| Background Mesangiasl cell (MC) proliferation and extracellular matrix ccumulation are the main causes of kidney diseases, such as IgA nephropathy, proliferative glomerulonephritis (GN), lupus nephritis, focal segmental glomerulosclerosis and diabetic nephropathy. MC) secrete cytokines and extracellular matrix, and induces glomerulosclerosis. Inhibiting MC proliferation and secretion is important in the therapy of proliferating glomerular disease. Saponin is the main active ingredient of polyphylla. It has multiple functions, such as anti-inflammatory immuno-regulation,anti-tumor,sedation,analgesic,hemostasis, etc. PolyphyllinⅡ(PPⅡ) is a strong immuno-modulator. Most researches focus on the effects of anti-tumor and immuno-regulation. It's effects on MC Apoptosis and secretion of extracellular matrix and inflammation factors is a research direction which has not been amply explored.Objective To investigate the effects of PPⅡon rat MC apoptosis and extracellular matrix, and explore the possible mechanism.Methods A range of PPⅡwas selected based on the results obtained after LDH and cytotoxicity activity tests. Rat mesangial cells (MC) were incubated with serum-free 1640 for 24h to synchronize cell growth. The cells were then divided into 6 groups:control group(10%FBS1640); LPS group(LPS10ug/ml+10%FBS1640); PPⅡ30ug/ml group(polyphyllinⅡ 30ug/ml +LPS10ug/ml+10%FBS1640); PPⅡ40ug/ml group (polyphyllinⅡ40ug/ml +LPS10ug/ml+10%FBS1640); PPⅡ50ug/ml group (polyphyllinⅡ50ug/ml +LPS10ug/ml+10%FBS1640); triptolide group(triptolide 48ug/ml+LPS10ug/ml+10%FBS1640) as the positive control group. The proliferation rate of MC was observed after 12 hours,24 and 36 hours with CCK-8 colorimetric assay respectively.Apoptosis of rat MC was detected by the Hoechst fluorescence staining method and flow cytometry. The cells were collected to extract total RNA and proteins 24 hours after incubation. The expressions of Bax, Bcl-2, FN, and collagenⅣ, were examined by Real-time-PCR and Western blot.Results (1) LDH and cytotoxicity activity tests showed:the LHD level of PPⅡ60ug/ml group (polyphyllinⅡ60ug/ml +LPS10ug/ml+10%FBS1640) was much higher than the other groups (P<0.01). Therefore,30ug/ml,40ug/ml,50ug/ml PPⅡconcentrations were selected in this experiment. (2) Compared with the blank group, the OD value of the LPS group at 12h,24h and 36h increased significantly (P<0.01). LPS promoted MC proliferation. At 12hours, there was no significant difference between the PPⅡ30ug/ml and LPS groups (P>0.05).There were significant differences at 24h and 36hbetween the two groups (P<0.01). Compared with the PLS group, OD values of the PPⅡ40ug/ml and PPⅡ50ug/ml groups at 12hours,24hours and 36hours decreased significantly (P<0.01). PPⅡinhibited MC proliferation in a dosage and time-dependent manner. Compared with the PPⅡ50ug/ml group, the inhibitory effect of the triptolide group at 12hours,24and 36hours were weakened (P<0.01). (3) The Hoechst fluorescence staining method showed:Cells in the control group were well circumscribed, and the nuclei hyper chromatic. Compared with the blank group, the cell quantity of the LPS group was much higher. Cells in the PPⅡgroup were not circumscribed, with nuclear hyperchromatism and apoptotic bodies were formed. (4) MC apoptosis was induced by PPⅡat 24hours in a dosage-dependent manner as shown by flow cytometry assay. The apoptosis rate of the triptolide group was lower than the PPⅡ50ug/ml group (P<0.01). (5) Compared with control group, the mRNA and protein expression of Bax of the PLS group increased. Compared with the LPS group, the mRNA and protein expression of Bax of different PPⅡgroups increased significantly in a dosage-dependent manner (P<0.01). Compared with the blank group, the mRNA and protein expression of Bcl-2 of the PLS group increased. Compared with the LPS group the mRNA and protein expression of Bax of different PPⅡgroups decreased significantly in a dosage-dependent manner (P<0.01). Compared with blank control group, the mRNA and protein expressions of FN and ColⅣof the PLS group increased (P<0.01). Compared with the LPS group, the mRNA and protein expressions of FN and ColⅣof different PPⅡgroups decreased significantly in a dosage-dependent manner (P<0.01) Conclusion 1,PPⅡinduces MC apoptosis by decreasing bax/bcl-2 level。2,PPⅡinhibits MC proliferation and decreases ECM,in a dosage-time-dependent manner.
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