *** at p < 0.05, p < 0.01 and p < 0.001, compared to the CML+PBS group; ns is a non-significant difference between the groups.AST refers to aspartate aminotransferase, ALT to alanine aminotransferase, and CML to carboxymethyl lysine.

Figure 13 .
Figure 13.Measurement of the zebrafish plasma hepatic enzyme levels (A) aspartate transaminase (AST) and (B) alanine transaminase (ALT) detected at 180 min post-injection of CML alone or in combination with apoA-I, CIGB-258 and apoA-I+CIGB-258.Statistical significance is denoted by *, ** and *** at p < 0.05, p < 0.01 and p < 0.001, compared to the CML+PBS group; ns is a nonsignificant difference between the groups.AST refers to aspartate aminotransferase, ALT to alanine aminotransferase, and CML to carboxymethyl lysine.

Table 1 .
Alternation in the median wavelength of maximum fluorescence (WMF) during urea-induced isothermal denaturation.

Table 1 .
Alternation in the median wavelength of maximum fluorescence (WMF) during urea-induced isothermal denaturation.