| Objective: To study the effect of Yin nourishing and blood circulation promoting formula on CsA, which activate TGF-β1 that regulate extracellular matrix(ECM) metabolism, and elucidate the anti-RIF(renal interstitial fibrosis) effect of Yin nourishing and blood circulation promoting formula.Methods : 1. Preparation for blood serum containing traditional Chinese medicine: Chose 6 true-bred male New Zealand rabbits, and randomly divide them into the following 6 groups during the experiment: Yin nourishing and blood circulation promoting formula (low-dose) group, Yin nourishing and blood circulation promoting formula( high-dose) group, Aweto group, Panax Notoginseng group, Rhizoma Anemarrhenae group, and control group. Rabbits were respectively given concentrated solutions of Yin nourishing and blood circulation promoting formula, Aweto, Panax Notoginseng, and Rhizoma Anemarrhenae, and distilled water, twice a day, continue for 10 days. Collect blood sample of rabbits 2 hours after gastric lavage. Separate and filtrate the blood sample, and store at—40℃.2. Preparation for cell: cultivate and proliferate LLC-PK1 cell in RPMI-1640 with 10%FCS and the sugar concentration of 5. 5mmol/l. During the experiment, LLC-PK1 cells were inoculated in 96-hole Tissue Culture Plates. As the cell conjugate to 80%-90% , change by serum-free medium, and cultivate for 24 hours, make cells still.3. Grouping: Divide cultivated cells into 6 experimental groups, in each of them, different reagents were added.①Traditional Chinese medicine group(CsA25mg/l+ low-dose blood serum containing Yin nourishing and blood circulation promoting formula );②Traditional Chinese medicine group(CsA25mg/l+ high-dose blood serum containing Yin nourishing and blood circulation promoting formula );③Aweto group (CsA25mg/l+ blood serum containing aweto ) ;④Panax Notoginseng group ( CsA25mg/l+blood serum containing Panax Notoginseng) ;⑤Rhizoma Anemarrhenae group (CsA25mg/l+blood serum containing Rhizoma Anemarrhenae);⑥Control group(CsA25mg/l+ blood serum without traditional Chinese medicine contained). In each group there are 16 wells, continue incubation for 24 hours, then cells were used for the following tests: detect the expression of collagen mRNA( type TGF-β1, uPA, tPA, PAI-1, FN, and IV) in each group with RT-PCR assay; detect the activity of tPA and uPA with fibrin zymography method; detect PAI-1 content with reverse-zymography method; detect FN content in culture supernatant with ELISA method. The above measurement data was expressed by mean±standard deviation. T-test was used in intergroup comparison. Data were analyzed statistically with software SPSS13.0. p<0.05 indicates significant contrast.Results: 1. TGF-β1 mRNA expression intensity by PCR detection indicated that the electrophoretic colour band and expression intensity of the control group and Rhizoma Anemarrhenae group intensified remarkably; the electrophoretic colour band and expression intensity of Panax Notoginseng group, Aweto group and traditional Chinese medicine(high-dose and low-dose) groups are weaker than control group; the high-dose traditional Chinese medicine has the weakest expression. The expression intensity of traditional Chinese medicine groups weaken as the dose increase. Expression intensity of type IVcollagen mRNA in each group indicated that the electrophoretic colour band and expression intensity of the Panax Notoginseng group, Aweto group and traditional Chinese medicine(high-dose and low-dose) groups are weaker than control group. The Aweto group and high-dose traditional Chinese medicine group have the lowest intensity of expression; the expression intensity of traditional Chinese medicine groups weaken as the dose increase. The electrophoretic colour band and expression of the control group have the strongest intensity of FN mRNA expression, while the Panax Notoginseng group, Rhizoma Anemarrhenae group, and traditional Chinese medicine(high-dose and low-dose) groups have weaker electrophoretic colour band and expression intensity; the high-dose traditional Chinese medicine group have the lowest intensity of expression; the expression intensity of traditional Chinese medicine groups weaken as the dose increase. In groups of uPA,tPA,PAI-1 which were generated in ECM regulation, there is no distinct contrast with the expression intensity. 2. Activity of tPA and uPA by fibrin zymography detection indicate that on tPA activity, high-dose traditional Chinese medicine groupis the highest, p<0.05, no.distinct contrast in the rest groups; UPA in the activity of each group in each group activity was not significantly different.3. PAI—1 content in supernatant by reverse-zymography detection indicate that Aweto group has the lowest PAI—1 content, p<0.05; no distinct contrast in the rest groups.4. ELISA detection of FN content in culture supernatant indicated that high-dose traditional Chinese medicine group and Aweto group have the lowest content, p<0.01, while low-dose traditional Chinese medicine group and Panax Notoginseng group have higher content.Conclusions: Yin nourishing and blood circulation promoting formula group, Aweto group, and Panax Notoginseng group can down-regulate TGF-βlmRNA expression induced by CsA, blockade ECM component induced by TGF-βin renal interstitium, and thus effect anti-fibrosis; dose-dependence exist in effect of Yin nourishing and blood circulation promoting formula on TGF-β1 down-regulation. However, the effect of Yin nourishing and blood circulation promoting formula on PA / PAI-1 system has not been improved in this experiment.
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