| Objective:The simiao powder containing serum is used to intervene uric acid induced HK-2fibrosis model, preliminary analyse the anti renal interstitial fibrosis mechanism with simiao powder.Methods:1.For the preparation of renal interstitial fibrosis model,the solution of concentration0.225mg/ml uric acid is adopted to stimulate human renal tubular epithelial cells for twenty four hours,then the reverse transcription-polymerase chain reaction (rt-pcr) is used to detect the cell expression of alpha SMA and E-CadherinmRNA.2.Different concentrations of simiao pawder containing medicated serum are used to intervene the model of renal interstitial fibrosis for twenty four hours. The cultivated cells can be divided into six groups,including the blank group for comparison,the uric acid model group,the saline group, and the low dose、midlle dose、high dose of the simiao pawer groups for intervention. assay is used to measure the cell proliferation of each group..The change of the cell morphology could be observed under the inverted microscope. Reverse transcription-polymerase chain reaction (rt-pcr) is used to detect the expression between the cell TGF-beta1, alpha SMA and E-CadherinmRNA.The Cell immunohistochemical method is used to detect the expression of protein between theTGF-beta1, alpha SMA, and the E-Cadherin.Results:1.The MTT results shows that0.225mg/ml of uric acid for tweenty four hours can remarkablly inhibit the activity of the human renal tubular epithelial cells (P<0.01).the cells activity of the human renal tubular epithelial induced by uric acid could be significantly promote by the simiao pawder appearing the dependence between the time and dose,in which of tweenty four hours at five percent drug serum of different simiao pawer is the most obvious (P <0.01).Under the observation of the inverted phase contrast microscope,the normal group of renal tubular epithelial cells appearing in paving stone, put in order.The number of model group cells are obviously reduced, the cell morphology is stretched and turns into spindle. after the intervention of simiao pawder in different concentrations of medicated serum, with the increasing of concentration of drug intervention, the adherent cell number increases, and the morphology of cells turn from the spindle into paving stone gradually.2.Compared with the normal group,tweenty four hours reverse transcription-polymerase chain reaction shows that alpha SMAmRNA expression increases of the model group, especially the0.225mg/ml of uric acid stimulation group (P<0.01). after the intervention of simiao pawder with different dose medicated serum at five percent the expression of alpha-SMAmRNA and TGF-beta one decrease, especially in the high dose(P<0.01). after the intervention of simiao pawder with drug-serum,the expression of E-CadherinmRNA was rises, especially in the high dose(P<0.01).Compared with the model group the difference was remarkable(P <0.05).The immunohistochemical results shows that,compared with the normal group, the target positive total area and IOD value of alpha-SMA and TGF-beta one in model group increase significantly (P<0.01). Compared with the model group, the target positive total area and IOD value of the TGF beta in simiao pawder drug serum of different dose was lower (P<0.05), and after the intervention of the high dose group, the decline was significantly (P<0.01).compared with the model group,the saline group has no difference, compared with the normal group,the target positive total area and IOD value of E-Cadherin in model group declines (P <0.05). Compared with model group, the target positive total area and IOD value of E-Cadherin in simiao pawder group at different dose increase (P <0.05), particularly in the high dose group (P <0.01).compared with the model group, the saline group has no difference. Immunohistochemical picture shows that, the protein expression of alpha SMA and TGF-beta at the normal group was a small amount of light yellow, while the brown positive area at model group was increase,and part of the area is dark brown, and the positive expression was strong. after the intervetion of simiao pawder in different dose,it is found that with the increasing of the drug dose,the expression of positive area decreases significantly, and the color becomes shallow.compared with the model group,the saline group has no difference. the protein expression of E-Cadherin in normal group was a small part, compared with the model group,the yellow positive area reduces, and the color becomes shallow.after adding the simiao pawder of different dose, it is found that with the increasing of the dosage,the positive expression area increase, and the color becomes brown gruadully. compared with the model group, the saline group has no difference.Conclusion:1.the uric acid solution is adopted to stimulate the human renal tubular epithelial cells (HK-2) and the renal interstitial fibrosis models are prepared, the mRNA expression of alpha SMA increase and the mRNA expression of E-Cadherin reduces at the model group.and the result illustrates that the renal interstitial fibrosis model is successful.The vitality of cells shows that, the cells vitality of human renal tubular epithelial was inhibited by the uric acid, and the cells vitality are promoted by the simiao pawder with drug serum.2.The cellular morphology shows that, the shapes of normal group cells appeare like the paving stones, and the shape of the model group cells appeare like fusiform or irregular.the shapes of the cells after adding the simiao pawder with drug serum turn from spindle into slabs stone gradually.it is a morphological basis that the renal interstitial fibrosis model could be improved by simiao pawder.3.The rt-pcr results shows that,the expression of TGF-beta mRNA and alpha beta SMA mRNA could be lowered partly,and the expression of E-Cadherin mRNA could be raised after adding the simiao pawder with drug serum of different doses, especially the high dose, then it could provide the basis for inhibiting the renal interstitial fibrosis by the simiao pawder.The immunohistochemical result shows that the protein expression of alpha SMA and TGF-beta could be declined and the protein expression of E-Cadherin could be raised by the simiao pawder drug serum in different dose.thus it can provide basis for inhibiting the renal interstitial fibrosis by the simiao pawder with drug serum. |
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