The Protective Effect Of Salidroside On Mouse Podocytes In High Glucose Environment And The Potential Mechanism

Part one The impact of salidroside on the apoptosis and oxidative stress of podocyte in high glucose environmentObjective: Currently,there are nearly 100 million Chinese people suffering from diabetics and China has become the country with the largest total population of diabetes.Therefore,more effective therapeutic measures are in crying need to prevent genesis and development of these complications.Diabetic nephropathy(DN)is one of the main microvascular diseases,even the lethal cause of diabetics,leading to end stage renal disease.To explore the pathogenesis of diabetic nephropathy and to seak effective treatment,has become a hot topic in Kidney and endocrine fields.So far,the pathogenesis of diabetic nephropathy is not clear.Research in recent years displays that injury and apoptosis of podocytes,is closely related to proteinuria.Chinese herbal medicine rhodiola rosea,with salidroside as one of its main effective constituents,possessing anti-aging,anti-hypoxic,and antioxidant activity.Some studies pointed out that,salidroside could enhance the activity of antioxidase,restrain oxidative stress,reduce proteinuria in diabetic rats.The present experiment aims to explore the impact of salidroside on podocyte oxidative stress and apoptosis through mouse podocytes in vitro in high glucose environment,with salidroside as the drug intervention.Method:1 The conditionally immortalized mouse podocytes were cultured and after synchronization,qualified podocytes were used in experiments.2 Differentiated podocytes were randomly divided into 3 groups: normal glucose group(NG,5.5mM),high glucose group(High glucose,HG,30mM)and high glucose+Salidroside(HG+Salidroside 50?M)group.We examined the effect of salidroside on ROS in podocytes using dichlorofluorescein diacetate(DCFH-DA,Molecular Probes)by fluorometric assay.MTT was used to evaluate the viability of podocytes in all groups.Western blot was used to evaluate the expression of Caspase-9 and Caspase-3.Results:1 morphology observation of podocytes: At 33 ? with ?–IFN(the permissive conditions),cells proliferated and maintained cobblestone-like morphology.When cultured at 37 ? without ?-IFN(nonpermissive condition),cells stretched out differently shaped long processes from cell bodies,arised many spindle-like projections from their primary processes and stopped proliferation.Specific protein Synaptopodin can be seen by fluorescent colouration in mature podocytes.2 The results of MTT:Copmared with normal glucose group,the podocyte viability of high glucose was lower;whlie copmared with high glucose group,the podocyte viability of high glucose+salidroside group was higher.The difference was statistically significant(P<0.05).3 The results of flow cytometry:Copmared with normal glucose group,the total ROS level of high glucose group was higher;whlie copmared with high glucose group,the total ROS level of high glucose+salidroside group was lower.The difference was statistically significant(P<0.05).4 The results of TUMEL:Copmared with normal glucose group,the apoptosis rate of high glucose group was higher;whlie copmared with high glucose group,the apoptosis rate of high glucose+salidroside group was lower.The difference was statistically significant(P<0.05).5 The results of Weternblot:Copmared with normal glucose group,the expression of Caspase-9 and Caspase-3 in high glucose group was higher;whlie copmared with high glucose group,the expression of Caspase-9 and Caspase-3 in high glucose+salidroside group was lower.The difference was statistically significant(P<0.05).Conclusion:1 High glucose reduced the viability of mice podocytes,enhanced ROS generation and promoted podocyte apoptosis.2 Salidroside increased the viability of mice podocytes in high glucose,and the mechanism may be due to decreasing the oxidative stress level.3 Salidroside treatment reduced podocyte apoptosis in high glucose invironment,and the mechanism may be reducing the expression of Caspase-9and Caspase-3.Part two The influence of salidroside on HO-1 and Nrf-2 expression in high glucose cultured mice podocytesObject: It was revealed in the former experiment that Salidroside could inhibit podocyte oxidative stress response and apoptosis in high glucose invironment.While the specific mechanism was not clear.Haem oxygenase(HO)is one of the body's important antioxidant systems,including three subtypes.Among them,HO-1 can induces a variety of products with effect of oxidation resistance and protection.Previous study revealed that HO-1expression increased in high glucose cultured podocytes.Nrf2/ARE pathway is the most important known antioxidant pathway.Some studies showed that,activating MAPK and Nrf2 could promote the expression of HO-1.The present research is aimed to verify whether salidroside could protect podocyte via modulating HO-1 and Nrf2.Method:1 The conditionally immortalized mouse podocytes were cultured and after synchronization,qualified podocytes were used in experiments.2 To explore the impact of salidroside with different concentration on HO-1 expression in podocytes cultured in normal glucose,the podocytes were divided into 4 groups,and respectively cultured in 0?10?20?50?M salidroside for 24 h.Then Western blot was used to evaluate HO-1 expression in all groups.To explore the impact of salidroside with different concentration on HO-1expression in podocytes cultured in high glucose,the podocytes were divided into 4 groups,and respectively cultured in 0?10?20?50?M salidroside for24 h.Then Western blot was used to evaluate HO-1 expression in all groups.To selcect the best intervention concontration of Salidroside as 50?M according to the experiment result.To explore the impact of salidroside with differentintervation time on HO-1 expression in podocytes cultured in high glucose,the podocytes were divided into 4 groups,and respectively harvest at 4?8?12?24h.Then Western blot was used to evaluate HO-1 expression in all groups.To selcect the best intervention concontration of Salidroside as 50?M according to the experiment result.3 The first grouping method: To explore the impact of salidroside on Nrf-2 expression,the podocytes were divided randomly into 3 groups: NG,HG,HG+SAL group.Then Western blot was used to evaluate Nrf-2expression 24 h later.The second grouping method: the podocytes were divided randomly into4 groups: NG,HG,HG+SAL,HG+SAL+SnPPIX(HO-1 inhibitor,10?M,1h).The total ROS was assessed by flow cytometry,and the expression of Caspase-9 and Caspase-3 were evalued by Western blot.The third grouping method: the podocytes were divided randomly into:NG,HG,HG+SAL,HG+SAL(50?M)+Scramble siRNA(10nM,12h),HG+SAL(50?M)+HO-1 siRNA(10nM,12h),HG+SAL(50?M)+Nrf2 siRNA(10nM,12h),and further experiments were done 24 h later.The podocytes disposed with siRNA were all transfected for 12 h and then were cultured in Salidroside for 24 h.The total ROS was assessed by flow cytometry,and the expression of Caspase-9 and Caspase-3 were evalued by Western blot.Results:1 Different concentration of Salidroside(0?10?20?50?M)was added to mouse podocytes in high glucose for 24 h.The results of Western blot revealed that the expression of HO-1were higher than the grous free of Salidroside.And HO-1 expression were the most at 50?M.2 At different time points(4?8?12?24h),the expression of HO-1 was assessed.The results of Western blot revealed that compared with high glucose group,HO-1 expression was higher,but the The difference was not statistically significant(P>0.05).While at 8h?12h?24h,HO-1 expression was higher,but the The difference was statistically significant(P<0.05).3 The results of Western blot revealed that,compared with high glucosegroup,the expression of Nrf2 in HG+SAL group was higher,and the difference was statistically significant(P<0.05).4 The results of flow cytometry revealed that compared with NG group,the total ROS was higher;compared with HG group,the total ROS was higher in HG+SAL+SnPPIX group.The difference was statistically significant(P<0.05).5 The results of Western blot revealed that,compared with NG group,the expression of Caspase-3 and Caspase-9 in HG was higher.Compared with HG group,the expression of Caspase-3 and Caspase-9 in HG was lower in HG+SAL group.Compared with HG+SAL group,the expression of Caspase-3and Caspase-9 in HG was higher in HG+SAL+SnPPIX group.The difference was statistically significant(P<0.05).6 The results of flow cytometry revealed that compared with NG group,the total ROS was higher in HG group;compared with HG group,the total ROS was lower in HG+SAL group.While compared with HG+SAL+scr siRNA,the total ROS in the HG+SAL+HO-1 siRNA group and the HG+SAL+Nrf2 siRNA group were higher.The difference was statistically significant(P<0.05).7 The results of Western blot revealed that,compared with NG group,the expression of Caspase-3 and Caspase-9 in HG was higher.Compared with HG group,the expression of Caspase-3 and Caspase-9 in HG+SAL was lower.While compared with HG+SAL+scr si RNA group,the expression of Caspase-3 and Caspase-9 in HG+SAL+HO-1 siRNA group and HG+SAL+Nrf2 siRNA group were increased.The difference was statistically significant(P<0.05).Conclusion:1 Salidroside could suppress oxidative stress level of podocytes in high glucose environment and protect podocytes.2 Salidroside could reduce podocyte apoptosis in high glucose environment,which may be related to its protective effect on podocyte.3 Salidroside could alleviate the oxidative stress and apoptosis ofpodocytes through upregulating Ho-1 and Nrf2 in high glucose.Part three Research on signal transduction mechanism of modulation of HO-1 by salidroside in high glucoseObject: The experiment in part two revealed that salidroside could alleviate the oxidative stress and apoptosis of podocytes through upregulating Ho-1 and Nrf2 in high glucose.While the specific mechanism was not clear.It was known that,PI3K/Akt took part in several biologic processions,and could involved in podocyte injury in diabetic patients.PI3K/Akt as a vital anti-apoptosis singnal pathway,could upregulate HO-1expression and protecet cells.ILK expression was abnormal in seveal glomerular diseases,which is closely related with PI3K/Akt.Studies showed that ILK had anti-apoptosis effect.MAPK has three subgroups: ERK,JNK and p38,which was involved in the modulation of oxidative stress and apoptosis.The present research is aimed to explore whether salidroside could promote HO-1 and Nrf2 expression by way of ILK/PI3K/Akt and MAPK family.Methods:1 The conditionally immortalized mouse podocytes were cultured and after synchronization,qualified podocytes were used in experiments.2 The first grouping method: podocytes were devided into NG,HG,HG+SAL 0.5h(50?M,0.5h),HG+SAL 1h(50?M,1h),HG+SAL 1.5h(50?M,1.5h),HG+SAL 2h(50?M,2h)group,p-ILK and p-Akt were assessed by Western blot.3 The second grouping method: podocytes were devided into NG,HG,HG+SAL 5min(50?M,5min),HG+SAL 10min(50?M,10min),HG+SAL15min(50?M,15min),HG+SAL 20min(50?M,20min)group,p-ERK,p-JNK and p-p38 were assessed by Western blot.4 The third grouping method: podocytes were devided into NG,HG,HG+SAL,HG+SAL+PD98059,HG+SAL+SP600125,HG+SAL+SB203580,HG+SAL+LY294002,HG+SAL+QLT0267,HO-1 and Nrf2 expressions were assessed by Western blot.Results:1 The results of Western blot revealed that,compared with NG group,p-Akt expression was lower than high glucose group.At 0.5,1,1.5 and 2h,p-Akt and p-ILK expression was lower than high glucose group when Salidroside was added to podocyte.Difference was statistically significant(P<0.05).2 The results of Western blot revealed that,compared with NG group,p-p38 expression of HG was higher with statistical significance,while p-JNK and p-ERK expression of HG were lower,but of no statistical significance.Compared with HG gropup,p-JNL and p-ERK expression were higher,while p-p38 was lower in HG+SAL 15 min,and HG+SAL 20 min groups(P<0.05).3 The results of Western blot revealed that,compared with NG group,HO-1 expression in HG was lower,but of no statistical significance(P>0.05)..Compared with HG group,HO-1 expression in HG+SAL was higher with statistical significance(P<0.05).Compared with HG+SAL group,HO-1expression in HG+SAL+SP600125 group was lower,while HG+ SAL+PD98059 group and HG+SAL+SB203580 group were higher.Difference was statistically significant(P<0.05).4 The results of Western blot revealed that,compared with NG group,HO-1 expression in HG was lower,but of no statistical significance.Compared with HG group,HO-1 expression in HG+SAL group was higher with statistical significance(P<0.05).Compared with HG+SAL group,HO-1expression in HG+SAL+QLT0267 group and HG+SAL+LY294002 group were lower wtih statistical significance(P<0.05).5 The results of Western blot revealed that,compared with NG group,Nrf2 expression was lower,but of no statistical significance.Compared with HG group,Nrf2 expression was higher in HG+SAL with statistical significance(P<0.05).Compared with HG+SAL group,Nrf2 expression in HG+SAL+SP600125 group and HG+SAL+LY294002 group were lower with statistical significance(P<0.05).While there were no significant change of Nrf2 expression in HG+SAL+PD98059 group and HG+SAL+SB203580group(P>0.05)Conlusions:1 Salidroside could promote HO-1 and Nrf2 expression through activating ILK and Akt pathway.2 Salidroside could promote HO-1 expression through JNK pathway.3 P38 MAPK and ERK could involve in the process of podocyte injury.To sum up,this study revealed that the oxidative stress level and podocyte apoptosis was increased in high glucose.While salidroside could relieve oxidative stress level and podocyte apoptosis.The mechanism may be related to upregulation of HO-1 and Nrf2 expression,and the signal transduction pathway ILK/PI3K/Akt and JNK could play a certain role.