Oleic Acid Protects against Hepatic Ischemia and Reperfusion Injury in Mice by Inhibiting AKT/mTOR Pathways

Hepatic ischemia-reperfusion (I/R) injury is a serious complication in patients who have undergone hepatic surgery such as orthotopic liver transplantation and partial hepatectomy. Recently, a new cytoprotective agent, ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), was reported to protect against hepatic I/R injury. However, the protective mechanism of UDCA-LPE is not fully understood. Therefore, we conducted this study to explore its underlying mechanism. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the liver lipid metabolism changes in mice during I/R. KEGG enrichment indicated that UDCA-LPE is likely to exert its protective role by regulating fatty acid (FA) metabolism. Further analysis found that UDCA-LPE significantly increased the ratio of oleic acid (OA) to palmitic acid (PA). We found that mice pretreated with OA improved tolerance to hepatic I/R injury. In addition, the phosphorylation level of AKT was markedly upregulated during oxidative stress to promote p65 nuclear translocation, triggering an inflammatory response that exacerbated cell damage and OA treatment significantly inhibited this process. Notably, OA was found to inhibit H2O2-induced oxidative stress, inflammation, and cell death in HepG2 cells. Furthermore, we found that OA supplementation to the medium did not result in a significant increase in intracellular OA, but marked increase in the ratio of OA to PA, which may be an important mechanism for the inflammatory response induced by oxidative stress during I/R. Finally, we demonstrated that OA increased the level of autophagy in HepG2 cells, which may be one of the protective mechanisms against oxidative stress. Collectively, this study revealed that FA metabolism functionally determines the oxidative stress-related inflammation caused by hepatic I/R. We hypothesize that OA treatment may be a promising strategy for preventing and treating I/R-induced liver damage.


Introduction
Hepatic ischemia-reperfusion (I/R) injury is a complication of hepatic surgery, and it can arise after liver resection and transplantation [1,2]. Hepatic I/R injury induces oxidative stress, inflammation, and other disorders in the liver, thus leading to the liver damage in patients requiring liver surgery [3][4][5][6]. However, the mechanisms underlying the I/R injury are not completely understood. So far, only a few effective protective strategies have been discovered [7]. Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a novel anti-inflammatory agent with hepatoprotective effects, was developed by Chamulitrat et al. by coupling UDCA with a phospholipid. This drug inhibits mitochondrial damage and apoptosis, induces the survival signaling pathway, and promotes the regeneration of hepatocytes [8]. The mechanisms underlying the protective effects of this drug include shifting FA pools toward monounsaturated fatty acids

Animal
Model. Eighty 18-week-old male C57/BL6 mice weighing 28 to 30 g were purchased from Beijing Vital River Laboratory Animal Technology Co. The animals were raised in cages under a 12/12-hour light/dark cycle at 25°C in the Animal Care Facility of Tongji Medical College.

Surgical Procedures.
After a one-week adaptive phase, the animals were divided into the following three groups: sham, I/R, and UDCA-LPE+I/R. Each group contained at least six mice. The surgical procedures were performed as previously described to induce hepatic I/R injury implicating 70% of the liver [20]. As shown in Figure 1(a), fasted mice were anesthetized with pentobarbital sodium (50 mg/kg) by an intraperitoneal injection and underwent a midline incision to expose the liver. An atraumatic clamp was placed across a branch of the portal triad to block the blood supply to the median and left lateral liver lobes to induce ischemia for 90 min. Following unclamping of the liver, hepatic reperfusion was allowed for 2 h, and this procedure represented the I/R group. UDCA-LPE stock was prepared at 5 mg/mL in 0.5% CMC. In the I/R group, the vehicle CMC was injected intraperitoneally. In the UDCA-LPE+I/R group, two doses of 50 mg/kg UDCA-LPE were injected intraperitoneally at 30 min prior to clamping and just prior to reperfusion (Figure 1(a)). The sham groups only received a switching abdominal surgery. After surgery, blood samples were collected and centrifuged at 3500 r/min for 15 min. Livers were harvested and fixed. Parts of the liver samples were sectioned into 1 mm slices, then stained with haematoxylin and eosin, photographed using a NanoZoomer S360 (Hamamatsu, Japan), and analyzed using NZAcquire software. The rest of the samples were stored at -80°C for subsequent tests.      . Mass tolerance of 5 ppm and 10 ppm was applied for precursor and product ions. Retention time shift of 0.25 min was performed in "alignment." M-score and chromatographic areas were used to reduce false positives.

Cell Lines and
2.8. Detection of Apoptosis. HepG2 cells were seeded at a density of 2 × 10 6 cells/mL of the DMEM medium with 10% FBS on 6-well plates to a final volume of 2 mL. After treatment with OA and H 2 O 2 , the cells were collected, washed twice with PBS, and then suspended in 1x Binding Buffer at a con-centration of 1 × 10 6 cells/mL. The cells were subjected to 5 μL of FITC-Annexin V and 5 μL propidium iodide (PI) staining using the Annexin V-FITC apoptosis kit (KeyGEN BioTECH, China). Next, 100 μL of the solution was transferred to a 5 mL culture tube and incubated for 30 min at RT (25°C) in the dark. The apoptosis ratio was quantified using flow cytometry. Data were presented by the system software (Cell Quest; BD Biosciences).  Anti-p-mTOR (ab109268, diluted 1 : 1000) was purchased from Abcam (CA, USA). 2.13. Data Analysis. The fluorescent density was analyzed with Image Pro Plus software. Densitometric analysis of Western blot was carried out with ImageJ software. Data are presented as means ± SD. Statistical analysis was performed using GraphPad PRISM. All data sets were tested for normality of distribution using the Shapiro-Wilk test. Comparison between two groups was assessed using twotailed unpaired Student's t-test or paired Student's t-test.

Gene Expression Analysis by
One-way/two-way ANOVA was used to perform the statistical analysis among more than two groups, followed by Tukey's post hoc test for multiple-group comparisons. A p value < 0.05 was considered statistically significant.

UDCA-LPE Protects
Mice from Hepatic Ischemia-Reperfusion Injury. Our previous study [20] has proved that UDCA-LPE was able to protect mice from liver I/R injury. However, we did not study the role of lipid metabolism in it before. Therefore, we repeated this experiment and confirmed the protective effect of UDCA-LPE. As described in Materials and Methods, our experimental design is depicted in Figure 1(a). The histological evaluation revealed that I/R caused massive hepatic necrosis. A significant improvement was observed in the UDCA-LPE+I/R group (Figure 1(b)) which is consistent with the observed necrosis during I/R showing in the TUNEL staining ( Figure 1(c)). I/R increased the levels of serum alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase ( Figure 1(d)). UDCA-LPE administration in mice undergoing I/R significantly inhibited the elevation with stronger effects in the UDCA-LPE+I/R group (Figure 1(d)).

UDCA-LPE Increases the Odds Ratio of Oleic Acid to
Palmitic Acid in the Liver of Mice. UDCA-LPE did not induce a significant change in lipid metabolism in mice during I/R injury. The TC, HDL-c, LDL-c, and TG levels in serum of mice did not seem to change significantly after treatment with UDCA-LPE before I/R injury (Figure 1(e)). Hence, we conducted lipidomic analysis of total mouse hepatic FA composition to understand the effects of lipid metabolism on the liver during I/R injury. KEGG enrichment (Figure 1(f)) analysis indicated that UDCA-LPE was most likely to participate in FA metabolism pathways during I/R injury. The heat map of changes in liver fatty acids in Figure 1(g) shows that UDCA-LPE can significantly increase the odds ratio of OA to PA.

Oleic Acid Alleviates Mouse Hepatic Ischemia-Reperfusion Injury.
To validate our hypothesis, we conducted an in vivo experiment. Adult male C57 mice were randomly divided into three groups. The sham group only received switching abdominal surgery. The I/R group underwent surgical procedures as previously described, while the OA+I/R group mice received OA intragastric administration (250 mg/kg) for two weeks before undergoing the same surgical procedures. By comparing H&E staining of mouse liver sections, we found that the OA+I/R group had less liver damage than the I/R group. Further biochemical tests were consistent with H&E staining of liver sections (Figure 2(a)). OA can reduce the level of liver enzymes during liver I/R in mice, especially ALT and LDH levels ( Figure 2(b), p < 0:05).
To investigate the effect of OA on cell lipid metabolism, HepG2 cells were treated with 100 μM OA for 24 h. The FA composition in HepG2 cells was analyzed by LC-MS/MS as described before. FA changes are displayed in the heat map ( Figure 2(e)). KEGG enrichment analysis (Figure 2(d)) indicated the pathways that OA is most likely to be involved in. It is worth noting that supplementation of OA did not significantly increase intracellular OA content, but increased the odds ratio of OA/PA (Figure 2(e)).

Oleic Acid Protects Hepatic Cells from H 2 O 2 -Induced
Inflammation. To mimic I/R injury in cell model, HepG2 cells were selected to undergo various concentrations of H 2 O 2 treatment. As a result, 100 μM H 2 O 2 was considered the best stimulation concentration (Fig. S1a). When pretreated with OA, the destructive effects of H 2 O 2 on cells were reduced. The cell viability in the OA group was higher than that in the vehicle group (Figure 3(a)). Consistently, results of FACS analysis (Figure 3(c)) demonstrated that as the concentration of H 2 O 2 increases, the number of apoptotic cells increases. However, it is worth noting that the OA group had lower number of apoptotic cells compared to the vehicle group. Western blot results also supported this observation. Cleaved caspase 3 level was markedly lower with OA treatment compared to groups without OA treatment ( Figure 3(d), p < 0:05).
HepG2 cells were pretreated with different concentrations of OA before undergoing oxidative stress. The expression of inflammation factors, such as TNF-α, IL-β, and IL-6, increased after H 2 O 2 treatment, which was consistent with the expression of inflammation factors after hepatic I/R injury (unpublished observations). However, inflammation factor expression in the OA+Oxi (100 μM) group sharply decreased compared with that in the Oxi group (Figure 3(b), p < 0:01). Western blot analysis identified that the PI3K/AKT/mTOR pathway was activated (Fig S2) under oxidative stress. The mRNA level of p65 expression showed the opposite tendency, which was consistent with the expression level of the inflammatory cytokines (Figure 3(e)). The mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) sharply decreased under oxidative stress (Figure 3(f)). However, OA does not seem to reverse the downregulation of peroxisome proliferator-activated receptor α (PPARα) caused by oxidative stress (Figure 3(g)).
3.5. Oleic Acid Protects Hepatic Cells via Alleviating H 2 O 2 -Induced Oxidative Stress. ROS fluorescence and immunoscore showed that OA was able to reduce the intercellular ROS production in HepG2 cells after H 2 O 2 treatment (Figure 4(a), p < 0:05). The MDA level increased during oxidation; however, the OA treatment (200 μM) significantly lowered the MDA level caused by H 2 O 2 (Figure 4(c), p < 0:05). The total GSH in HepG2 cells was measured. After H 2 O 2 treatment, the GSH level decreased, while the degree of decline was significantly lower in the OA (100 μM)+Oxi group (Figure 4(d), p < 0:05). Western blot analysis demonstrated that the protein expression level of renalase was elevated during oxidation. In the OA-treated group, the degree of increase was not as high as that compared with the vehicle group (Figure 4(b)).
3.6. OA Inhibits the Nuclear Translocation of p65, Resists Apoptosis, and Enhances Autophagy during Oxidative Stress in HepG2 Cells. Western blot analysis of the cytoplasm and nuclear p65 protein demonstrated that there was no significant change in cytoplasmic p65 protein levels in HepG2

Discussion
Our experiments demonstrated that UDCA-LPE protects the liver from I/R injury in mice, which is consistent with a previous study [20]. We used lipid metabolomics combined with bioinformatics techniques to demonstrate that the protective mechanism of UDCA-LPE is closely related to FA metabolism. However, our results indicated that UDCA-LPE has no significant effect on lipid metabolism in serum, but can significantly alter the lipid composition of the liver. Our study also identified that UDCA-LPE can significantly increase the ratio of OA/PA in the liver. This may be one of the important mechanisms of protective effect of UDCA-LPE on the liver against I/R injury. In agreement with this view, OA supplementation was shown to attenuate liver I/R injury in vivo, since PA is generally considered harmful to the liver [20][21][22]  A large body of research [25][26][27] has proved that inflammation and oxidative stress play an important, adverse role in hepatic I/R injury. Recently, studies reported that supplementation with EVOO prevents oxidative stress [28,29]. As OA is one of the main components of EVOO, we explored the association between OA and EVOO. According to our findings, OA was able to decrease hepatic expression of TNF-α, IL-1β, IL-6, and p65 to levels comparable to the control group. As reported, liver inflammation induced by oxidative stress is closely related to the activity of Nrf2 and    PPARα. Ligand activation of PPARα induces antioxidant, metabolic, and anti-inflammatory responses. Our study proved that OA supplementation only upregulates Nrf2 mRNA expression during oxidative stress. OA also tends to reduce the mRNA expression level of PPARα. The mechanisms of I/R injury are diverse, but the emergence of ROS is one of the most critical factors [30][31][32]. The sources of ROS are xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase and have become the current priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage [30,33]. ROS leads to cell death by mediating apoptosis, mitoptosis, necrosis, and necroptosis [32]. In our study, we found that OA can significantly reduce the amount of intracellular ROS produced during oxidative stress. This clearance effect is partly due to the enhancement of intracellular glutathione (GSH) production induced by OA (Fig S1. b). Malondialdehyde (MDA), produced from PUFAs by both chemical reactions and reactions catalyzed by enzymes, represents the intracellular lipid peroxidation level [34]. Our study also confirmed that OA was able to limit intracellular lipid peroxidation levels induced by H 2 O 2 . Our previous study found that renalase is highly sensitive and responsive to oxidative stress in vitro and in vivo [35]. In this study, we found OA decreased the expression of renalase induced by H 2 O 2 . Previous studies have confirmed that OA is effective in the prevention of diverse types of digestive disorders such as inflammatory bowel disease and cardiovascular disease due to its antioxidant capacity [36][37][38]. The antioxidant effect of OA in liver I/R injury is consistent with the existing literature. The nuclear transcription of NF-κB is activated via AKT signaling pathway under oxidative stress [39], which is independent of PI3K phosphorylation. The activation of these signaling pathways leads to the production of various proinflammatory mediators, such as TNF-α, IL-1β, IL-6, induced nitric oxide synthase (iNOS), and cyclooxygense-2 (COX-2), which are capable of amplifying the process of inflammation [40]. NF-κB is a highly inducible transcription factor that plays an important role in the hepatic acute phase response, innate/adaptive immunity, and cellular survival through the induction of genetic networks [41,42]. RELA, also known as p65, is a REL-associated protein involved in NF-κB heterodimer formation, nuclear translocation, and activation. In addition, phosphorylation of AKT (Ser473) is associated with p65 [43]. We identified an increase in nuclear protein level of p65 under oxidative stress conditions, and OA significantly inhibited this process. It is worth mentioning that in the cytoplasm, we did not detect changes in protein expression level of p65. However, at the mRNA level, we found that OA can significantly reduce the mRNA transcription level of p65. This may be due to the certain time lag between protein expression and mRNA expression. Combining previous reports with our research results, we hypothesize that OA reduces nuclear transcription of p65 by inhibiting phosphorylation of AKT, thereby reducing inflammatory factor expression. Additionally, oxidative stress-induced ROS and inflammatory factors lead to apoptosis, which is highly associated with AKT phosphorylation. OA is able to clear ROS and downregulate the expression of inflammation factors by blocking AKT phosphorylation in an unknown manner, thus protecting cells from apoptosis.
Autophagy is a biological process in which macromolecules and damaged organelles in the cytoplasm are degraded [32]. It is a self-degrading system that keeps normal cells in a homeostatic environment. Autophagy-associated cell death is considered an important mechanism for nonapoptotic cell death [44]. However, the role of autophagy in liver I/R injury remains controversial. Moderate autophagy levels are beneficial for hepatocytes to maintain homeostasis, but enhanced autophagy may further aggravate the damage [45,46]. In either case, autophagy is closely related to liver I/R injury. Autophagy is regulated by autophagy-related genes (ATG) and can be induced by a variety of factors. LC3-II is produced during autophagy and thus can be used as an autophagosomal marker [47,48]. The insulin-like growth factor I-(IGF-I-) AKT-mTOR pathway (IIS) is involved in multiple bioprocesses like aging, longevity, and cell survival/death signaling [49]. IIS integrates a wide array of metabolic signals, cross-talk with p53, NF-κB, or ROS, and influences gene expression to shape the cellular metabolic profile and stress resistance [49][50][51][52][53]. mTOR is located downstream of PI3K-AKT signaling, regulates cell growth, and inhibits the initial process of autophagy [45,46,54]. In this study, we identified for the first time that OA reduces the expression of p-mTOR protein by inhibiting the phosphorylation of AKT, thereby abolishing the inhibition of autophagy by mTORC1 and thus promoting autophagy. This finding is consistent with the findings of Qin et al. and provides a rationale for a novel therapeutic strategy for managing liver I/R injury [54].
The mechanism of OA inhibiting the phosphorylation of AKT remains unclear. Activation of PI3K by extracellular stimuli results in activation of AKT in almost all cells and tissues. PI3K and its lipid products are generally considered to be obligate and rate limiting for proper AKT activation [55]. However, our study demonstrated that OA blocks the AKT phosphorylation in a PI3K-independent manner. This may be due to the increase in p-PI3K level when OA was introduced (Fig. S4), while p-AKT level was suppressed. The phosphorylation level of AKT is positively correlated with the number of PIP3, which is mainly phosphorylated by PIP 2 in the cell and nuclear membranes [55,56]. The molecular structure of OA has great similarity with the lipid part of PI-4,5-P 2 and PI-3,4,5-P 3 , which means it is likely to competitively combine with AKT, thereby inhibiting its phosphorylation. We will explore its mechanism in depth in a subsequent study.

Conclusions
In this study, we found that UDCA-LPE can significantly increase the ratio of OA/PA in the liver, which may account for its protective effect against the liver I/R injury. OA supplementation demonstrated the alleviation of mouse liver I/R injury. A relatively low dose of OA protects against oxidative stress, inflammation, and apoptosis and enhances autophagy in HepG2 cells. Furthermore, OA supplementation reversed I/R-induced hepatocyte death. As a MUFA, OA can eliminate ROS and MDA produced during hepatic I/R injury and suppress the expression of inflammatory factors by inhibiting nuclear transcription of p65 by suppressing AKT phosphorylation. In addition, OA induces the enhancement of autophagy by suppression of AKT/mTOR pathways, thus protecting cells from oxidative stress. Considering all these positive effects, OA supplementation represents a potential suitable therapeutic strategy preventing liver I/R injury. It is reasonable to expect that there will continue to be many important mechanistic and medical insights regarding lipid metabolism, which could lead to potentially beneficial, novel therapeutic strategies to many patients.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.