Effect Of Chongcaoyishen Particle On Proliferation And Oxidative Stress Of Human Glomerular Mesangial Cells In High-glucose Environment

Objective:To observe the effect of Chongcaoyishen Particle on proliferation and oxidative stress of human glomerular mesangial cells(HMCs)cultivated in high-glucose environment and to explore its renoprotective mechanism.Methods:According to Serum pharmacological method,we first got the rats serum at different concentration of Chongcaoyishen Particle and Fosinopril,and then studied the effect of the serum on HMCs.The HMCs cultivated in high-glucose environment in vitro were randomly divided into six groups:the control group,the high-glucose group,the Chongcaoyishen Particle of low,medium and high dose groups and Fosinopril group.After 24 h,48 h and 72 h exposure,the supernatants and HMCs were collected separately.The proliferation of HMCs was tested with CCK-8 at 24 h,48 h and 72 h time points.The levels of intracellular reactive oxygen species(ROS)were determined by flow cytometry at 48 h and 72 h time points.The activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalese(CAT)and the levels of malondialdehyde(MDA)were respectively determined by colorimetric method at 48 h and 72 h time points.Expressions of p22 phox and p47 phox mRNA were detected by Real-time PCR at 48 h and 72 h time points.The expression levels of p22 phox and p47 phox protein were measured by Western blot.Results:1.The results of HMCs proliferation detected via CCK-8 method:Compared with the control group,the high-glucose group(30 mmol/L)displayed a significant increase in the proliferation of HMCs at 24 h,48 h,72 h time points(P<0.01).Compared with high-glucose group,when treated with Chongcaoyishen Particle and Fosinopril at 48 h and 72 h time points,the cell proliferation was inhibited at varying degrees(P<0.01 or P<0.05).2.Comparison of intracellular ROS production in HMCs:Cells cultured in high-glucose environment for 48 h or 72 h showed a significant increase in the intracellular level of ROS when compared with that in the control group(P<0.05).When HMCs were treated with Chongcaoyishen Particle and Fosinopril,the intracellular ROS levels decreased significantly compared with that of the high-glucose group(P<0.01).3.Comparison of the activities of SOD,GSH-PX,CAT and MDA level in the supernatant in HMCs in different groups:At the same time,compared with the control group,the activities of SOD,GSH-PX,CAT significantly decreased in glucose group,while the level of MDA greatly increased(P<0.01 or P<0.05).When treated with Chongcaoyishen Particle and Fosinopril at the same time points,the activities of SOD,GSH-PX,CAT increased and the level of MDA decreased at varying degrees(P<0.01 or P<0.05).4.Comparison of the expression levels of p22 phox and p47 phox mRNA in HMCs in different groups:Compared with the control group at the same time point(48 h or 72 h),the expression levels of p22 phox and p47 phox mRNA were significantly upregulated(P<0.01);Compared with the high-glucose group at the same time point(48 h or 72 h),when HMCs were treated with Chongcaoyishen Particle and Fosinopril,the expression levels of p22 phox and p47 phox mRNA were downregulated(P<0.01).5.Comparison of the expression levels of p22 phox and p47 phox protein in HMCs in different groups:HMCs in the control group expressed p22 phox and p47 phox at a low level.At the same time point(48 h or 72 h),the protein expression levels of p22 phox and p47 phox in the high-glucose group was significantly higher than that in the control group(P<0.01);Compared with the high-glucose group at the same time point(48 h or 72 h),Chongcaoyishen Particle and Fosinopril could markedly suppress the expression levels of p22 phox and p47 phox protein(P<0.01).Conclusions:1.Extracellular high glucose can promote the proliferation of HMCs,increase the level of ROS and MDA,and suppress activities of SOD,GSH-PX,CAT in the supernatant in HMCs;p22 phox and p47 phox mRNA and protein expression can be also increased by high glucose,thus intensifying oxidative stress reaction.2.Chongcaoyishen Particle can inhibit the proliferation of HMCs cultured in high glucose medium3.Chongcaoyishen Particle can reduce oxidative stress reaction of HMCs in the following ways:reducing the expression levels of ROS and MDA,increasing the activities of SOD,GSH-PX,CAT,decreasing the expression levels of p22 phox and p47 phox mRNA,and suppressing the expression of p22 phox and p47 phox protein.4.Chongcaoyishen Particle can protect the kidney and delay DKD progression.Maybe the mechanism of control and protection of DKD is that Chongcaoyishen Particle inhibit proliferation of HMCs and oxidative stress reaction.Meanwhile,the therapeutic effect of Chongcaoyishen Particle has some time and dose effect dependence.