Mechanism Study On The Effects Of The Effective Components Of Astragalus And Leech And Their Compatibility On P38-MAPK,NF-κB And IκB In Rat Mesangial Cells

Purpose:1.As a separate experiment,using molecular biology method,by exploring different effective components of astragalus root,leech and components compatibility of the lipopolysaccharide(LPS)induced proliferation of rat glomerular mesangial cells(GMC)related factor wire P38 lightning split the original activated protein kinase(P38 lightning MAPK),nuclear transcription factors-kappa B(nf-kappa B)predominate,nuclear growth factor kappa B predominate inhibitory factor(I kappa alpha B predominate)expression and the effect of combination of wire P38 lightning split the original activated protein kinase/nuclear transcription factor(-kappa B)predominate(P38MAPK/nf-kappa B)signaling pathway,analysis of GMC proliferation and extracellular matrix(ECM)the possible mechanism of the deposit,comparative yiqi blood compatibility of traditional Chinese medicine(TCM)different component and component strength,so as to explore Chinese medicine from yiqi possible work mechanism to protect the kidney blood levels.Discover yiqi blood feasibility scheme of traditional Chinese medicine used in clinical treatment,provides the pharmacological basis for the modem traditional Chinese medicine treating children MsPGN.2.Based on the experimental results and continuity discovers the experiment scheme,perfect the relevant experimental data,for further and confirmed between index and index,the correlation between indicators and pathways,combining seemingly "independent" the experiment index into a "line","by the point and the surface of the further system to clarify the mechanism and treatment of disease mechanism.Method:in vitro culture of rat GMC,CCK 8 method is used to test compatibility of astragalus root,leech the effective components and component of rat mesangial cells HBZY best medicinal concentration.Experiments were randomly divided into normal control group,model group,the hirudin group and astragalus polysaccharides,astragalus glycosides group,calycosin separately,hirudin and astragalus polysaccharide group,hirudin&astragalus glycosides group,hirudin group&calycosin separately.Adding different effective components and components compatibility respectively to the LPS stimulate cell proliferation after 12 h of HBZY intervene in 24h,respectively using Western blotting method and cell chemical immunofluorescence detection P38MAPK glomerular cells,the nf-kappa B,I kappa B protein expression changes predominate.Results:1.Western blotting method:Model group rats after LPS induced proliferation of mesangial cells P38MAPK,nf-kappa B protein expression quantity more than the normal group,compared two groups(P<0.05),the experimental group compared with model group,in addition to calycosin separately,hirudin calycosin separately outside protein quantity of weak reduction(P>0.05),and the rest of the group P38MAPK and nf-kappa B protein expression were significantly decreased(P<0.05),indicating that astragalus root,leech effective components and component compatibility to different degree by P38MAPK,nf-kappa B protein expression.Compared with P>0.05,there was no statistically significant difference in protein content between the two groups.2.Western blotting method showed that the model group after rat mesangial cell proliferation induced by LPS,I kappa B protein expression quantity than the normal group significantly reduced predominate,eompared two groups(P<0.05),except slight increase in hirudin calycosin separately group(P<0.05),the experimental group I kappa B protein predominate volume were increased significantly compared with model group(P<0.05),the difference between the treatment group(P>0.05,no statistical significance.The results showed that the effective components and their compatibility inhibited the phosphorylation of I B and upregulated the expression of I B protein.3.Cytochemical immunofluorescence showed that there was little green fluorescence expression in the normal group nucleus,indicating low NF-B protein expression in the normal HBZY nucleus.Compared with the LPS group,the green fluorescence intensity was higher,suggesting that LPS induced HBZY cell proliferation and then caused the high expression of NF-B protein in the nucleus.Except for hirudin astragaloside iv group and hirudin follicular isoflavone group,the green fluorescence intensity in HBZY cell nucleus was slightly lower than that in LPS group(P>0.05),the green fluorescence intensity in other treatment groups was significantly lower,suggesting that the active components and component compatibility of astragalus membranaceus and hirudin significantly down-regulated the high expression of P38MAPK protein(P<0.05).Comparison among groups:the down-regulation effect of hirudin group on the high expression of NF-B in the nucleus was significantly higher than that of each effective component group of Astragalus and the compatibility group of components of astragalus and hirudin(P<0.05),with statistical difference.The effect of hirudin was significantly better than that of each effective component group and component formula group of Astragalus.4.Chemical immunofluorescence method Cells showed:normal group green fluorescent expression intensity is low,prompt P38MAPK protein expression with the normal HBZY cells,but the expression quantity is low,compared with normal group,LPS group cells green fluorescence intensity is high,the prompt after LPS effect cause HBZY cell proliferation,resulting in high P38MAPK protein expression in cells,in addition to the hirudin astragalus armour glycosides,hirudin astragalus polysaccharides group weak decrease(P>0.05),The green fluorescence intensity in the hirudin group,ASTRagalus polysaccharide group,Astragaloside IV group,fururelin isoflavone group and hirudin follicular isoflavone group was significantly lower than that in the LPS group(P<0.05),suggesting that qi-and blood-stasis-promoting drugs had a significant down-regulation effect on the high expression of P38MAPK protein,and there was a statistical difference.P>0.05 was compared between the groups of astragalus menbranaceus and the compatibility group of each component,indicating that there was no significant difference in the down-regulation effect of astragalus membranaceus and the compatibility group of each component on P38MAPK protein expression.Compared with the hirudin group,the fluorescence expression of P38MAPK in the hair isoflavone group,the hirudin APS group,the hirudin ASTRagalus glycoside group and the hirudin hair isoflavone group were P<0.05,indicating that the down-regulation effect of hirudin on the expression of P38MAPK protein was better than that of the hair isoflavone group and the combination group of each component.5.Cytochemical immunofluorescence showed that the expression intensity of green fluorescence was high in the normal group,indicating the high expression of I B protein in the normal HBZY cells.Low green fluorescence intensity in LPS group suggested that LPS induced proliferation of HBEH cells,which in turn resulted in low expression of I B protein in cells.The green fluorescence intensity in the hirudin group,APS group,ASTRagaloside IV group,Mauri isoflavone group,hirudin APS group,and HIRudin ASTRagaloside IV group was significantly higher than that in the LPS group(P<0.01),indicating that the above drugs significantly upregulated the low expression of I B protein,with statistically significant differences.I kappa B protein predominate in hirudin calycosin separately group compared with model group,although have increased but weak effect(P>0.05)s the comparison between each group:hirudin group of intracellular I raised the effect of low kappa B predominate protein expression was obviously higher than that of astragalus effective into the group and astragalus root,leech components compatibility group(P<0.05),statistieally difference,the control effect of hirudin astragalus is obviously better than the effective grouping and components compatibility groups.Conclusion:Hirudin,astragalus polysaccharides,astragalus glycosides,calycosin separately,hirudin astragalus polysaccharides,hirudin astragalus glycosides,hirudin calycosin separately to different extent reduce the induced by LPS HBZY rises the nf-kappa B cell proliferation,P38MAPK protein expression,hirudin,astragalus polysaccharides,astragalus glycosides,calycosin separate.ly,hirudin astragalus polysaccharides group,hirudin astragalus glycosides,hirudin calycosin separately set can be increased by LPS induced HEZY proliferation and reduced the I kappa B protein expression,predominate The results showed that the effective components and the compatibility of components of hirsch could affect the expression of P38MAPK,NF-B and I B factors by regulating the activity of P38MAPK/NF-B pathway,so as to reduce GMC proliferation,inhibit EMC sedimentation and delay the progression of MsPGN disease.