三七总皂苷对低氧状态下大鼠PASMCs增殖、凋亡及Notch3通路的影响

Effects of Panax notoginseng saponins on proliferation,apoptosis and Notch3 pathway of rat PASMCs under hypoxia

目的:探讨在低氧(hypoxia, Hypo)条件下三七总皂苷(Panax notoginseng saponins, PNS)对大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)增殖和凋亡及其Notch3通路的影响。方法:以大鼠PASMCs为研究对象,饥饿24 h后将细胞分为6组:正常对照(control, Con)组、Hypo组、Hypo+PNS组、Hypo+DAPT{N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester;Notch3通路抑制剂}组、Hypo+过表达Notch3(Hypo+Notch3)组和Hypo+Notch3+PNS组。Con组置于常氧环境(21%O_(2)、5%CO_(2)、74%N_(2))下培养48 h,其余5组预先进行相对应的干预后置于低氧条件(5%O_(2)、6%CO_(2)、89%N_(2))下培养48 h。造模结束后用细胞计数试剂盒8(Cell Counting Kit-8, CCK-8)测各组细胞活力;EdU染色法检测细胞增殖;TUNEL染色法检测细胞凋亡;RT-qPCR法检测PASMCs中Notch3、Hes-1、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)和caspase-3的mRNA表达水平;Western blot检测PASMCs中Notch3、Hes-1、PCNA和caspase-3的蛋白表达水平;通过在细胞中过表达Notch3,探讨PNS是否通过Notch3影响PASMCs的增殖。结果:EdU和TUNEL结果表明,与Con组相比,Hypo组PASMCs增殖能力增强,且细胞凋亡显著减少(P<0.01),Notch3、Hes-1和PCNA的mRNA和蛋白表达水平显著升高,而caspase-3的mRNA和蛋白表达水平显著降低(P<0.05);给予PNS干预后,Notch3、Hes-1和PCNA的mRNA和蛋白表达水平显著降低(P<0.05),而caspase-3的mRNA和蛋白表达水平显著升高(P<0.05);给予Notch3通路抑制剂DAPT干预后,Notch3、Hes-1和PCNA的mRNA蛋白表达水平显著降低(P<0.05),而caspase-3的mRNA和蛋白表达水平显著升高(P<0.05)。进一步结果显示,在PASMCs中过表达Notch3可逆转PNS对Hypo诱导的PASMCs增殖的抑制作用(P<0.05)。结论:PNS可通过降低Notch3表达,抑制Hypo诱导的大鼠PASMCs增殖并促进PASMCs凋亡。

AIM:To investigate the impact of Panax notoginseng saponins(PNS)on the Notch3 pathway and its subsequent effects on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs)under hy‐poxic conditions.METHODS:Rat PASMCs were subjected to a 24-hour starvation period and then categorized into 6 groups:control(Con)group,hypoxia(Hypo)group,Hypo+PNS group,Hypo+DAPT{N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester;Notch3 pathway inhibitor}group,Hypo+Notch3 overexpression(Hypo+Notch3)group,and Hypo+Notch3+PNS group.The cells in Con group were cultured for 48 h in a normal oxygen environment(21%O_(2),5%CO_(2) and 74%N_(2)).Meanwhile,the other five groups were treated with respective intervention treatment and cultured under hypoxic conditions(5%O_(2),6%CO_(2) and 89%N_(2))for 48 h.The cell viability was assessed by CCK-8 as‐say,the cell proliferation was detected by EdU staining,and cell apoptosis was determined using TUNEL staining.The mRNA expression levels of Notch3,Hes-1,proliferating cell nuclear antigen(PCNA)and caspase-3 were detected by RT-qPCR,and corresponding protein levels were measured by Western blot.In addition,the effect of PNS on the proliferation of PASMCs with Notch3 overexpression was also investigated.RESULTS:The EdU and TUNEL assays revealed that com‐pared with Con group,the rat PASMCs in Hypo group showed increased proliferation and reduced apoptosis(P<0.01).Both mRNA and protein levels of Notch3,Hes-1 and PCNA were elevated(P<0.05),while caspase-3 mRNA and protein levels were decreased(P<0.05).After PNS treatment,a decline in Notch3,Hes-1 and PCNA expression occurred(P<0.05),and caspase-3 levels increased(P<0.05).After Notch3 pathway inhibitor DAPT intervention,decreased levels of Notch3,Hes-1 and PCNA(P<0.05),and increased caspase-3 levels were observed(P<0.05).Subsequent findings demonstrated that Notch3 plasmids could counteract the anti-proliferative effect of PNS under Hypo(P<0.05).CON-CLUSION:Panax notoginseng saponins may reduce Hypo-induced proliferation of rat PASMCs and bolster the apoptosis by suppressing the Notch3 expression.