Effect Of Tangshenning On TGF-?1 And P38MAPK Signal Pathway In Mouse Mesangial Cells Under High Glucose Condition

?Objective?: To observe the expression of high glucose cultured mouse mesangial cells TGF-beta 1,p38 MAPK,CREB and FN protein at the cell molecular level,to discuss the mechanism of effect of Tangshenning recipe inhibits the proliferation of extracellular matrix,and provide a new way of thinking and the starting point for the prevention and treatment of traditional Chinese medicine DN.?Methods?: 1.SV40 MES 13 cultured mouse mesangial cells at 37 DEG C and 5%CO2 cell incubator,containing 10% fetal bovine serum DMEM low sugar medium suspension cells inoculated in 25 ml cell culture flask experiments from 3-5 cells.2.Using 20 healthy male SD rats randomly into experimental animal were randomly divided into 2 groups,serum group(15 rats)and control group(5 rats),rats dose according to animal and human body surface area reduced rat dosage was 8 times the human equivalent dose 61.2 g·kg-1·d-1 gavage,the control group treated with normal saline,daily gavage 1 times(2.25 m L / 250g),continuous gavage for 7 days,the blood serum was collected from.3.The cultured SV40 MES 13 mouse glomerular mesangial cells were divided into two groups and the serum was added.Experimental groups: normal control group(low glucose DMEM 5.5mmol/L D-glucose medium N group);high glucose group(30mmol/L glucose containing DMEM medium H group,high glucose +);Tangshenning group(including 30mmol/L glucose DMEM medium T group,61.2 g·kg-1·d-1? 30.6 g·kg-1·d-1? 7.65 g·kg-1·d-1 three dose 10% Tangshenning medicated serum,were named T group,MT group,LT group);high glucose + valsartan group(V group,30mmol/L glucose +10 mol/L valsartan)intervention for 24 hours.4.Packet cell at 37 DEG C,5 CO2% of conventional cell culture incubator after 24 h by enzyme-linked immunosorbent assay(ELISA)detection in the supernatant of FN in screening of drug concentration,and the effect of Tangshenning on mesangial cells secrete FN protein expression.5.By using Western blotting(Western blot)protein was detected in the glomerular mesangial cells of p38 MAPK,p-p38 MAPK,TGF-beta 1,CREB,the cells were at 37 DEG C,5 CO2% of conventional cell culture incubator after 24 h with 2.5X107/L were inoculated into 60 mm culture dish,when cell growth to logarithmic 24 hours after the intervention period,termination of culture,discard the culture medium in the bottle,rinse 2 times,dry by PBS liquid,adding cell lysates(RIPA and PMSF mixed liquid)80ul,put on the ice by cell scratch from 3 minutes,suction flasks and scraping on the liquid to 1.5ml EP tube was measured,protein content,protein separation with 10% SDS-PAGE electrophoresis,the total protein content of each hole on 40 ug,with electrophoresis protein transferred to PVDF membrane,TBS-T membrane washing three times,every 5 minutes,5% skim milk powder closed 1 For hours,TGF-beta 1 and p38 MAPK,an anti anti phosphorylated p38 MAPK antibody,CREB antibody,4 C were incubated overnight,then wash the TBS-T film three times,each time for 5 minutes,after adding two anti HRP incubated at room temperature for 1 hours,three TBS-T wash the membrane every 5 minutes after washing,the membrane with chemiluminescent agent film exposure chamber.?Results?: 1.Effect of Tangshenning on expression of FN in mouse mesangial cells induced by high glucose in the supernatant of each group: compared with normal control group,the content of FN in the supernatant of mesangial cells in high glucose group increased significantly(P < 0.05);compared with high glucose group,Tangshen Ning high dose group in the supernatant FN content significantly decreased(P < 0.05),in a dose-dependent manner(P < 0.05),no significant differences between the content of FN in low dose group and high glucose group(P > 0.05),Tangshenning high dose group was determined as the final dosage.2.The expression level of Tangshenning on high glucose induced glomerular mesangial cells p38 MAPK,p-p38 MAPK,TGF-beta 1,CREB: compared with normal control group,significantly increased expression of p-p38 MAPK in high glucose group,TGF-beta 1,CREB protein level(P < 0.05),and high glucose group Tangshenning the expression of p-p38 MAPK group,valsartan group,TGFbeta 1 and CREB proteins were significantly reduced(P < 0.05),non phosphorylated p38 MAPK expression had no significant difference in each group.?Conclusion?: 1.High glucose environment can significantly promote the expression of TGF-1,pp38 MAPK,CREB in glomerular mesangial cells,and promote the secretion of FN protein in glomerular mesangial cells.2.It can significantly reduce the expression of TGF-1,p-p38 MAPK,CREB protein in mesangial cells under high glucose,and inhibit the secretion of FN protein in mesangial cells under high glucose.