Wenyang-Huoxue-Lishui Formula Protects And Enhances Recovery Of Cultured Murine Podocytes Via Dynamin Up-regulation

Objective: To observe the effect of Wenyang Huoxue Lishui Formula on the expression of dynamin in podocyte of mice induced injury by aminonucleoside puromycin.Hence,in this study,we attempt to explore the underlying mechanism by which Wenyang Huoxue Lishui Formula reduce proteinuria of primary nephrotic syndrom.Methods: 1.Preparation of Wenyang Huoxue Li Formula containing serum.2.Cell culture,passaged and induced differentiation of mature podocytes,with the concentration of 45mg/L puromycin damage podocytes,and the use of warm yang and blood saliva Chinese medicine serum intervention to glucocorticoid dexamethasone intervention as a positive control,and A: normal control group,B: puromycin(45mg/L)group/model group,C: dexamethasone(1?mol/L)group,D: 10% of traditional Chinese medicine Serum group,E: 20% serum group of Chinese medicine,F: serum white control group.3.The relative expression of catin m RNA and dynamin m RNA was detected by RT-PCR.4.Western blotting was used to detect the expression of Cat L and dynamin in each group.5.Immunofluorescence staining combined with laser confocal microscopy was used to observe the morphological changes of podocyte cytoplasmic microfilaments,microtubule and the expression of Cat L and dynamin in podocyte injury model induced by puromycin in Chinese medicine.Results:1.RT-PCR showed that Cat L m RNA expression in the model group was higher than that in the normal group(P<0.01).Compared with the model group,the expression of Cat L m RNA in the Chinese medicine group and dexamethasone group decreased(P<0.01).The expression of Cat L m RNA in 10% Chinese medicine group was higher than that in 20% Chinese medicine group(P<0.01).In the experiment,the dynamin m RNA in podocyte among six gourp couldn't be found significant difference(P > 0.01).2.Immunofluorescence assay showed,Compared with the normal group,the fluorescence intensity of the Cat L in the model group was enhanced and the Dynamin fluorescence intensity was weakened.Compared with the model group,the fluorescence of Cat L protein in the serum group and the dexamethasone group was weakened,while the Dynamin fluorescence enhanced.3.Western-blot showed,Compared with the normal group,the expression of Cat L in the model group was higher than that in the normal group while the expression of dynamin was lower(P<0.01).Compared with the model group,the expression of Cat L in the Chinese medicine group and the dexamethasone group decreased and the expression of Dynamin increased(P < 0.01).Compared with dexamethasone group,there was no significant difference in the expression of Cat L between the two groups(P>0.01),while the expression of dynamin in 10% traditional Chinese medicine group is lower,and the 20% traditional Chinese medicine group is higher.Compared with the 20% traditional Chinese medicine group,the expression of Cat L in the 10% Chinese medicine group was higher and the Dynamin expression was lower(P<0.01).There was no significant difference between in serum control group and normal group with the expression of Cat L and Dynamin(P > 0.01).4.Microfilament and microtubule immunofluorescence results: Compared with the normal group,the model group cell body shrinkage,pseudopodia retraction,cytoskeleton structure of microfilament formation of tension fibers appeared by the coordinated and orderly parallel arrangement of patterns into a disorder The number of microfilaments decreases,and tends to marginalize the formation of the outer ring,at the same time the cytoskeleton of microtubules appears to reduce the number of depolymerization,disorder,but in the podocyte primary protrusions The number of cells increased,and the podocyte morphology and skeletal protein structure were improved in the serum group and dexamethasone group.Conclusion: After podocytes were challenged using PAN,the expression of cathepsin Cat L was elevated and the dynamin expression was reduced.However,the m RNA expression of dynamin wasn't affected.The actin microfilaments and microtubules were reorganized and became disorder.We demonstrated that treatment of cultured murine podocytes with the Wenyang Huoxue Lishui Formula protected and enhanced recovery from PAN-induced injury.Wenyang Huoxue Lishui Formula stabilized actin microfilaments and microtubules against disruption by PAN,and induced a significant increase in dynamin expression and decrease Cat L expression.