Blackberry-Loaded AgNPs Attenuate Hepatic Ischemia/Reperfusion Injury via PI3K/Akt/mTOR Pathway

Liver ischemia-reperfusion injury (IRI) is a pathophysiological insult that often occurs during liver surgery. Blackberry leaves are known for their anti-inflammatory and antioxidant activities. Aims: To achieve site-specific delivery of blackberry leaves extract (BBE) loaded AgNPs to the hepatocyte in IRI and to verify possible molecular mechanisms. Methods: IRI was induced in male Wister rats. Liver injury, hepatic histology, oxidative stress markers, hepatic expression of apoptosis-related proteins were evaluated. Non-targeted metabolomics for chemical characterization of blackberry leaves extract was performed. Key findings: Pre-treatment with BBE protected against the deterioration caused by I/R, depicted by a significant improvement of liver functions and structure, as well as reduction of oxidative stress with a concomitant increase in antioxidants. Additionally, BBE promoted phosphorylation of antiapoptotic proteins; PI3K, Akt and mTOR, while apoptotic proteins; Bax, Casp-9 and cleaved Casp-3 expressions were decreased. LC-HRMS-based metabolomics identified a range of metabolites, mainly flavonoids and anthocyanins. Upon comprehensive virtual screening and molecular dynamics simulation, the major annotated anthocyanins, cyanidin and pelargonidin glucosides, were suggested to act as PLA2 inhibitors. Significance: BBE can ameliorate hepatic IRI augmented by BBE-AgNPs nano-formulation via suppressing, oxidative stress and apoptosis as well as stimulation of PI3K/Akt/mTOR signaling pathway.


Introduction
Liver ischemia-reperfusion injury (IRI) occurs when the blood flow interrupted following liver surgery which is accompanied by restoration. Liver IRI is also a significant complication of hepatic tumor resection, transplantation, hypovolemic shock, and other hepatic surgeries [1]. Liver I/R causes hepatocyte necrosis and results in structural damage to the liver [2,3]. Several mechanisms have been proposed to describe the pathogenesis of liver I/R injury including adenosine triphosphate (ATP) depletion, reactive oxygen species (ROS) overproduction, macrophage activation, inflammation, and apoptosis [4,5].
Liver I/R injury consists of complex processes [6]. Adenosine triphosphate (ATP) depletion interferes with metabolic and transport processes dependent on cellular energy [6,7]. The mechanism of reperfusion consists of two phases: in the initial step, activated resident

Preparation of Blackberry Extract
Blackberry leaves were collected on Jan. 2020 from El-Orman Botanical Garden (Giza, Egypt). Plant was kindly identified by Eng. Trease Labib (former-Head of El-Orman Botanical Garden) and Prof. Nasser Barakat (Faculty of Science, Minia University, Egypt). A voucher specimen (2020-Der 05) was deposited at the Department of Pharmacognosy (Faculty of Pharmacy, Deraya University, New Minia, Egypt). Blackberry leaves were gathered and cleaned, rinsed with deionized water, and dried for four days before being ground thoroughly. The resulting powder (10 g) was thoroughly mixed with absolute ethanol (99%; 50 mL). The supernatants were taken and rotary evaporated at 40 • C. The dried extract was used for dissolving in deionized water, filtered through a 25 µm filter paper, and lyophilized to get dried active constituents. The dried active ingredient was then re-dissolved in deionized water (200 mg/mL), and kept at −20 • C for further characterization. The resulting blackberry extract solution was termed BBE [41].

LC-HR-ESI-MS Metabolic Profiling of Blackberry Leaves Extract
Metabolomics analysis using LC-HR-ESI-MS were carried out as described by Abdelmohsen et al. [42]. In brief, plant samples (1 mg/mL in methanol) was analyzed using an Accela HPLC (Thermo Fisher, Dreieich, Germany) equipped with an ACE C18, 75 mm 3.0 mm, 5 m column (Hichrom Limited, Berkshire, UK) and an Exactive (Orbitrap) mass spectrometer (Thermo Fisher). The gradient elution technique was applied at 300 L/min with purified water [TOC was 20 ppb] and acetonitrile, each containing 0.1% formic acid. The elution started with 10% acetonitrile and was gradually increased to 100% acetonitrile within 30 min, followed by an isocratic period of 5 min before returning to 10% acetonitrile for 1 min. The injection volume was 10 µL, and the column temperature was set at 20 • C. With a spray voltage of 4.5 kV and a capillary temperature of 320 • C, HR-ESI-MS was available in both negative and positive ionization modes. Using the insource collisioninduced dissociation process, the ESI-MS mass range was assigned at m/z 100-2000 and m/z 50-1000. The raw data were imported and analyzed in MZmine 2.12, 27 for differentiation of the HR-MS data, as previously described in detail in Abdelmohsen et al. [42]. A chemotaxonomic filter was applied to the resulting hits to reduce the number of identities per metabolite and include only those that were relevant. As a result, 13 metabolites (1-13) were described using Dictionary of Natural Products and METLIN databases [43].

Solid State Synthesis of AgNPs
AgNPs were prepared adopting the solid state technique. Briefly, pre-determined grinded pectin, stabilizer and reducing agent, was mixed with 0.02 g NaOH (Table 1). After further grinding of the mixture, silver nitrate (AgNO 3 ) was added and grinding was maintained for another 3 min. The formation of AgNPs was confirmed when the color of the mixture was changed from colorless to yellowish color. Formed mixture was kept under continuous grinding for 10 min till the color was changed to deep reddish brown. The final product of AgNPs was kept at room temperature for further characterization and application [37]. To prepare blackberry silver nitrate nanoparticles (BBE-AgNPs), 0.2 g of the formed AgNPs was dissolved in 160 mL of deionized water. To ensure complete dissolution of the mixture, magnetic stirring then sonication was maintained for 20 min. Then, BBE (40 mL; 10 mg/mL) was added gradually to the prepared solution with stirring for another 30 min. prepared BBE-AgNPs were kept for characterization prior to administration to rats [37].

Characterization of the Prepared Metal Nanoparticles
To gain more consent on the formation of AgNPs, spectrophotometric scanning, using UV-vis spectrophotometer (Spectronic Genesys ® , with Winspec Software, Spectronic, (Pittsford, NY, USA) was carried out. Briefly, a diluted sample (0.001 g/20 mL) was scanned at a wavelength ranging from 250 to 700 nm. In addition, "Transmission electron microscopy"; "TEM; JEOL JEM-2100" operating at 200 kV was utilized for imaging of the prepared BBE-AgNPs. Particle size and zeta potential of diluted BBE-AgNPs (0.001 g/20 mL) were determined by a "ZETASIZER Nano series; Nano ZS Malvern Instrument Ltd., Malvern, UK" [44].

Entrapment Efficiency
Entrapment efficiency of prepared silver nanoparticles was determined indirectly as described by Hussein, Jihan, et al. [37] Briefly, 1 mL of the prepared solution of BBE-AgNPs was suspended in 10 mL of deionized water. Absorbance was measured using UV-vis spectrophotometer (Spectronic Genesys ® , with Winspec Software, Spectronic, (Pittsford, Metabolites 2023, 13, 419 5 of 24 NY, USA) at 515 nm using water as blank. The amount of the free BB extract (un-loaded) in the supernatant was determined. The percentage of the entrapped BBE (EE %) was calculated from Equation (1) as follows: The experiment was carried out in triplicate and the average was calculated.

Hepatic Ischemia/Reperfusion Surgery Model
Male wistar rats weighing 200-240 g were obtained from the Animal Facility, Nahda University in Egypt. "The Commission on the Ethics of Scientific Research", Faculty of Pharmacy, Minia University approved the research (License No. ES02/2021). As previously explained, hepatic ischemia was established [22]. Briefly, Wistar rats were anesthetized with (1 g/kg) i.p injection of urethane hydrochloride and a midline laparotomy was done to identify and clamp the portal vein, hepatic artery, and hepatic duct. After 30 min, the clip was withdrawn to begin hepatic reperfusion, which lasted 2 h.

Experimental Design
The study was performed on 70 male wistar rats randomly allocated to seven groups of ten rats each, (n = 10) (

Preparation of Tissue Homogenate
Liver tissues were cut into small pieces and homogenized in 5 mL phosphate buffer containing [0.5 g of Na2HPO4 in addition to 0.7 g of NaH2PO4 per 500 mL of deionized water (pH 7.4) per gram tissue] followed by centrifugation at 4000 rpm for 10 min at 4 °C. Supernatant was separated to evaluate oxidant and antioxidant markers.

Determination of Liver Function Parameters
The collected sera were used for the estimation of ALT and AST according to previous methods of Reitman and Frankel [49] using the corresponding Biodiagnostic Laboratory colorimetric assay kits (Giza, Egypt).

Assessment of Oxidant and Antioxidant Markers
The tissue levels of reduced GSH, SOD, CAT and MDA were estimated using Biodiagnostic Laboratory colorimetric assay kits (Giza, Egypt). All assessments were performed according to the manufacturers' protocols. Group I (Sham): sham-operated control group, animals subjected to the surgery procedures but without clamping.
Group IV (AgNPs): animals treated with empty silver nanoparticles (100 mg/kg) orally for 14 days then animals had hepatic IRI.
When the experimental procedures were completed, rats was sacrificed, blood was collected by cardiac puncture for serum analysis and liver tissue was harvested; both were stored at −80 • C until used.

Preparation of Tissue Homogenate
Liver tissues were cut into small pieces and homogenized in 5 mL phosphate buffer containing [0.5 g of Na 2 HPO 4 in addition to 0.7 g of NaH 2 PO 4 per 500 mL of deionized water (pH 7.4) per gram tissue] followed by centrifugation at 4000 rpm for 10 min at 4 • C. Supernatant was separated to evaluate oxidant and antioxidant markers. The collected sera were used for the estimation of ALT and AST according to previous methods of Reitman and Frankel [49] using the corresponding Biodiagnostic Laboratory colorimetric assay kits (Giza, Egypt).

Assessment of Oxidant and Antioxidant Markers
The tissue levels of reduced GSH, SOD, CAT and MDA were estimated using Biodiagnostic Laboratory colorimetric assay kits (Giza, Egypt). All assessments were performed according to the manufacturers' protocols.

Histopathological Study
To assess the histological alterations, Liver tissue samples were fixed in 10% buffered formalin solution for 24 h before being dehydrated in escalating degrees of ethanol, cleaned in xylene, and fixed with paraffin. These samples were cut into 4 µm thick slices and stained with hematoxylin and eosin (H & E), after which the pathological alterations were observed under a microscope (scale bar 200 µm) and evaluated by a professional observer who was not informed of the identity of the studied specimens. Histological changes were scored in a blind fashion from 0, no injury; 1, mild injury (25%); 2, moderate injury (50%); 3, severe injury (75%); and 4, very severe injury (almost 100%) based on the degree of central vein congestion, sinusoidal congestion, parenchymal cells necrosis, cytoplasmic vacuolization, and inflammatory cells infiltration using modified Suzuki classification [50]. The overall histology score was calculated by adding the scores for all parameters. Each rat had three slides made for examination. All photomicrographs in this work were obtained with an Olympus (U.TV0.5XC-3) light microscope and digital camera.

Western Blotting
For evaluation of cleaved capsase-3, p-PI3K, p-mTOR and relative p-Akt/Akt expression in liver tissue, immunoblotting was conducted as described before [51]. In brief, liver tissue was homogenized in RIPA lysis buffer, which comprised 25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS and 1% Protease/phosphatase Inhibitor cocktail. The supernatant was produced by centrifuging the homogenate for 20 min at 4 • C and then storing it at −80 • C. After boiling for 5 min at 95 • C, samples (30 µg/lane) were run through a 10% SDS-PAGE gel. The nitrocellulose membranes were blocked in 7.5% skimmed milk in TBS-T (0.05% Tween-20 Tris-buffered saline) for 2 h at room temperature before being incubated with primary antibodies diluted at 1:1000 against cleaved caspase-3, p-PI3K, p-Akt, Akt, and p-mTOR overnight at 4 • C. Following that, the membranes were washed and probed for 1 h at room temperature with secondary alkaline phosphatase-conjugated Mouse/Rabbit IgG antibody, followed by repeated washing. Protein bands were eventually observed using a colorimetric detection approach based on 5-bromo-4-chloro-3-indolyphosphate (BCIP)/nitro-blue tetrazolium (NBT). Image-J (NIH, Bethesda, MD, USA) software was used to examine the quantification of the identified bands. As a loading control, protein loading was adjusted for β-actin.

Immunohistochemistry
Immunohistochemical expression (IHC) was performed using polyclonal antibody for Bax and Cleaved Caspase-9. Liver tissue samples were fixed in 10% buffered formalin solution and embedded in paraffin. Deparaffinized and well hydrated sections (4 µm) were cooked for 10 min in a 25 mM citrate buffer solution (pH 6.0), then passed to boiling deionized water and allowed to cool for 20 min. Tissue slices were treated with 3% H 2 O 2 to inhibit endogenous peroxidase activity. Slides were treated with 1% fetal bovine serum and 10% rat serum for 1 h at room temperature to inhibit non-specific immune-staining. The slides were treated overnight at 4 • C with anti-Bax and anti-Cleaved Caspase-9 antibodies. Subsequently, the slices were then stained with a solution of DAB (3-3-diaminobenzidine) after being treated for 30 min at 37 • C with biotinylated goat anti-rabbit IgG secondary antibody (Invitrogen). After 10 s of hematoxylin staining, the slides were mounted. Negative controls were used to assess the technique's specificity (canceling the incubation with the primary antibody and incubating it with non-immune sera). To measure the positive areas, the programme Image J (NIH, Bethesda, MD, USA) was employed. Results were expressed as the percentage of stained cells in each field [52].

Ischemia Potential Protein Targets Determination
As a way to propose protein targets for the BBE-identified compounds, we ran inverse docking simulations on each chemical against every protein in the Protein Data Bank. In order to accomplish this, we made use of the idTarget platform (http://idtarget.rcas.sinica. edu.tw/; accessed on 15 September 2022). This structure-based screening platform employs a novel docking strategy, called divide-and-conquer docking, which adaptively constructs small overlapping grids to limit the search space on protein surfaces. As a result, it can conduct a large number of high-quality docking experiments in a significantly shorter amount of time [53]. The data were compiled as a table of binding affinity scores, which were then ranked from the highest negative value to the lowest one.
Using a cutoff of −10.0 kcal·mol kcal/mol for binding affinity, we determined which targets were optimal for each molecule annotated in BBE. A total of 11 protein targets were identified for compounds 1-13 of which PLA 2 (PDB: 5WZS) was found to be the most relevant target to ischemia-reperfusion.

Molecular Dynamic Simulation and Binding Free Energy Calculation
Previous report for calculating the binding free energy (∆G) and performing molecular dynamic simulations were followed [54]. These procedures are described in detail in the Supplementary File.

Statistical Analysis
All numerical results were presented as mean ± SEM for 6 rats in each group and analyzed with GraphPad prism version 9 (San Diego, CA, USA). To analyze differences across all experimental groups, One-way ANOVA test was employed, followed by a Tukey-Kramer post hoc test. The significance level was set at p < 0.05.

LC-HR-ESI-MS Chemical Profiling of BBE
Metabolomic profiling of BBE using LC-HR-MS for dereplication purposes has led to the identification of a number of natural products (Table 1 and Figure 2), of which Flavonoids were identified to predominate (1-6) followed by phenolic acids (9)(10)(11)(12). LC-HRMS analysis was performed using both positive and negative ionization modes to cover a wide range of compounds in BBE ( Figures S1 and S2). In addition, two anthocyanins (6 and 7) and one alkaloids (13) were also identified as major compounds in BBE. All of these annotated natural products have been previously reported from black berry [55].
Flavonoids were identified to predominate (1-6) followed by phenolic acids (9)(10)(11)(12). LC-HRMS analysis was performed using both positive and negative ionization modes to cover a wide range of compounds in BBE ( Figures S1 and S2). In addition, two anthocyanins (6 and 7) and one alkaloids (13) were also identified as major compounds in BBE. All of these annotated natural products have been previously reported from black berry [55].

Characterization of the Prepared BBE-AgNPs Nanoparticles
Silver nitrate nanoparticles of blackberry extract (BBE-AgNPs) were successfully prepared. That was obvious due to the yellow then reddish discoloration of the colorless pectin on reducing silver ions. Moreover, the given UV peak at 410 nm due to surface plasmon resonance confirmed the formation of AgNPs. (Figure 3) shows the spherical Nano-structure of the formed nanoparticles. Formulated nanoparticles were homogenously distributed within Nano-sized range (37.2 nm to 300.8 nm). The homogenous distribution could be attributed to the reducing power of PVP allowing the formation of tiny clusters of AgNPs. This obvious variation in particle size of different formulations could be attributed to the different formulation parameters. Increasing the amount of pectin, relatively to AgNO3, in F2 compared to F1 has resulted in reduced particle size from 190.3 ± 5.6 to 37.2 ± 1.3, respectively ( Table 2). This could be due to the dispersing effect of pectin that prevents the agglomeration of AgNPs. On the other hand, increasing the amount of pectin above certain limit may hinder the formation of small nanoparticles as reported by Natsuki et al. [56].

Characterization of the Prepared BBE-AgNPs Nanoparticles
Silver nitrate nanoparticles of blackberry extract (BBE-AgNPs) were successfully prepared. That was obvious due to the yellow then reddish discoloration of the colorless pectin on reducing silver ions. Moreover, the given UV peak at 410 nm due to surface plasmon resonance confirmed the formation of AgNPs. (Figure 3) shows the spherical Nano-structure of the formed nanoparticles. Formulated nanoparticles were homogenously distributed within Nano-sized range (37.2 nm to 300.8 nm). The homogenous distribution could be attributed to the reducing power of PVP allowing the formation of tiny clusters of AgNPs. This obvious variation in particle size of different formulations could be attributed to the different formulation parameters. Increasing the amount of pectin, relatively to AgNO3, in F2 compared to F1 has resulted in reduced particle size from 190.3 ± 5.6 to 37.2 ± 1.3, respectively ( Table 2). This could be due to the dispersing effect of pectin that prevents the agglomeration of AgNPs. On the other hand, increasing the amount of pectin above certain limit may hinder the formation of small nanoparticles as reported by Natsuki et al. [56].

Entrapment Efficiency of the Prepared BBE-AgNPs Nanoparticles
The amount of the free BBE in the supernatant was determined. The percentag the entrapped BBE (EE %) was calculated from (Equation (1)) as showed in Table 2. F2 was selected for biological study due to its small particle size which allow hanced cellular uptake to hepatic cells. Zeta potential of the selected formula wasmv which indicates the improved physical stability of the prepared AgNPs and that loaded BBE did not affect the charge or the stability of the prepared nanoparticles.

Entrapment Efficiency of the Prepared BBE-AgNPs Nanoparticles
The amount of the free BBE in the supernatant was determined. The percentage of the entrapped BBE (EE %) was calculated from (Equation (1)) as showed in Table 2. F2 was selected for biological study due to its small particle size which allow enhanced cellular uptake to hepatic cells. Zeta potential of the selected formula was-32.5 mv which indicates the improved physical stability of the prepared AgNPs and that the loaded BBE did not affect the charge or the stability of the prepared nanoparticles.

Serum ALT and AST Activities
It was revealed that the hepatic I/R-induced injury is associated with a substantial elevation in serum ALT (3.9 fold) ( Figure 4A) and AST (4.7 fold) ( Figure 4B), as compared to the sham rats. However, treatment with 200 BBE significantly leveled off these markers. Additionally, pretreatment with 200 BBE loaded AgNPs and 50 BBE loaded AgNPs has prevented the I/R effect and returned these enzymes at their normal level with the lower dose showing a better effect to match that of Silymarin. This drug targeting effect of nanoparticles superimposed that BBE treatment alone. The results are presented in Tables S1 and S2 in the Supplementary Data.

Hepatic Lipid Peroxidation and Antioxidant Markers
As shown in Figure 5, the hepatic I/R-induced injury is associated with a signific decrease in antioxidant parameters ( Figure 5A) GSH (80%), ( Figure 5B) SOD (50 ( Figure 5C) CAT (63%) and increase in ( Figure 5D) MDA (1.8 fold) which is a marker lipid peroxidation, oxidative stress, and tissue injury, as compared to that of the sh rats. However, treatment with 200 BBE significantly normalized these markers. Ad tionally, pretreatment with 200 BBE-AgNPs and 50 BBE-AgNPs has inhibited the I/R fect and kept these parameters at their normal level, with the lower dose showing a b

Hepatic Lipid Peroxidation and Antioxidant Markers
As shown in Figure Figure 5C) CAT (63%) and increase in ( Figure 5D) MDA (1.8 fold) which is a marker f lipid peroxidation, oxidative stress, and tissue injury, as compared to that of the sha rats. However, treatment with 200 BBE significantly normalized these markers. Add tionally, pretreatment with 200 BBE-AgNPs and 50 BBE-AgNPs has inhibited the I/R fect and kept these parameters at their normal level, with the lower dose showing a b ter effect to match that of Silymarin. The results are presented in Tables S3-S6

Histopathological Analysis
The microscopic examination ( Figure 6) of (A) sham rats showed normal hepa architecture (non-dilated and non-congested central veins (arrow) surrounded hepatocytes arranged in normal cords and separated by hepatic sinusoids without va olation (arrow head) (grade 0). Nonetheless, sections of the hepatic IRI group reveals focal pale eosinophilic area with small dark pyknotic nuclei represent 25% of liver tis (arrow), peripheral dilated and congested central vein surrounded by dilated and c gested sinusoids (arrow head), (C) marked dilated and congested central vein and

Histopathological Analysis
The microscopic examination ( Figure 6) of (A) sham rats showed normal hepatic architecture (non-dilated and non-congested central veins (arrow) surrounded by hepatocytes arranged in normal cords and separated by hepatic sinusoids without vacuolation (arrow head) (grade 0). Nonetheless, sections of the hepatic IRI group reveals (B) focal pale eosinophilic area with small dark pyknotic nuclei represent 25% of liver tissue (arrow), peripheral dilated and congested central vein surrounded by dilated and congested sinusoids A better effect is detected in sections of 50 BBE AgNPs and 200 BBE AgNPs treated groups with the lower dose showing a better effect comparable to silymarin which reveals mild histopathological alterations ( Figure 6 and Table 3).  A better effect is detected in sections of 50 BBE AgNPs and 200 BBE AgNPs treated groups with the lower dose showing a better effect comparable to silymarin which reveals mild histopathological alterations ( Figure 6 and Table 3).

Effect of BBE-AgNPs on Cleaved Caspase-3, p-PI3k, p-Akt, p-mTOR Protein Expression
As depicted in western blotting, induction of hepatic I/R sharply reduced the hepatic protein expression of phosphorylated PI3K, Akt and mTOR, by 85.8%, 74.5% and 81.2%, respectively, compared to those of sham group, as well as elevated the protein expression of casp-3 by (4.2 fold). Pre-administration of BBE promoted the phosphorylation PI3K, Akt and mTOR, an effect that was further augmented to reach almost 4.7, 2.1 and 3.1 folds respectively by 200 BBE-AgNPs and 6.1, 3.5 and 4.3 folds respectively by 50 BBE-AgNPs, compared to the IRI group. BBE caused a subtle yet significant decrease in the protein expression of Casp-3, while both doses of BBE-AgNPs revealed a drug targeting effect showing better effects, compared to the IRI, BBE and empty AgNPs pretreated groups. Collectively, results explain that the nano formulation enhanced the drug targeting effect of BBE and maintained the protein expressions at the normal level (Figures 7 and 8

Effect of BBE-AgNPs on Cleaved Caspase-3, p-PI3k, p-Akt, p-mTOR Protein Expressio
As depicted in western blotting, induction of hepatic I/R sharply reduced th patic protein expression of phosphorylated PI3K, Akt and mTOR, by 85.8%, 74.5% 81.2%, respectively, compared to those of sham group, as well as elevated the pr expression of casp-3 by (4.2 fold). Pre-administration of BBE promoted the phospho tion PI3K, Akt and mTOR, an effect that was further augmented to reach almost 4.

Effect of BBE-AgNPs on Bax and Cleaved Caspase-9 Protein Expression
As observed in (Figure 9), the immunohistochemical imaging revealed that the Sham group shows negative hepatic Bax and caspase-9 immunoreactivity contrary to those of the I/R and AgNPs groups that have strong intensity of cytoplasmic staining revealing strong immune expression (8.5 and 7.8 folds) respectively for Bax and (12.4 and 11 folds) and for cleaved caspase-9 compared to Sham group. Nonetheless, a moderate Bax and cleaved caspase-9 expression is seen in section of BBE treated rats, whereas weak expressions are recorded on 200 or 50 BBE-AgNPs pretreated groups that simultaneously decreased Bax and caspase-9 expressions by 64%, 60% for 200 BBE-AgNPs treated group and by 75%, 66.6% for 50 BBE-AgNPs treated group. The results are presented in Tables S7 and S8 in the Supplementary Data.

Effect of BBE-AgNPs on Bax and Cleaved Caspase-9 Protein Expression
As observed in (Figure 9), the immunohistochemical imaging revealed that the Sham group shows negative hepatic Bax and caspase-9 immunoreactivity contrary to those of the I/R and AgNPs groups that have strong intensity of cytoplasmic staining revealing strong immune expression (8.5 and 7.8 folds) respectively for Bax and (12.4 and 11 folds) and for cleaved caspase-9 compared to Sham group. Nonetheless, a moderate Bax and cleaved caspase-9 expression is seen in section of BBE treated rats, whereas weak expressions are recorded on 200 or 50 BBE-AgNPs pretreated groups that simultaneously decreased Bax and caspase-9 expressions by 64%, 60% for 200 BBE-AgNPs treated group and by 75%, 66.6% for 50 BBE-AgNPs treated group. The results are presented in Tables S7 and S8 in the Supplementary Data. those of the I/R and AgNPs groups that have strong intensity of cytoplasmic staining revealing strong immune expression (8.5 and 7.8 folds) respectively for Bax and (12.4 and 11 folds) and for cleaved caspase-9 compared to Sham group. Nonetheless, a moderate Bax and cleaved caspase-9 expression is seen in section of BBE treated rats, whereas weak expressions are recorded on 200 or 50 BBE-AgNPs pretreated groups that simultaneously decreased Bax and caspase-9 expressions by 64%, 60% for 200 BBE-AgNPs treated group and by 75%, 66.6% for 50 BBE-AgNPs treated group. The results are presented in Tables  S7 and S8 in

In Silico Study
Identifying the Likely Molecular Target of BBE's Dereplicated Natural Products The modelled structures of the dereplicated compounds (Table 1, Figure 2) in BBE was put through a docking-based virtual screening to identify the likely molecular target(s) via which it can mediate its liver protective effect. You can access the idTarget online system at https://idtarget.rcas.sinica.edu.tw (accessed on 14 February 2023) [53]. The vast majority of the proteins available through the Protein Data Bank (PDB;

In Silico Study Identifying the Likely Molecular Target of BBE's Dereplicated Natural Products
The modelled structures of the dereplicated compounds (Table 1, Figure 2) in BBE was put through a docking-based virtual screening to identify the likely molecular target(s) via which it can mediate its liver protective effect. You can access the idTarget online system at https://idtarget.rcas.sinica.edu.tw (accessed on 14 February 2023) [53]. The vast majority of the proteins available through the Protein Data Bank (PDB; https://www.rcsb.org/ (accessed on 22 November 2021) can be screened virtually using this platform's inverse docking method. An Excel file was generated from the obtained results, with the binding affinity scores ordered from highest negative value to lowest. The optimal result was chosen using a cut-off value of −10.0 kcal/mol.
Human Phospholipase A2 (PLA2) (PDB code: 5WZS) [57] was found to be the most relevant target associated to the liver dysfunction upon induction of ischemia-reperfusion [58][59][60]. Both cyanidin and pelargonidin glucosides (the major anthocyanins of BBE; Table 1 and Figure 2) were the compounds that showed the best docking scores with this target protein (−12.67 and −12.58 kcal/mol). PLA2 over expression occurred in liver cells as a result of ischemia-reperfusion indirectly induce the activity of the oxidant enzymes COX and LOX that in turn, increase the intracellular oxidative stress. From another side PLA2 induce cardiolipin hydrolysis that in turn, leads to mitochondrial dysfunction and also increased intracellular oxidative stress ( Figure 10). Besides their powerful intrinsic antioxidant effect, black berry's anthocyanins have been shown to be effective PLA2 inhibitors [61][62][63][64].
Metabolites 2023, 13, x FOR PEER REVIEW 16 of 2 results, with the binding affinity scores ordered from highest negative value to lowest The optimal result was chosen using a cut-off value of −10.0 kcal/mol. Human Phospholipase A2 (PLA2) (PDB code: 5WZS) [57] was found to be the mos relevant target associated to the liver dysfunction upon induction of ische mia-reperfusion [58][59][60]. Both cyanidin and pelargonidin glucosides (the major antho cyanins of BBE; Table 1 and Figure 2) were the compounds that showed the best docking scores with this target protein (−12.67 and −12.58 kcal/mol). PLA2 over expression oc curred in liver cells as a result of ischemia-reperfusion indirectly induce the activity of the oxidant enzymes COX and LOX that in turn, increase the intracellular oxidative stress From another side PLA2 induce cardiolipin hydrolysis that in turn, leads to mitochon drial dysfunction and also increased intracellular oxidative stress ( Figure 10). Beside their powerful intrinsic antioxidant effect, black berry's anthocyanins have been shown to be effective PLA2 inhibitors [61][62][63][64]. To further validate the preliminary docking findings, the generated docking pose of both cyanidin and pelargonidin glucosides inside the PLA2's active site were subjected to 50 ns-long MDS experiments to study their dynamic modes of interaction and to es timate their binding affinities (i.e., absolute binding free energy, ΔGbinding). As shown in (Figure 11), Both cyanidin and pelargonidin glucosides were able to achieve stable bindings inside the human PLA2's active site via forming three stable H-bonds with ASN-1, ASP-47, and GLY-30 similarly to the co-crystalized inhibitor. In addition, they established π-staking interactions with PHE-5 together with co-ordinate interaction with Ca 2+ through their phenolic hydroxyl groups. As a result, their deviations (i.e. To further validate the preliminary docking findings, the generated docking poses of both cyanidin and pelargonidin glucosides inside the PLA2's active site were subjected to 50 ns-long MDS experiments to study their dynamic modes of interaction and to estimate their binding affinities (i.e., absolute binding free energy, ∆G binding ). As shown in (Figure 11), Both cyanidin and pelargonidin glucosides were able to achieve stable bindings inside the human PLA2's active site via forming three stable H-bonds with ASN-1, ASP-47, and GLY-30 similarly to the co-crystalized inhibitor. In addition, they established π-staking interactions with PHE-5 together with co-ordinate interactions with Ca 2+ through their phenolic hydroxyl groups. As a result, their deviations (i.e., RMSD) from the initial docking poses were only about~2.52 Å (RMSD of the co-crystalized inhibitor~2.76 Å), and their calculated absolute binding free energy (∆G binding ) were −9.56 and −9.58 kcal/mol, respectively (∆G binding of the co-crystalized inhibitor = −8.78 kcal/mol) indicating significant affinity to the enzyme's active site. Previously, a number of anthocyanins and anthocyanidines were found to act as potent PLA2 inhibitors both in vitro and in vivo [61,62]. Accordingly, the previous in silico-based investigation suggested that BBE-derived anthocyanins (i.e., cyanidin and pelargonidin glucosides) are a promising scaffold for developing potent anti-inflammatory and antioxidant agents targeting PLA2. However, careful in vitro and in vivo testing of the plant's isolated anthocyanins for their PLA2's inhibitory activity should be carried out in future biological investigations of this plant.

Discussion
Hepatic IRI, in addition to hemorrhagic shock, is a condition in which cellular damage occurs frequently during liver transplantation, partial hepatic resection, and trauma circumstances [65]. The current emphasis on herbal supplements motivates scientists to research natural agents in a variety of illnesses. Several researches have demonstrated that natural drugs can play an important role in human diseases prevention. Among these is blackberry extract, which has been shown to be anti-carcinogenic, anti-inflammatory, antimicrobial anti-diabetic, and antiviral [66,67]. Blackberry leaf extract is a natural therapeutic medication with many regulatory actions, including improved microcirculation, suppression of apoptosis, and inhibition of neutrophil infiltration and inflammation [68]. For many years, blackberry-based therapy has been utilized for hepatic disorders and as a potent antioxidant [45,69]. Unfortunately, it is only effective at high doses which limited its clinical utility [45].
The optimization of a nano-sized drug delivery system has the ability to significantly improve the therapeutic efficacy of the loaded drug [70][71][72][73][74]. The goal of this work was to minimize particle size and improve the EE% and medication targeting action of BBE-AgNPs nano-formulation. To improve therapeutic efficacy, blackberry-based therapy is implemented by incorporating the extract into AgNPs nano-architecture to accomplish targeted therapy. It should be mentioned that, depending on the AgNPs quantities and schedule of administration, silver nanoparticles can have both protective and harmful effects [75][76][77]. Therefore, the therapeutic window for I/R injury in the liver Accordingly, the previous in silico-based investigation suggested that BBE-derived anthocyanins (i.e., cyanidin and pelargonidin glucosides) are a promising scaffold for developing potent anti-inflammatory and antioxidant agents targeting PLA2. However, careful in vitro and in vivo testing of the plant's isolated anthocyanins for their PLA2's inhibitory activity should be carried out in future biological investigations of this plant.

Discussion
Hepatic IRI, in addition to hemorrhagic shock, is a condition in which cellular damage occurs frequently during liver transplantation, partial hepatic resection, and trauma circumstances [65]. The current emphasis on herbal supplements motivates scientists to research natural agents in a variety of illnesses. Several researches have demonstrated that natural drugs can play an important role in human diseases prevention. Among these is blackberry extract, which has been shown to be anti-carcinogenic, anti-inflammatory, antimicrobial anti-diabetic, and antiviral [66,67]. Blackberry leaf extract is a natural therapeutic medication with many regulatory actions, including improved microcirculation, suppression of apoptosis, and inhibition of neutrophil infiltration and inflammation [68]. For many years, blackberry-based therapy has been utilized for hepatic disorders and as a potent antioxidant [45,69]. Unfortunately, it is only effective at high doses which limited its clinical utility [45].
The optimization of a nano-sized drug delivery system has the ability to significantly improve the therapeutic efficacy of the loaded drug [70][71][72][73][74]. The goal of this work was to minimize particle size and improve the EE% and medication targeting action of BBE-AgNPs nano-formulation. To improve therapeutic efficacy, blackberry-based therapy is implemented by incorporating the extract into AgNPs nano-architecture to accomplish targeted therapy. It should be mentioned that, depending on the AgNPs quantities and schedule of administration, silver nanoparticles can have both protective and harmful effects [75][76][77]. Therefore, the therapeutic window for I/R injury in the liver should be thoroughly investigated. It should be taken into consideration that reported studies have already found that liver accumulated the highest level of AgNPs in animals which make it an ideal nano-carrier to the liver [78,79].
In this study, we developed a BBE Loaded AgNPs (BBE-AgNPs) formulation that contained the antioxidant blackberry leaves extract constituents to protect the liver from I/R injury. Besides, silymarin, a natural hepato-protective agent, was used as a positive control because it was reported that silymarin protects liver tissue in hepatic I/R injury in rats by regulating the apoptotic pathway and modulating the antioxidant status [80]. Current results demonstrated that BBE could significantly decrease the elevated level of serum hepatic enzymes (ALT&AST) in IRI-induced in rats. Additionally, BBE-AgNPs showed restoration of hepatic function similar to that of silymarin, and with lower dose. In hepatic cells, when the cellular membrane of the liver cells was damaged, the permeability of the membrane would be increased, resulting in the leakage of the liver enzyme (ALT) in hepatic cytoplasm into circulation and a rapid raise of serum ALT level [20]. The unremitting hepatic injury also leads to a discharge of AST in hepatic cytoplasm and the mitochondria into the circulation [81]. Parallel with the elevated ALT and AST serum levels, Suzuki's score were increased in hepatic IRI as showed severe hepatic necrosis, vacuolar degeneration and inflammatory cellular infiltrates that accompanied by congested and dilated hepatic central vein and sinusoids. All aforementioned damages were reduced by BBE-AgNPs pretreatment with a posterior effect for the lower dose. These results suggest that BBE loaded AgNPs may exert hepatoprotective effect after I/R injury.
The process of I/R injury disrupts redox balance and damages normal tissue functions and structure, culminating in the accumulation of ROS. As a result, the most common category liver ischemia-reperfusion injury mechanism is the formation of ROS and oxidative stress [82]. The role of oxidative stress in ischemia and reperfusion injury is widely acknowledged. Here, IRI group exhibited a significant decrease of antioxidants (SOD, CAT, and GSH) with elevation of MDA as a lipid peroxidation marker. SOD has the ability to increase the conversion of O 2 − to O 2 and H 2 O 2 . Under CAT catalysis, H 2 O 2 can also be degraded into H 2 O and O 2 . As a result, both enzymes were found to be helpful by hastening the detox of ROS in IRI [69]. The initial line of defense against oxidative stress is antioxidants such as SOD, CAT, and GSH [83]. This study found that BBE-AgNPs mediated the aforementioned antioxidant markers linked with IRI and had a mitigative effect against oxidative stress-induced damage which is observed by decreased MDA level. The BBE components SOD/CAT-like activities may scavenge O 2 − and H 2 O 2 to create O 2 by employing the excessive ROS generated during hepatic ischemia/reperfusion as a trigger. Such a cascade reaction efficiently performed ROS scavenging (O 2 − and H 2 O 2 ) and reducing oxidative stress during hepatic ischemia and reperfusion [84]. Numerous cellular pathways, including the protective (PI3K/Akt/mTOR) pathway, are suppressed during hepatic I/R shock, while several harmful processes, such as oxidative stress and apoptosis, are activated [22].
As illustrated in (Figure 12), The Phosphatidylinositol-3-kinase (PI3K) pathway, which includes Akt, mTOR, is activated by the dimerization of tyrosine kinase receptor that activated by growth factors. It represents an important component in regulating apoptosis and pro-inflammatory cytokines [85]. Hepatic I/R damage is linked to a number of signaling pathways. Recent study found that activated phosphorylated Akt (p-Akt) dramatically mitigate I/R injury to the liver. Apoptosis, also known as type I programmed cell death, is linked to hepatic I/R injury. PI3K/Akt signaling activation protects hepatic cells from apoptosis [86]. cleaved to release the active caspases. Therefore, cleaved caspase-3 is considered a reliable biomarker for apoptosis [89]. The expression of Bax, cleaved casp-9 and casp-3 proteins was elevated in the IRI rats. Bax alone has been found to be adequate for apoptotic induction [90]. Apoptosis was observed to be related with caspase-3 activation and Bax expression during graft cold storage and after cold I/R in rat liver transplantation [91]. Apoptosis of hepatocytes and sinusoidal endothelial cells is an important cause of liver I/R injury [92]. Figure 12. A hypothetical scheme of BBE-loaded AgNPs protection to hepatic ischemia/reperfusion injury (IRI). When the liver is subjected to ischemia/reperfusion, the expression of Bax is strongly induced, which leads to release of cytochrome c from mitochondria and formation of Apaf-1 which activate Caspase-9 and then Caspase-3 in hepatocytes leading to apoptosis. On the other hand, the inhibited PI3K/Akt/mTOR pathway inhibits cell growth and cell survival. Therefore, the PI3K/Akt/mTOR pathway activation by BBE-AgNPs components may regulate the expression of genes closely associated with apoptosis such as Bax, Caspase-9 and Caspase-3 leading to attenuation of liver injury after IR (created with BioRender.com).
Interestingly, Bax, casp-9 and casp-3 proteins were down-regulated after the treatment with BBE-AgNPs in both doses more than BEE alone. Taken together, we demonstrated that BBE-AgNPs in both doses mitigated the hepatic ischemic damage and limited apoptosis by suppressing Bax, casp-9 and casp-3 proteins expression.
Furthermore, it can be deduced that using 50 BBE-AgNPs yielded better outcomes for all treatment groups of experimental rats than the 200 BBE-AgNPs. This may be due to the hepatotoxic effect of relatively higher doses of AgNPs as explained by previous studies [75,93,94]. Vasquez et al. measured the effects of AgNPs on hepatic enzymes after a single dose of Paracetamol (2 g/kg BW) and revealed that the smaller dose of AgNPs (100 mg/kg BW) are less toxic than larger dose of AgNPs (200 mg/kg BW) [75]. Also Hamad et al. observed that sections of the livers of larger dose AgNPs (200, 300 mg/kg) treated mice showed vascular congestion and scattered inflammatory cells (lymphocytes) which indicate the initiation of the inflammation. On contrast smaller dose AgNPs (50, 100 mg/kg) administration showed no histopathological changes [93]. Figure 12. A hypothetical scheme of BBE-loaded AgNPs protection to hepatic ischemia/reperfusion injury (IRI). When the liver is subjected to ischemia/reperfusion, the expression of Bax is strongly induced, which leads to release of cytochrome c from mitochondria and formation of Apaf-1 which activate Caspase-9 and then Caspase-3 in hepatocytes leading to apoptosis. On the other hand, the inhibited PI3K/Akt/mTOR pathway inhibits cell growth and cell survival. Therefore, the PI3K/Akt/mTOR pathway activation by BBE-AgNPs components may regulate the expression of genes closely associated with apoptosis such as Bax, Caspase-9 and Caspase-3 leading to attenuation of liver injury after IR (created with BioRender.com).
The current findings revealed that BBE-AgNPs in multiple doses upregulated the phosphorylation of PI3K, Akt, and mTOR proteins of the protective signaling pathway, and reduced the ischemic injury more effectively than BBE alone. Previous studies showed that the overproduction of ROS and inhibition of PI3K/Akt/mTOR pathway led to the up-regulation of Bax, Casp-9 and Casp-3 expressions, resulting in apoptosis [86,87]. Bcl-2 associated X (Bax) proteins are proapoptotic molecules that induce mitochondrial cytochrome C release, forming apoptotic protease activating factor 1 (APAF-1) and hence boosting caspase-9 and caspase-3 production [88]. Caspase-3 is one of the main molecules involved in signaling apoptosis. When the apoptotic cascade is triggered, procaspases are cleaved to release the active caspases. Therefore, cleaved caspase-3 is considered a reliable biomarker for apoptosis [89]. The expression of Bax, cleaved casp-9 and casp-3 proteins was elevated in the IRI rats. Bax alone has been found to be adequate for apoptotic induction [90]. Apoptosis was observed to be related with caspase-3 activation and Bax expression during graft cold storage and after cold I/R in rat liver transplantation [91]. Apoptosis of hepatocytes and sinusoidal endothelial cells is an important cause of liver I/R injury [92].
Interestingly, Bax, casp-9 and casp-3 proteins were down-regulated after the treatment with BBE-AgNPs in both doses more than BEE alone. Taken together, we demonstrated that BBE-AgNPs in both doses mitigated the hepatic ischemic damage and limited apoptosis by suppressing Bax, casp-9 and casp-3 proteins expression.
Furthermore, it can be deduced that using 50 BBE-AgNPs yielded better outcomes for all treatment groups of experimental rats than the 200 BBE-AgNPs. This may be due to the hepatotoxic effect of relatively higher doses of AgNPs as explained by pre-vious studies [75,93,94]. Vasquez et al. measured the effects of AgNPs on hepatic enzymes after a single dose of Paracetamol (2 g/kg BW) and revealed that the smaller dose of AgNPs (100 mg/kg BW) are less toxic than larger dose of AgNPs (200 mg/kg BW) [75]. Also Hamad et al. observed that sections of the livers of larger dose AgNPs (200, 300 mg/kg) treated mice showed vascular congestion and scattered inflammatory cells (lymphocytes) which indicate the initiation of the inflammation. On contrast smaller dose AgNPs (50, 100 mg/kg) administration showed no histopathological changes [93].

Conclusions
Finally, we conclude that by using nanotechnology, BBE herbal extract can be loaded to nanoparticles, that ultimately enhances absorbability together with bioavailability toward hepatic IRI and enhances therapeutic outcome with a relatively low dosage, which is likely attributed to improved pharmacokinetics and tissue distribution [95]. Our findings suggest that BBE-AgNPs can protect against mitochondrial apoptosis and oxidative stress caused by liver I/R, which make BBE-AgNPs a potential therapeutic candidate. In-depth, further understanding of the molecular pathways of IRI is critical for improving ongoing therapeutic regimens and developing novel appropriate interventions. Furthermore, attempts should be made to load other natural extracts into nano-carriers, which may enhance effectiveness and patient survival rate. Collectively, this study offers a viable approach of multifunctional nano-therapeutics for IRI treatment, and it also highlights the dual protection mechanism of BBE via ROS clearance and apoptosis regulation.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/metabo13030419/s1 [96][97][98][99][100], Figure S1: Total ion chromatogram of crude extract of Blackberry leaves (Positive mode); Figure S2: Total ion chromatogram of crude extract of Blackberry leaves (Negative mode); Table S1: Effect of different treatments on serum ALT level; Table S2: Effect of different treatments on serum AST level; Table S3: Effect of different treatments on hepatic MDA level; Table S4: Effect of different treatments on hepatic GSH level; Table S5: Effect of different treatments on hepatic SOD level; Table S6: Effect of different treatments on hepatic CAT level; Figure S3: Western blotting of p-mTOR original images; Figure S4: Western blotting of p-Akt original images; Figure S5: Western blotting Akt original images; Figure S6: Western blotting of p-PI3k original images; Figure S7: Western blotting of Cleaved Caspase-3 original images; Figure S8: Western blotting of B-Actin original images; Table S7: Effect of different treatments on percentage area of positive Bax staining; Table S8: Effect of different treatments on percentage area of positive Caspase-9 staining.