| Part I The protective effects of Klotho on diabetes-induced myocardial injury in diabetic mice ObjectiveTo investigate the myocardial protective effect and mechanism of Klotho on diabetic mice MethodsForty 6-week-old C57BL/6 mice(weighing 18–22 g)were purchased from the Laboratory Animal Unit of Nanchang University.After a week of adaptive feeding,diabetes was induced in the animals by intraperitoneal(i.p.)injection with a single dose of freshly prepared streptozotocin(130 mg/kg).One week later,blood glucose levels were measured using a glucometer after the mice had fasted overnight.Only mice with a fasting blood glucose concentration >16.8 mmol/L were considered to be diabetic.Mice were divided randomly into 3 groups,control group(Con),diabetes group(DM),and diabetes+Klotho group(DM+KL),Mice in DM+KL group were treated with KL protein(0.01 mg/kg i.p.every 48 h)for 12 weeks.Mice in DM group were simply treated with vehicle.Blood glucose and body weight level were measured regularly.After 12 weeks of administration,the following experiments were conducted:(1)body weight,heart weight and tibia length of the mice were measured,and fasting blood glucose of the mice were measured;(2)Echocardiography was performed to assess LVEF,LVFS,E'/A';(3)Serum IL-18 ? IL-1? ? TNF-? concentration was measured by ELISA;(4)Serum levels of LDH,CK-MB were measured;(5)The changes of myocardial tissue structure in each group were observed by HE staining;(6)The contents of myocardial collagen fibers in each group were compared by masson staining and the collagen volume fraction was analyzed;(7)The apoptosis of myocardial cells in each group was compared by TUNEL staining;(8)The expression levels of NLRP3 and Il-1? in myocardium of rats in each group were detected by immunohistochemistry;(9)RT-qPCR was performed to compare the levels of ANP,BNP,TXNIP,NLRP3,caspase-1 and ASC genes in the myocardial tissues of each group;(10)Western blot to determine each myocardial tissue collagen I?collagen III??-SMA?cleaved-caspase 3,TXNIP,NLRP3,mIL-1?,cleaved caspase-1,ASC relative changes of protein expression and comparative analysis.Results1.Compared with the control group,1 week after STZ injection in the DM group,the blood glucose level significantly increased and the body weight gradually decreased,while Klotho had no significant changes in the blood glucose and body weight of the mice.2.Compared with Con group,the ratio of heart weight/body weight(HW/BW)and heart weight/tibial length(HW/TL)increased in DM group,and the HW/BW and HW/TL decreased significantly after KL administration(P<0.05).3.Compared with the control group,the mRNA expressions of ANP and BNP in DM group were increased,while the mRNA expressions of ANP and BNP were decreased after Klotho treatment(p<0.05).4.Compared with the control group,serum LDH and CK-MB in the DM group increased,while KL treatment prevented the increased CK-MB and LDH levels in DM.5.Echocardiography results showed that compared with the control group,LVEF,FS and E '/A' ratio decreased in the DM group.After Klotho administration,LVEF,FS and E '/A' ratio were increased.6.HE staining revealed that compared with the control group,myocardial fiber was disordered in the DM group,which was significantly improved after Klotho treatment.The results of Masson staining showed that collagen fiber deposition was decreased after Klotho treatment,and collagen volume fraction was significantly decreased compared with the DM group(P<0.05).Immunohistochemical results showed that the myocardial tissue of DM group showed obvious brown-yellow area,which was significantly improved after Klotho treatment.TUNEL staining results showed that compared with the DM group,the number of apoptotic cells was significantly reduced in the Klotho group,with statistically significant difference(P<0.05).7.Compared with control group,the serum level of IL-1?,TNF-?,and IL-18 in DM mice were elevated,while Klotho administration reversed it.8.RT-qPCR results of myocardial tissue showed that compared with the control group,TXNIP,NLRP3,caspase-1 and ASC levels were increased in the DM group,and Klotho treatment significantly reversed these changes.9.Western blot analysis showed that the collagen I?collagen III??-SMA?cleaved-caspase-3?TXNIP?NLRP3?mIL-1??cleaved-caspase-1?ASC expression increased in DM group and Klotho treatment reversed these changes.Conclusions1.Klotho improved myocardial hypertrophy,reduced pathological damage of myocardial tissue and inhibited myocardial inflammation in diabetic mice.2.The protective mechanism of Klotho on diabetic mice may be related to its inhibition of the activation of NLRP3 inflammasome.Part ? Protective effect and mechanism of Klotho on cardiomyocytes injury induced by high glucose Objective1.To explore the effects of Klotho on high glucose-induced NLRP3 inflammasome activation in H9C2 cells.2.To investigate the mechanism by which Klotho regulates NLRP3 inflammasome in high glucose-induced H9C2 cells.3.To explore the effect and mechanism of Klotho on high glucose-induced cardiomyocyte injury.Methods1.To investigate the effects of Klotho on high glucose-induced inflammasome activation.The H9C2 cells were cultured and divided into three groups:(1)control group(Con): the medium contained 5.5 mmol/L of D-glucose);(2)high glucose group(HG): cells were cultured with 35 mmol/L of D-glucose for 48 h);(3)HG +Klotho group(HG+KL): cells were pretreated with KL(400pM)for 1 h and then co-incubated with 35 mmol/L of D-glucose for 48 h);Then perform next experiments:(1)RT-qPCR was used to detect mRNA expression of NLRP3?ASC?IL-1??caspase-1 in each group.(2)The relative changes of NLRP3,ASC,mIL-1??cleaved-caspase-1 protein expression in each group were observed by Western blot.(3)ELISA was used to measure the supernatants IL-1?,IL-18,TNF-? level in each group.2.To investigate the mechanism by which Klotho regulates NLRP3 inflammasome in high glucose-induced H9C2 cells.The H9C2 cells were cultured and divided into four groups:(1)control group(Con): the medium contained 5.5 mmol/L of D-glucose);(2)high glucose group(HG): cells were cultured with 35 mmol/L of D-glucose for 48 h);(3)HG +Klotho group(HG+KL): cells were pretreated with KL(400pM)for 1 h and then co-incubated with 35 mmol/L of D-glucose for 48 h);(4)HG+N-acetyl-L-cysteine group(HG+NAC): cells were pretreated with NAC(5mM)for 1 h and then co-incubated with 35 mmol/L of D-glucose for 48 h);Then perform next experiments:(1)ROS levels were detected with the peroxide-sensitive fluorescent probe DCFH-DA;(2)The relative changes of TXNIP in each group was observed by Western blot;(3)Detection of IL-1? in each group by ELISA.3.To explore the effect and mechanism of Klotho on high glucose-induced cardiomyocyte injury.NLRP3 siRNA was used to explore the mechanism by which KL regulated cardiomyocyte injury,Then H9C2 cells were cultured and divided into four groups::(1)control group(Con): the medium contained 5.5 mmol/L of D-glucose);(2)high glucose group(HG): cells were cultured with 35 mmol/L of D-glucose for 48 h);(3)HG+NLRP3 siRNA group(HG+siNLRP3): H9C2 cells were transfected transiently with NLRP3 siRNA for 24 h,and then incubated with 35 mmol/L of D-glucose for 48);(4)HG +Klotho group(HG+KL): cells were pretreated with KL(400pM)for 1 h and then co-incubated with 35 mmol/L of D-glucose for 48 h).Then perform next experiments:(1)Cell apoptosis was detected by flow cytometry;(2)The expression of cleaved-caspase-3,cytochrome C protein were examined by western blot;(3)??m was assessed by JC-1.Results1.KL suppressed NLRP3 inflammasome activation in HG-induced H9C2 cells.The RT-qPCR results also indicated that the mRNA expression levels of NLRP3,IL-1?,caspase-1,and ASC were elevated in H9C2 cells treated with HG,while the expression of NLRP3 inflammation was inhibited by KL treatment.The immunoblotting assay results revealed that high-glucose-induced protein expression of NLRP3,ASC,cleaved-caspase-1(p20),mIL-1?,and TXNIP were suppressed by KL.Moreover,ELISA results revealed that IL-1?,TNF-?,and IL-18 protein levels in the supernatants of H9C2 cell medium were dramatically reduced by KL.2.KL inhibited HG-induced ROS generation in H9C2 cellsA significant increase in ROS level in HG-treated H9C2 cells,and KL inhibited HG-induced ROS generation,which was comparable to the effect induced by treatment with NAC,an ROS scavenger.Then we found that pretreated with NAC and Klotho decreased the TXNIP expression and IL-1? secretion in HG-cultured H9C2 cells.3.KL attenuated HG-induced apoptotic death in cardiomyocytes by inhibiting NLRP3 inflammation in H9C2 cells.Successful transfection was proved by western blotting,the protein levels of cleaved-caspase-1 and mIL-1? were inhibited in HG induced H9C2 cells,suggesting the inactivation of inflammasome.Flow cytometry revealed that NLRP3 knockdown significantly decreased the apoptotic cells in HG-induced H9C2 cells,which was comparable to the effect induced by treatment with KL.Moreover,pretreatment of H9C2 cells with siNLRP3 or KL significantly inhibited the HG-induced upregulation of cleaved-caspase-3,cytochrome C in H9C2 cells.JC-1 staining revealed that HG significantly decreased ??m.Pretreatment of H9C2 cells with siNLRP3 or KL effectively nullified the HG-induced drop in ??m.Conclusions1.KL suppressed NLRP3 inflammasome activation in HG-induced H9C2 cells.2.KL inhibited HG-induced ROS/TXNIP/ NLRP3 activation in H9C2 cells.3.KL and NLRP3 knock down reduced cardiomyocyte apoptosis by inhibiting Cyto c/caspase-3 pathway induced by high glucose. |
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