Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells

AIM: To investigate the roles of ATP-sensitive potassium (K_ATP) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide (H₂S) on the cardiomyocyte injury. METHODS: The expression level of K_ATP channel protein was tested by Western blot. The cell viability was measured by CCK-8 assay. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was examined by JC-1 staining. RESULTS: After the H9c2 cells were treated with 35 mmol/L glucose (high glucose, HG) for 1~24 h, the protein level of K_ATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h. Pretreatment of the cells with 400 μmol/L NaHS (a donor of H₂S) prior to exposure to HG for 12 h considerably blocked the down-regulation of K_ATP channels induced by HG. Pretreatment of the cells with 100 μmol/L mitochondrial K_ATP channel opener diazoxide, 50 μmol/L non-selective K_ATP channel opener pinacidil or NaHS obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss. Pretreatment with 100 μmol/L mitochondrial K_ATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective K_ATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS. CONCLUSION: K_ATP channels mediate the inhibitory effect of H₂S on HG-induced cardiac injury.     

Estrogen affects reactivity of rat mesenteric artery through ATP-sensitive potassium channel

AIM: To observe the expression of ATP-sensitive potassium (K_ATP) channel mRNA in mesenteric artery smooth muscle of rats with estrogen administration, and to evaluate the role of K_ATP channel in the effects of estrogen on the reactivity of mesenteric artery in rats. METHODS: Forty-eight female Sprague-Dawley rats, weighing (100?10) g, were randomly divided into 3 groups: sham operation (sham) group, ovariectomy (Ovx) group and ovariectomy with estrogen administration (Ovx+E) group. The mRNA expression of K_ATP subunits in mesenteric artery smooth muscle was detected by quantitative real-time PCR. Artery reactivity in the rats was observed by norepinephrine-induced pressor response. RESULTS: Compared with sham group, the mRNA expression of Kir6.1 and SUR2B was significantly decreased in Ovx group (P<0.05), but increased in Ovx+E group (P<0.05). Compared with sham group and Ovx+E group, the pressor response induced by norepinephrine in the rats were enhanced in Ovx group (P<0.05). No significant difference between sham group and Ovx+E group was observed. After administered with glibenclamide (a K_ATP channel blocker), the pressor response induced by norepinephrine was enhanced in sham group and Ovx +E group, and no changes in Ovx group. Meanwhile, no difference among the 3 groups was found. CONCLUSION: Estrogen up-regulates the expression of K_ATP channel subunits, which may be involved in estrogen-reduced pressor response to norepinephrine.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced inflammation mediated by necroptosis in H9c2 cardiac cells

AIM: To investigate the role of ATP-sensitive potassium (K_ATP) channels in the inhibitory effect of hydrogen sulfide (H₂S) on high glucose(HG)-induced inflammation mediated by necroptosis in H9c2 cardiac cells.METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and cyclooxyge-nase-2 (COX-2) were determined by Western blot. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by ELISA.RESULTS: After H9c2 cardiac cells were treated with 35 mmol/L glucose (HG) for 24 h, the expression of RIP3 was significantly increased. Pre-treatment of the cells with 100 μmol/L diazoxide (DZ; a K_ATP channel opener) or 400 μmol/L NaHS (a donor of H₂S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG. Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a K_ATP channel blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3. On the other hand, co-treatment of the cells with 100 μmol/L necrostatin-1 (a specific inhibitor of necroptosis) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses, evidenced by decreases in the expression of COX-2 and secretion levels of IL-1β and TNF-α. However, pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION: K_ATP channels play an important role in the inhibitory effect of H₂S on HG-induced inflammation mediated by necroptosis in H9c2 cardiac cells.     

Inhibitory effect of hydrogen sulfide (H2S) on cultured rat aortic smooth muscle cells

AIM:To explore the effects of hydrogen sulfide (H₂S) on proliferation of vascular smooth muscle cells (VSMC) stimulated by endothelin (ET-1, 10^-7mol/L) and mitrogen-activated protein kinase (MAPK) activity in VSMCs.METHODS:Cultured VSMCs were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS groups, (5) serum+NaHS group, and (6) endothelin+NaHS group. VSMC proliferation was measured by[³H]-TdR incorporation and MAPK activity in VSMC was determined by radioactivity assay.RESULTS:ET-1 increased VSMC[³H]-TdR incorporation by 2.39 times (P＜0.01) and MAPK activity by 1.62 times(P＜0.01), as compared with control. H₂S (5×10^-5-5×10^-4mol/L) decreased VSMC[³H]-TdR incorporation and MAPK activity by 16.8%-37.4% and 7.4%-33.6%, respectively (P<0.05 or P＜0.01).CONCLUSION:This study demonstrates that H₂S inhibits ET-1-induced proliferation of VSMC, which might be mediated by the inhibition of MAPK.     

Influence of hydrogen sulfide on ileal epithelial cell apoptosis in rats with intestinal ischemia-reperfusion injury

AIM: To investigate the influence of hydrogen sulfide (H₂S) on intestinal epithelial cell mitochondrial morphology and function and the expression of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax in rats with intestinal ischemia-reperfusion (I/R) injury. METHODS: Wistar rats (n=24) were randomly divided into 3 groups (8 in each group): sham group, I/R group and I/R+sodium hydrosulfide (NaHS) group. The animal model of intestinal I/R injury was established. The rats in I/R+NaHS group received NaHS (100 μmol/kg bolus +1 mg·kg^-1·h^-1 infusion) 10 min prior to the onset of reperfusion, whereas the rats in I/R group and sham group received equal volume of normal sodium. Ileum epithelial mitochondrial morphology and function were measured. Plasma H₂S was detected by sensitive sulfide electrode. The expression of Bcl-2 and Bax mRNA was studied by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax were tested by Western blot.RESULTS: The area, volume density, maximum diameter, minimum diameter and equivalent diameter of mitochondria, and the expression of cleaved caspase-3, Cyt C and Bax in I/R group were significantly higher than those in I/R+NaHS and sham groups (P<0.01). The mitochondrial count, circumference, specific surface area, area density and population density, plasma H₂S, respiratory control rate (RCR), the ratio of P/O, R₃ , R₄, and the expression of Bcl-2 in I/R group were sharply lower than those in I/R+NaHS and sham groups (P<0.01). H₂S was negatively correlated with caspase-3, cleaved caspase-3, Cyt C and Bax (P<0.01), and was positively correlated with Bcl-2 (P<0.01). CONCLUSION: H₂S has a protective effect on mitochondrial morphology and function in rats with intestinal I/R injury by down-regulating cleaved caspase-3, Cyt C and Bax and up-regulating Bcl-2.     

Endogenous hydrogen sulfide regulates oxidant/antioxidant balance in rat with chronic obstructive pulmonary disease

AIM: To investigate the role of hydrogen sulfide (H₂S) in the protection against oxidative stress in rats with chronic obstructive pulmonary disease (COPD).METHODS: The rat model of COPD was established by cigarette smoking (CS) combined with lipopolysaccharide (LPS) instillation. Thirty-two healthy male Sprague-Dawley rats were randomly divided into 4 groups: control group, CS+LPS group, CS+LPS+NaHS (H₂S donor) group and CS+LPS+PPG (DL-propargylglycine, an inhibitor of cystathionine-γ-lyase) group. After 30 days, the lung functions of the rats were measured, the histological changes of lungs were observed under light microscope and the pathological scores were calculated. The H₂S level in plasma and the protein expression of cystathionine-γ-lyase (CSE) in the lung tissues were measured. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and catalase (CAT) were detected to reflect oxidative stress.RESULTS: Compared with control group, the peak expiratory flow (PEF) decreased by 24% and intra-pressure (IP) increased by 66% in CS+LPS group. The pathological scores of the lung tissues also increased. Compared with CS+LPS group, no change in the lung function was observed after given NaHS or PPG, but the pathological scores decreased in CS+ LPS+ NaHS group. Compared with control group, the content of H₂S in plasma was increased by 26% on day 16. Compared with CS+LPS group, the content of H₂S in plasma of CS+LPS+PPG group was decreased by 22% after 30 days. Compared with control group, the protein expression of CSE increased, and no statistical difference among CS+LPS group, CS+ LPS+ NaHS group and CS+LPS+PPG group was observed. Compared with control group, MDA content in the lung tissues was increased by 24% in CS+LPS group, the activity of SOD was increased by 47% and the activity of CAT was increased by 52%. Compared with CS+LPS group, the MDA content in CS+LPS+NaHS group was decreased by 21%, and no statistical difference in the activity of SOD and CAT was observed. The activity of SOD decreased by 33% after given PPG.CONCLUSION: H₂S plays a role as anti-oxidant in the rats with COPD. The CSE/ H₂S pathway may be involved in the development of COPD.     

Role of hydrogen sulfide in acute lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM: To explore the role of endogenous and exogenous hydrogen sulfide (H₂S) in acute lung injury (ALI) induced by ischmia-reperfusion (IR) of hind limbs in rats.METHODS: A Sprague-Dawley rat model of acute lung injury was induced by ischemia of the hind limbs for 4 h and reperfusion for another 4 h. The rats (n=120) were randomly divided into 4 groups: control, IR, NaHS (H₂S donor)+IR, and propargylglycine +IR. The animals were sacrificed after reperfusion. Lung weight/body weight ratio (LW/BW) was measured and calculated. Morphological changes of the lung tissues were observed. The concentrations of H₂S, nitric oxide (NO) and carbon monoxide (CO) in plasma were tested. The content of malondialdehyde (MDA), the activity of CSE, inducible nitric oxide synthase (iNOS) and hemeoxygenase (HO) in the lungs were determined. The polymorpho-nuclear neutrophils(PMN) and protein content in bronchoalveolar lavage fluid(BALF) were also measured. The correlation of H₂S content with the above indices was analyzed.RESULTS: Compared with control group, severe injuries of the lung tissues, raised LW/BW, MDA concentration, PMN and protein contents in BALF were observed in IR group. Limb IR also made a drop in the concentration of plasma H₂S and the activity of lung CSE, while the activity of iNOS and HO in the lung tissues and the levels of plasma NO and CO increased. Administration of NaHS before IR attenuated the changes induced by IR, while pre-administration of PPG exacerbated the IR injuries and increased the plasma NO level and lung iNOS activity. The H₂S content was positively correlated with CSE activity, CO content and HO-1 activity (P<0.01), and negatively correlated with the other indices (P<0.01).CONCLUSION: Down-regulation of H₂S/CSE is involved in the pathogenesis of acute lung injury induced by IR. Endogenous and exogenous H₂S protects against lung injuries. The anti-injury effects of H₂S are related with its anti-oxidative activity to attenuate the inflammatory over-reactions in the lung induced by PMN. Down-regulation of NO/iNOS system and up-regulation of CO/HO-1 system by H₂S are also involved in the process of anti-injury to ALI.     

Neuroprotective effect of H2S by inhibiting autophagy after restoration of spontaneous circulation in rats with cardiac arrest

AIM: To investigate the neuroprotective effect of hydrogen sulfide (H₂S) after cardiopulmonary resuscitation in rats with cardiac arrest (CA), and to explore the effects of H₂S on neuron autophagy. METHODS: The CA model was established through asphyxia. Male Wistar rats were randomly divided into sham group, model group and NaHS group. The levels of beclin-1 and LC3 II/I were measured by Western blot at 2 h, 4 h, 12 h and 24 h after the restoration of spontaneous circulation (ROSC). At 12 h after ROSC, the formation of autophagic vacuole with LC3 dots was determined by immunohistochemical (IHC) method. The phenomenon of neuron autophagy was observed under transmission electron microscope. The numbers of apoptotic neurons were counted by TUNEL staining at 72 h after ROSC. The neurolo-gic deficit score (NDS) was used to evaluate the neurologic function after ROSC. RESULTS: The level of beclin-1 was gradually increased in model group, but it was increased and then gradually recovered in NaHS group (P < 0.05). The conversion of LC3 II in the cerebral cortex was the same as beclin-1. The results of IHC showed that LC3-positive nuclei in model group were more than those in NaHS group (P < 0.05). The number of autophagic vacuole in model group was more than that in NaHS group (P < 0.05). The number of the TUNEL-positive cells in model group was more than that in NaHS group (P<0.05). The NDS of the animals in NaHS group after ROSC was lower than that in model group(P < 0.05). CONCLUSION: H₂S inhibits neuronal autophagy, decreases apoptosis and improves neurologic function in CA rats after ROSC.     

Effects of hydrogen sulfide on impaired wound healing in ob/ob mice

AIM: To investigate the role of hydrogen sulfide(H₂S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS: The ob/ob mice were randomly divided into 3 groups, including vehicle, insulin and NaHS for treatment. C57BL/6 mice were treated with vehicle as control. Full-thickness punch biopsy wounds were created on the mice. Firstly, H₂S concentrations in the skins and granulation tissues were measured. The mRNA expression of cystathionine γ-lyase(CSE) was detected by RT-qPCR. The protein expression of CSE and MMP-9 were determined by Western blot. The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod. The levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS: The H₂S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice(P<0.05). CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice(P<0.05). The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group(P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group. The neutrophil and monocyte/macrophage infiltration, and TNF-α and IL-6 levels were significantly increased in ob/ob groups, but were decreased in NaHS group(P<0.01 or P<0.05). Meanwhile, NaHS increased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION: H₂S/CSE down-regulation contributes to impaired wound healing in diabetes, which is alleviated by exogenous H₂S possibly through anti-inflammation.     

Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous H2S production aggravate intestinal injury in severe acute pancreatitis

AIM:To investigate the potential role of endogenous hydrogen sulphide (H₂S) in severe acute pancreatitis (SAP). METHODS:A rat model of SAP was used to evaluate the role of H₂S on intestinal motility by counting the number of fecal pellets and the effect of H₂S on the expression of inflammation-related molecule in intestine was investigated. The colonic muscle cells (CMCs) were treated with plasma of SAP rats, tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6), and the expression of cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), Sp1 and PI3K/Akt related proteins at mRNA and protein levels were determined by RT-qPCR, Western blot and immunohistochemical staining,respectively. The PI3K inhibitor LY294002 and the siRNA-Sp1 were used to suppress the activity of PI3K/Akt/Sp1 signaling pathway. RESULTS:H₂S facilitated an inhibitory effect on the intestinal motility and enhanced the inflammatory responses in SAP (P<0.05). The expression of CSE and CBS in CMCs was significantly increased after treatment with TNF-α or IL-6 (P<0.05). Blockage of the PI3K/Akt/Sp1 signaling pathway remarkably inhibited the synthesis of CSE and CBS in CMCs(P<0.05). CONCLUSION:Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous production of H₂S play a vital role in the pathogenesis of SAP.     

Mutation of ATP-sensitive K+ channels and neonatal diabetes iDEND syndrome

Activating mutation in the KCNJ11 gene encoding Kir6.2 subunit of adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channel gives rise to intermediate developmental delay, epilepsy and neonatal diabetes (iDEND) syndrome, a rare hereditary endocrine metabolic disorder characterized by neonatal diabetes accompanied by developmental delay and muscle weakness, but no epilepsy. The Kir6.2 Val⁵⁹→Met⁵⁹ (V59M) activating mutation is the common cause of iDEND syndrome (>50%). Activating mutation causes iDEND syndrome by inhibiting normal closure of ATP-sensitive K⁺ channel, which leads to reduce insulin secretion. Most of such patients are more sensitive to sulfonylurea. High blood-brain barrier permeability and sulfonylurea receptor 1 (SUR1)-specific drugs are expected to become a major therapy.     

Hydrogen sulfide suppresses the increase in reactive oxygen species in neurons induced by angiotensin II via ERK1/2 signal pathway

AIM: To investigate the effect of hydrogen sulfide (H₂S) on the reactive oxygen species (ROS) level in medullary neurons induced by angiotensin II (Ang II). METHODS: Primarily cultured rat medullary neurons were divided into 5 groups as follows: control group, Ang II group, sodium hydrosulfide(NaHS) group, NaHS with Ang II group, and PD98059 (an inhibitor of p-ERK1/2) with Ang II group. ROS production was measured with dihydroethidium (DHE) staining. The expression of p-ERK1/2 and ERK1/2 was determined by Western blotting. The activity of neurons was detected by CCK-8 assay. RESULTS: Ang II at concentration of 100 nmol/L significantly increased ROS level in the neurons, but the effect was inhibited by NaHS at concentrations of 50~200 μmol/L, while NaHS alone had no influence on the ROS level in neurons. Additionally, PD98059 also depressed the ROS level in neurons induced by Ang II. Furthermore, the enhanced expression of p-ERK1/2 in the neurons induced by Ang II was significantly reduced by NaHS. CONCLUSION: H₂S remarkably inhibits the ROS level in the neurons induced by Ang II via activation of MAPK signal pathways, especially p-ERK1/2, indicating that H₂S rescues neurons from oxidative stress through declining the enhanced expression of p-ERK1/2.     

Effect of exogenous hydrogen sulfide on expression of NLRP3 inflammasome in hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) on the expression of NLRP3 inflammasome in hepatocytes.METHODS: The hepatocytes L02 and SMMC-7721 were used to establish the model of inflammation by stimulating with lipopolysaccharide (LPS) at different concentrations in vitro. The expression of NLRP3 inflammasome in the hepatocytes was detected by Western blot and the cell viability was measured by MTT assay for determining appropriate concentration of LPS. The hepatocytes were divided into 4 groups:the cells in control group were incubated with normal medium for 18.5 h; the cells in LPS group were incubated with normal medium for 0.5 h followed by 100 μg/L LPS for 18 h; the cells in LPS+H₂S group and H₂S group were incubated with 200 μmol/L sodium hydrosulfide hydrate (NaHS) for 0.5 h followed by 100 μg/L LPS or normal medium for 18 h, respectively. The protein expression of NLRP3 and caspase-1 in the cells of every group was determined by Western blot. RESULTS: Compared with control group, the protein expression of NLRP3 and caspase-1 increased significantly in LPS group (P<0.05) and had no significant change in H₂S group. Compared with LPS group, the protein expression of NLRP3 and caspase-1 in LPS+H₂S group decreased significantly (P<0.05). CONCLUSION: In hepatocytes, exogenous H₂S suppresses the expression of NLRP3 inflammasome.     

Exogenous hydrogen sulfide attenuates obstructive kidney injury in mice

AIM:To investigate the protective effect of exogenous hydrogen sulfide (H₂S) on obstructive renal injury in mice, and to explore the possible potential mechanisms involved in this animal model. METHODS:Male C57BL/6 mice (8 weeks old) were randomly divided into sham group, operation group and H₂S group, with 5 rats in each group. The model of obstructive renal injury was induced by unilateral ureteral obstruction (UUO). The mice in H₂S group were intraperitoneally injected with NaHS daily, while the mice in sham group and operation group were administered with the same volume of saline intraperitoneally. After 7 d, the mice were executed and the renal tissues were taken out for experiments. RNA was extracted to detect the mRNA expression of H₂S catalytic enzymes in the mice of 3 groups. HE staining was performed to observe the structural changes of renal tissues in the mice. Renal fibrosis in the mice of 3 groups was evaluated by Masson staining. The content of cystatin C in the plasma was detected to reflect glomerular filtration ability. The protein expression of LC3, beclin-1 and fibronectin (FN) in the mice of 3 groups was determined by Western blot. RESULTS:Compared with sham group, the mRNA expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) in operation group decreased significantly. The collagen fiber content in operation group was increased significantly, while collagen fiber content in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of FN in operation group was increased significantly, while the protein expression of FN in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of LC-Ⅱ and beclin-1 in operation group was increased significantly, while the protein expression of LC-Ⅱ and beclin-1 in H₂S group was increased significantly as compared with the operation group. CONCLUSION:Exogenous H₂S possibly mitigates renal fibrosis in UUO mice by up-regulating autophagy.     

Effect of hydrogen sulfide on airway inflammation induced by ozone in mice

AIM: To investigate the effect of hydrogen sulfide (H₂S) on airway inflammation induced by ozone (O₃) exposure and its mechanisms.METHODS: C57BL/6 mice (n=32) were randomly divided into control group, O₃ group, NaHS+O₃ group and NaHS group. The mice in O₃ group and O₃+NaHS group were exposed to 2.14 mg/m³ O₃ for 3 h on days 1, 3 and 5, while the mice in control group and NaHS group were exposed to filtered air. NaHS (14 μmol/kg) was administered intraperitoneally to the mice in NaHS group and O₃+NaHS group 30 min before each exposure. After the last exposure for 24 h, the airway responsiveness was determined, and bronchoalveolar lavage fluid (BALF) was collected for counting inflammatory cells and measuring total protein concentration. The lung tissues were collected for observing the morphological changes with HE staining. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), malondialdehyde (MDA) and NF-κB p65 protein in the lungs were determined.RESULTS: Compared with control group, the airway responsiveness, inflammatory cells, protein concentration, inflammation score, levels of IL-6, IL-8, MDA and NF-κB p65 in O₃ group increased significantly, but these in NaHS+O₃ group decreased compared with O₃  group.CONCLUSION: The present findings suggest that H₂S attenuates O₃ induced airway inflammation by inhibiting NF-κB expression and preventing lipid peroxidation.     

Hydrogen sulfide protects H9c2 cardiomyocytes against high glucose-induced injury by inhibiting necroptosis

AIM: To study whether hydrogen sulfide(H₂S) protects H9c2 cardiomyocytes against high glucose(HG)-induced injury by inhibiting necroptosis. METHODS: The protein levels of RIP3(an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot. The cell viability was measured by CCK-8 assay. The intracellular le-vels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP) was examined by rhodamine 123 staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cells were treated with HG(35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h. Pretreatment of the cells with 400μmol/L NaHS(a donor of H₂S) or co-treatment of the cells with necrostatin-1(Nec-1; a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG. Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the generation of ROS and MMP loss. On the other hand, pretreatment with NaHS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes. CONCLUSION: H₂S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.     

Hydrogen sulfide inhibits adenosine triphosphate-induced activation and IL-1β releases in rat microglia

AIM: To investigate the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), on the membrane permeability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of IL-1β induced by adenosine triphosphate (ATP) in rat microglia, and to explore the effect of H₂S on ATP-P2X purinergic signaling pathway and the molecular mechanism of its neuroprotective effect. METHODS: Rat microglia in logarithmic growth phase were used in the study. The[Ca²⁺]_i was detected by Fura-2/AM staining. Fluorescent dye YO-PRO-1 was used to observe the membrane permeability. Interleukin-1β (IL-1β) was measured by rat IL-1β ELISA kits. RESULTS: The YO-PRO-1 fluorescence intensity was obviously elevated by ATP induction in a dose-dependent manner in the rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.05).[Ca²⁺]_i rapidly increased and then decreased slowly, forming a stable platform for a long time when rat microglia were treated with ATP. Ca²⁺ spike activity induced by ATP had no change, but the platform disappeared (P<0.05) after NaHS pretreatment. The ATP and LPS together facilitated the release of IL-1β, but the phenomenon was inhibited by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide may decrease the membrane permeability, calcium inflow and IL-1β release in rat microglia activated by high dose of ATP. The cytoprotection of hydrogen sulfide may be mediated by purinergic signaling pathway.     

Hydrogen sulfide attenuates urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway

AIM:To explore the effect of hydrogen sulfide (H₂S) on urosepsis-induced acute kidney injury. METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, NaHS treatment (NaHS) group, and NaHS combined with TAK-242 (a TLR4 inhibitor) treatment (NaHS+TAK-242) group. After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer. The serum levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), procalcitonin (PCT), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot. RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with NaHS. The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-α in the septic rabbits were higher than those in control group, and decreased significantly in NaHS group and NaHS+TAK-242 group. The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group. However, NaHS or NaHS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits. CONCLUSION:H₂S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.