| Objective: To investigate the effect of Yiqihuoxuejiedu herbs drug serum,Adrenomedullin(ADM)inhibit autophagy in the human proximal tubular epithelial cell(HK2 cells)induced with aldosterone(ALD)in vitro.Method: HK2 cells were induced by 1μmol/L aldosterone and treated with10% drug serum,Adrenomedullin 100nmol/L,and were divided into the following four groups: control group,aldosterone group,the aldosterone+drug-containing serum group and aldosterone+ Adrenomedullin group.After 48 hours,observe the expressions of microtubuleassociated protein 1 light chain 3,MAP1-LC3(LC3),Beclin1,Autophagy related genes5(Atg5),mammalian target of rapamycin(mTOR),p-mTOR,Extracellular signaling regulates kinase(ERK),P-ERK,Serum and glucocorticoid induced kinase-1(SGK-1)were determined by western blot and immunofluorescence.Results:1 The expression of SGK-1,P-ERK1/2 and P-mTOR in autophagy by ImmunofluorescenceThe expression of SGK-1,P-ERK1/2 and P-mTOR in HK2 cells was observed by laser confocal microscopy.The results showed that the expression of SGK-1 was significantly increased after aldosterone stimulation,mainly in the cytoplasm,which was significantly different from that control group.;The expression of TCM,adrenomedulin in the treatment group was significantly lower than that in the aldosterone group.The expression of P-ERK1/2 in aldosterone group was significantly higher than that in control group,and the expression of P-ERK1/2 was found in cytoplasm and nucleus.After treated by TCM or adrenomedulin,the expression decreased significantly.P-mTOR was a negative regulatory factor of autophagy.The expression of P-mTOR in aldosterone group was lower than in control group,mainly in cytoplasm,.The expression of P-mTOR was increased after TCM or adrenomedulin treatment.2 The expression of autophagy associated protein Atg5,Beclin1 and LC3 in HK2 cells by immunofluorescenceThe results showed that the number of autophagy associated protein(Atg5,Beclin1)in aldosterone group was significantly higher than that in control group,and it was mainly distributed in the cytoplasm,and that in TCM and adrenomedulin group was significantly lower than that in aldosterone group.LC3 was distributed in the cytoplasm in control group,which was induced by aldosterone,autophagy membrane was formed and fused with lysosome to form autophagy lysosome,which was distributed in the cytoplasm with grininess.With treament with TCM,adrenomedulin,the grininess in the cytoplasm was decreased and LC3 was dispersed throughout the cytoplasm.3 The expression of SGK-1,P-ERK1/2,Atg5,Beclin1 and LC3Ⅱ/Ⅰ in HK2 cells by Western blotThe results showed that the expression of SGK-1,P-ERK/ERK in aldosterone group was significantly higher than that in control group,and the difference was statistically significant.(P < 0.01)TCM,adrenomedulin could inhibit its expression,but there was no significant difference between the two groups.(P > 0.05)The expression of mTOR,which is a autophagy inhibitor protein,and P-mTOR in aldosterone group were lower than those in control group,and the difference was statistically significant.(P < 0.01)TCM,adrenomedulin can upregulate its expression,but there was no significant difference between the two groups.(P > 0.05)Atg5,Beclin1,LC3,which is the most important positive regulatory factor in autophagy,were involved in the formation of autophagy.The expression of Atg5,Beclin1,LC3 in aldosterone group was higher than that in control group,and the difference was statistically significant.(P < 0.01)TCM and adrenomedulin group were significantly lower than aldosterone group,and the difference was statistically significant.(P < 0.05)Conclusion:1 Aldosterone induces autophagy in human proximal tubular epithelial cells2 Adrenomedullin antagonizes renal tubular epithelial cell injury induced by activation of mineralcorticoid receptor complex by regulating mitogen-activated protein kinase(MAPK)pathway,which can protects renal tubular epithelial cells.3 Yiqi Huoxue jiedu herbs can inhibit autophagy of human proximal renal tubular epithelial cells,which may be related to down-regulation of SGK-1,P-ERK1/2 expression. |
