三七总皂苷通过抑制ADAM10/Notch3信号通路改善野百合碱诱导的大鼠肺动脉高压

Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway

本研究旨在探讨三七总皂苷(Panax notoginseng saponins, PNS)对肺动脉高压(pulmonary arterial hypertension, PAH)肺血管重构的作用及对ADAM10/Notch3通路的影响。建立PAH大鼠模型,随机将雄性Sprague Dawley (SD)大鼠分为对照组、野百合碱(monocrotaline, MCT)组、MCT+PNS组,每组各10只。对照组每日腹腔注射等体积生理盐水;MCT组第一天腹腔注射60 mg/kg MCT,之后每日腹腔注射等体积生理盐水;MCT+PNS组第一天腹腔注射60 mg/kg MCT,之后每日腹腔注射50 mg/kg PNS,各组造模时间持续21天。造模结束后,采用右心导管插管技术测量肺动脉平均压(meanpulmonaryarterialpressure,mPAP),计算右心室肥厚指数(right ventricular hypertrophy index, RVHI),HE和Masson染色观察肺血管壁微观形态与变化,Western blot和qPCR检测大鼠肺血管组织ADAM10、Notch3、Hes-1、P27、PCNA、Caspase-3蛋白及mRNA的表达,免疫荧光染色检测Notch3和α-SMA蛋白表达和定位,免疫组织化学染色检测ADAM10蛋白表达量。结果显示:与对照组相比,MCT组mPAP、RVHI明显升高,肺血管明显增厚以及胶原纤维显著增多,ADAM10、Notch3、Hes-1、PCNA mRNA及蛋白的表达显著升高;同时P27、Caspase-3 mRNA及蛋白的表达则明显降低;与MCT组相比,MCT+PNS组mPAP、RVHI降低,肺血管得到明显改善以及胶原纤维显著减少,ADAM10、Notch3、Hes-1、PCNA mRNA及蛋白的表达有所下降,同时P27、Caspase-3 mRNA及蛋白的表达则轻微升高。免疫荧光染色结果显示Notch3和α-SMA染色可以重叠,证明Notch3表达于平滑肌细胞,MCT组Notch3表达量明显增加,给予PNS干预后,Notch3的表达量低于MCT组。免疫组织化学染色结果显示,与对照组对比,MCT组ADAM10表达量明显增多,与MCT组相比,MCT+PNS组ADAM10表达量减少。这些结果表明,PNS可改善MCT诱导的大鼠PAH,其机制可能是通过抑制ADAM10/Notch3信号通路而实现的。

In this study, we investigated the effects of Panax notoginseng saponins(PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension(PAH). PAH rat model was established, and male Sprague Dawley(SD)rats were randomly divided into control group, monocrotaline(MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure(mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index(RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1,P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI,pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1,and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10,Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.