α-Lipoic Acid Protects against Cyclosporine A-Induced Hepatic Toxicity in Rats: Effect on Oxidative Stress, Inflammation, and Apoptosis

The clinical application of cyclosporine A (CsA) as an immunosuppressive agent is limited by its organ toxicity. We aimed to evaluate the effectiveness of α-lipoic acid against CsA-induced hepatotoxicity and to delineate the underlying molecular mechanisms. Male Wistar rats (n = 24, 8 per each group) received the vehicle, CsA (25 mg/kg) and/or ALA (100 mg/kg, p.o.) for 3 weeks. Biochemical markers of liver function (serum ALT, AST, ALP < GGT), oxidative stress (MDA, TAC, SOD, GSH, Nrf2/HO-1), inflammation (NF-κB, CD68, iNOS, NO, COX-2), and apoptosis (caspase-3) were assessed in serum and tissue. Liver histological analysis using H&E and Sirius red was performed. The development of liver injury in CsA-treated animals was indicated by elevated levels of liver enzymes, oxidants/antioxidants imbalance, inflammatory cells infiltration, up-regulated expression of inflammatory mediators, and apoptosis. These changes were associated with altered architecture of hepatic cells and fibrous connective tissue. ALA co-administration protected against CsA-induced liver damage and ameliorated biochemical changes and cellular injury. In conclusion, ALA demonstrated hepatoprotective potential against CsA-induced liver injury through combating oxidative stress, inflammation, and apoptosis, highlighting ALA as a valuable adjunct to CsA therapy.


Introduction
The increased rate of organ transplantation and incidence of autoimmune diseases worldwide have resulted in wide application of immunosuppressive agents for better clinical outcomes [1]. Cyclosporine A (CsA) is an immunosuppressant peptide widely used

Sampling
At the end of the experimental protocol duration, after 12 h overnight fasting, a blood sample was withdrawn under completely aseptic conditions from the retro-orbital venous plexus using a disposable plastic syringe. The samples were collected into plastic containers and sera were collected after centrifugation at 15,000 rpm for 20 min, then divided into aliquots and stored at −80 • C until used for biochemical investigations: liver function tests (ALT, AST, ALP, and GGT) and oxidative stress markers (MDA, TAC, and NO).
All rats from each group were sacrificed under diethyl ether anesthesia, and the liver was removed for molecular studies. The liquid nitrogen frozen liver tissue samples (30-50 mg in weight) were used for total RNA extraction and real-time qRT-PCR analysis of expression of SOD, Nrf-2, TNF-α, NF-KB and Caspase-3.
Oxidative stress markers were measured in the sera and tissues of all rats. Estimation of the lipid peroxidation product, Malondialdehyde (MDA), was done based on the thiobarbituric acid reaction [18], and the results were expressed as µmol MDA/L. The total antioxidant capacity (TAC) was estimated by the commercially available colorimetric kits (Cat.No. # TA 25 12), which were supplied by Bio-Diagnostics, Dokki, Giza, Egypt and used according to the method previously reported by Koracevic et al. [19]. The results were expressed as mmol/L. The serum nitric oxide (NO) was measured by the colorimetric method (Nitric Oxide Assay Kit, Abcam co. ab272517), and the results were expressed as µmol/L. Additionally, activity of superoxide dismutase (SOD) and catalase (CAT) enzymes and the level of reduced glutathione (GSH) were evaluated in hepatic tissues by colorimetric methods using commercially available kits (Bio-Diagnostics, Dokki, Giza, Egypt).

Histopathological Examination
Two different sections of the liver from each animal in the control and treatment groups were collected (16 sections/group), fixed in 10% neutral buffered formalin, and then the specimens were dehydrated in an ascending series of ethyl alcohol, cleared in xylene, and embedded in paraffin wax. The blocks were prepared to contain single section/block (2 blocks/animal, making total 16 blocks/group). Tissue blocks were sectioned into approximately 5 µm thick slices using a rotary microtome. Sections were routinely stained using hematoxylin and eosin (H&E) for histological studies. Evaluation of liver fibrosis was conducted using Sirius red-stained liver sections. Slides were incubated overnight with 0.1% Sirius red (Sigma-Aldrich, UK), treated with 0.01 M hydrochloric acid, and followed by dehydration in serial ethanol concentrations without water. Slides stained with H&E and Sirius red were inspected and captured using ×10 and ×40 objective lens with a camera-aided light microscope. An Olympus®CX41 light microscope was used for examining the sections, which were photographed by its digital camera, Olympus ® SC100.
Image J software was used to analyze the area percentage of fibrosis in Sirius redstained sections [25]. Calibrations were performed with Image J using a straight-line tool at the appropriate calibration (pixel to µm ratio). Measurements were done in the pictures captured by the objective lens of magnification 10X to create images that cover the entire area to be examined. Briefly, to isolate red-stained collagen, we changed the image type to RGB Stack, which yields the gray-scale images of the channels (Image → Type → RGB Stack). In the Green channel, the threshold was set at 0-87 (Image → Adjust → Threshold). We recorded the area, area fraction, limit to threshold, and display label (Analyze → Measure). Two different sections from each animal were analyzed, and from each section, (6-10) different non-overlapping fields were examined (12-20 fields/animal) [26].

Immunohistochemical Studies
For the immunohistochemical study, paraffin sections of 5 µm thickness were prepared and the staining was performed using the labeled Streptavidin-Biotin immunoperoxidase technique according to the manufacturer's instructions. For IHC quantitative assessment, the Allred score was used. It provides a scale of 0-8 representing the Allred index (0-1 = negative, 2-3 = mild, 4-6 = moderate, and 7-8 = strongly positive). Allred is obtained by the sum of staining intensity grading (0-3) and the positive cell proportion grading (0-5) [27] and is quantified using the QuPath program (0.1.2) [28].

Statistical Analysis
Graphpad prism 8 software was used for statistical analyses and the graphical presentation of data. Quantitative data were initially tested for normality using Shapiro-Wilk's test, with data being normally distributed if p > 0.050. Data are expressed as the mean ± Standard error (SE). Bartlett's test was used for examining the homogeneity of variances. One-way analysis of variance (ANOVA) was used to compare the quantitative data between the studied groups. Tukey post hoc assessment was used for multiple comparisons when homoscedasticity was met, whereas Games-Howell adjustment was used when homoscedasticity was not met. Significance was considered at p values less than 0.05.

Effect of α-LA on CsA-Induced Changes in Liver Function
As shown in Table 1, rats treated with α-LA demonstrated no changes in liver function indices, indicating the safety of α-LA on normal rats at the selected dose. However, the CsAtreated group showed substantial alterations in liver function indices (ALT, AST, GGT, and ALP) compared to the normal control group. In contrast, administration of α-LA normalized these biochemical indicators of liver function compared to the CsA-treated group.

Effect of α-LA on CsA-Induced Hepatic Cellular Injury and Fibrosis
H&E staining was used to investigate if α-LA has a protective impact on liver tissue damage induced by CsA. As indicted in Figure 1A-C, hepatic sections from the normal group demonstrated normal arrangement and morphology of the liver cells. The liver cells of the CsA-treated group showed cirrhotic nodules, fibrous connective tissue, inflammatory cells, dilated lymphatics, and proliferated biliary epithelium, Figure 1D,E. However, milder lesions were observed in hepatic sections from αLA + CsA characterized by thin fibrous connective tissue deposition with fewer inflammatory cells, Figure 1F,G.
The Sirius red stain was used to observe fibrosis in the different experimental groups. As shown in Figure 2A-C, no fibrous tissue deposition was observed in the normal control group. However, hepatic sections from the CsA-treated group showed excessive connective tissue deposition, Figure 2D,E. On the other hand, administration of α-LA resulted in mild fibrosis, as indicated by thin red-stained fibrous connective tissue deposition in hepatic sections from the CsA + α-LA group, Figure 2F,G.

Effect of α-LA on CsA-Induced Oxidative Stress in Rat Serum and Liver Tissues
Tables 2-4 and Figure 3 demonstrate the results of oxidative stress markers in serum and hepatic tissues, respectively from various experimental groups. The CsA-treated group showed a marked increase in the serum and tissue oxidative stress marker MDA, accompanied by a marked reduction in TAC, hepatic tissue mRNA expression of Nrf2 and SOD, SOD and CAT activity, GSH content, and immunostaining of HO-1 when compared to the control group. Treatment with α-LA dampened serum and tissue MDA and restored serum TAC, mRNA expression of Nrf2 and SOD, tissue activity of SOD and CAT, GSH content, and immunostaining of HO-1 in hepatic tissue when compared to the CsA-treated group.  The Sirius red stain was used to observe fibrosis in the different experimental groups. As shown in Figure 2A-C, no fibrous tissue deposition was observed in the normal control group. However, hepatic sections from the CsA-treated group showed excessive connective tissue deposition, Figure 2D,E. On the other hand, administration of α-LA resulted in mild fibrosis, as indicated by thin red-stained fibrous connective tissue deposition in hepatic sections from the CsA + α-LA group, Figure 2F,G.

Effect of α-LA on CsA-Induced Oxidative Stress in Rat Serum and Liver Tissues
Tables 2-4 and Figure 3 demonstrate the results of oxidative stress markers in serum and hepatic tissues, respectively from various experimental groups. The CsA-treated group showed a marked increase in the serum and tissue oxidative stress marker MDA, accompanied by a marked reduction in TAC, hepatic tissue mRNA expression of Nrf2 and SOD, SOD and CAT activity, GSH content, and immunostaining of HO-1 when compared to the control group. Treatment with α-LA dampened serum and tissue MDA and restored serum TAC, mRNA expression of Nrf2 and SOD, tissue activity of SOD and CAT, GSH content, and immunostaining of HO-1 in hepatic tissue when compared to the CsAtreated group.

Effect of α-LA on CsA-Induced Inflammation in Rat Liver Tissues
As shown in Table 5 and Figures 4-6, CsA treatment is associated with hepatic inflammation, as indicated by a significant increase in the mRNA expression of TNF-α and NF-κB. This was accompanied by a marked increase in CD68, COX-2 and iNOS immunostaining in hepatic tissue and a decrease in serum NO in CsA-treated rats. On the other hand, administration of α-LA along with CsA ameliorated hepatic inflammation, as indicated by a significant downregulation of the mRNA expression of TNF-α and NF-κB, accompanied by marked increase in CD68, COX-2, and iNOS immunostaining in hepatic tissue and restoration of serum NO. Data are expressed as mean ± SE, * the CsA-treated group versus the control group at p < 0.05, # α-LA + the CsA-treated group versus the CsA-treated group at p < 0.05.
in CsA-treated animals.

Effect of α-LA on CsA-Induced Inflammation in Rat Liver Tissues
As shown in Table 5 and Figures 4-6, CsA treatment is associated with hepatic inflammation, as indicated by a significant increase in the mRNA expression of TNF-α and NF-κB. This was accompanied by a marked increase in CD68, COX-2 and iNOS immunostaining in hepatic tissue and a decrease in serum NO in CsA-treated rats. On the other hand, administration of α-LA along with CsA ameliorated hepatic inflammation, as indicated by a significant downregulation of the mRNA expression of TNF-α and NF-κB, accompanied by marked increase in CD68, COX-2, and iNOS immunostaining in hepatic tissue and restoration of serum NO.   Data are expressed as mean ± SE, * the CsA-treated group versus the control group at p ˂ 0.05, # α-LA + the CsA-treated group versus the CsA-treated group at p ˂ 0.05.

Effect of α-LA on CsA-Induced Apoptosis in Rat Liver Tissues
CsA administration induced apoptosis in rats' hepatic tissue as indicated by a significant increase in caspase-3 mRNA expression and immunostaining in hepatic tissue compared to the control group. In contrast, administration of α-LA to CsA-treated rats significantly down-regulated caspase-3 mRNA expression and immunostaining in hepatic tissues, Figures 7 and 8.

Effect of α-LA on CsA-Induced Apoptosis in Rat Liver Tissues
CsA administration induced apoptosis in rats' hepatic tissue as indicated by a significant increase in caspase-3 mRNA expression and immunostaining in hepatic tissue compared to the control group. In contrast, administration of α-LA to CsA-treated rats significantly down-regulated caspase-3 mRNA expression and immunostaining in hepatic tissues, Figures 7 and 8.

Discussion
The present study demonstrated protective efficacy of α-LA in CsA-induced liver injury. α-LA administration decreased oxidative stress and restored antioxidant element levels in serum and liver tissues, downregulated inflammatory cell recruitment and inflammatory markers, and suppressed apoptosis.
Clinical and experimental studies demonstrated that CsA treatment is associated with functional and morphological changes [1,29,30]. CsA is a prototypical cholestasisproducing agent. It affects hepatocytes, the mitochondrial function canalicular system, causing liver injury [1]. Hyperbilirubinemia, elevated serum transaminases, ALP, and GGT characterize functional alterations, while mononuclear cell infiltrations, congestion, and hepatocytes' degenerative changes with nodular cirrhosis identify morphological changes [31]. Consistently, the results of the present study reported impaired liver function in parallel with microscopic lesions in the CsA-treated group.
Oxidative stress has been studied as the underlying pathogenic pathway in CsAinduced toxicity by several studies [32,33]. Indeed, CsA causes intramitochondrial Ca ++ disturbance, disrupts mitochondrial oxidative phosphorylation, triggers ROS production, and induces oxidative damage to macromolecules [34]. These events were coupled with impaired intracellular antioxidant systems. This pro-oxidants/antioxidants imbalance disrupts normal cellular functioning and membrane integrity. In line, the present study demonstrated increased MDA and NO concomitant with compromised TAC in serum and SOD gene expression in the hepatic tissue of CSA-treated animals. This was associated with increased hepatic tissue MDA content along with reduced GSH levels, SOD, and CAT activity in CsA-treated animals.
Nrf2 is a transcription factor that regulates several genes encoding for detoxifying enzymes and antioxidant proteins such as HO-1, glutathione S-transferase, glutathione peroxidase modulation of NADPH oxidase, and the Nrf2/HO-1 pathway by vanillin in cisplatin-induced nephrotoxicity in rats [35,36]. Moreover, Nrf2 regulates the balance of apoptotic/antiapoptotic proteins, controlling cellular apoptosis [37]. HO-1 is one of the classical Nrf2 controlled genes and the Nrf2/HO-1 axis has been considered as a crucial antioxidant target. Further, it has been reported that activation of the Nrf2/HO-1 axis is accompanied by NF-κB inhibition in various models of liver toxicity [38,39]. Indeed, CsA-induced hepatorenal injury was accompanied by a decrease in Nrf2 tissue expression, along with its target antioxidant proteins, including HO-1 [40]. Similarly, our data showed reduced Nrf2 gene expression and HO-1 immunostaining in the liver tissue of CSA-treated animals.
Downregulation of Nrf-2 activates NF-κB with subsequent activation of pro-inflammatory mediator production, including COX-2 and inflammatory cytokines. On the other hand, activation of NF-κB disrupts Nrf-2 signaling [41]. Indeed, previous studies demonstrated increased hepatic NF-kB p65 and inflammatory cytokines levels in CsA-treated rats [42].
Constitutive production of NO plays a crucial role in hepatic perfusion. NO is produced by NOS, which possess three subtypes, inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) [1]. Studies have shown that baseline production of NO by eNOS is hepatoprotective, however increased NO levels by iNOS in an inflammatory environment is damaging to hepatic tissues [43]. Of note, macrophages are considered the major source of iNOS. In this context, CsA treatment is accompanied by increased inflammatory cell infiltration along with upregulated iNOS in hepatic tissue. Interestingly, this was concomitant with a decrease in NO level, which can be explained by consumption of produced NO by oxidative stress [1]. In addition to iNOS, inflammatory cells also produce COX-2. Increased COX-2 expression triggers the production of vasoconstrictive agents, leading to a reduction in blood flow to tissues. Additionally, COX-2 and its products are generally considered potent proinflammatory mediators [44]. CsA treatment is accompanied by increased COX-2 levels in various tissues [45,46]. In agreement with these reports, the IHC findings of the present study demonstrated inflammatory cell infiltration as reflected by increased immunostaining of CD68, in addition to increased iNOS and COX-2 immunostaining in rat hepatic tissues following CsA administration.
Oxidative stress and inflammation are well-known inducers of apoptotic changes [47]. Previous reports indicated induction of cellular apoptosis by CsA treatment [48]. Caspase-3 is a main protease implicated in cellular apoptosis, where it is considered the last signal of cell death. CsA has been reported to enhance the expression and activation of caspase-3 leading to cell apoptosis [4,49,50]. In line, the CsA-treated group in the present study demonstrated increased hepatic mRNA levels and immunostaining of caspase-3.
Based on the above discussion, oxidative stress, inflammation, and apoptosis are considered key pathogenic pathways in CsA-induced toxicity to various body organs including the liver. Thereby, targeting these pathways could be an effective strategy to combat CsA-accompanied toxicity.
ALA and its reduced/dihydro metabolite have been reported as powerful inhibitors of lipid and protein oxidation and free-radical quenching agents. Interestingly, ALA is both fat and water soluble; allowing it to act as an antioxidant in both fatty and watery parts of cells [51]. In addition to its antioxidant efficacy, ALA has been widely studied for its anti-inflammatory and anti-apoptotic impacts in various disease models [13,[52][53][54].
ALA protective efficacy has been attributed to its effect on energy metabolism and/or redox status. ALA enhances glucose uptake and protects mitochondrial function. In addition, ALA is a direct scavenger of reactive oxygen species, metal chelator and stabilizer, and inducer of cellular antioxidants [7,59]. Herein, we demonstrated that ALA could attenuate CsA-induced oxidative stress. However, the potential effect of ALA on energy metabolism needs to be investigated.
Previous studies have reported a high liver capacity for the uptake and accumulation of ALA. Administration of ALA has been associated with amelioration of energy-impaired and redox-unbalanced diseases [60]. On the other hand, Silvestri et al. reported that α-lipoic acid might have a double-edged behavior in terms of its oxidative state that may vary according to the biological compartment considered. Nevertheless, an ALA-associated increase in ROS level did not reach an extent able to promote oxidative DNA damage [61]. Furthermore, excessively high doses of ALA have been reported to induce cellular mitochondrial damage. This was attributed to the acceleration of aerobic respiration at high ALA doses, leading to the heating up of the mitochondria with subsequent breakdown of their membranes [62]. Therefore, ALA at high doses may be detrimental due to its deleterious effects on energy metabolism and redox status, whether this effect is a direct effect or due to the increased endogenous ALA synthesis needs further investigation.

Conclusions
In conclusion, the present study showed that the antioxidant, anti-inflammatory, and anti-apoptotic activities of ALA prevented CsA-induced liver injury. Thus, co-administration of ALA might represent an effective therapeutic strategy to ameliorate liver damage induced by CsA. However, further studies are needed to investigate the effect of ALA on mitochondrial function and energy consumption in CsA-induced liver injury. Additionally, whether this effect is a direct effect of administered ALA, or due to an enhanced endogenous production of ALA warrants further investigation.