The Study On The Mechanism Of The Effects Of Nephrin And Podocin On Human Podocyte Slit Diaphragm Which Caused By Active Principle Of Astragalus Membranaceus

Objective:To analysis the changes of expression of associated proteins which in human podocytes that treated with different concentrations of astragalus polysaccharides(APS)and astragaloside ?(AS?)after being jnjured by puromycin aminonucleoside(PAN).A comparison of the treatment effects of different concentrations seeks whether there is a correlation between curative effect and concentration.To explore the role of PKC/MAPK pathway in it,and guess the possible mechanism of action of APS and AS? to protect podocytes.That provides a new thinking for clinical targeted therapy.Method:To confirm the appropriate concentration and time of PAN that caused injury of human podocyte slit diaphragm and the maximum nontoxic concentration of APS and astragaloside IV by using cytotoxicity test CCK8.The differentiation of human podocytes must be completed before establishing the damage model of human pdocytes.Then dealing these cells with different concentrations of APS and ASIV and solvent(deal cells in normal group and model group with maintenance media).Testing the mRNA levels of nephrin and podocin by RTPCR as where as the protein levels of nephrin,podocin,PKC,JNK and p38 by Western blotting.Finally,conjecturing the relationship between the indexes,the indexes and the disease by means of analysing the change regulations of related proteins.Result:1.RTPCR method showed that:the mRNA expression levels of nephrin and podocin in injured human podocytes were decreased significantly in modelgroup than those in normal group(P<0.05).However,after treating with different concentrations of APS,the expressions were increased in varying degrees.There have differences that nephrin in high concentration group and podocin in medium concentration group and high concentration group compare with modelgroup(P<0.05).What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that APS can increase the mRNA expression of nephrin and podocin in injured human podocytes.2.RTPCR method showed that:the mRNA expression levels of nephrin and podocin in injured human podocytes were decreased significantly in model group than those in normal group(P<0.05).However,after treating with different concentrations of AS?,the expressions were increased in varying degrees.There have differences that nephrin and podocin in high concentration group compare with model group(P<0.05).What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that AS? can increase the mRNA expression of nephrin and podocin in injured human podocytes.3.Western blotting methods howed that:the protein expression levels of nephrin and podocin in injured human podocytes were decreased significantly in model group than those in normal group(P<0.05).However,after treating with different concentrations of APS,the expressions were increased in varying degrees.There have differences that nephrin and podocin in high concentration group compare with model group(P<0.05)What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that APS can increase the expression of nephrin and podocin in injured human podocytes.4.Western blotting method showed that:the protein expression levels of nephrin and podocin in injured human podocytes were decreased significantly in model group than those in normal group(P<0.05).However,after treating with different concentrations of AS?,the expressions were increased in varying degrees.There have differences that nephrin and podocin in both medium concentration group and high concentration group compare with model group(P<0.05).What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that AS? can increase the expression of nephrin and podocin in injured human podocytes.5.Western blotting method showed that:the protein expression levels of PKC,JNK and p38 in injured human podocytes were increased significantly in model group than those in normal group(P<0.05).However,after treating with differentconcentrations of APS,the expressions were decreased in varying degrees.There have differences that PKC,JNK and p38 in both medium concentration group and high concentration group compare with model group(P<0.05).What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that APS can increase the expression of PKC,JNK and p38 in injured human podocytes.6.Western blotting method showed that:the protein expression levels of PKC,JNK and p38 in injured human podocytes were increased significantly in model group than those in normal group(P<0.05).However,after treating with different concentrations of AS?,the expressions were decreased in varying degrees.There have differences that JNK and p38 in both medium concentration group and high concentration group and PKC in high concentration group compare with model group(P<0.05).What's more,these differences display concentration dependent and have nothing to do with solvent(P>0.05).Therefore,it can be considered that AS? can increase the expression of PKC,JNK and p38 in injured human podocytes.Conclusion:APS and AS? could upregulate the expression of nephrin,podocin and downregulate the expression of PKC,JNK and p38 which changed in injured podocytes caused by PAN,and the regulation showed concentration dependent.lt was indicated that APS and AS? can influence the express of nephrin and podocin which are specific proteins in podocyte slit diaphragm by adjusting the activity of PKC/MAPK pathway.All of these achieve the goal that maintain the integrity of slit diaphragm,protect the podocytes and postpone the progression of kidney disease.