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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of hydrogen sulfide on high glucose-induced injury of mouse glomerular podocytes

AIM: To investigate the effect of hydrogen sulfide (H2S) on high glucose （HG）-induced injury of the mouse podocyte cell line MPC5. METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group. After treated for a certain time, the cells were collected for further detection. The expression of zonula occludens-2 (ZO-2), nephrin, β-catenin and cystathionine γ-lyase (CSE) was determined by Western blotting. RESULTS: High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner. NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner. HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-catenin in HG+NaHS group was observed as compared with HG group. Compared with NG group, the expression of ZO-2 and nephrin was decreased obviously, and the level of β-catenin was increased in HG+NaHS group. CONCLUSION: Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury. Exogenous H2S protects against hyperglycemia-induced podocyte injury, possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway.     

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.     

Effects of hydrogen sulfide on wound healing of skin ulcer in diabetic rats

AIM: To study the effect of hydrogen sulfide on wound healing of skin ulcer in diabetic rats.METHODS: Male SD rats were randomly divided into 3 groups, including non-diabetic control(NDC) group, untreated diabetic control(UDC) group, and treated diabetic administration(TDA) group. Diabetic rats were induced by intraperitoneal injection of streptozotocin(STZ). After 1 week, wound healing model was prepared by making a round incision(2.0 cm in diameter) on the dorsal skin in full thickness. The rats from TDA group received 2% sodium bisulfide ointment on the skin ulcer wound, and the animals from UDC and NDC groups received control cream. After 21 d of treatment with sodium bisulfide, blood samples were collected for biochemical analysis, including prothrombin time(PT), thrombin time(TT), and fibrinogen(FIB) in plasma, as well as the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA) in the serum. White blood cells(WBC) and lymphocytes were also counted. Granulation tissues from the wound were processed for histological examination and Western blot analysis was used to detect heme oxygenase-1(HO-1) and tumor necrosis factor α(TNF-α) expression.RESULTS: Compared with UDC group, sodium bisulfide treatment accelerated wound healing of skin ulcer(P<0.01), and increased the activity of SOD in serum(P<0.01) in the diabetic rats. The declined number of WBC and lymphocytes, prolonged PT and TT, and decreased FIB levels in rats treated with sodium bisulfied were also confirmed. Pathological section showed that there were inflammatory cell infiltration, and irregular and loose fibril alignment in the granulation tissue of rats from the UDC group, but there were regular fibril alignment and increased angiogenesis in the granulation tissue of rats from the TDA group(P<0.05). Furthermore, sodium bisulfide treatment raised HO-1 protein expression, and decreased TNF-α protein expression in the diabetic rats.CONCLUSION: Hydrogen sulfide accelerates the wound healing of skin ulcer in the rats with diabetes. The beneficial effect of H₂S may be associated with formation of granulation, anti-inflammation, and antioxidation.     

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.     

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 H2S and ATP-sensitive potassium channels on colonic hypermotility in a rat model of chronic stress

AIM: To investigate the potential role of exogenous hydrogen sulfide (H₂S) and ATP-sensitive potassium (K_ATP) channels in chronic stress-induced colonic hypermotility.METHODS: Male Wistar rats were divided into water avoidance stress (WAS) group and sham WAS (SWAS) group. Organ bath recordings were used to test the contractile activity of colonic strips. The effects of H₂S donor NaHS and pretreatment with glibenclamide on the contractions of colonic smooth muscle were studied and the IC₅₀ of NaHS was calculated. The localization and expression of the subunits of K_ATP channels were determined by the methods of immunohistochemistry and Western blotting.RESULTS: WAS increased contractile activity of colonic strips. NaHS concentration-dependently inhibited the spontaneous contractions of strips from the SWAS and WAS rats. The IC₅₀ of NaHS for longitudinal muscle (LM) and circular muscle (CM) of the WAS rats was 0.2033 mmol/L and 0.1438 mmol/L, significantly lower than those of the SWAS rats. Glibenclamide significantly increased the IC₅₀ of NaHS for LM and CM from the SWAS and WAS rats. In both SWAS and WAS rat colon, Kir6.1, Kir6.2 and SUR2B were expressed on the plasma membrane of the smooth muscle cells. WAS treatment resulted in up-regulation of the expression of Kir6.1 and SUR2B in the colon devoid of mucosa and submucosa.CONCLUSION: The increased expression of Kir 6.1 and SUR2B in colonic smooth muscle cells may be a defensive response to chronic WAS. H₂S donors may have potential clinical effect on treating chronic stress-induced colonic hypermotility.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

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.     

LBP regulates PI3K/Akt/eNOS signaling pathways in ovariectomized rat myocardium to exert antioxidative effect

AIM: To observe the influence of Lycium barbarum polysaccharide (LBP) on the PI3K/Akt/eNOS signaling pathways in ovariectomized rat myocardium. METHODS: Female SD rats (n=30) were divided into sham operation group, ovariectomized group, progynova group, high-dose LBP group and low-dose LBP group. The serum levels of estradiol, lactate dehydrogenase (LDH) and creatine kinase (CK) were measured by ELISA. The myocardial contents of H₂S and oxidative stress injury-related indicators were also detected. The morphological changes of the myocardium were observed with HE staining. The expression of eNOS and PI3K/Akt pathway-related proteins in the myocardium was determined by Western blot. RESULTS: Compared with sham operation group, the serum level of estradiol, the content of H₂S, the activity of GSH-Px, and the expression of eNOS and PI3K/Akt pathway-related proteins in the myocardium in ovariectomized group were all decreased, and the levels of ROS and MDA in the myocardium were increased (P<0.05). The serum levels of LDH and CK were also increased. The arrangement of the myocardial cells was disordered, and the intercellular space was also increased in the ovariectomized group. Compared with ovariectomized group, the serum level of estradiol, the myocardial levels of H₂S and GSH-Px, and the protein levels of eNOS and phosphorylated Akt were all increased in high dose group, while the levels of ROS and MDA in the myocardium were decreased (P<0.05). The serum levels of LDH and CK were also decreased. The morphological changes of the rat myocardium were improved in high dose group. CONCLUSION: LBP prevents and treats postmenopausal cardiovascular lesions through regulating PI3K/Akt/eNOS signaling pathways in ovariectomized rats.