| Part 1The expression of TGF 1 in exosomes in the urine of rats with IgA nephropathy and the protective mechanism of Dioscorea nipponica Makino on the kidney Objective:Dioscorea nipponica Makino reduce the proteinuria of IgA nephropathy rats,pathological damage to the kidney,kidney tissues Col ?,Fibronectin(FN),Laminin(LN),and a-smooth muscle actin(a-sma),the effect of transforming growth factor betal(TGF? 1)signaling pathway,and to explore the expression of TGF? 1 in urinary exosomes of IgA nephropathy model rats.Method:Healthy SD rats were divided into blank group(N group),model group(M),telmisartan group(T group),yam high dose group(DH),dose group(DM group)in yam,yam,low dose group(DL),adaptive feed a week later,the group M,T,DH group,DM group,the DL group building in rats,after the success of the building were given drug treatment accordingly.Normal saline was given to N group and M group,telmisartan solution was given to T group,high dose of pangolin total saponins was given to DH group,medium dose of pangolin total saponins was given to DM group,and low dose of pangolin total saponins was given to DL group.After 8 weeks of treatment,urine,serum and kidney tissue samples of each group were collected.Serum creatinine(Scr),Blood urea nitrogen(BUN),Serum albumin(ALB),Glutamic-pyruvic transaminase(ALT),and Glutamic oxalacetic transaminase(AST)were determined.HE staining,PASM staining and Masson staining were used to observe the glomerular changes under light microscope.The ultrastructure of renal tissue was observed under transmission electron microscope.Using immunohistochemical method,the Western Blot method(Western Blot)on kidney alpha SMA,col ?,FN,LN,TGF beta 1,smad3 for testing.Exosomes were extracted from rat urine by overspeed centrifugation,exosome morphology was observed by transmission electron microscopy,exosome marker protein was detected,and particle size was determined to identify the extracted substance as exosome.Western Blot was used to detect the expression of TGF 1 protein in urinary exosomes.Results:1.The efect of pangolin total saponins on proteinuria:compared with group N,the 24-hour urinary protein quantification was significantly increased in group M(P<0.05).Compared with the M group,the 24-hour urinary protein quantitation in T group,DH group,DM group and DL group was significantly reduced(P<0.05).There was no significant difference among T group,DH group,DM group and DL group(P>0.05).2.Blood biochemical indexes:compared with N group,Scr,BUN,ALB,ALT and AST levels of M group,T group,DH group,DM group and DL group showed no significant difference(P>0.05),and no significant difference between groups(P>0.05).3.Renal specimens stained with HE,PASM and Masson were observed under light microscope:group N:normal glomerular structure,clear vascular lumen,no proliferation,fibrosis or hyaline lesion in mesangial cells and mesangial matrix,smooth balloon wall.Group M:the glomeruli showed compensatory dilatation and atrophy,with swollen and hyperemic vascular globules,and some glomerular capillary globules swelling,from obvious lobulated and nodular lesions to compressive atrophy.Local mesangial stromal hyperplasia,basement membrane thickening,balloon wall narrowing.Group T:the degree of glomerular dilatation was slightly reduced,the vascular bulb was slightly congested,the mesangial matrix hyperplasia was not obvious,and no adhesion was observed on the balloon wall.DH group,DM group and DL group:the lesion was significantly reduced compared with that of M group,showing mild vascular bulb,mild proliferation of mesangial matrix,and slight thickening of basement membrane without balloon adhesion.DH group and DM group were better than DL group.4.Ultrastructure of renal tissue observed under electron microscope:no electronic dense matter was found in the mesangial area of group N,the foot processes were orderly arranged,the size was uniform,and the thickness of basement membrane was consistent;The M mesangial area was scattered in a large area of high-density electronic dense matter,the foot processes were fused in large areas,and local abscissions were observed.The mesangial region of group T was scattered in a small amount of high-density electronic dense matter,local fusion of foot processes was observed,and the thickness of basement membrane was consistent.In DH group,DM group and DL group,mesangial areas were scattered in a very small amount of high-density electronic density,foot processes were fused in a small amount,and the thickness of basement membrane varied.5.Kidney immunohistochemical detection results show that compared with the N group,group M kidney tissues of alpha SMA,col IV,FN,LN,the expression of TGF beta 1 and smad3 levels(P<0.05);Compared with M,T,DH group,DM group,DL alpha SMA,col ?,FN,LN,the expression of TGF beta 1 and smad3 level decreased(P<0.05);T group,the DH group,DM group,the comparison between DL way alpha SMA,col ?,FN,LN,the expression of TGF beta 1 and smad3 level no difference(P>0.05).6.Kidney western blot test results show that compared with the N group,group M kidney tissues of alpha SMA,col ?,FN,LN,the expression of TGF beta 1 and smad3 levels(P<0.05);Compared with M,T,DH group,DM group,DL alpha SMA,col ?,FN,LN,the expression of TGF beta 1 and smad3 level decreased(P<0.05);T group,the DH group,DM group,the comparison between DL way alpha SMA,col ?,FN,LN,the expression of TGF beta 1 and smad3 level no difference(P>0.05).7.Western Blot analysis of TGF 1 in urinary exosomes showed that the expression level of TGF 1 in renal tissues of group M was higher than that of group N(P<0.05).Compared with M,the expression levels of TGF 1 were decreased in T group,DH group,DM group and DL group(P<0.05).There was no difference in the expression level of TGF 1 between T group,DH group,DM group and DL group(P>0.05).Conclusion:1.Pangolin total saponin can reduce the proteinuria of IgA nephropathy rats and has safety.2.Pangolin saponins can reduce the deposition of extracellular matrix in kidney and reduce the degree of renal fibrosis.3.TGF 1 in urinary exosomes can reflect the degree of disease change.Part 2Effects of lps-induced exosomes released by mesangial cells on normal mesangial cellsObjective:1.To investigate the effects of lipopolysaccharide(LPS)on mesangial exosomes.2.To investigate the effect of TGF 1 in exosomes released by mesangial cells in LPS environment on the activation,proliferation and production of extracellular matrix of normal mesangial cells.Method:Mesangial cells can be divided into blank group(NL),the LPS group and LPS+TGF beta 1 sirna group were given normal culture medium containing LPS culture medium,the medium containing LPS+TGF beta 1 sirna after 48 hours,the detection of three groups of cells proliferation by CCK8 method,using Western Blot method to detect three groups of cells in the extracellular matrix of alpha SMA,FN,LN,and TGF beta 1,smad3 and p-smad3 expression.Exosomes in cell culture medium of each group were extracted,exosomes were identified,and TGF 1 content in exosomes of each group was detected.The blank group,the LPS group and LPS+TGF beta 1 sirna group is an equal amount of cells to produce secrete body in normal mesangial cells,divided into secrete body outside the blank group(exobiology+NL group),secrete body outside LPS group(group exobiology+LPS),secrete body outside LPS+TGF beta 1 sirna group(exobiology+LPS+TGF beta 1 sirna group)and no outside secrete group(outside the normal training,not to join secrete,marked as NEXO group),CCK8 method is used to detect four groups of cell proliferation,The expression of extracellular matrix-sma,FN,LN,Col IV,TGF 1,smad3 and p-smad3 were detected by Western Blot.Results:1.Cell proliferation in the NL group,LPS group and LPS+TGF 1siRNA group was detected by CCK8 assay.The results showed that compared with the NL group,the LPS group showed significant cell proliferation(P<0.05).Compared with NL group,LPS+TGF 1 siRNA group showed slightly more cell proliferation,but no statistical difference(P>0.05).Compared with LPS group,LPS+TGF IsiRNA group showed significantly lower cell proliferation(P<0.05).2.Western Blot analysis of ECM in cells of NL group,LPS group and LPS+TGF 1 siRNA group showed that compared with NL group,the expressions of SMA,FN,LN,Col IV,TGF 1,smad3 and p-smad3 were all increased in LPS group(p<0.05).Compared with LPS group,LPS+TGF 1siRNA group-sma,FN,LN,ColIV,TGF 1,smad3 and p-smad3 were all decreased(p<0.05).3.Exosome identification:CD9 and CD81(the marker protein of exosome)were positive in the vesicle samples isolated after ultracentrifugation,while Calnexin(the marker protein of endoplasmic reticulum)was negative.The exosomes were found to be typical circular vesicles by electron microscopy.Particle size analysis detected that the vesicle size peak was between 30 and 150nm,which proved that the extracted vesicles were exosomes without cell contamination.4.Western Blot analysis of TGF 1 levels in exosomes of NL group,LPS group and LPS+TGF 1siRNA group showed that compared with NL group,TGF 1 levels in exosomes of LPS group were significantly increased(P<0.05).Compared with the LPS group,the content of TGF 1 in exosomes in the LPS+TGF 1 siRNA group was decreased(P<0.05).5.Cell proliferation in EXO+NL group,EXO+LPS group,EXO+LPS+TGF 1siRNA group and NEXO group was detected by CCK8 assay.The results showed that cell proliferation in EXO+LPS group was significantly higher than that in EXO+NL group(P<0.05).Compared with the EXO+LPS group,cell proliferation was significantly reduced in the EXO+LPS+TGF 1siRNA group(P<0.05).6.Western Blot analysis of ECM in EXO+NL group,EXO+LPS group,EXO+LPS+TGF 1siRNA group and NEXO group showed that:compared with EXO+NL group and NEXO group,the expressions of SMA,FN,LN,Col IV,TGF 1,smad3 and p-smad3 in EXO+LPS group were all increased(p<0.05).Compared with the EXO+LPS group,the EXO+LPS+TGF 1siRNA group decreased in-sma,FN,LN,Col IV,TGF 1,smad3 and p-smad3(p<0.05).Conclusion:1.LPS can increase TGF 1 content in mesangial exosomes.2.TGF 1 in exosomes promotes proliferation of mesangial cells and increases extracellular matrix deposition.Part 3Dioscorea nipponica Makino total saponin reduce the production of extracellular matrix by interfering with TGF? 1 protective mesangial cells in exosomesObjective:It was proved that in LPS environment,Dioscorea nipponica Makino total saponin could regulate the activation,proliferation and extracellular matrix of mesangial cells by intervening TGF? 1 in the exosomes released by mesangial cells.Method:The mesenchymal cells were divided into blank group(NL group),LPS group,LPS+TGF 1siRNA group,LPS+pangolin TGF?1siRNA group(LPS+CSL group),LPS+TGFB 1siRNA group and pangolin TGF? 1siRNA group(LPS+CSL+TGFB 1siRNA group).Normal medium containing LPS medium,medium containing LPS+TGF? 1siRNA,LPS+pangolin TGF? 1siRNA,LPS+The proliferation of cells in the five groups was detected by CCK8 assay,and the expression of extracellular matrix-sma,FN,LN,Col IV,TGF? 1,smad3 and p-smad3 in the five groups was detected by Western Blot,Exosomes in cell cultures of each group were extracted,exosomes were identified,and TGF 1 content in exosomes of each group was detected by Western Blot.The blank group,the LPS group and LPS+TGF? beta 1 sima,LPS+yam total saponins group and LPS+TGF? beta 1 sirna+yam total saponins group the same number of cells secrete outside body respectively add to the number of normal mesangial cells,divided into secrete body outside the blank group(exobiology+NL group),secrete body outside LPS group(group exobiology+LPS),secrete body outside LPS+TGF beta 1 sirna group(exobiology+LPS+TGF beta 1 sirna group),secrete body outside LPS+CLS group(exobiology+LPS+CSL group),Exosomes in the LPS+TGF?1siRNA group+pangolin TGF?1siRNA+CSL group(EXO+LPS+TGF?1siRNA+CSL group)and non-exosome group(i.e.,normal culture without exosome.labeled as NEXO group)were used to detect the proliferation of six groups of cells,and Western Blot was used to detect the extracellular matrix-sma,FN,LN,Col IV,and the expression of TGF?1,smad3 and p-smad3 in the six groups of cells.Results:1.CCK8 assay of cell proliferation in NL group,LPS group,LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group showed that:compared with NL group,LPS group showed significant cell proliferation(P<0.05).Compared with LPS group,LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group showed decreased cell proliferation with statistical difference(P<0.05),but there was no statistical difference between LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group(P>0.05).2.Western Blot analysis of ECM in cells of NL group,LPS group,LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group showed that the expressions of a-sma,FN,LN,Col IV,TGF 1,smad3 and p-smad3 in LPS group were increased compared with NL group(p<0.05).Compared with LPS group,LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group,a-sma,FN,LN,Col ?,TGF?1,smad3 and p-smad3 were all decreased(p<0.05).There were no significant differences in LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group,a-sma,FN,LN,Col IV,TGF?1,smad3 and p-smad3(p>0.05).3.Exosome identification:CD9 and CD81 were positive and Calnexin was negative in vesicle samples isolated after high-speed centrifugation.The exosomes were found to be typical circular vesicles by electron microscopy.Particle size analysis detected that the vesicle size peak was between 30 and 150nm,which proved that the extracted vesicles were exosomes without cell contamination.4.Western Blot analysis of TGF?1 levels in exosomes of NL group,LPS group,LPS+TGF?1siRNA group,LPS+CSL group,LPS+CSL+TGF?1siRNA group showed that compared with NL group,TGF?1 levels in exosomes of LPS group were significantly increased(P<0.05).Compared with the LPS group,the content of TGF 1 in the LPS+TGF?1siRNA group,LPS+CSL group and LPS+CSL+TGF?1siRNA group decreased(P<0.05).5.Cell proliferation in the EXO+NL group,EXO+LPS group,EXO+LPS+TGF?1 siRNA group,EXO+LPS+CSL group,EXO+LPS+CSL+TGF?1siRNA group and NEXO group was detected by CCK8 assay.Compared with the EXO+LPS group,cell proliferation was significantly reduced in the EXO+LPS+TGF 1 siRNA group,EXO+LPS+CSL group,and EXO+LPS+CSL+TGF?1 siRNA+group(P<0.05).6.Western Blot analysis of ECM in EXO+NL group,EXO+LPS group,EXO+LPS+TGF?1 siRNA group,EXO+LPS+CSL group,EXO+LPS+CSL+TGF?1 siRNA group,and NEXO group showed that:compared with EXO+NL group and NEXO group,the expression of a-sma,FN,LN,Col IV,TGF?1,smad3 and p-smad3 in EXO+LPS group were increased(p<0.05).Compared with the EXO+LPS group,the EXO+LPS+TGF?1siRNA group,the EXO+LPS+CSL group,the EXO+LPS+CSL+TGF?1siRNA+group,the a-sma,FN,LN,Col IV,TGF 1,smad3 and p-smad3 groups were all decreased(p<0.05).Conclusion:Dioscorea nipponica Makino total saponin can inhibit proliferation of mesangial cells and alleviate extracellular matrix deposition by interfering with TGF?1 in extracellular exosomes... |
PDF Full Text Request