Beneficial Role of Hydrogen Sulfide in Renal Ischemia Reperfusion Injury in Rats

PURPOSE: Hydrogen sulfide (H2S) is an endogenous gaseous molecule with important physiological roles. It is synthesized from cysteine by cystathionine γ-lyase (CGL) and cystathionine β-synthase (CBS). The present study examined the benefits of exogenous H2S on renal ischemia reperfusion (IR) injury, as well as the effects of CGL or CBS inhibition. Furthermore, we elucidated the mechanism underlying the action of H2S in the kidneys. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly assigned to five groups: a sham, renal IR control, sodium hydrosulfide (NaHS) treatment, H2S donor, and CGL or CBS inhibitor administration group. Levels of blood urea nitrogen (BUN), serum creatinine (Cr), renal tissue malondialdehyde (MDA), and superoxide dismutase (SOD) were estimated. Histological changes, apoptosis, and expression of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) were also evaluated. RESULTS: NaHS attenuated serum BUN and Cr levels, as well as histological damage caused by renal IR injury. Administration of NaHS also reduced oxidative stress as evident from decreased MDA, preserved SOD, and reduced apoptotic cells. Additionally, NaHS prevented renal IR-induced MAPK phosphorylation. The CGL or CBS group showed increased MAPK family activity; however, there was no significant difference in the IR control group. CONCLUSION: Exogenous H2S can mitigate IR injury-led renal damage. The proposed beneficial effect of H2S is, in part, because of the anti-oxidative stress associated with modulation of the MAPK signaling pathways.

Optic Nerve Crush Induces Changes of Hydrogen Sulfide Synthases Expression in the Rat Retina / 法医学杂志

Objective To investigate the expression changes of the hydrogen sulfide synthases cystathionineγ-lyase (CSE), cystathionineβ-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST), after optic nerve crush (ONC) in rat the retina.Methods The rat model of ONC was established.Rats were divided into normal control, ONC, and sham control groups.Histopathologic changes in retina, the number of retinal ganglion cells (RGC) and retinal thickness of inner part (RTIP) were measured.The changes of CSE, CBS and 3-MST mRNA expression were detected with quantitative real-time PCR.Results The retinal histostructure was normal in normal controls and with minor changes in sham controls, respectively.Compared with sham group, significant retina damages were found in the ONC group:a time-dependent reduction of RGC number and RTIP.Expressions of CSE, CBS and 3-MST mRNA in rat retina were detected in normal control.Compared with normal controls, the expressions of CSE, CBS and 3-MST mRNA did not show any significant changes in the sham controls.Compared with sham controls, CBS mRNA expressions showed a time-dependent increase at 3 d, 7 d and 14 d after crush in the ONC group;CSE mRNA expressions increased to the peak at 3 d and then slightly reduced at 14 d after crush;3-MST mRNA expressions showed the trend of increase at 3 d and 7 d and then enhanced remarkably at 14 d after crush.Conclusion Hydrogen sulfide synthases CSE, CBS and3-MST expressions were up-regulated in rat retina following ONC, which may cause an increase in local endogenous hydrogen sulfide production in the retina and a compensatory protective effect.

Effect of amygdala H2S system on depression-like behavior in posttraumatic stress disorder rats / 中国病理生理杂志

AIM:To explore the depression-like behavior and cystathionine β-synthase (CBS)/hydrogen sulfide (H2S) levels of amygdala in posttraumatic stress disorder (PTSD) rats and to study the effect of exogenous H2S on PTSD rats.METHODS:Single prolonged stress paradigm was adopted to prepare PTSD animal model.Forced swimming test and sucrose preference test were used to evaluate the depression-like behavior.The content of CBS/H2S in amygdala tissue was measured by Western blot and methylene blue method.In vivo extracellular single unit recordings was used to examine the frequency of spontaneous discharges of amygdala neurons.RESULTS:The immobility time in forced swimming test of PTSD group increased and sucrose preference in sucrose preference test of PTSD group decreased compare with normal group (P<0.01).CBS/H2S level in amygdala tissue of PTSD group decreased compared with normal group (P<0.01).The immobility time of the rats in forced swimming test of NaHS+PTSD group decreased and the sucrose prefe-rence in sucrose preference test of NaHS+PTSD group increased compare with PTSD group (P<0.01).L-cysteine increased the frequency of spontaneous discharges of amygdala neurons (P<0.01).CONCLUSION:Depression-like behavior is aggravated in PTSD model rats owing to the inhibition of CBS/H2S content in amygdala tissue.The mechanism of behavior-improving effect of H2S on PTSD model rats is possibly related to excitating amygdala neurons and increasing the frequency of spontaneous discharges.

Correlation between the gene polymorphism of homocysteine metabolic enzyme cystathionine β-synthase, N5,10-methylenetetrahydrofolate reductase and chronic pulmonary heart disease / 新乡医学院学报

Objective To investigate the correlation between the gene polymorphism of homocysteine metabolic enzyme cystathionine β-synthase(CBS) 844ins68,N5,10-methylenetetrahydrofolate reductase(MTHFR) C677T and chronic pulmonary heart disease(CPHD).Methods A total of 230 patients with CPHD in observation group were selected from January 2014 to November 2016 in the Second People's Hospital of Xinxiang City,and 235 healthy subjects in healthy control group were selected at the same time.The lung function test was performed with lung function instrument,and the percentage of the forced expiratory volume in one second to predicted value(FEV1％ pred) and the forced expiratory volume in one second to forced vital capacity(FEV1％) value were recorded in the two groups.The fasting ulnar venous blood was collected from the patients in the observation group on the next morning after hospitalization and the subjects in the control group on the morning of health examination.The levels of plasma homocysteine (Hcy),fasting blood glucose (FBG),triacylglycerol (TG),total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were detected.The DNA was extracted from the whole blood cells.The CBS 844ins68 polymorphism was detected by polymerase chain reaction genotyping.The MTHFR C677T polymorphism was detected by restriction fragment length polymorphism polynerase chain reaction.Results There was no significant difference in the FBG level between the two groups (P ＞ 0.05).The levels of Hcy,TG,TC and LDL-C in the observation group were significantly higher than those in the healthy control group (P ＜ 0.05),and the FEV1 and FEV1％ pred were significantly lower than those in the healthy control group (P ＜ 0.05).There were two genotypes of CBS 844ins68 in the two groups.The genotype frequencie of DD and DI in the observation group was 91.74％ and 8.26％,and the allele frequency of D and I was 95.87％ and 4.13％ respectively.The genotype frequency of DD and DI in the healthy control group was 94.04％ and 5.96％,and the allele frequency of D and I was 97.02％ and 2.98％ respectively.There was no significant difference in genotype and allele frequency distribution between the two groups (x2 =0.935,0.901;P ＞ 0.05).Three genotypes of MTHFRC677T were detected after enzyme digestion in the two groups.The genotype frequency of CC,CT and TT in the healthy control group was 27.66％,48.94％ and 23.40％;and the allele frequency of C and T was 52.13％ and 47.87％ respectively.The frequency of TT genotype and T allele in the observation group was significantly higher than that in the healthy control group (x2 =7.730,7.326;P ＜ 0.05).Conclusions Hcy level increasing may be a risk factor for CPHD.The polymorphisms of CBS 844ins68 gene may be unrelated to the occurrence of CPHD.The polymorphism of the MTHFR C677T gene may contribute to CPHD by affecting Hcy level.The T allele of MTHFR C677T may be a risk factor for CPHD,and the MTHFRC677T gene may be a genetic predisposition to CPHD.

Changes in Hydrogen Sulfide in Rats with Hepatic Cirrhosis in Different Stages / 华中科技大学学报（医学）（英德文版）

This study aimed to observe changes in the hydrogen sulfide (H2S) system in the blood and liver tissue of rats with hepatic cirrhosis at different stages by studying the effect of H2S on the course of hyperdynamic circulation in rats with hepatic cirrhosis.H2S concentration in the blood from the portal vein and inferior vena cava of hepatic cirrhosis rat model induced with carbon tetrachloride was detected on the 15th,30th,and 52nd day.The expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) protein,and CBS and CSE mRNA in the liver was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR),respectively.The results indicated that H2S concentration in the blood from the portal vein and inferior vena cava of rats with hepatic cirrhosis was significantly lower than that in the control group.H2S was gradually decreased with the development of the disease and significantly lower in the blood from portal vein than in the blood of inferior vena cava at the mid-stage and the late stage groups.The expression levels of CBS and CSE protein,and CBS and CSE mRNA in the livers with hepatic cirrhosis at different stages were all higher than those in the control group,and the expression gradually increased with the development of the disease.The expression of CBS was lower than CSE in the same stages.The results indicated that the CSE mRNA was expressed predominantly in the cirrhosis groups as compared with CBS rnRNA.Among experimental rats,the H2S system has an important effect on the occurrence and development of hyperdynamic circulation in rats with hepatic cirrhosis.This finding adds to the literature by demonstrating that H2S protects vascular remodelling in the liver,and that CSE is indispensable in this process.

Dynamic change of cystathionine β-synthase during cerebral ischemia-reperfusion and its effect in rats / 中国免疫学杂志

Objective:To observe the dynamic change of cystathionine β-synthase during cerebral ischemia-reperfusion and its effect in rats.Methods:The ischemic model was established with line embolism to block the middle cerebral artery .The reverse tran-scription-polymerase chain reaction ( RT-PCR) and Western blot assay were used to assess the expression of cystathionine β-synthase (CBS) in SHAM group,I group,and IR group.ELISA assay was performed to detected the homocysteine (HCY) level in plasma.After treating with the inhibitor of cystathionine β-synthase called hydroxyla mine(HA),the expression of hemeoxygenase 1(HO-1) and the pathologic change of the brain was evaluated .Results:As compared to sham group ,the expression of CBS was significantly up-regulated in ischemia-reperfusion group at 12 h post-reperfusion.Meanwhile,it existed the lowest level of HCY at 12 h post-reperfusion,comparing to sham grouzp ( 5.73 ±1.17 vs 2.88 ±0.93 , F=25.56 , P=0.001 ) .When inhibited the activity of CBS via using HA , the down-regulation of HO-1 protein and further damage in neuron were observed .Conclusion:Cystathionine β-synthase serves as an protective factor during cerebral ischemia-reperfusion.

Changes of CBS expression in brain following ischemia and reperfusion of limbs / 中国病理生理杂志

AIM:To detect the changes of cystathionine β-synthase (CBS) expression in the brain following ischemia-reperfusion of hind limbs in the rats and to elucidate their significance .METHODS:Hind limb ischemia was in-duced by clamping infrarenal aorta with a microvascular clip and brain injury was made by following reperfusion .The brain tissue was obtained from the animals subjected to sham operation , 4 h of ischemia without reperfusion and 3 h, 6 h, 12 h, 24 h of reperfusion following 4 h of ischemia .The expression of CBS at mRNA and protein levels was measured at different time points by reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting .The CBS activity and hy-drogen sulfide ( H2 S) concentration in the brain were determined by a universal microplate reader .The observation of path-ologic changes of the brain was made following the inhibition of CBS by hydroxylamine .RESULTS:The relative expression of CBS at mRNA and protein levels in IR group significantly increased compared with sham group .It reached a peak at 12 h (P0.05).This time course correlated with increased CBS activity and H2 S concentration .No statistically significant difference in the above indexes between sham operation group and ischemia group was observed (P>0.05).Inhibition of CBS activity induced more severe brain injury in IR group .CONCLUSION:Up-regulation of CBS/H2 S pathway is involved in the protection for neurons during the ischemia-reperfusion of hind limbs .

Changes of endogenous cystathionine-β-synthase/hydrogen sulfide system in PC12 cells injury induced by rotenone / 中国药理学通报

Aim To research dynamically the changes of endogenous cystathionine- β-synthase/hydrogen sul-fide system in PC12 cells injury induced by rotenone. Methods Rotenone-induced injury in PC12 cells ( characteristic of dopaminergic neurons) was used as a PD cell model. The expression of CBS was evaluated by Western blot. Intracellular CBS activity and H2 S production were detected by Methylene blue spectro-phot-ometric method. The viability of PC12 cells was measured by CCK-8 assay. GSH detection kit was used to detect the intracellular GSH content. Results In the groups of 6 and 12 hours, the expression and activ-ity of CBS were elevated, and the production of H2 S was increased. In the groups of 24 and 48 hours, CBS expression and activity were significantly decreased, and the amount of H2 S was significantly reduced. Ap-plication of 1. 5 μmol·L-1 rotenone for different time (6-48h) could decrease the cell viability and intra-cellular GSH contents in a time-dependent manner. Conclusions The expression and activity of endoge-nous CBS, stimulated by rotenone, are elevated firstly and then decreased. The generation of H2 S, stimulated by rotenone, is increased and then reduced significant-ly, which may be related to PC12 cells against oxida-tive stress damage induced by rotenone.

Biochemical Effects of Some Peroxisome Proliferator Activated Receptor Ligands in Hyperhomocysteinemic-rats.

Hyperhomocysteinemia (HHcy) is implicated in peripheral vascular, cerebrovascular and coronary heart disease. This study was conducted to investigate the effects of pioglitazone (PPAR-γ agonist) and gemfibrozil (PPAR-α agonist), either alone or in combination, in HHcy-rats. HHcy was induced by keeping rats on high methionine diet (8% w/w) for 6 weeks. Rats were divided into two major groups; normal fed rats and methionine overload rats. Each group was further subdivided into four subgroups: a) control group, b) pioglitazone (10 mg/kg), c) gemfibrozil (100 mg/kg), and d) combination of the chosen PPAR ligands. The drugs were given orally daily for consecutive six weeks. The results showed that the great reduction of HHcy was obtained by the drug combination. Induction of HHcy in rats resulted in elevation of LDL-cholesterol and suppression of HDLcholesterol. Hepatic paraoxonase-1 activity was inhibited in all HHcy-rat groups. RT-PCR for liver cyclooxygenase-2 (COX-2) showed marked expression in HHcy-rats, which was attenuated by treatment with PPAR agonists. RT-PCR for cystathionine β-synthase (CBS) showed positive expression in all HHcy-rat groups except that treated with combination of PPAR ligands. These results suggest that pioglitazone-gemfibrozil combination showed ameliorative effect on hyperhomocysteinemia and its consequences in rats.

Homocysteine, Hydrogen sulfide (H2S) and NMDA-Receptor in Heart Failure.

Mitochondrial mechanism of oxidative stress and matrix metalloproteinase (MMP) activation was unclear. Our recent data suggested that MMPs are localized to mitochondria and activated by peroxynitrite, which causes cardiovascular remodeling and failure. Recently, we have demonstrated that elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy) increase oxidative stress in the mitochondria. Although HHcy causes heart failure, interestingly, it is becoming very clear that Hcy can generate hydrogen sulfide (H2S), if the enzymes cystathionine β-synthase (CBS) and cystathionine -lyase (CGL) are present. H2S is a strong anti-oxidant and vasorelaxing agent. Paradoxically, it is interesting that Hcy, a precursor of H2S can be cardioprotective. The CGL is ubiquitous, while the CBS is not present in the vascular tissues. Therefore, under normal condition, only half of Hcy can be converted to H2S. However, there is strong potential for gene therapy of CBS to vascular tissue that can mitigate the detrimental effects of Hcy by converting it to H2S. This scenario is possible, if the activities of both the enzymes (CBS and CGL) are increased in tissues by gene therapy.