|Year : 2023 | Volume
| Issue : 4 | Page : 239-247
Pachymic acid protects hepatic cells against oxygen-glucose deprivation/reperfusion injury by activating sirtuin 1 to inhibit HMGB1 acetylation and inflammatory signaling
Chengbiao Xue1, Zhigao Xu1, Zhongzhong Liu1, Cheng Zeng1, Qifa Ye2
1 Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, National Quality Control Center for Donated Organ Procurement, Hubei Key Laboratory of Medical Technology on Transplantation, Hubei Clinical Research Center for Natural Polymer Biological Liver, Hubei Engineering Center of Natural Polymer-based Medical Materials, Wuhan, Hubei, China
2 Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, National Quality Control Center for Donated Organ Procurement, Hubei Key Laboratory of Medical Technology on Transplantation, Hubei Clinical Research Center for Natural Polymer Biological Liver, Hubei Engineering Center of Natural Polymer-based Medical Materials, Wuhan, Hubei; Research Center of National Health Ministry on Transplantation Medicine Engineering and Technology, The 3rd Xiangya Hospital of Central South University, Changsha, Hunan, China
|Date of Submission||03-Nov-2022|
|Date of Decision||07-Apr-2023|
|Date of Acceptance||17-Apr-2023|
|Date of Web Publication||22-Jun-2023|
Dr. Qifa Ye
Zhongnan Hospital of Wuhan University, No. 169, Donghu Road, Wuchang, Wuhan, Hubei 430071
Source of Support: None, Conflict of Interest: None
Ischemia-reperfusion injury is an important cause of liver injury occurring during liver transplantation. It is usually caused by inflammatory response and oxidative stress-induced oxidative damage. Pachymic acid (PA) has various biological activities such as anti-inflammatory, antioxidant and anti-cancer. However, the action mechanism of PA in hepatic ischemia-reperfusion injury is currently unknown. In this study, liver cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) to simulate a hepatic ischemia-reperfusion injury model. The binding relationship between PA and sirtuin 1 (SIRT1) was analyzed by molecular docking. Cell viability was detected by Cell Counting Kit-8. Expression levels of SIRT1 and high mobility group box 1 (HMGB1) were detected by western blot. Subsequent levels of inflammatory factors were detected by related kits and western blot. Meanwhile, related kits were used to examine levels of oxidative stress markers including reactive oxygen species, malondialdehyde, superoxide dismutase and cytotoxicity-associated lactate dehydrogenase. Finally, cell apoptosis was detected by flow cytometry and western blot. The results showed that PA significantly ameliorated OGD/R-induced decrease in SIRT1 expression, increase in HMGB1 acetylation and HMGB1 translocation. Moreover, the elevated levels of inflammatory factors, oxidative stress indexes and cell apoptosis upon exposure to OGD/R were reversed by PA treatment. Moreover, the addition of SIRT1 agonist and inhibitor further demonstrated that PA exerted the aforementioned effects in OGD/R-exposed cells by targeting SIRT1. Thus, the present study revealed the mechanism by which PA ameliorated OGD/R-induced hepatic injury via SIRT1. These results might provide a clearer theoretical basis for the targeted treatment of OGD/R-induced hepatic injury with PA.
Keywords: HMGB1 acetylation, liver injury, oxygen-glucose deprivation/reperfusion, pachymic acid
|How to cite this article:|
Xue C, Xu Z, Liu Z, Zeng C, Ye Q. Pachymic acid protects hepatic cells against oxygen-glucose deprivation/reperfusion injury by activating sirtuin 1 to inhibit HMGB1 acetylation and inflammatory signaling. Chin J Physiol 2023;66:239-47
|How to cite this URL:|
Xue C, Xu Z, Liu Z, Zeng C, Ye Q. Pachymic acid protects hepatic cells against oxygen-glucose deprivation/reperfusion injury by activating sirtuin 1 to inhibit HMGB1 acetylation and inflammatory signaling. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:239-47. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/239/379404
Chengbiao Xue and Zhigao Xu contributed equally to the study.
| Introduction|| |
Ischemia-reperfusion injury is an important cause of liver injury that occurs during liver transplantation and is a potential cause of posttransplant graft dysfunction. It usually results from inflammatory response and oxidative damage. Pachymic acid (PA), a lanostane-type triterpene found in Poria cocos, has various biological activities including anti-inflammatory, antioxidant, and anti-cancer., The research has found that PA attenuates sepsis-induced acute kidney injury and has anti-inflammatory and antioxidant effects. In addition, PA attenuates acute lung injury in septic mice and protects against brain ischemia/reperfusion injury via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway., Although PA has been reported to exhibit positive anti-inflammatory effects, its mechanism of action in hepatic ischemia/reperfusion injury has not been investigated.
High mobility group box 1 (HMGB1) is an important inflammatory mediator that plays an important role in liver diseases, including ischemia-reperfusion injury, liver transplantation, viral hepatitis, liver fibrosis and liver cancer.,, It has been found that HMGB1 released by damaged hepatocytes can increase the inflammatory response and affect the progression of liver diseases. Moreover, the transfer of HMGB1 from the nucleus to the cytoplasm may be affected by post-translational modifications, including acetylation, methylation and phosphorylation., Of interest, sirtuin 1 (SIRT1)-mediated HMGB1 deacetylation has been found to attenuate the inflammatory response.,
Since HMGB1 is a key protein mediating the pathogenesis of chronic and acute liver disease, the purpose of the present study is to investigate whether PA ameliorates oxygen-glucose deprivation/reperfusion (OGD/R)-evoked hepatocyte injury through SIRT1-mediated HMGB1 deacetylation by analyzing the levels of inflammation, oxidative stress, and apoptosis in OGD/R-injured hepatocytes.
| Materials and Methods|| |
Cell culture and OGD/R model
Immortalized human hepatocyte MIHA (Accession No. CVCL_SA11) was purchased from Hunan Fenghui Biotechnology Co., Ltd. MIHA cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution and placed in a humidified incubator with 5% CO2 at 37°C. Glucose-free DMEM medium was used instead of normal medium. Cells were transferred to a hypoxic incubator (1% O2, 5% CO2, 94% N2) at 37°C and received OGD treatment for 4 h. PA (white crystalline powder) was purchased from Merck (CAS. 29070-92-6) and dissolved in dimethyl sulfoxide at 40 mM and stored away from light at -20°C. To investigate the effect of PA, cells were treated with 80 μM PA for 2 h before OGD treatment. Following OGD treatment, cells were incubated in an incubator containing 5% CO2 at 37°C for 6 h. Then, subsequent experiments were performed. In addition, SIRT1 inhibitor EX527 (1 μM) and SIRT1 agonist SRT1720 (1 μM) were obtained from Merck in reference to the available report.
Cell viability was measured with Cell Counting Kit-8 (CCK-8) (Solarbio Biotechnology Co., Ltd.). After cells were inoculated into 96-well plates, they were pretreated with PA at concentrations of 20, 40 and 80 μM for 2 h. The drug stock solution was diluted with DMEM medium. Cells in the control group were cultured with blank culture medium. Subsequently, OGD/R exposure was performed. When OGD/R model was successfully constructed, 10 μl CCK-8 solution was added to each well and incubated continuously for 4 h. The absorbance was measured using an enzyme marker (Thermo Fisher Scientific, Inc.).
Western blotting analysis
Cells were harvested, and nuclear-cytoplasmic fractionation was conducted using the nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology Inc.) according to the manufacturer's instructions. The protein concentrations were detected by using the bicinchoninic acid protein assay kit. Total proteins were separated by electrophoresis on 8%–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4°C with the corresponding primary antibodies. After washing, the membranes were incubated with the secondary antibody for 1 h at room temperature. After washing, the blots were visualized by enhanced chemiluminescence. The intensity of the bands was quantified using ImageJ software. The primary antibodies used and the dilution rates were as follows: Anti-SIRT1 (1:2000, Proteintech™), anti-HMGB1 (1:1000, Proteintech™), anti-acetylated (Ac)-HMGB1 (1:1000, Abcepta), anti-phosphorylated (p)-nuclear factor-kappaB (NF-κB) p65 (1:1000, Sigma-Aldrich), anti-NF-κB p65 (1:1000, Sigma-Aldrich), anti-B-cell lymphoma 2 (Bcl2; 1:2000, Abcam), anti-BCL-2 associated X (Bax; 1:1000, Abcam), anti-cleaved-caspase 3 (1:5000, Abcam), anti-caspase 3 (1:2000, Abcam), anti-Histone H3 (1:5000, Proteintech™), anti-beta-actin (β-actin; 1:1000, Proteintech™).
Analysis of inflammatory factors and oxidative stress indexes
Cells are inoculated on 6-well plates to 80%–90% confluence. The cell culture supernatant was collected at 4°C following 3000 × g centrifugation for 5 min to remove debris. The expression levels of inflammatory factors tumor necrosis factor-alpha (TNF-α; Cat. No. SEKH-0047), interleukin-1beta (IL-1β; Cat. No. SEKH-0002) and IL-6 (Cat. No. SEKH-0013) were detected by enzyme-linked immunosorbent assay kits (Beijing Solarbio Science Technology Inc.). The expression levels of reactive oxygen species (ROS; Cat. No. E004-1-1), malondialdehyde (MDA; Cat. No. A003-1-2), lactate dehydrogenase (LDH; Cat. No. A020-2-2), and superoxide dismutase (SOD; Cat. No. A001-3-2) were measured using the corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute). It should be noted that when the cell plasma membrane ruptures, the cytosolic LDH will be released into the culture medium. Therefore, LDH is also often used as an index for cell death.
Cell apoptosis analysis
Cell apoptosis levels in different groups were analyzed using flow cytometry. Cells were inoculated on 6-well plates, which were subsequently subjected to indicated treatment. After 24 h of incubation, cells were treated with Dead Cell Apoptosis Kit with Annexin V FITC and propidium iodide (PI; Invitrogen™, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, followed by the adoption of flow cytometry to detect apoptosis in each group.
Molecular docking analysis
The SIRT1 structure (PDBID: 5BTR) used for the docking study was obtained from the RCSB PDB web page (https://www.rcsb.org/) and cleaned in the PyMOL software (v2.2), retaining the protein structure only. The structure of PA was imported into OpenBabel software (v2.4) for hydrogenation. Subsequently, molecular docking was run in AutoDock (v4.2) and docking energy values were displayed.
All experiments in this study were repeated three times, and data are expressed as mean ± standard error. Data were statistically analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) software and compared using one-way analysis of variance and Tukey's post hoc test. P < 0.05 was considered statistically significant.
| Results|| |
Pachymic acid pretreatment activates sirtuin 1 to inhibit HMGB1 acetylation in OGD/R-exposed cells
To evaluate the role of PA in SIRT1-mediated HMGB1 deacetylation in OGD/R-exposed cells, normal hepatocytes were firstly treated with different concentrations of PA and then cell viability was examined using CCK-8. And SIRT1 and HMGB1 protein expression were detected by western blotting. No significant changes in hepatocyte viability were observed following incubation of 20–80 μM PA for 12–24 h [Figure 1]a. Correlation between PA and SIRT1 was verified by molecular docking. Multiple intermolecular forces were noticed between PA ligand and SIRT1 receptor that stabilized them together. Results showed that PA shared the H-bonds to ILE-210 from SIRT1 and other ligand parts bound via hydrophobic contacts [Figure 1]b. SIRT1 and HMGB1 protein levels were measured. OGD/R induction inhibited SIRT1 protein level and promoted HMGB1 acetylation as compared to the control group. Moreover, following pretreatment with PA, the protein level of SIRT1 was significantly increased and the acetylated HMGB1 expression level was significantly decreased compared with the OGD/R group, and dose-dependent changes in these protein levels were shown following PA treatment [Figure 1]c, [Figure 1]d, [Figure 1]e. Similarly, OGD/R induction decreased the expression level of HMGB1 in the nucleus and increased the expression level of HMGB1 in the cytoplasm in comparison to the control group. HMGB1 expression in the nucleus was increased and HMGB1 expression in the cytoplasm was decreased with increasing PA dosage as compared to OGD/R group [Figure 1]f, [Figure 1]g, [Figure 1]h. These indicated that PA could potentially inhibit HMGB1 acetylation by activating SIRT1.
|Figure 1: PA pretreatment activated SIRT1 to inhibit HMGB1 acetylation in OGD/R-exposed cells. (a) Cell viability was detected by CCK-8. (b) The correlation between PA and SIRT1 was verified by molecular docking. (c) HMGB1 acetylation and SIRT1 expression levels detected by western blot. (d) Quantification of SIRT1 expression. (e) Quantification of HMGB1 acetylation. (f) HMGB1 expression levels in the nucleus and cytoplasm were detected by western blot. (g) Quantification of HMGB1 expression in the nucleus. (h) Quantification of HMGB1 expression in the cytoplasm. The experiments were performed in triplicate. N = 3–5. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to Control; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to OGD/R. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion, HMGB1: High mobility group box 1, CCK-8: Cell counting Kit-8.|
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Pachymic acid pretreatment activates sirtuin 1 to improve the viability of hepatocytes following OGD/R injury
To validate that PA modulated HMGB1 acetylation via activating SIRT1, further studies were conducted using EX527 as a SIRT1 inhibitor and SRT1720 as a SIRT1 activator. Cell viability was significantly increased in OGD/R-exposed cells pretreated by 80 μM PA versus OGD/R group. In the 80 μM PA + OGD/R group, the addition of SIRT1 inhibitor (EX527) diminished cell viability and the addition of SIRT1 agonist (SRT1720) increased cell viability [Figure 2]a. Subsequently, changes in SIRT1/HMGB1 signaling were investigated by detecting the levels of related proteins. Compared to the 80 μM PA + OGD/R group, EX527 significantly inhibited SIRT1 expression and significantly increased HMGB1 acetylation levels. Similarly, SRT1720 activated SIRT1 expression and further inhibited HMGB1 acetylation in comparison to the 80 μM PA + OGD/R group [Figure 2]b, [Figure 2]c, [Figure 2]d. Besides, in the 80 μM PA + OGD/R + EX527 group, HMGB1 expression level in the nucleus was significantly decreased and HMGB1 expression in the cytoplasm was significantly increased by EX527. In contrast, in the 80 μM PA + OGD/R + SRT1720 group, HMGB1 expression level in the nucleus was increased, while HMGB1 expression in the cytoplasm was significantly decreased [Figure 2]e, [Figure 2]f, [Figure 2]g. Overall, activation of SIRT1 by PA could inhibit HMGB1 acetylation.
|Figure 2: PA pretreatment activated SIRT1 to improve the viability of hepatocytes following OGD/R injury. (a) Cell viability was detected by CCK-8. (b) HMGB1 acetylation and SIRT1 expression levels detected by western blot. (c) Quantification of SIRT1 expression. (d) Quantification of HMGB1 acetylation. (e) HMGB1 expression levels in the nucleus and cytoplasm were detected by western blot. (f) Quantification of HMGB1 expression in the nucleus. (g) Quantification of HMGB1 expression in the cytoplasm. The experiments were performed in triplicate. N = 3–5. ***P < 0.001 compared to Control; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to OGD/R; &P < 0.05, &&P < 0.01 and &&&P < 0.001 compared to OGD/R + 80 μM PA. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion, HMGB1: High mobility group box 1, CCK-8: Cell counting Kit-8.|
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Pachymic acid pretreatment activates sirtuin 1 to attenuate inflammation and oxidative stress in OGD/R-induced hepatocytes
To confirm the impacts of PA on inflammatory response and oxidative stress in OGD/R-exposed hepatocytes via activating SIRT1, inflammatory factors and oxidative stress-related indicators were investigated. The results showed that the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 were significantly increased after OGD/R induction compared with the control group; while the expression levels of these factors were decreased in a dose-dependent manner by the pretreatment with increasing doses of PA compared with the OGD/R group. In contrast to OGD/R-treated cells pretreated by 80 μM PA, EX527 increased SIRT1 expression and inhibited the expression of inflammatory factors, while SRT1720 suppressed the expression levels of inflammatory factors and activated SIRT1 expression [Figure 3]a, [Figure 3]b, [Figure 3]c. Besides, the changes in the inflammatory signal showed the same trend as those in inflammatory factors. Briefly, OGD/R treatment increased p-p65/p65 expression, which was then inhibited by PA pre-treatment. Furthermore, EX527 again elevated p-p65/p65 expression in PA-pretreated cells exposed to OGD/R and SRT1720 further declined p-p65/p65 expression in PA-pretreated cells exposed to OGD/R [Figure 3]d and [Figure 3]e.
|Figure 3: PA pretreatment activated SIRT1 to attenuate inflammation in OGD/R-induced hepatocytes. (a) TNF-α, (b) IL-1β and (c) IL-6 levels were measured by relevant kits. (d) NF-κB expression was detected by western blot. (e) Quantification of NF-κB expression. The experiments were performed in triplicate. N = 3–5. ***P < 0.001 compared to Control; ##P < 0.01 and ###P < 0.001 compared to OGD/R; &P < 0.05, &&P < 0.01 and &&&P < 0.001 compared to OGD/R + 80 μM PA. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin, NF-κB: Nuclear factor-kappaB.|
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In regard to detection of oxidative stress, ROS level was significantly increased after OGD/R interference compared to the control group. However, the presence of PA significantly reduced ROS level compared to the OGD/R group. SRT1720 further lessened ROS level in PA-pretreated cells upon exposure to OGD/R, while EX527 raised ROS level in PA-pretreated cells upon exposure to OGD/R [Figure 4]a. Likewise, the trends of the effects of PA, EX527 and SRT1720 on the changes of MDA and LDH levels in OGD/R-exposed cells were quite consistent with the trends on ROS level [Figure 4]b and [Figure 4]c. Additionally, SOD level was significantly decreased after OGD/R injury. After the addition of PA, SOD level was significantly back-regulated, and SRT1720 enhanced SOD level again, while EX527 abolished the stimulated SOD level imposed by PA [Figure 4]d. These results demonstrated that PA could reduce OGD/R-induced inflammatory response and oxidative stress by activating SIRT1.
|Figure 4: PA pretreatment activated SIRT1 to attenuate oxidative stress in OGD/R-induced hepatocytes. (a) ROS level was measured by the kit. The levels of (b) MDA, (c) LDH and (d) SOD were measured by related kits. The experiments were performed in triplicate. N = 3–5. ***P < 0.001 compared to Control; ###P < 0.001 compared to OGD/R; &P < 0.05 and &&&P < 0.001 compared to OGD/R + 80 μM PA. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion, ROS: reactive oxygen species, MDA: Malondialdehyde, LDH: Lactate dehydrogenase, SOD: Superoxide dismutase.|
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Pachymic acid pretreatment activates sirtuin 1 to reduce OGD/R-induced hepatocyte apoptosis
In succession to the above studies, the effects of PA on OGD/R-exposed hepatocyte apoptosis through targeting SIRT1 were also explored. Cell apoptosis was detected by flow cytometry and the expression levels of apoptosis-related proteins were assayed. Quantification of cell apoptosis indicated that apoptosis was significantly increased after OGD/R injury compared to the control group. In contrast, the rate of apoptosis was reduced in a dose-dependent manner by PA treatment versus the OGD/R group. Also, SRT1720 could further inhibit OGD/R-stimulated apoptosis in the presence of PA. However, EX527 evidently reversed the inhibitory effect of PA on cell apoptosis in OGD/R + 80 μM PA + EX527 group versus OGD/R + 80 μM PA group [Figure 5]a and [Figure 5]b. The expression level of Bcl-2 was inhibited and the expression levels of Bax and cleaved-caspase 3/caspase 3 were increased in the OGD/R group compared with the control group. With the increase of PA dosage, these protein expression levels in the OGD/R group were significantly back-regulated. Meanwhile, in PA-pretreated cells subjected to OGD/R, SRT1720 further promoted Bcl-2 expression and inhibited Bax and cleaved-caspase 3/caspase 3 expression, while the appearance of EX527 suppressed Bcl-2 expression and elevated Bax and cleaved-caspase 3/caspase 3 expression [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d. The results also demonstrated that SIRT1 might be involved in the protective role of PA against OGD/R-induced hepatocyte injury.
|Figure 5: PA pretreatment activated SIRT1 to reduce OGD/R-induced hepatocyte apoptosis. (a) Cell apoptosis was detected by flow cytometry. (b) Quantification of cell apoptotic rate. The experiments were performed in triplicate. N = 3–5. ***P < 0.001 compared to Control; ###P < 0.001 compared to OGD/R; &&&P < 0.001 compared to OGD/R + 80 μM PA. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion.|
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|Figure 6: PA pretreatment activated SIRT1 and reversed OGD/R-induced alternations in the levels of apoptosis-related proteins in hepatocytes. (a) The expression levels of Bcl-2, Bax, cleaved-caspase 3/caspase 3 were detected by western blot. (b) Quantification of Bcl2 expression. (c) Quantification of Bax expression. (d) Quantification of cleaved-caspase 3/caspase 3 expression. The experiments were performed in triplicate. N = 3–5. ***P < 0.001 compared to Control; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to OGD/R; &P < 0.05 and &&&P < 0.001 compared to OGD/R + 80 μM PA. PA: Pachymic acid, SIRT1: Sirtuin 1, OGD/R: Oxygen-glucose deprivation/reperfusion.|
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| Discussion|| |
Ischemia-reperfusion injury is a critical condition with severe clinical manifestations, including brain dysfunction, acute heart failure, gastrointestinal dysfunction, systemic inflammatory response syndrome, and multi-organ dysfunction syndrome.,, In hepatic transplantation, when the blood supply returns after ischemia, the liver is prone to further injury, aggravating the damage caused by ischemia and eventually leading to microcirculatory failure. Therefore, studying the mechanism of liver ischemia-reperfusion injury may be of great significance in protecting against tissue damage. PA has excellent anti-inflammatory and antioxidant effects., Meanwhile, inflammatory and oxidative damage are the main causes of hepatic ischemia-reperfusion injury. However, studies concerning the mechanism of PA in hepatic ischemia-reperfusion injury have not been reported. In the present study, the potential mechanisms of PA in liver ischemia-reperfusion injury were investigated in OGD/R-induced cell model.
Of interest, the finding in the previous report has uncovered that PA attenuates injury and apoptosis in ischemia-reperfusion. Similarly, through estimating cell apoptosis in different groups, the present study revealed that OGD/R induction increased apoptosis, while PA pretreatment markedly reduced the apoptosis rate. Also, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase 3/caspase 3 were examined and it was discovered that OGD/R exposure inhibited Bcl-2 expression and increased Bax and cleaved-caspase 3/caspase 3 expression. PA treatment raised Bcl-2 expression while suppressed Bax and cleaved-caspase 3/caspase 3 expression in OGD/R-treated hepatocytes.
SIRT1 is a nicotinamide adenine dinucleotide-dependent deacetylase that is mainly involved in cellular metabolism, stress response and aging., Recent studies have demonstrated that ischemia-reperfusion can affect the expression level of SIRT1., In the same way, in this study, a significant inhibition of SIRT1 expression levels was detected after OGD/R interference. Intriguingly, the correlation between PA and SIRT1 was successfully verified by molecular docking analysis and their stable binding was demonstrated. The expression level of SIRT1 in OGD/R cells was significantly upregulated with increasing PA concentrations.
HMGB1 is the deacetylation target of SIRT1. During the process of inflammation, acetylated HMGB1 released from stress cells are translocated from nucleus to cytoplasm, and activates NF-κB signaling., In our study, the down-regulated SIRT1 expression imposed by OGD/R induction was accompanied by a significant increase in acetylated HMGB1 expression and the stimulated translocation of HMGB1 from nucleus to cytoplasm. In contrast, when cells were pretreated with PA, HMGB1 acetylation expression and translocation from nucleus to cytoplasm were inhibited with increasing doses of PA. In addition, the SIRT1 inhibitor EX527 showed an inhibitory effect on the functions of PA, while SIRT1 agonist SRT1720 further enhanced the effects of PA. Thus, it was evident that PA exerted its effect on HMGB1 through regulation of SIRT1.
Through detection of levels of inflammatory factors TNF-α, IL-1β, and IL-6, PA was found to significantly inhibit OGD/R-induced inflammatory response, as well as to inhibit the p-p65/p65 expression. This was consistent with the previously reported anti-inflammatory effects of PA through inhibition of inflammatory signaling., EX527 and SRT1720 were used to modulate the SIRT1 expression in hepatocytes. EX527 has been widely used as a selective SIRT1 inhibitor and was > 200 times more selective for SIRT1 than SIRT2 and SIRT3. SRT1720 is a selective activator of human SIRT1 and shows less potent activities for SIRT2 and SIRT3, and has been reported to be 1000 fold more potent than resveratrol. The regulatory effects of EX527 and SRT1720 on inflammatory factors and signaling demonstrated that PA functioned by promoting the expression of SIRT1. On the other hand, the detection of oxidative stress-related indicators revealed that PA also played an antioxidant role in OGD/R-induced hepatocytes, which was consistent with the inhibition of MDA and activation of SOD expression due to PA in acute lung injury., Notably, the protective effect of PA on OGD/R-evoked oxidative stress was significantly altered by the addition of EX527 or SRT1720, suggesting that PA protected against OGD/R-induced oxidative stress by targeting SIRT1 in hepatocytes.
| Conclusion|| |
Collectively, this study revealed the mechanism by which PA ameliorated OGD/R-induced hepatocyte injury via SIRT1. It was noticed that PA activated SIRT1 to inhibited HMGB1 acetylation, attenuated the inflammatory response and oxidative stress, and suppressed cell apoptosis. These results might provide a more clarified theoretical basis for treatment of OGD/R-induced hepatocyte injury with PA.
This article does not contain any studies with human participants or animals performed by any of the authors.
Financial support and sponsorship
This project was supported by Hubei Provincial Natural Science Foundation of China (Grant No. 2020CFB652) and Wuhan University Zhongnan Hospital medical science and technology innovation platform support project (Grant No. PTXM2020029).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]