Based On Endoplasmic Reticulum Stress Signaling Pathway To Analyze The Molecular Mechanism Of Hypoxia/Reoxygenation Injury Model Of Hk-2 Cells Induced By Shen Shuai Kang Enema

Objectives: Based on endoplasmic reticulum stress signaling pathway to analyze the molecular mechanism of hypoxia / reoxygenation damage model of HK-2 cells induced by Shen Shuai Kang enema.Methods: Six white rabbits of New Zealand were randomly divided into 3 groups(2 rats in each group),which were divided into blank group,PBS group and Shen Shuai Kang enema group.The normal feeding of the blank group was not treated with experimental treatment.PBS group was treated with PBS,and Shen Shuai Kang enema group to give Shen Shuai Kang enema.Blood was taken after the rabbits were enema in the fourth day.The serum of PBS group was added to the PBS serum of hypoxia / reoxygenation injury group,the serum of the low dose group which including the serum of the Shen Shuai Kang enema group 25% and blank group 75% was added to the low dose serum of hypoxia / reoxygenation injury group,the serum of the middle dose group which including the serum of the Shen Shuai Kang enema group 50% and blank group 50% was added to the middle dose serum of hypoxia / reoxygenation injury group,the serum of the high dose group which including the serum of the Shen Shuai Kang enema group 100% was added to the high dose serum of hypoxia / reoxygenation injury group.The changes of apoptosis in the 4 groups of HK-2 cell hypoxia / reoxygenation injury models were detected by CFSF/PI staining.The flow cytometry was used to detect the changes of apoptosis rate in the 4 groups of HK-2 cells hypoxia / reoxygenation injury model group.RT-PCR was used to detect the expression of CHOP and GRP78 mRNA in the 4 groups of HK-2 cells hypoxia / reoxygenation injury model group.The Western blot method was used to detect the expression of CHOP,GRP78 and Caspase-12 protein in the 4 groups of HK-2 cells hypoxia / reoxygenation injury model group.Results:1.According to the results of CFSE/PI staining showed that at 4h the number of 4 groups of cells apoptosis increased significantly,indicating that the model of hypoxia / reoxygenation injury of HK-2 cells was established successfully.Shen Shuai Kang enema fluid medicated can reduce the apoptosis of HK-2 cells caused by hypoxia / reoxygenation injury and the high dose group had the best efficacy.2.Annexin V/PI staining was used to detect the apoptosis / death rate of HK-2 cells in hypoxia / reoxygenation injury model.0 hour after hypoxia/reoxygenation injury,the rate of cells apoptosis in each group was quite low,all of them were at a lower level.The apoptosis rate of cells in each group increased significantly in 4h-12 h.The data of 4h and 12 h showed that the apoptosis rate of high dose,middle dose and low dose enema serum containing hypoxia/reoxygenation injury model was significantly lower than that of PBS group,and the difference was statistically significant(P<0.05).Middle dose serum culture group,inhibition of apoptosis is most obvious,the apoptosis rate of model group were lower than PBS group cell apoptosis in hypoxia/reoxygenation injury model group,indicating Shen Shuai Kang serum can alleviate ischemia reperfusion injury.And at 24 h,the apoptosis rate of HK-2 cells in the 4 groups was significantly lower than that before.3.RT-PCR was used to detect the expression of CHOP and GRP78 mRNA in the model group of HK-2 cells hypoxia / reoxygenation injury.In 0h-24 h,GRP78 and CHOPmRNA were expressed.In 4h and 8h,the expression of GPR78 mRNA in serum group containing Shen Shuai Kang enema was lower than than that of PBS group at the same time point,and there was a statistical difference compared with the same time point PBS group(P<0.05).At 12 h,the expression of GPR78 mRNA in high dose group containing Shen Shuai Kang enema was lower than that of PBS group at the same time point,and there was a statistical difference compared with the same time point PBS group(P<0.05).At 4h,the expression of CHOP mRNA in the middle dose group and the high dose group decreased significantly,and there was a statistical difference compared with the same time point PBS group(P<0.05).At 12 h,the expression of CHOP mRNA in the middle dose group was the highest,and there was a statistical difference compared with the same time point PBS group(P<0.05).The results showed that the model induced endoplasmic reticulum stress and the expression of endoplasmic reticulum marker GRP78 mRNA and CHOP mRNA was influenced by Shen Shuai Kang enema.4.Western blot method was used to detect HK-2 hypoxia / reoxygenation injury model group CHOP、caspase-12 and GRP78 protein expression.At 8h,the expression of Caspase-12 protein and CHOP protein in the three groups of Shen Shuai Kang enema was lower than that in group PBS,while there was no difference in other time compared with that in the PBS group.The expression of GRP78 protein in the three groups of Shen Shuai Kang enema was equivalent to that in the PBS group.Conclusions: The molecular mechanism of Shen Shuai Kang enema may be through inhibit the up regulation of CHOP and Caspase-12 at 8h to inhibit of endoplasmic reticulum stress reduced the apoptosis of HK-2 cells.