Effect Of Hydrogen Sulfide On The Endothelium Dysfunction Of Hypertension And The Underlying Mechanism

Part one Hydrogen sulfide improves the endothelium dysfunction by the pathway of PPARδ/PeNOS in hypertensive patientsObjective: Hydrogen sulfide has been confirmed to be the third gaseous signal molecule. As a biologically active mediator, hydrogen sulfide exhibits potent cardiovascular protective effect. PPARδ, one of the nuclear receptor superfamily, plays the crucial role in cadiovascular system and has the potential therapeutic utilization for treating cardiovascular disease. Based on the effect of H2 S and PPARδ, in this study, we evaluate the interaction of them on the effect of renal artery in hypertensive patients.Methods: Normotensive and hyptensive renal artery tissues were collected from 46 patients suffered from Nephron sparing surgery. Among these patients, 20 cases were with normal blood pressure and 26 suffer from hypertension. The inclusion criteria of the cases was that systolic pressure was above 140 mmHg or diastolic pressure was above 90 mmHg. All of them were without diabetes. The ages from two groups had no significant difference(64.00±2.892 vs 59.92±2.058). The renal arteries from two groups were cultured with or without NaHS 50 μmol/L, and then fixed into organ bath to be detected the endothelium dependent relaxation. Western blot was used to observed the protein expressions of AT1, CSE, PPARδ, peNOS, et al. Human umbilical vein endothelial cell were treated with AngII, and then the effect of H2 S on the generation of NO were observed. The level of NO in plasma was obtained by laser condocal scanning microscope.Results:1 Exogenously administrating Na HS lowered AT1 protein expression, upregulated the expression of CSE.2 In renal arteries of hypertension patients, treated with NaHS enhanced ACh induced endothelium dependent dilations.3 Compared with normotensive patients, there showed that the decreased expression of PPARδ and peNOS. Chronicly cultured with NaHS, these two proteins were upregulated.4 Chronicly cultured with NaHS, the espressions of pAkt and pAMPK in hypertension patients.were incresed5 In cultured Human Umbilical Vein Endothelial Cell, NaHS increased the production of NO treated with AngII. Whereas the effect of NaHS was partially inhibited by GSK0660(antagonist of PPARδreceptor, 1 μmol/L); Compoud C(AMPKα inhibitor, 10 μmol/L); LY294002(PI3k inhibitor, 5 μmol/L); Wortmanin(PI3k inhibitor, 0.1 μmol/L). Losartan(AT1 receptor inhibitor, 3 μmol/L) generated the similar effect of H2 S.Conclusion: Our present results firstly demonstrated that treated with NaHS for 12 h, there showed that the endothelium dependent relaxation was enhanced and then the endothelial function was improved in hypertensive patients. The effect of H2 S may be generated by rectify the unbalance of endogenous CSE/AT1 system, and partially through activating the pathway of PPARδ/peNOS/NO. Part two Hydrogen sulfide exhibites protective effect on endothelium through activating PPARδ in renal vascular hypertensive ratsObjective: Renal vacular hypertension was second hypertension. As a curative hypertension, more and more studies focused on its pathomechanism. RAS was over activated and then caused endothelium dyfunction. H2 S which was confirmed to be the third gaseous signal molecular could be producted endogenously in mammaliam and played significant role on maitaining the homeostasis of cardiovascular system. PPARδ, which was closely related to metabolism, also possessed protective effects in cardiovascular diseases. Our experiment was to reveal whether hydrogen sulfide attenuated endothelium dysfunction through PPARδ pathway.Methods: Healthy male Sprague-Dawley rats were randomly divided into 4 groups: Sham operation group(Sham), Sham+NaHS group, 2K1 C group, 2K1C+NaHS group. sodium hydrosulfide(56 μmol/kg) daily intraperitoneal injection was administrated in Sham+NaHS and 2K1C+NaHS groups; Other groups were given daily intraperitoneally injected with saline solution started from the fourth week after 2K1 C operation. Rats from four groups drank water freely, was fed with a standard laboratory diet, and housed in a temperature-controlled room with a 12 h/12 h day/night cycle. Mean arterial pressure(MAP) was measured by a tail-cuff technique using a noninvasive blood pressure measurement system every four weeks. Microvessel recording technique was used to observe the effect of NaHS on endothelium-dependent or independent relaxation induced by ACh or SNP in isolated rena artery rings of rats. GSK0660(antagonist of PPARδ, 1 μmol/L); Compoud C(AMPKα inhibitor, 10 μmol/L); LY294002(PI3k inhibitor, 5 μmol/L); Wortmanin(PI3k inhibitor, 0.1 μmol/L); GW0742(agonist of PPARδ, 0.1 μmol/L); L-NAME(nitric oxide synthase inhibitor, 100 μmol/L) were used to make sure the mechanism of H2 S. Monobromobimane method was used to detect the level of H2 S in plasma. Western blot was adminstrated to observe the protein level of PPARδ, CSE, peNOS, pAkt, pAMPK, et al. Renal blood flow volume was observed by small animal ultrasound. The morphological kidney manifestation was showed by HE stain. The level of NO in plasma was tested by Nitric Oxide assay kit.Results:1 The general conditions of four groups were observed as followed. The weight in 2K1 C and 2K1C+NaHS groups increased slowly from 12 weeks after 2K1 C operation until the end of the experiment compared with Sham group. The water and food intake had no significant difference among these four groups. The urine volume increased in 2K1 C group.2 Exogenously administrating NaHS lowered blood pressure, increased the level of plasma AngII, downregulated AT1 protein expression in renal vascular hypertensive rats.3 Chronicly using NaHS decreased the ratio of left kidney to right kidney, attenuated kidney function and alleviated the kidney damage.4 Treated with NaHS did not chang the blood flow volume in left renal artery, but significantly increased the right blood flow volume in 2K1 C renal vascular hypertension.5 In renal arteries of 2K1 C grouup, treated with NaHS enhanced ACh induced endothelium dependent relaxation, but had no effect on endotheliun independent relaxation.6 Treated with NaHS in 2K1 C rats improved the level of H2 S and NO, also upregulated the expression of CSE.7 Compared with Sham group, there showed the decreased expression of PPARδ and peNOS. Chronicly treated with NaHS upregulated these two proteins.8 Chronicly administrating NaHS incresed the espressions of pAkt and pAMPK in 2K1 C renal vasdular hypertension.9 In cultured 2K1 C and 2K1C+Na HS renal arteries, 12 h incubation with GSK0660(antagonist of PPARδ, 1 μmol/L); Compoud C(AMPKα inhibitor, 10 μmol/L); LY294002(PI3k inhibitor, 5 μmol/L); Wortmanin(PI3k inhibitor, 0.1 μmol/L); GW0742(agonist of PPARδ, 0.1 μmol/L); 30 min incubation with L-NAME(nitric oxide synthase inhibitor, 100 μmol/L) partly inhibited the effect of NaHS on endothelium-dependent relaxation(EDR) and the expression of related proteins.Conclusion: Our present results firstly revealed that exogenously administrating NaHS exhibited protective effect on endothelium through inhibiting RAS, activating PPARδ and then upregulating the expression of peNOS, increasing NO level in renal vascular hypertension rats. Part three Hydrogen sulfide improves endothelial dysfunction via down regulating BMP4/COX2 pathway in 2K1 C hypertensive rats Objective: Bone morphogenetic protein 4(BMP4), a member of the TGF-β superfamily, activates Smads as the immediate downstream molecules, following binding to BMP receptors. Previous studies found that BMP4 as a protein mediated bone growth and embryonic development. Recent reports showed that BMP4 played an important role on cadiovascular system. BMP4 activated the NADPH oxidase, increased the level of ROS, upregulated the expression of COX2, and then decresed the endothlium dependent relaxation, and resulted in hypertension. Our study was to elucidate wether NaHS-induced endothelial protective function is mediated by BMP4(bone morphogenetic protein 4) and oxidative stress-dependent upregulation of cyclooxygenase(COX)-2 in renal hypertension.Methods: Healthy male Sprague-Dawley rats were randomly assigned into 4 groups, namely: Sham operation group(Sham), Sham+NaHS group, 2K1 C group, 2K1C+NaHS group. Sham+NaHS and 2K1C+NaHS used sodium hydrosulfide(56 μmol/kg) daily intraperitoneal injection; Other set of groups used daily intraperitoneal injection of saline solution which was started from the fourth week after 2K1 C operation. Rats from four groups drank water freely, was fed a standard laboratory diet, and housed in a temperature-controlled room with a 12 h/12 h day/night cycle. Mean arterial pressure(MAP) was measured by a tail-cuff technique using a noninvasive blood pressure measurement system every four weeks. Microvessel recording technique was used to observe the effect of NaHS on endothelium-dependent contraction induced by ACh in isolated rena artery rings of rats at the end of 20 th week. Monobromobimane method was used to detect the level of H2 S in plasma. Western blot was adminstrated to observe the protein level of CSE, BMP 4, COX2, et al. and the expressions of proteins related to oxidative stress. The levels of SOD and MDA in plasma were detected by SOD and MDA kit.Results:1 Exogenously administrating NaHS lowered blood pressure, increased the level of plasma H2 S and CSE protein expression downregulates AT1 protein expression in renal vascular hypertension rats.2 Exogenously administrating NaHS ameliorated EDCs(endothelium dependent contractions)in renovascular hypertension rats.3 Treated with NaHSN reduced the protein expressionS of BMP4 and AT1.4 Exogenously administrating NaHS decreased the upregulated protein expression of enzymes related to oxidative stress.5 Moreover, exogenously administrating NaHS increased the activity of the major antioxidant enzyme SOD, decreased MDA level6 Exogenously administrating NaHS prevented vascular dysfunction through downregulating MAPK and Cyclooxygenase-2 protein expression in 2K1 C rats.Conclusion: Our present results firstly demonstrated that NaHS was effective on inhibiting the AT1 receptor-mediated cellular signaling cascade, suppressing BMP4 expression and vascular oxidative stress and on ameliorating EDCs and endothelial dysfunction in renovascular hypertension.