Study On The Effect Of The Traditional Chinese Medicine Compound Tangshen An On The Nrf2/ARE Pathway Of Human Renal Mesangial Cells Under High Glucose Environment

Purpose:To explore whether the renal protective effect of Tangshen’an is related to the activation of Nrf2/ARE pathway,antioxidant stress,and reduction of inflammatory response.Material and method:Male Wistar rats were randomly divided into 6 groups: normal group,model group,irbesartan group,and three Tangshen’an groups with different dosage.Preparation of drug-containing serum.The serum of normal group was added into mesangial cells cultured in normal concentration of glucose,and the other 5 groups were cultured in high concentration of glucose cultured mesangial cells.The expression of m RNA and protein of neclear factorerythroid 2-related factor 2(Nrf2)and its downstream factorγ-glutamylcysteinesynthetase(γ-GCS)were detected by RT-PCR and Western blot.The levels of antioxidant stress indicators Nrf2 and γ-GCS in the supernatant of each group were detected by ELISA.The levels of inflammatory-related factors interleukin-6(IL-6)and tumor necrosis factor-a(TNF-α)in the supernatant of each group were detected by ELISA.Results:1.The expression of m RNA and protein of Nrf2 and γ-GCS in human mesangial cells cultured in high concentration of glucose.The normal serum of normal group and model group was respectively added to human mesangial cells cultured in normal concentration of glucose and that cultured in high concentration of glucose.After 24 hours,human mesangial cells were collected,then detected by RT-PCR and Western blot.Compared with the normal group,the expression of m RNA and protein of Nrf2 and γ-GCS in human mesangial cells cultured in the model group increased(P<0.01).2.Tangshen’an can up-regulate the expression of m RNA and protein of Nrf2 and γ-GCS in human mesangial cells cultured in high concentration of glucose.The model group cultured normal serum into mesangial cells cultured in high concentration of glucose,the drug intervention group was divided into four kinds of drug-containing serums,low,medium and high doses of glucose of Tangshen’an and irbesartan,and cultured in human mesangial cells cultured in high concentration of glucose.After 24 hours,human mesangial cells were collected,then detected by RT-PCR and Western blot.Compared with the model group,the drug intervention group can increased the expression of m RNA and protein of Nrf2 and γ-GCS in human mesangial cells(P<0.01).3.The protein expression of Nrf2 and γ-GCS in the supernatant of human mesangial cells cultured in high concentration of glucose.By detectiong protein expression of Nrf2 and γ-GCS in human mesangial cells,we figure that high concentration of glucose up-regulates the protein expression of Nrf2 and γ-GCS in human mesangial cells.To further investigate the effects of high concentration of glucose on Nrf2 and γ-GCS in human mesangial cells supernatant,after culturing the glomerular mesangial cells in the normal group and the model group for 24 hours,the supernatant was taken,then detected by ELISA.Compared with the normal group,the expression of Nrf2 andγ-GCS m RNA and protein in the supernatant of human mesangial cells cultured in the model group were increased(P<0.01).4.Tangshen’an can reduces the content of Nrf2 and γ-GCS in the supernatant of human mesangial cells cultured in high concentration of glucose.To investigate the effect of Tangshen’an on the content of Nrf2 and γ-GCS in human mesangial cells supernatant,we cultured the glomerular mesangial cells of the model group and each administration group for 24 hours,and collected the supernatant for detection.Compared with the model group,the expressions of m RNA and protein of Nrf2 and γ-GCS in human mesangial cells cultured in each treatment group were decreased to different degrees(P<0.01).There was no significant difference between the middle dose group and the high dose group of Tangshen’an and the irbesartan group(P>0.05).5.High concentration of glucose can increase the protein expression of IL-6 and TNF-α in the supernatant of human mesangial cells.The normal serum of normal group and model group was respectively added to human mesangial cells cultured in normal concentration of glucose and that cultured in high concentration of glucose.After 24 hours,the supernatant of human mesangial cells were collected,then detected by ELISA.Compared with the normal group,the levels of IL-6 and TNF-α in the supernatant of human mesangial cells in the model group were increased(P<0.01).6.Tangshen’an can reduces the content of IL-6 and TNF-α in the supernatant of human mesangial cells cultured in high concentration of glucose.The model group cultured normal serum into mesangial cells cultured in high concentration of glucose,the drug intervention group was divided into four kinds of drug-containing serums,low,medium and high doses of glucose of Tangshen’an and irbesartan,and cultured in human mesangial cells cultured in high concentration of glucose.After 24 hours,the supernatant of human mesangial cells were collected,then detected by ELISA.Compared with the model group,The levels of IL-6 and TNF-α in the supernatant of human mesangial cells were down-regulated in each drug intervention group(P<0.01).Conclusion:1.Cultured in high concentration of glucose environment,the expression of Nrf2/ARE signaling pathway in human mesangial cells will increase compensatoryly,and the content of IL-6 and TNF-α will also increase.2.Tangshen’an can activate Nrf2/ARE pathway,antioxidant stress,reduce inflammation,and eventually achieve the role of renal protection.3.Tangshenan may protect the mesangial cells of kidney by promoting the transfer of Nrf2/ARE pathway-related factors in the cell supernatant into the cells.