Exogenous Hydrogen Sulfide Inhibits High Glucose-induced Injuries Via Regulation Of Leptin/LEPR-JAK/STAT Signal Pathway In HUVECs

Chapter 1 Exogenous hydrogen sulfide inhibits high glucose-induced injuries via regulation of leptin/LEPR signaling pathway in HUVECs[Background]In China,there are hundreds and thousands of diabetes mellitus patients,the amount of which has been more than one hundred million.Hyperglycaemia,which is an important characteristics of diabetes mellitus,is the most direct factor that can result in nearly all the complications including macrovascular disease,microvascular disease,neuropathy et al.Hyperglycaemia can cause many kinds of complications of diabetes mellitus by many kinds of pathophysiology mechanism,just like vascular endothelial dysfunction et al.Leptin is a hormone mainly secreted by adipocytes,exerting its biological activity through the leptin receptor(LEPR).As cytokine,leptin can exert pleiotropic biological effects such as the regulation of inflammation,reproduction,and angiogenesis.The leptin/LEPR signal pathway have been studied extensively and characterized at biochemical and molecular levels in many systems.Emerging evidence indicates that leptin signaling may be a potential new target in diabetic vascular disease treatment.Hydrogen sulfide(H2S)is a new type of gas signal molecule.H2S can protect many types of cells by its antioxidant effect and its role on regulating intracellular signaling pathways.Recently,the effect of H2S on vascular diseases caused by DM has attract lots of attention.However,whether H2S can protect endothelial cell from HG-induced injury by regulating the leptin/leptin receptor pathway remains to be researched.[Objective]1.To explore whether the leptin/LEPR signal pathway mediates HG-induced injury in human umbilical vein endothelial cells(HUVQCs);2.To investigate whether hydrogen sulfide(H2S)inhibits the HG-induced injury by modulating the leptin/LEPR signal pathway in HUVECs.[Methods]1.HUVECs were cultured with 40 mmol/L glucose(high glucose,HG)for 24 h to establish the HG-induced injury model;2.Cell viability was examined by cell counter kit-8(CCK-8);3.The expression levels of leptin and leptin receptor protein were measured by Western blot assay;4.Apoptotic cells were assessed by Hoechst 33258 nuclear staining followed by photofluorography;5.The judgement of intracellular reactive oxygen species(ROS)level was detected by DCFH-DA staining followed by photofluorography;6.Mitochondrial membrane potential(MMP)was performed by Rhodamine 123(Rh123)staining followed by photofluorography.[Results]1.HUVECs were treated with 40 mmol/L glucose for 3-24 h,respectively.The expression of leptin began to significantly increase at 3 h after exposure to HG and peaking at 9 h(p<0.01);2.HUVECs were treated with 40 mmol/L glucose for 3-24 h,respectively.The expression of lepfin receptor(LERP)began to significantly increase at 3 h after exposure to HG and peaking at 9h(p<0.01);3.Pretreatment of HUVECs with 400 umol/L sodium hydrosulfide(NaHS,donor for H2S)for 30 min inhibited HG-induced an increase in the expression levels of leptin in HUVECs(p<0.01);4.Pretreatment of HUVECs with 400 umol/L NaHS for 30 min inhibited HG-induced an increase in the expression levels of leptin receptors in HUVECs(p<0.01);5.Pretreat HUVECs with 400umol/l NaHS for 30min alleviated HG-induced injury:the rate of cell viability increased,the amount of apoptotic cell decreased and accumulation of intracellular ROS and loss of MMP were attenuated significantly(p<0.01);6.Pretreatment of HUVECs with 50 ng/ml leptin antagonists(LA),had cardiomyocyte protection similar to the one of NaHS,leading to an increase in cell viability,decreases in amount of apoptotic cells,generation of intracellular ROS,and a loss of MMP(p<0.01).[Conclusion]1.The leptin/LEPR signal pathway mediated the HG-induced injury in HUVECs;2.Exogenous hydrogen sulfide protects against the HG-induced injury by inhibiting leptin/LEPR pathway in HUVECs.Chapter 2 Exogenous hydrogen sulfide inhibits high glucose-induced injuries via regulation of JAK/STAT signal pathway in HUVECs[Background]In the first chapter,we have confirmed that H2S can attenuate high glucose induced damage in HUVECs by inhibiting leptin/LEPR signal pathway.Janus kinase/Signal transducer and activator of transcription(JAK/STAT)pathway was confirmed earliest as the most important downstream signaling pathways of leptin.After binding with its receptor,leptin can change the receptor’s conformation,and then activate the JAK proteins(especially include JAK2),the activated state JAK2 can then activate STAT(STAT3)pathway.Some studies have pointed out that JAK/STAT pathway was involved in leptin-induced cardiomyocyte hypertrophy.It was also reported that high glucose can cause diabetic nephropathy by activating the JAK/STAT pathway.However,wether JAK/STAT pathway mediate high glucose-induced vascular endothelial cell injury is not yet clear.there are less research that study this issue which worth being exploring.[Objective]1.To explore whether the JAK/STAT signal pathway mediates HG-induced injury in HUVECs;2.To investigate whether H2S inhibits the HG-induced injury by modulating JAK/STAT signal pathway in HUVECs.[Methods]1.HUVECs were cultured with 40 mmol/L glucose(high glucose,HG)for 24 h to establish the HG-induced injury model;2.Cell viability was examined by cell counter kit-8(CCK-8);3.The expression levels of p-JAK2 and p-STAT3 protein were measured by Western blot assay;4.Apoptotic cells were assessed by Hoechst 33258 nuclear staining followed by photofluorography;5.The judgement of intracellular reactive oxygen species(ROS)level was detected by DCFH-DA staining followed by photofluorography;6.Mitochondrial membrane potential(MMP)was performed by Rhodamine 123(Rh123)staining followed by photofluorography.[Results]1.Pretreatment of HUVECs with 400 umol/L NaHS for 30 min inhibited HG-induced an increase in the expression levels of p-JAK2 in HUVECs(p<0.01);2.Pretreatment of HUVECs with 400 umol/L NaHS for 30 min inhibited HG-induced an increase in the expression levels of p-STAT3 in HUVECs(p<0.01);3.Pretreat HUVECs with 400umol/I NaHS for 30min alleviated HG-induced injury:the rate of cell viability increased,the amount of apoptotic cell decreased and accumulation of intracellular ROS and loss of MMP were attenuated significantly(p<0.01);4.Pretreatment of HUVECs with 20 μmol/L the inhibitor of JAK/STAT pathway(AG490)for 30 min,had cardiomyocyte protection similar to the one of NaHS,leading to an increase in cell viability,decreases in amount of apoptotic cells,generation of intracellular ROS,and a loss of MMP(p<0.01).[Conclusion]1.The JAK/STAT signal pathway mediated the HG-induced injury in HUVECs;3.Exogenous hydrogen sulfide protects against the HG-induced injury by inhibiting JAK/STAT signal pathway in HUVECs.