Mechanism Of Cortex Mori Radicis On Prevention Of High Glucose-induced Myocardial Microvascular Endothelial Cells Injury In Mice Based On TRPC6-ERK Pathway

Part I Protective effect of CMR on myocardium and myocardial microvascular injury in diabetic miceObjective In the present study,type 1 diabetes mellitus model was induced on mice.We aimed to investigate the potential effects related to the early effects of CMR on myocardial and myocardial microvascular injury in diabetic mice.Methods Twelve mice were selected from 60 SPF male C57 BL / 6 mice as a control group.The remaining 48 mice were injected with 50 mg / kg STZ for 5 consecutive days.Three days after the end of STZ injection,fasting blood glucose was tested.Blood glucose levels were measured 72 h after STZ injection.Diabetic mice were diagnosed by fasting glucose levels ≥ 15 mmol/L.Diabetic mice were randomly divided into the model group and the CMR treatment group(low dose,high dose),with 12 mice in each group.CMR was treatmented for 4 weeks.After treatment,OGTT was performed,and then the mice were sacrificed.Observe myocardial tissue morphology by HE staining.CD31 immunohistochemical staining was used to observe myocardial microvessel density.Western blot was used to detect the expression of TRPC6-ERK signaling pathway-related proteins in myocardial tissue.Results(1)The blood glucose of mice in the model group was higher than that of the control group at all time points in the OGTT.The blood glucose of mice treated with high and low concentration CMR was significantly lower than that of the model group at each time point.(2)In the model group,myocardial fibers were disordered and enlarged.Compared with mice in the diabetes model group,myocardial fiber disorder and enlargement were alleviated in the low-and high-concentration treatment group.(3)The density of myocardial microvessels in the model group was significantly reduced,and the density of myocardial microvessels in the high-concentration CMR treatment group was higher than that in the model group.(4)The expression level of TRPC6 in myocardial tissue of the model group increased,and the ratio of p-ERK / ERK increased.Compared with the model group,the expression of TRPC6 and the ratio of p-ERK / ERK in myocardial tissue of the mice treated with high and low CMR were reduced.Conclusion(1)Early treatment of CMR can reduce blood glucose in type 1 diabetic mice,and can improve myocardial damage and decrease in myocardial microvessel density in type 1 DM mice.(2)Early treatment of CMR can inhibit the overexpression of TRPC6 and the phosphorylation of ERK in myocardial tissue of type 1 DM mice.Part 2: Protective effect of CMR-containing drug serum on mouse myocardial microvascular endothelial cells and its possible mechanismObjective In order to study the effect of Sangbaipi on myocardial microvascular endothelial cells(CMECs),in this section,primary cultured CMECs were used as experimental objects.The CMECs were given high glucose treatment to observe the effect of high glucose on cells.And then investigate the protective effect and mechanism of CMR drug-containing serum on CMECs.Methods CMECs were cultured by enzyme digestion.The cell activity and expression of TRPC6,p-ERK and ERK in CMECs stimulated by high glucose at 30 mmol/L for different durations were detected by MTT assay and Western blot.CMECs were treated with high-glucose DMEM containing 10% different dose gradient CMR drug-containing serum(5 times,10 times,15 times,and 20 times dose CMR respectively).MTT assay and Western blot were used to detect the activity of endothelial cells and the expression of TRPC6,p-ERK and ERK to determine the optimal dose of CMR drug-containing serum.Set up 5 groups: control group,high glucose treatment group,optimal multiple of CMR drug-containing serum+high glucose treatment group,TRPC Inhibitor(2-APB)+high glucose treatment group,optimal dose of CMR drug-containing serum+TRPC6 agonist(Hyperforin)+ high glucose treatment group.Western Blot was used to detect the expression levels of p-ERK,ERK,Bax,Bcl-2,Cleaved Caspase-3 and GAPDH in each group.Results(1)After 24 hours of 30mmol/L glucose stimulation,the cell viability of CMECs was reduced.And after 48 hours of high glucose stimulation,cell viability decreased significantly.TRPC6 expression levels and p-ERK/ERK ratios were increaswas selected as the intervention conditions for subsequent experiments.(2)Compared with the high glucose group,the cell viability of the 10-fold and 15-fold dose CMR-containing serum group was increased.Compared with the model group,the expression ed in the 24 and 48 hours of HG treatment group.And 24 hours of high glucose treatment levels of TRPC6 and the ratio of p-ERK / ERK decreased.The reduction was more pronounced in the 15-fold dose group.(3)Compared with the high glucose treatment group,the p-ERK /ERK ratio,Bax / Bcl-2 ratio,and Cleaved Caspase-3 expression were reduced in the15-times CMR drug-containing serum group,TRPC6 inhibitor group.The TRPC6 agonist reversed the effect of CMR drug-containing serum to a certain extent.Conclusions(1)TRPC6 expression level and ERK phosphorylation level of myocardial microvascular endothelial cells increased and cell viability decreased after 24 hours of high glucose treatment.(2)CMR may reduce ERK phosphorylation and inhibit apoptosis by down-regulating high glucose-induced overexpression of TRPC6 in myocardial microvascular endothelial cells.