Bufalin On Rat Mesangial Cell Proliferation And Apoptosis

Pathological proliferation of mesa ngial cells and increase of mesa ngial matrixare the most common pathologica l changes in glomerulonephritis. Most ofglomerulonephritis have the trend of chronic progressive development. In the processof the progress, me sangia l cells are not only victims, but also actively participate inthe development of glomerular infla mma tion and promote glomerulosclerosis throughthe secretion of infla mma tory cytokines and extracellula r matrix. One of theimportant factors that promote fibrosis is transforming growth factorβ1 (TGF-β1),which ma y stimula te the secretion of extracellular matrix. Its activation can stimula temesa ngial cell synthesis of type II and IV colla gen, FN and other importantextracellular matrix, increase the accumula tion of mesa ngial extracellular matrix inmesa ngium, and accelera te the progression of glomerular sclerosis. At the same time,apoptosis also pla ys an important role in the formation and dissipation of glomerularproliferative lesion. In recent years, studies have shown that in some self-limitingproliferative glomerular diseases, proliferative mesa ngial cells can relieve themselvesthrough apoptosis mecha nism and restore norma l structure of glomerular. In theaspect of the treatment of proliferative glomerulonephritis, currently the best drug isadrena l glucocorticoids. However, there are a considerable number of patients whohave generated dependence, resista nce, or even serious side effects on it.Subsequently, the immunosuppressive agents emerged such as Cyclo sphospha mide,Chlora mbucil, Cycloporin A.Nevertheless, the kind of drugs do not have satisfactoryefficacy on some patients. Therefore, it was becoming one of hot spots to develop andexcavate new drug coming from natural pla nt to treat proliferousglomerulonephritis. A monomer of tranditiona l chinese med icine, Bufalin, is derivedfrom gla ndular secretion of retroauricular and skin gla nds in Bufo toads and black toads. The molecular formula is C24 H34 O4 and molecular weight is 386.5. Researchesshow that such ingredient has anti-cancer, cardiac, toxic and narcotic effects. Sucheffects are probably because of inhibition of leukemia cell proliferation throughinterfering cell cycle, induction of tumor cell differentiation and apoptosis throughchanging apoptosis-related genes such as bcl-2 family, and abnorma lly activation of avariety of pathways such as MAPK. However, little is known about the role of Bufalinon glomerular mesa ngial cells. We speculate that Bufalin ma y have a similar role onmesa ngial cells. To that end, on the one hand we observed the influence of Bufalin onGMC abnorma l proliferation and extracellular matrix secretion; on the other hand, weobserved the influence of Bufalin on the apoptosis of abnorma l proliferative GMC.This study provides a theoretica l basis for the future development and treatment ofclinica l proliferative glomerulonephritis. ParPart one: Effect of Bufalin on Rat's Glomerular MesangialCell Proliferation and Extracellular Matrix HyperplasiaObjective: To investigate the effect of Bufalin on Rat's glomerular mesa ngial cells(GMC) proliferation and hyperplastic extracellular matrix (EMC) induced bylipopolysaccharide(LPS).Methods: Rat's GMC lines were cultivated.GMC in vitro were divided into 3groups:control group;LPS group( LPS 5mg/L); Bufalin group(LPS 5mg/L andBufalin 1×10-8mol/L). Proliferation of GMC was assessed by MTT assay and flowcytometry.The mRNA of IL-6 and TGF-β1 were measured by RT-PCR ,mea nwhilethe expression of Fibronectin(FN) and TGF-β1 in the conditioned med ium weremeasured by ELISA.Results: The GMC proliferation was induced in LPS group, but that was inhibited inBufalin groups in a dose-dependent ma nner.The percentage of cells in S Phase inBufalin group were significa ntly less tha n that in LPS group(P<0.01).The expressionof IL-6 and TGF-β1 mRNA were significa ntly inhibited in Bufalin group(P<0.01).The expression of FN and TGF-β1 protein in Bufalin group weresignifica ntly less tha n those in LPS group(P<0.01).Conclusion: Bufalin could inhibit the GMC proliferation and the hyperplastic ECMcomponents induced by LPS. ParPart two:Effect of Bufalin on Apoptosis of Rat's GlomerularMesangial Cells induced by lipopolysaccharideObjective: To investigate the effects of Bufalin on glomerular mesa ngial cells(GMC)apoptosis induced by lipopolysaccharide(LPS) .Methods: Rat's GMC lines were cultivated.GMC in vitro were divided into 3groups:control group;LPS group( LPS 5mg/L); Bufalin group(LPS 5mg/L andBufalin 1×10-8mol/L).Cell structure was observed by electron microtrop.Thetranscriptiona l level of Bax and bcl-2 gene were detected by RT-PCR .The proteinlevel of Bax and bcl-2 were detected by Western blot method.Result: The morphology of GMC presented changes of apoptosis with electronmicrotrop.The expression of Bax mRNA and protein in Bufalin group weresignifica ntly more tha n that in LPS group(P<0.01),but the expression of bcl-2 mRNAand protein were less tha n that in LPS group(P<0.01).Conclusion: Bufalin ma y induce the apoptosis of lipopolysaccharide-ind uced GMCby modulate modulateing genes Bax and bcl-2.