丹皮酚联合三七总皂苷对AngⅡ诱导的心脏成纤维细胞增殖及TGF-β/Smads信号通路的影响

Effects of paeonol combined with Panax notoginseng saponins on the proliferation of angiotensin-induced cardiac fibroblasts and TGF-β/Smads signal pathway

目的探讨丹皮酚联合三七总皂苷应用对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导新生大鼠的心脏成纤维细胞(cardiac fibroblasts,CFs)增殖及TGF-β/Smads信号通路的影响。方法采用胰酶消化法分离培养的CFs随机分为6组:正常对照组,模型组,三七总皂苷组,丹皮酚组,丹皮酚与三七总皂苷联合用药大、小剂量组。采用MTT比色法测定各组培养液中CFs增殖情况,免疫细胞化学法检测α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平,RTPCR法检测转化生长因子-β1(transforming growth factor-β1,Tgfb1)的mRNA表达水平,Western blot法检测TGF-β1、磷酸化的Smad2/3(p-Smad2/3)的蛋白表达水平。结果与正常对照组比较,模型组大鼠CFs增殖明显增高,Tgfb1的mRNA表达显著增加,α-SMA及TGF-β1、p-Smad2/3的蛋白表达显著增加;与模型组比较,丹皮酚组、三七总皂苷组、联合用药的大剂量和小剂量组均可抑制由AngⅡ诱导的CFs的增殖,Tgfb1的mRNA表达显著减少,α-SMA及TGF-β1、p-Smad2/3的蛋白表达显著减少;与丹皮酚组、三七总皂苷组比较,联合用药大、小剂量组在抑制CFs增殖的作用上显著增强,抑制Tgfb1的mRNA表达效果显著增强,抑制α-SMA及TGF-β1、p-Smad2/3的蛋白表达效果显著增强;与联合用药小剂量组比较,联合用药大剂量组对以上指标的抑制效果更佳,显示一定的剂量依赖性。结论丹皮酚与三七总皂苷联合应用对抑制CFs增殖,呈现明显的协同作用。其作用机制与抑制TGF-β/Smads信号通路的传导有关。

AIM To investigate the effects of combining paeonol（ Pae） with Panax notoginseng saponins（ PNS） on the proliferation of cardiac fibroblasts（ CFs） induced by angiotensinⅡ（ AngⅡ） and TGF-β/Smads signal pathway. METHODS The CFs of neonate rat were isolated and cultured by trypsin digestion and randomly divided into six groups： normal control group,model group,PNS group,Pae group,the combination of Pae and PNS high-dose and low-dose groups. The cell proliferations were measured with MTT. The expression of α-smooth muscle actin（ α-SMA） protein was detected by immunocytochemistry method. The mRNA expression of transforming growth factor-β1（ Tgfb1） was detected by RT-PCR. The protein levels of TGF-β1 and p-Smad2/3 were determined by Western blot. RESULTS Compared with the control group,the rat CFs proliferation,Tgfb1 mRNA,α-SMA protein,TGF-β1 and p-Smad2/3 protein expression increased obviously in the model group. Compared with the model group,the CFs proliferation,Tgfb1 mRNA,α-SMA protein,TGF-β1 and p-Smad2/3 protein expression decreased significantly in PNS group,Pae group,the combination of Pae and PNS high-dose and small-dose groups. Compared with PNS group and Pae group,the CFs proliferation,Tgfb1 mRNA,α-SMA protein,TGF-β1 and p-Smad2/3 protein expression decreased significantly in combination of Pae and PNS high-dose and low-dose groups. With regard to the inhibition of abovementioned indicators,the effect of Pae combined with PNS high-dose group were better than that of Pae combined with PNS low-dose group in a dose-dependent manner.CONCLUSION The combination of Pae and PNS can inhibit CFs proliferation and show significant synergistic effects related with the suppression of TGF-β/Smads signal transduction pathway.