2018 Volume 41 Issue 2 Pages 229-238
Maintaining a certain level of hydrogen sulfide (H2S) in ischemia–reperfusion (I/R) is essential for limiting injury to the liver. Exogenous H2S exerts protective effects against this injury, but the mechanisms remain unclear. Liver injury was induced in Wistar rats undergoing hepatic I/R for 30 min, followed by a 3-h reperfusion. Administration of GYY4137 (a slow-releasing H2S donor) significantly attenuated the severity of liver injury and was reflected by reduced inflammatory cytokine production and cell apoptosis, the levels of which were elevated by I/R, while DL-propargylglycine (PAG, an inhibitor of cystathionine γ-lyase [CSE]) aggravated liver injury. Delivery of GYY4137 significantly elevated the plasma levels of H2S and upregulated the expression of microRNA-21 (miR-21), leading to the activation of the Akt pathway, in rat livers subjected to I/R. To further investigate the protective mechanisms of H2S during liver I/R injury, we established a cell model of hypoxia/reoxygenation (H/R) by incubating Buffalo rat liver (BRL) cells under hypoxia for 4 h followed by normoxia for 10 h. The regulatory effect of miR-21 on the Akt pathway by downregulating phosphatase and tensin homolog (PTEN) was validated by luciferase assays. Incubation of sodium hydrosulfide (NaHS), an H2S donor, increased the expression of miR-21, attenuated the reduced cell viability and the increased apoptosis by H/R, in BRL cells. Anti-miR-21 abolished the protective effects of NaHS by inactivating the Akt pathway. In conclusion, the present results indicate the activation of the Akt pathway regulated by miR-21 participates in the protective effects of H2S against I/R-induced liver injury.
Liver ischemia–reperfusion (I/R) is very common during major liver surgeries, hepatic trauma and circulatory shock, and often causes damage to the liver by inducing the generation of reactive oxygen species and the release of pro-inflammatory cytokines.1) Thus, exploring the underlying mechanisms and seeking effective agents remain an urgent priority for coping with this damaging process.
As a gaseous mediator, hydrogen sulfide (H2S) has been recognized as a crucial signaling molecule with a wide range of physiological functions.2) In mammalian cells, H2S is produced enzymatically through the reverse trans-sulfuration mainly by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which utilize the substrates homocysteine and L-cysteine.2) Recently the biological importance of H2S has shed light on the pathogenesis of various human diseases, paving the avenue for therapeutic interventions.2) H2S demonstrates a potential role in preserving function and limiting injury in multiple organs induced by I/R.3–8) We have previously reported that administration of sodium hydrosulfide (NaHS), a donor of H2S, attenuated the severity of liver injury in a rat model of hepatic I/R.9) However, the underlying mechanisms accounting for the activities of H2S against hepatic I/R-induced injury remain unclear.
Several studies suggest that H2S may activate the Akt pathway,5,8) which has been previously demonstrated to participate in the mechanisms of hepatic I/R-induced injury.10,11) On the other hand, microRNA-21 (miR-21) serves as an activator for the Akt pathway by regulating the expression of phosphatase and tensin homolog (PTEN),12,13) and also exhibits anti-inflammatory and anti-apoptotic effects.14,15) Nevertheless, the role of miR-21 in mediating the protective effects of H2S in hepatic I/R-induced injury is unknown.
Based on the above compelling findings, we hypothesized that H2S may protect against liver I/R-induced injury by activating the Akt pathway through a miR-21-dependent mechanism. We, therefore, addressed this question by utilizing a warm liver I/R model in rats, in which GYY4137 [a water-soluble and slow-releasing H2S donor16)] and DL-propargylglycine (PAG) [an irreversible inhibitor of CSE6,9,17)] were applied. We also examined the regulatory participation of miR-21 on the protective activity of H2S in a cell model of hypoxia/reoxygenation (H/R).
Male Wistar rats (200–250 g) were supplied by the Animal Research Center of Harbin Medical University, China. This study was approved by the Animal Ethics Committee of Harbin Medical University, in compliance with Experimental Animal Regulations by the National Science and Technology Commission in China (permit SYXK20020009). Forty-eight rats were randomly assigned into one group of sham operation and three liver I/R groups (each group had 12 rats). Sham-operated rats and rats in one group of I/R received an injection of 1.0 mL of normal saline. Rats in the other two I/R groups received an injection of GYY4137 (133 µmol/kg) or PAG (50 mg/kg) (Sigma-Aldrich, Shanghai, China), dissolved in 0.2 mL of normal saline.6,9,16) A group of 6 healthy untreated rats and two groups of 6 sham-operated rats receiving an injection of GYY4137 or PAG as above were included in this study as controls. Saline, GYY4137 and PAG were administered intraperitoneally 1 h prior to the commencement of surgery.
Surgical Procedures and SamplingAll surgical procedures were performed under aseptic conditions as previously reported.9–11,18) Briefly, rats underwent a midline laparotomy under general anesthesia by continuous isoflurane inhalation. The portal triad was isolated and clamped, and then the abdominal cavity was temporally closed with metal clips. The clips were removed 30 min later, and reperfusion was initiated. The abdominal wall was closed with a continuous suture, and reperfusion was allowed for 3 h. In preliminary experiments, we found that damage to the livers induced by I/R reached a peak at 3 h and then declined in this model. Thus a 3-h reperfusion was chosen for the formal experiments. Sham-operated rats underwent the same surgical procedures except for clamping the portal triad.
At the end of reperfusion, a median sternotomy was performed. Blood samples were collected from the right ventricle, and centrifuged at 1000×g for 10 min, and the plasma decanted and stored at −80°C. Livers were harvested and divided into two halves. The left half was fixed in 10% buffered formalin, while the right half was snap-frozen in liquid nitrogen and stored at −80°C.
In situ Hybridization for Detecting miR-21Double digoxigenin (DIG)-labeled locked nucleic acid microRNA probes for rno-miR-21 (TCA ACA TCA GTC TGA TAA GCT A, RNA-Tm 84°C) and a scrambled sequence (GTG TAA CAC GTC TAT ACG CCC A, RNA-Tm 87°C) as a negative control (Exiqon, Vedbaek, Denmark) were used for in situ hybridization of liver sections according to the manufacturer’s manual. Formalin-fixed liver tissues were paraffin embedded, sectioned (5 µm) and mounted on SuperFrost slides. Slides were deparaffinized, and then incubated with Proteinase-K (15 µg/mL) for 10 min at 37°C. Slides were washed twice in phosphate buffered saline (PBS), dehydrated in ethanol, and hybridized with hybridization mix (1 : 500 dilution of probe) for 1 h at 49°C. They were then washed in standard saline citrate buffer and blocked. Positive signals were developed with an anti-DIG primary mouse monoclonal antibody, followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody.
Cell Culture and H/R Cell ModelBuffalo rat liver (BRL) cells were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, U.S.A.) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 units/mL) in a humidified incubator containing 5% (v/v) CO2. The in vitro H/R cell model was induced by culturing cells under hypoxia (1% O2, 5% CO2, and 94% N2) for 4 h, followed by culturing them under normoxia for 10 h.11)
Transfection of OligonucleotidesMiR-21 mimics (5′-AAC AUCAGUCUGAUAAGCUAUU-3′), anti-miR-21 (5′-UCAACAUCAGUCUGAUAAGCUA-3′) and the negative control oligonucleotides (5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from GenePharma Co., Ltd., Shanghai, China). Cells were grown to 60–70% confluence, and incubated with oligonucleotides at a final concentration of 0.1 µM by using Lipofectamine™ 2000 (Invitrogen, Beijing, China) in a serum-free medium for 48 h and then subjected to assays.
Caspase-3 Activity AssayCells were lysed and 100 µg of lysates were incubated with caspase-3 colorimetric Aspartate-Glutamate-Valine-Aspartate-p-nitroaniline (DEVD-pNA) substrate at 37°C. The resulting colorimetric product was measured with a microplate spectrophotometer at 402 nm according to the instructions, supplied in the caspase-3 activity kit (ab39401, Abcam, Shanghai, China). Caspase-3 activity was calculated as a fold change in comparison to cells without treatment. All conditions were run in duplicate and three independent experiments were performed.
Luciferase Reporter AssayTo evaluate the function of miR-21, the 3′UTR of PTEN with a miR-21 targeting sequence was cloned into a pMIR-REPORT luciferase reporter vector (Ambion). The assay was performed as described previously.19) Briefly, the reporter vector plasmid or a luciferase reporter vector without miR-21 target was transfected into cells using Lipofectamine 2000. Luciferase activities in cells were measured by using a luciferase assay kit (Promega, Madison, WI, U.S.A.), and the regulatory function of miR-21 was expressed as a percentage of the luciferase activity of the reporter vector with miR-21 target over the one without miR-21 target.
Biochemical Analysis and Measurement of Cytokines in the Plasma, Histological Examination, Measurement of Plasma H2S, H2S Synthesizing Activity and Myeloperoxidase (MPO) Activity in Liver Tissues, in Situ Terminal Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Staining, Cell Viability Analysis, Quantitative (q)RT-PCR for Detecting PTEN mRNA and miR-21, in Vitro Apoptosis Assay and Immunoblotting AnalysisThese methods have been described previously.6,9) Further details are provided in the Supplementary Materials (online only).
Statistical AnalysisAll experimental data were expressed as mean values±standard error of the mean (S.E.M.). Statistical difference was determined by using One-way ANOVA followed by Dunnett’s post hoc test. “p<0.05” was considered statistically significant.
Groups of rats were either untreated, sham-operated or subjected to liver I/R, and pre-administered with saline, GYY4137 or PAG. The plasma levels of H2S in untreated healthy rats ranged from 17.2 to 26.8 (22.6±3.7) µM, and sham-operated rats had a slightly increased level of H2S (24.3±4.1 µM) (Fig. 1A). However, saline-treated I/R rats had a higher level of H2S (41.3±4.9 µM) than sham-operated rats. Administration of GYY4137 significantly increased the levels of H2S in sham-operated (53.4±7.5 µM) and I/R rats (78.2±9.1 µM), but PAG treatment reduced H2S levels in sham-operated (17.7±2.9 µM) and I/R rats (26.9±4.2 µM) (Fig. 1A).
Blood and liver samples were harvested from healthy control and sham-operated rats, and rats undergoing liver I/R and pre-administered with saline, GYY4137 or PAG. The plasma levels of H2S (A) and H2S synthesizing activity in livers (B) were examined. (C) Liver tissues were subjected to immunoblotting to detect the expression of CBS and CSE proteins. Band densities were normalized to β-actin. n, number of samples per group. “*” (p<0.05) and “**” (p<0.001) indicate a significant difference. “#” (p<0.05) indicates a significant increase from sham-operated rats treated with saline.
Hepatic H2S synthesizing activity in sham-operated rats treated with either saline, GYY4137 or PAG remained low and was not significantly different from that in healthy controls (Fig. 1B). Liver I/R increased H2S synthesizing activity in rat livers, compared with sham operation (Fig. 1B). This increase was diminished significantly by PAG, but GYY4137 had little effect on H2S synthesizing activity (Fig. 1B). Although CSE and CBS are both expressed in livers, CSE plays a dominant role in H2S synthesis as the expression levels of CSE were 60-fold higher than CBS,20) which was also observed in the present results (Fig. 1C). Sham operation had little effect on hepatic expression of CSE or CBS. Livers subjected to I/R had a significantly higher level of CSE than sham-operated livers, but I/R had little effect on CBS expression (Fig. 1C). Neither GYY4137 nor PAG had a significant effect on the expression of CBS or CSE (Fig. 1C).
Exogenous H2S Delivered by GYY4137 Attenuates Liver I/R-Induced Injury by Inhibiting InflammationHistopathological analysis showed that saline-treated I/R rats had significantly higher histological scores (Table 1), and the histological alteration was characterized as inflammatory cell infiltration, hemorrhagic change and focal tissue destruction (Fig. S1A). Exogenous H2S delivered by GYY4137 attenuated, while PAG treatment aggravated, the severity of liver injury induced by I/R (Table 1, Fig. S1A). The histological observations were supported by the results of liver function biochemistry, which showed that saline-treated I/R rats had significantly higher plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); pre-administration of GYY4137 reduced, while PAG further increased, the levels of ALT and AST elevated by I/R (Table 1). The plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β were increased by liver I/R; but administration of GYY4137 attenuated, while PAG further increased, the levels of these three cytokines in I/R rats (Table 1). The results of hepatic inflammation were further supported by the activities of MPO, a marker of neutrophil infiltration (Table 1).
| Group | Sham | Hepatic I/R | ||
|---|---|---|---|---|
| Treatments | Saline | Saline | GYY4137 | PAG |
| Biochemical analysis | ||||
| ALT (U/L) | 102.3±15.8 | 325±37.6a | 185.6±30.8b | 513.5±51.4b |
| AST (U/L) | 30.8±6.7 | 165.1±22.9a | 86.2±23.4b | 186.4±23.5 |
| Histological scores | 1.2±0.6 | 11.7±3.8a | 7.6±2.4c | 14.9±3.1 |
| Liver MPO (U/mg) | 8.9±1.3 | 15.2±2.7a | 11.3±1.5c | 17.5±2.6 |
| Plasma cytokines | ||||
| TNF-α (ng/L) | 42.9±5.6 | 223.7±22.3a | 167.4±17.9b | 264.7±30.5c |
| IL-6 (ng/L) | 22.8±5.7 | 187.7±25.3a | 129.4±18.6c | 256.4±34.7c |
| IL-1β (ng/L) | 13.5±3.6 | 31.5±4.2a | 21.8±5.6c | 38.7±4.5 |
Notes: Abbreviations: ALT, aminotransferase; AST, aspartate aminotransferase; I/R, ischemia reperfusion; IL, interleukin; PAG, DL-propargylglycine; SOD, superoxide dismutase, TNF-α, tumor necrosis factor-α. Each group had 12 rats. Data are expressed as mean values±standard deviation. “a” (p<0.001) indicates a significant difference vs. Sham; “b” (p<0.001) and “c” (p<0.05), a significant difference vs. saline-treated hepatic I/R rats.
Administration of GYY4137 attenuated, while PAG further increased, programmed cell death in liver sections from I/R rats, as examined by TUNEL staining (Fig. S1B, E), in accordance with our previous report.9) We then detected the expression of caspase-3, and its upstream regulator caspase-9 by immunohistochemistry. The results showed that liver sections from saline-treated I/R rats had stronger staining of both cleaved caspase-9 and caspase-3; pre-administration of GYY4137 attenuated, while PAG further increased, their expression (Fig. S1C and D).
H2S Regulates the Expression of miR-21 and the PTEN/Akt Pathway in LiversIt has been reported that exogenous H2S induces the expression of miR-21 and attenuates myocardial ischemia injury.21) Therefore, we applied an in situ hybridization assay to detect and localize the expression of miR-21 in livers. I/R elevated the in situ signals in rat livers compared with sham operation; GYY4137 further significantly increased, while PAG reduced, the in situ signals in I/R livers (Fig. S1E). We further quantified the expression levels of miR-21 by using qRT-PCR, and the results (Fig. 2A) were consistent with the findings of in situ hybridization. We have previously reported that the activation of Akt contributes to the protective mechanisms of I/R-induced liver injury.11) Akt activation is negatively controlled by PTEN, which is a known target of miR-21.13,19) By using immunoblotting, we found that the above results of miR-21 expression were accordance with the patterns of PTEN expression and Akt phosphorylation, and further supported by the expression of downstream molecules of this pathway, including GSK-3β (glycogen synthase kinase-3β) and S6K (ribosomal protein S6 kinase). The expression patterns of caspase-9 and caspase-3 by immunoblotting (Fig. 2B) were also consistent with the results of apoptosis (Fig. S1B), and the expression of cleaved caspase-9 and caspase-3 as detected by immunohistochemistry (Fig. S1C, D).
Liver samples were harvested from sham-operated rats and rats undergoing liver I/R and pre-administered with saline, GYY4137 or PAG. (A) The expression levels of miR-21 in liver tissues were measured by qRT-PCR. (B) Liver tissues were subjected to immunoblotting. Band density was measured and normalized to β-actin. “*” (p<0.05) and “**” (p<0.001) indicate a significant difference from sham-operated rats. “#” (p<0.05) and “##” (p<0.001) indicate a significant increase; while “†” (p<0.05) and “††” (p<0.001), a significant reduction, from saline-treated rats undergoing liver I/R.
To investigate the regulatory function of miR-21 on PTEN, we transfected a luciferase reporter containing the 3′-UTR of PTEN with a miR-21 seed site (Fig. 3A) into BRL cells. This binding site for miR-21 at PTEN 3′-UTR is highly conserved across species (Fig. 3B). Co-transfection of anti-miR-21 increased, while miR-21 mimics decreased, the luciferase activity in BRL cells (Fig. 3C). We next examined whether targeting miR-21 could regulate PTEN expression and the sequential Akt activation. Neither anti-miR-21 nor miR-21 mimics affected the expression of PTEN mRNA (Fig. 3D). However, anti-miR-21 significantly increased the expression of PTEN protein and inhibited the activation of Akt, while miR-21 mimics showed an opposite effect (Fig. 3E). The results suggest that miR-21 may negatively regulate the expression of PTEN mainly by a mechanism preventing mRNA from being translated in this cell model.22)
(A) Predicted paring of rno-miR-21 to the 3′-UTR of rat PTEN gene, and the diagram of a pMIR-REPORT luciferase reporter vector (pMIR-luc-PTEN3’UTR-miR21) containing the 3′-UTR of PTEN with miR-21 seed site. (B) The potential binding site of miR-21 in the PTEN 3’UTR region is highly conserved in various species. Hsa, Homo sapiens; Rno, Rattus norvegicus; Mmu, Macaca mulatta; Ggg, Gorilla gorilla gorilla; Bta, Bos Taurus. (C) BRL cells were transfected with pMIR-luc-PTEN3’UTR-miR21 or a control vector, and co-transfected with negative control, anti-miR-21 or miR-21 mimic oligonucleotides. Relative luciferase activity was calculated as the percentage of luciferase activity in pMIR-luc-PTEN3’UTR-miR21-transfected cells over those with the control vector. (D, E) BRL cells were transfected with negative control, anti-miR-21 or miR-21 mimics as indicated. (D) The expression of PTEN mRNA was detected by qRT-PCR. (E) The expression of PTEN, p-Akt and Akt proteins was detected by immunoblotting. The density of PTEN band was normalized to β-actin, and the ratio of p-Akt/Akt band was calculated. “##” (p<0.001) indicates a significant increase; while “†” (p<0.05) and “††” (p<0.001), a significant reduction, from mock cells.
We first examined the effects of exogenous H2S on the viability of BRL cells by incubating them with different concentrations of NaHS. Compared with untreated cells, the viability of NaHS-treated cells remained almost unchanged when the concentration of NaHS was not greater than 50 µM, but was significantly reduced when the concentration of NaHS was greater than 50 µM (Fig. 4A). H/R exposure markedly reduced cell viability, and NaHS at concentrations of 3.125–25 µM significantly attenuated this reduction (Fig. 4B). Based on the results, a concentration of NaHS at 20 µM was selected for the following experiments. Incubation with NaHS had little effects on CSE expression, but H/R slightly increased CSE expression in BRL cells incubated in the presence or absence of NaHS (Fig. 4C). However, incubation with NaHS induced upregulation of miR-21 by ca. 2 folds in cells under normal condition or exposed to H/R, and H/R slightly increased miR-21 expression in BRL cells incubated in the presence or absence of NaHS (Fig. 4D). Negative control oligonucleotides had little effect on cell viability; but anti-miR-21 significantly abolished, while miR-21 mimics slightly enhanced, the ability of NaHS in restoring the viability of cells exposed to H/R (Fig. 4E).
(A) BRL cells were incubated with different concentrations of NaHS for 24 h. (B) Cells incubated with different concentrations of NaHS were exposed to H/R. (C, D) Cells were incubated under normal conditions or exposed to hypoxia/reoxygenation (H/R) in the presence or absence of NaHS (20 µM). The expressions of CSE (C) and miR-21 (D) and were detected by immunoblotting and qRT-PCR, respectively. The density of CSE band was normalized to β-actin. (E) Cells transfected with negative control (N.C.), anti-miR-21 or miR-21 mimic oligonucleotides were incubated under normal conditions or exposed to H/R in the presence or absence of NaHS (20 µM). Cell viability was examined. Open bars represent cells cultured under normal conditions, while solid bars, cells subjected to H/R. “*” (p<0.05) and “**” (p<0.001) indicate a significant difference. “†” (p<0.05) and “††” (p<0.001) indicate a significant reduction from untreated controls.
Untransfected BRL cells and the same cells transfected with anti-miR-21 were incubated in the presence and absence of NaHS and exposed to H/R. NaHS significantly inhibited cell apoptosis induced by H/R, and anti-miR-21 transfection could diminish this effect of NaHS (Figs. 5A, S2). Furthermore, H/R exposure significantly increased the level of caspase-3 activation, and NaHS significantly attenuated this increase and anti-miR-21 could abolish this effect of NaHS (Fig. 5B). The above results were consistent with the activation of caspse-3 as detected by immunoblotting (Fig. 5C). These observations were also supported by the patterns of PTEN expression and p-Akt activation and their downstream molecules, p-GSK-3β (Fig. 5C).
Untransfected or anti-miR-21-transfected BRL cells were incubated in the presence or absence of NaHS (20 µM) and exposed to hypoxia/reoxygenation (H/R). Untreated cells served controls. (A) Cells were subjected to flow cytometry to measure apoptosis rates (%). (B, C) Cells were lysed and subjected to caspase-3 activity assays (B) and immunoblotting (C). The density of each band in (C) was measured and normalized to β-actin. “*” (p<0.05) and “**” (p<0.001) indicate a significant difference from untreated controls. “††” (p<0.001) indicates a significant difference from saline-treated cells subjected to H/R; while “#” (p<0.05) and “##” (p<0.001), from NaHS-treated cells subjected to H/R.
H2S has emerged as a crucial signaling molecule participating in many physiological and pathological processes. In the present study, the levels of H2S in plasma were elevated in rats afflicted with liver I/R. This may have been due to a self-protective response of the host against I/R-induced injury.21) This view is supported by previous studies, in which myocardial I/R-induced injury and acute lung injury induced by infrarenal aortic cross-clamping (IAC) increased the plasma levels of H2S in rats.6,17,21) Several H2S-releasing drugs have demonstrated a considerable promise because of its safety and effectiveness in the management of many disorders in animal experiments, and have been evaluated in clinical trials.2) In agreement with the present results, H2S has displayed protective activities against the injury to several organs including the heart,17,21) liver9) and brain,4) induced by I/R, and lung injury induced by various factors.23) We have recently reported that GYY4137 administration attenuated acute lung injury induced by IAC in rats by inhibiting inflammation and angiopoietin-2 release.6)
Leukocytic infiltration and the release of pro-inflammatory cytokines are considered critical factors contributing to liver I/R-induced injury.1) H2S has been recognized as an anti-inflammatory mediator by inhibiting neutrophil accumulation, leukocyte adherence at the leukocyte–endothelium interface, and reducing the production of inflammatory cytokines.7,24) Here we showed that exogenous H2S delivered by GYY4137 resulted in reduced activity of MPO and lower levels of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β, which were elevated by liver I/R. These results are supported by several previous reports.5,6,9,17) Because of its lipophilic structure as a gasotransmitter,25) it is likely that H2S may have many variable targets without a confirmed receptor.
MiR-21 has been implicated in endogenous mechanisms of organ and cell protection after ischemic or pharmacological preconditioning by its anti-inflammatory potential.21,26) Here, we have shown that exogenous H2S induced upregulation of miR-21 in rat I/R livers and cells. It would be worthwhile to confirm the involvement of miR-21 in the protective mechanism of H2S against liver I/R-induced injury in an in vivo model by delivering anti-miR-21 or mimics specifically targeting livers in situ. Unfortunately, we were unable to do so due to technical challenge. Alternatively, we employed an in vitro cell model to explore the underlying mechanisms. PTEN, a known miR-21 target, is a well characterized phosphatase that inhibits Akt activation.21) By downregulating PTEN, miR-21 promotes the activation of the Akt pathway.13,19) However, exogenous H2S could restore the activation of Akt pathway by downregulating PTEN via increasing the expression of miR-21. The regulatory effect of miR-21 on the expression of PTEN has been validated by using luciferase reporter assays in BRL cells.
It has long been recognized that liver I/R induces apoptosis of hepatocytes, as a part of the process of liver injury.1,9,11) The Akt pathway plays important roles in maintaining cell survival against I/R-induced injury by regulating the apoptosis signaling transduction pathways.27–29) The activated Akt pathway exerts an inhibitory effect on cell apoptosis by suppressing the activation of caspase-9 through GSK3β. The cleaved caspase-9 functions as an initiator of the mitochondrial apoptotic pathway and induces sequential activation of caspase-3, the primary activator of apoptotic DNA fragmentation.30) In accord, we showed herein that exogenous H2S reduced the cleavage of caspase-9 and caspase-3 by activating the Akt pathway.
The present study has investigated the potential mechanisms underlying the protective effects of H2S against liver I/R (Fig. 6). Administration of GYY4137, a water-soluble and slow-releasing H2S donor,16) acts as an exogenous H2S donor to the rats, leading to the activation of Akt pathway through inhibiting PTEN in a miR-21-dependent manner,12,13) since anti-miR-21 could partially abolish the protective effects of H2S against liver I/R. Activated Akt inhibits the activation of GSK3β through increasing its phosphorylation,31) leading to reduced caspase-9 activation and sequential caspase-3 inhibition. Inhibition of GSK3β suppresses the production of inflammatory cytokines by activating transcription factors, thought to be involved in the regulation of the inflammatory responses.31,32) In contrast, PAG, a CSE inhibitor,6,9,17) reduces the endogenous production of H2S, aggravating the severity of liver injury induced by I/R. Liver I/R can also upregulate the expression of CSE, which may represents a self-defense mechanism by maintaining a certain level of H2S. In summary, the present results indicate that miR-21-mediated activation of Akt pathway participates in the protective activity of H2S against liver I/R-induced injury.
“→” indicates promotion, positive regulation or activation; “⊥,” inhibition, negative regulation or blockade. The dotted line indicates a subordinate function. Abbreviations: CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; GSK-3β, glycogen synthase kinase-3β; PAG, DL-propargylglycine; PTEN, phosphatase and tensin homolog; S6K, ribosomal protein S6 kinase.
This study was supported by Grants from the National Key Research and Development Program of China (2017YFC1308602), the National Natural Scientific Foundation of China (81472321, 81272467 and 81703055), Heilongjiang Natural Scientific Foundation (C201310) and Fundamental Research Funds for Heilongjiang Provincial Universities (2017LCZX06). We thank Dr. Shiva Reddy (University of Auckland, New Zealand) for revising the manuscript.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
