Effects Of Modified Hangqichifeng Decoction On Apoptosis Of Mice Mesangial Cells Undergoing Inflammatory Proliferation

Research Background:Abnormal glomerular mesangial cells proliferation is the main pathological change of glomerular diseases such as IgA nephropathy, membrane hyperplastic nephritis, lupus nephritis, focal segmental glomerular sclerosis and diabetic glomerular sclerosis-Abnormal proliferation of glomerular mesangial cells can cause excessive generation and accumulation of extracellular matrix, mesangial cells in the process of progress is not only the victims, and through the secretion of inflammatory factors and extracellular matrix actively participates in the development of glomerular inflammation.And ultimately lead to glomerular sclerosis, renal fibrosis and end-stage renal failure. So try to control the mesangial cell proliferation, prevention of renal glomerular damage for the reverse is of great significance to the evolution of chronic disease. Apoptosis in the process of formation and dissipation of glomerular hyperplastic lesions also plays a very important role. In recent years, according to a study in some self-limited hyperplastic glomerular disease, the proliferation of glomerular mesangial cells can relieve themselves and make structure of glomerular returned to normal by apoptosis mechanism.Modified Hangqichifeng Decoction (MHD) is the commonly used formula for treating chronic nephritis by Zhang Yu, previous studies have shown that MHD medicated serum can inhibit the proliferation of mesangial cells in mice induced by LPS, matrix Col-IV in mesangial cells and the expression of LN and FN. But whether it can promote the mesangial cells to apoptosis, is still poorly understood. This experiment by studying the MHD medicated serum on mouse mesangial cell apoptosis induced by LPS, provide experimental and theoretical basis for its clinical application. Objective:Research the effects of Modified Hangqichifeng Decoction on apoptosis of mouse mesangial cells undergoing inflammatory proliferation.Methods:Modified Hangqichifeng Decoction and telmisartan in mice after lavage, preparation of medicated serum, with DMEM containing10%medicated serum culture medium in vitro cultivation of MMCs, LPS as a stimulating factor, cells can be divided into the control group, the LPS group, the telmisartan group(TM group), MHD with high, medium and low dose group. Use1. TEM observation of MMCs ultrastructural changes.2. Flow cytometry instrument to detect apoptosis.(AnnexinV-FITC/PI staining)3. Western Blot method to detect the Bcl-2,Bax,Caspase-3protein expression in MMCs.Results:1. Under the observation of TEM, cells of the control group and the LPS group are normal and contain abundant cell organelle. The majority of the cells showed the round or elliptic morphology,with clear nucleolus,smooth and complete nuclear membrane edge and without apoptotic body and margination of chromosome.Cells in Telmisartan group cell are shrinking,with round morphology,cell membrane protrusions, more organelles within the wrapped slurry is slightly reduced and crowded.The nucleolus is clear and the nuclear membrane is thickness. Cells in MHD groups showed that nucleolus are disappeared, nuclear membrane thickness, chromatin pyknosis and margination.2. Apoptosis rate of MHD groups, compared to the LPS group were significantly increased (p<0.01); Telmisartan group compared to the LPS group, increased apoptosis and has statistical significance (p<0.05); And comparisons between groups in MHD, the difference had statistical significance (p<0.05), and have a certain effect; Telmisartan group compared with MHD low dose group, no significant difference (p>0.05), but compared with the MHD mid and high dose, the difference is significantly (p<0.05, p<0.01respectively)3. The control group, lipopolysaccharide group with telmisartan group between the Bcl-2, no significant differences between relative expression of Bax protein (p>0.05), while the MHD groups and LPS stimulation and telmisartan group compared to the Bcl-2protein expression significantly reduced (p<0.01), the expression of Bax protein increased significantly (p<0.01); MHD in every dose group, the Bcl-2protein expression with the higher dose of MHD and decreasing, and high dose group of MHD, compared low dose group, the Bcl-2protein expression significantly reduced (p<0.01).4. Telmisartan, MHD each dose group and the control group, lipopolysaccharide group between the relative expression of Caspase3protein have significant difference (p<0.01); MHD groups compared with telmisartan group, Caspase-3protein levels in MMCs have significantly higher (p<0.01); MHD groups between each dose groups, with the increase of dose, Caspase3in MMCs between groups in protein expression in turn increases, and there are statistically significant (MHD in high dose group and low dose, dose, compared (p<0.01);MHD mid dose and low dose compared (p<0.05);5. Compared with the control group and LPS group, the Bcl-2/Bax telmisartan group no significant change (p>0.05); MHD in each dose group and control group, LPS group, compared the Bcl-2/Bax have statistical significance (MHD low dose group compared with control group or LPS group,p<0.05; MHD, high dose group or LPS group compared with control group, p<0.01); Chinese traditional medicine group compared with telmisartan group, significantly reduce the Bcl-2/Bax ratio (MHD low dose group of the Bcl-2/Bax ratio compared with telmisartan group,(p<0.05);MHD mid dose and high dose group the Bcl-2/Bax ratio compared with telmisartan group, p<0.01). Pairwise comparison between MHD in each dose group was statistically significant (p<0.05), and with the increase of the dose, the Bcl-2/Bax ratio gradually reduce.Conclusions.Compared with Telmisartan, MHD can significantly promote the apoptosis of mouse mesangial cells induced by LPS and may have certain dose-effect relationship.2.MHD can promote cell apoptosisby by raising Bax, Caspase3protein expression level, decreasing the Bcl-2protein expression level and the Bcl-2/Bax ratio.