前列消汤干预TGF-β1/Smad4、Smad 7信号通路调控实验性自身免疫性前列腺炎小鼠前列腺成纤维细胞增殖与凋亡的研究

Regulatory effect of Qianliexiao Decoction on TGF-β1, Smad4 and Smad 7 signaling pathways and the proliferation and apoptosis of prostate fibroblasts in mice with experimental autoimmune prostatitis

目的:观察中药方前列消汤干预TGF-β1/Smad4、Smad 7信号转导通路对实验性自身免疫性前列腺炎(EAP)小鼠前列腺成纤维细胞(PrF)增殖与凋亡的调控作用。方法:采用C57BL/6小鼠制备前列消汤含药血清,根据细胞毒性实验结果,以5%、10%、20%的含药血清分别作为低、中、高剂量组进行干预实验。采用"免疫诱导+中医病因造模法"建立湿热证EAP小鼠模型,无菌条件下取出前列腺组织,分离培养PrF,采用差速贴壁法纯化PrF,免疫荧光法检测PrF纯度,纯度达90%以上即以5 ng/ml TGF-β1浓度的培养液刺激进行建模。取建模后对数生长期的PrF随机分为空白组(无血清培养液),模型组,阳性对照组(含TGF-β1浓度5 ng/ml的培养液),前列消汤低、中、高剂量组,每组6个复孔;干预培养24 h后检测各组PrF增殖情况,采用Western印迹检测TGF-β1的表达水平和Smad4、Smad7、p-Smad4、p-Smad7的表达水平,qPCR检测TGF-β1、Smad4、Smad7 mRNA的表达,流式细胞术检测PrF细胞凋亡情况。结果:经TGF-β1诱导后,与模型组比较,阳性对照组PrF细胞增殖明显(P<0.05),前列消汤低、中剂量组PrF细胞增殖受抑制(P<0.05),前列消汤高剂量组PrF细胞增殖受明显抑制(P<0.01);与阳性对照组比较,前列消汤各剂量组PrF细胞增殖明显受到抑制(P<0.01),且呈明显的量效关系。与模型组比较,阳性对照组Smad4、p-Samd7和TGF-β1蛋白表达显著增加(P<0.05);经前列消汤含药血清干预后,与阳性对照组比较,前列消汤含药血清能显著下调PrF细胞内p-Smad4,TGF-β1蛋白的表达(P<0.01),而增加p-Samd7的表达水平(P<0.01)。qPCR结果显示,与模型组相比,前列消汤高剂量组Smad4基因表达有显著差异(P<0.05),前列消汤低、中、高剂量组Smad7基因表达有显著差异(P<0.05);前列消汤低、中、高剂量组TGF-β1基因表达水平显著上调(P<0.01);与阳性对照组相比,前列消汤高剂量组Smad4基因表达有显著差异(P<0.05),模型组、前列消汤低、中、高剂量组Smad7基因表达有显著差异(P<0.01),前列消汤低、中、高剂量组TGF-β1基因表达水平显著下降(P<0.01)。与模型组比较,阳性对照组PrF细胞凋亡率无明显差异(P>0.05),前列消汤各剂量组凋亡率均显著提高(P<0.05),且呈明显的量效关系。结论:经TGF-β1诱导可刺激PrF细胞增殖,上调TGF-β1、p-Smad4的表达,下调p-Smad7的表达。前列消汤可以抑制PrF增殖,且呈明显的量效关系,其机制可能与其降低TGF-β1和p-Smad4的表达、提高p-Smad7的表达从而阻断TGF-β1通过Smad4途径诱导PrF细胞增殖相关。

Objective:To study the effect of intervening in the signal transduction pathways of TGF-β1,Smad4 and Smad7 with Qianliexiao Decoction(QLX)on the proliferation and apoptosis of prostate fibroblasts(PrF)in mice with experimental autoimmune prostatitis(EAP).Methods:A model of EAP with damp-heat syndrome was established in C57 BL/6 mice by immunization induction combined with the TCM modeling method.The prostate tissue of the mice was harvested for isolation,culturing and purification of PrFs and detection of their purity.After modeling by stimulation with a medium containing>90%-purity or 5 ng/ml TGF-β1,the PrFs in the logarithmic growth phase were obtained and randomly divided into a blank control(serum-free medium),a model control,a positive control(medium containing 5 ng/ml TGF-β1),a low-dose QLX(serum containing 5%QLX),a medium-dose QLX(serum containing 10%QLX),and a high-dose QLX group(serum containing 20%QLX).After 24 hours of intervention,the proliferation of the PrFs was measured,the protein expressions of TGF-β1,Smad4,Smad7,p-Smad4 and p-Smad7 detected by Western blot,their mRNA expressions determined by qPCR,and the apoptosis of the PrFs examined by flow cytometry.Results:After induction with TGF-β1,the proliferation of the PrFs was significantly increased in the positive control(P<0.05),but inhibited in the medium-and low-dose QLX groups(P<0.05)and even more significantly in the high-dose QLX group as compared with that in the model control(P<0.01).The expressions of Smad4,p-Samd7 and TGF-β1 proteins in the PrFs were remarkably higher in the positive control than in the model control group(P<0.05),while those of p-Smad4 and TGF-β1 markedly lower(P<0.01)and that of p-Smad7 dramatically higher in the QLX intervention groups than in the positive control(P<0.01),in an evident dose-dependent manner.In comparison with the model control group,the high-dose QLX group exhibited a significant decrease in the mRNA expression of Smad4(P<0.05)but all the three QLX groups showed a dramatic increase in those of Smad7(P<0.05)and TGF-β1(P<0.01).The mRNA expression of Smad4 was markedly down-regulated in the high-dose QLX group compared with that in the positive control(P<0.05),that of Smad7 up-regulated in the model control and QLX groups(P<0.01),and that of TGF-β1 down-regulated in the three QLX groups(P<0.01).The apoptosis rate of the PrFs was significantly higher in the QLX groups than in the model control(P<0.05)in a dose-dependent manner,but showed no statistically significant difference between the model control and the positive control groups(P>0.05).Conclusion:TGF-β1 can stimulate the proliferation of PrFs,up-regulate the expressions of TGF-β1 and p-Smad4,and down-regulate that of p-Smad7,while QLX can inhibit the proliferation of PrFs in a dose-dependent manner by decreasing the expressions of TGF-β1 and p-Smad4,increasing that of p-Smad7,and thereby suppressing TGF-β1-induced proliferation of PrFs.