Effects Of Compatibility Of Salvianolic Acid A And C On The Endoplasmic Reticulum Stress Related Apoptosis Protein Induced By HSA In HK-2 Cells

Objective:To study the effect of Salvianolic acid A and C compound on the endoplasmic reticulum stress related apoptotic proteins in HK-2 cells induced by human serum albumin.Methods:HK-2 cells were cultured in vitro and randomly divided into 5 groups: normal group,model group,Salvianolic acid A group(group SalA),Salvianolic acid C group(group SalC),Salvianolic acid A+C group(compatibility group,salvianolic acid A,C ratio of 1: 1).In addition to the normal group,the other groups were induced by HSA after the synchronization of 24 h,and the three drug groups were given corresponding drug to co-culture.The cells were collected at 24 h,48h and 72 h interval after co-culture.The quantitative expression of GRP78,ATF-4,Caspase-12 and CHOP protein was detected by Western blot(WB).Immunofluorescence technique was used to measure the determined expression of CHOP and ATF-4 protein.Flow cytometry(FCM)was used to detect the apoptosis rate and activation rate of Caspase-3.Results:1.The apoptosis rate of various groups by FCM: Compared with the normal group,the apoptosis rate of model group was significantly higher in three time period(P < 0.05).Compared with the model group,the apoptosis rate of each drug group was significantly lower in three time period(P < 0.05).Compared with the drug groups,the apoptosis rate of compatibility group was significantly lower than that in the SalA group and the SalC group in three time period(P < 0.05),SalA,C group had no significant difference in 24 h and 48 h compared with each other(P > 0.05).2.The activation rate of Caspase-3 in each time group was detected by FCM: Compared with the normal group,the activation rate of Caspase-3 in the model group was significantly higher in three time period(P < 0.05).Compared with the model group,the Caspase-3 activation rate of each drug group was significantly lower in three time period(P < 0.05).Compared with the drug groups: There was no significant difference in the activation rate of Caspase-3 between the drug group in 24h(P > 0.05).The activation rate of Caspase-3 in compatibility group and SalA group was significantly lower than that in SalC group in 48h(P <0.05),but there was no significant difference in the activation rate of Caspase-3 between SalA group and compatibility group(P > 0.05).The Caspase-3 activation rate of the compatibility group was significantly lower than that of SalA group and SalC group in 72h(P < 0.05).3.The expression of GRP78 protein in each time group was detected by WB: Compared with the normal group,the relative expression of GRP78 in the model group was significantly increased in three time period(P <0.05).Compared with the model group,the relative expression of GRP78 in each drug group was significantly reduced in three time period(P < 0.05).Compared with the drug groups: The relative expression of GRP78 in SalC group was significantly lower than that in other drug groups in 24h(P < 0.05).There was no significant difference in the relative expression of GRP78 in each drug group in 48h(P > 0.05).In 72 h,the relative expression of GRP78 in the compatibility group was significantly lower than that in other drug groups(P < 0.05).4.Detection of Caspase-12 protein expression in each time group of cells by WB: Compared with the normal group,the relative expression of Caspase-12 in the model group was significantly increased in three time period(P < 0.05).Compared with the model group,the relative expression of Caspase-12 in each drug group was significantly reduced in three time period(P < 0.05).Compared with the drug groups,the relative expression of Caspase-12 in the compatibility group was significantly lower than that in the other groups in three time period(P < 0.05).5.Detection of CHOP protein expression in each time group of cells:(1)WB detection results: Compared with the normal group,the relative expression of CHOP in the model group was significantly increased in three time period(P < 0.05).Compared with the model group,the relative expression of CHOP in each drug group was significantly reduced in three time period(P < 0.05).Compared with the drug groups,the relative expression of CHOP in the compatibility group was significantly lower than that in the other groups in three time period(P < 0.05).(2)Results of cell immunofluorescence assay: The positive expression of CHOP was a green fluorescent signal,which was located in the nucleus of HK-2 cells.6.Detection of ATF-4 protein expression in each time group of cells:(1)WB detection results: Compared with the normal group,the relative expression of ATF-4 in the model group was significantly increased in three time period(P < 0.05).Compared with the model group,the relative expression of ATF-4 in each drug group was significantly reduced in three time period(P < 0.05).Compared with the drug groups,the relative expression of ATF-4 in the compatibility group was significantly lower than that in the other groups in three time period(P < 0.05).(2)Results of cell immunofluorescence assay: The positive expression of ATF-4 was a green fluorescent signal,which was located in the nucleus of HK-2 cells.Conclusion:Salvianolic acid A,C and molecule drugs compound could inhibit the activation of Caspase-3 and the expression of GRP78,Caspase-12,CHOP,ATF-4 protein in HK-2 cells induced by HSA.The results suggesting that salvianolic acid A,C and molecule drugs may affect the expression of endoplasmic reticulum stress related apoptosis proteins in HK-2 cells,interfere with the endoplasmic reticulum stress related apoptosis pathway,reduce the apoptosis of renal cells,and thus play a role in delaying the process of renal fibrosis.