Effect Of Traditional Chinese Medicine On The Autophagy - Related Protein Of RSC96 Schwann Cells

Objective:Exploring the molecular mechanism of the treatment with Tang-Tong-Fang(TTF) prescription of diabetic peripheral neuropathy(DPN),from the expression of autophagy related protein of RSC96,which will provide new evidence for the effectiveness of utilizing TTF to prevent and treat DPN.Method:Establishing the apoptosis model of RSC96 cells, which are immortalized cells transformed from Schwann Cells spontaneously, after a intervention of 72 hours with 125mM high glucose DMEM. Detecting the low/middle/high level of TTF concentrations dissolved in 125mM high glucose DMEM with CCK-8 method, which are adopted to intervene RSC96 cells’apoptosis damage. This experiment was set up 5 groups, including control group(con), high glucose model group(Model), Tang-Tong-Fang in low dose group(LTTF), Tang-Tong-Fang in medium dose group(MTTF), Tang-Tong-Fang in high dose group(HTTF).Detecting the expression changes of autophagy related protein, like LC3,Beclin l,Atg5,Atg7,FoxO3a,TAK1 with Western bolt method. Detecting the mRNA content changes of autophagy related protein, like LC3,Beclin 1,Atg5,Atg7,FoxO3a,Bcl-2 with RT-PCR method. This experiment mainly explored the intervention effect of Tang-Tong-Fang reducing RSC96 cells’apoptosis damage by high glucose from autophagy.Results:1. High glucose inhibited the proliferation of RSC96 cells, and also,the inhibition and the high glucose concentration were positively correlated.2. RSC96 cells were damaged in apoptosis after 72 hours intervention by 100mM or 125mM glucose, and the apoptosis rate was 7.457±0.963 or 16.530±1.005, compared with Con, P＜0.01.3. Identified 3 effective concentration of TTF, which improved the inhibition of RSC96 cells proliferation caused by high glucose, respectively were low(0.5mg/ml)、medium(1.0mg/ml)、high(1.5mg/ml) level of concentration.4. RSC96 cells cultured by high glucose after 72 hours, its expression of autophagy related protein, like LC3,Beclin 1,Atg5,Atg7,FoxO3a,TAK1, was higher than Con, P<0.05,however, after the intervention of different concentration of TTF, its expression decreased,P<0.05.5. RSC96 cells cultured by high glucose after 72 hours, its mRNA content of autophagy related protein, like LC3,Beclin 1,Atg5,Atg7,FoxO3a, was higher than Con and the Bcl-2 mRNA content was lower,P<0.05,however, after the intervention of different concentration of TTF, the mRNA content of autophagy related protein decreased and the Bcl-2 mRNA content increased,P<0.05.Conclusion and significance:High glucose inhibited the proliferation of RSC96 cells, and it was most obvious that the inhibition of 125mM glucose. RSC96 cells would be damaged at apoptosis obviously after 72 hours intervention by 125mM glucose. After 72 hours intervention by high glucose, the bcl-2 mRNA content of RSC96 cells decreased, the expression and mRNA content of autophagy related protein, like LC3,Beclin 1,Atg5,Atg7,FoxO3, increased than Con, as well as, the expression of TAKl,one of upstream kinases located at AMPK pathway, increased than Con, which confirmed that cells apoptosis associated with excessive activation of cells autophagy. However, after the intervention of TTF, the bcl-2 mRNA content of RSC96 cells increased, and the expression and mRNA content of autophagy related protein, like LC3,Beclin 1,Atg5,Atg7,FoxO3, decreased, which showed that TTF can inhibit excessive activation of RSC96 cells autophagy through reducing the expression of autophagy related protein. That could be one of the molecular mechanisms of Tang-Tong-Fang’s treatment of diabetic peripheral neuropathy. This results above provided new theoretical evidence for the effectiveness of prevention and treatment of DPN with Tang-Tong-Fang.