黄芪多糖通过抑制钙蛋白酶1/NF-κB信号通路减轻低氧诱导的肺动脉高压小鼠肺炎症反应和纤维化

Astragalus polysaccharides alleviates pulmonary inflammation and fibrosis in mice with hypoxia-induced pulmonary arterial hypertension by inhibiting calpain-1/NF-κB signaling pathway

目的探讨黄芪多糖(APS)对低氧诱导的肺动脉高压(PAH)模型小鼠产生的肺炎症和纤维化的保护作用及机制。方法①体内实验:采用低氧舱(10%O_(2),90%N_(2))内饲养C57BL/6小鼠制备肺动脉高压模型。雄性野生型C57BL/6小鼠分为正常对照组、低氧模型组、模型+APS 200和400 mg·kg^(-1)组(每日ig给予)、模型+BAY11-70825 mg·kg^(-1)组(NF-κB抑制剂,ip给予,每周3次);钙蛋白酶1敲除(Capn1 EK684 FN-/-)雄性小鼠分为敲除对照组和敲除低氧组;28 d后,测定右心室压(RVSP)和右心室肥厚指数(RVHI);HE染色观察肺组织病理形态并检测血管壁厚度比(WTR)和血管壁面积比(WAR);Masson染色观察肺组织胶原纤维沉积;ELISA检测血清白细胞介素1β(IL^(-1)β)和肿瘤坏死因子α(TNF-α)水平及肺组织中羟脯氨酸(HYP)水平;Western印迹法检测肺组织钙蛋白酶1、基质金属蛋白酶9(MMP-9)和转化生长因子β_(1)(TGF-β_(1))蛋白表达水平以及NF-κB P65蛋白磷酸化水平。②体外实验:原代培养肺动脉平滑肌细胞(PASMC)分为细胞对照组、低氧组、低氧+APS 0.6 g·L^(-1)、低氧+BAY11-708210μmol∙L^(-1)和低氧+MDL-28170(钙蛋白酶1抑制剂)10μmol∙L^(-1)组。除细胞对照组外,其余组加药后直接置低氧培养箱(3%O_(2)、97%N_(2))培养24 h;CCK-8法检测细胞存活率;ELISA检测细胞中IL^(-1)β和TNF-α水平;Western印迹法检测细胞钙蛋白酶1、MMP-9和TGF-β_(1)蛋白表达水平以及NF-κB P65蛋白磷酸化水平。结果①体内实验:与野生型正常对照组比较,低氧模型组小鼠RVSP、RVHI、WAR、WTR、肺组织胶原纤维沉积面积、小鼠血清中IL^(-1)β和TNF-α水平、肺组织中HYP水平、钙蛋白酶1、MMP-9、TGF-β_(1)蛋白表达水平及NF-κB P65磷酸化水平显著升高(P<0.05);与低氧模型组相比,模型+APS组、敲除低氧组和模型+BAY11-7082组小鼠的RVSP、RVHI、WAR、WTR、肺组织胶原纤维沉积面积、血清中IL^(-1)β和TNF-α水平、肺组织中HYP水平、钙蛋白酶1、MMP-9和TGF-β_(1)蛋白表达水平及NF-κB P65磷酸化水平显著降低(P<0.05)。②体外实验:与细胞对照组相比,低氧组细胞存活率、细胞中IL^(-1)β和TNF-α水平、钙蛋白酶1、MMP-9、TGF-β_(1)蛋白表达水平及NF-κB P65磷酸化水平显著升高(P<0.05);与低氧组相比,低氧+APS组、低氧+BAY11-7082组、低氧+MDL-28170组细胞存活率、细胞中IL^(-1)β和TNF-α水平、钙蛋白酶1、MMP-9、TGF-β_(1)蛋白表达水平及NF-κB P65磷酸化水平显著降低(P<0.05)。结论APS对低氧诱导的小鼠PAH具有改善作用,可通过抑制钙蛋白酶1/NF-κB信号通路减轻肺组织炎症反应和纤维化反应。

OBJECTIVE To investigate the protective effect and mechanism of Astragalus polysac⁃charide(APS)on pulmonary inflammation and a fibrosis in mice with hypoxia-induced pulmonary arterial hypertension(PAH).METHODS①In vivo:pulmonary hypertension model was established by feeding C57BL/6 mice in a hypoxicchamber(10%O_(2),90%N_(2)).Male wild-type C57BL/6 mice were divided into the normal control group,model group,model+APS 200,400 mg·kg^(-1) group(daily ig given)and the model+BAY11-70825 mg·kg^(-1) group(NF-κB inhibitor,ip given,3 times per week),while calpain-1 knockout(CAPN1 EK684 FN-/-)male mice were divided into the knockout control group and knockout hypoxia group.After 28 d,the right ventricular pressure(RVSP)and right ventricular hypertrophy index(RVHI)were measured.The pathological morphology of the lung was observed by HE staining,and the vascular wall thickness ratio(WTR)and vascular area ratio(WAR)were detected.Deposition of collagen fibers in lung tissues was observed by Masson staining.ELISA was performed to detect interleukin-1(IL^(-1)β)and tumor necrosis factor(TNF-α)levels in serum and hydroxyproline(HYP)levels in lung tissue.The protein expressions of calpain-1,matrix metalloproteinase-9(MMP-9),transforming growth factorβ1(TGF-β1)and phosphorylated NF-κB P65 in lung tissue were assayed by Western blotting.②In vitro:primarily cultured pulmonary artery smooth muscle cells(PASMCs)were divided into the cell control group,hypoxia group,hypoxia+APS 0.6 g·L^(-1),hypoxia+BAY11-708210μmol·L^(-1) and the hypoxia+MDL-2817010μmol·L^(-1) group(calpain-1 inhibitor).Except the cell control group,these groups were immediately put into the hypoxia incubator(3%O2,97%N2)after dosing for 24 h.The viability of cells was determined using the CCK-8 method.ELISA was performed to detect the expressions of IL^(-1)βand TNF-αin cells.The protein expressions of calpain-1,MMP-9,TGF-β1 and phosphorylated NF-κB P65 in cells were assayed by Western blotting.RESULTS①In vivo:compared with the wild normal control group,the RVSP,RVHI,WAR,WTR,collagen fiber deposition areas in the lung and IL^(-1)β,TNF-αlevels in serum,HYP level of the lung tissue,the protein expressions of calpain-1,MMP-9,TGF-β1 and phos⁃phorylated NF-κB P65 in lung tissues were increased significantly in the hypoxia model group(P<0.05).Compared with the hypoxia model group,the RVSP,RVHI,WTR,WAR,collagenfiber deposition areas in the lung tissue and IL^(-1)β,TNF-αlevels in serum,HYP levels in lung tissue,the protein expressions of calpain-1,MMP-9,TGF-β1 and phosphorylated NF-κB P65 in lung tissues were decreased signifi⁃cantly in the model+APS 0.6 g·L^(-1) group,knockout hypoxia group and model+BAY11-708210μmol·L^(-1) group,respectively(P<0.05).②In vitro:compared with the normal control group,the viability of cells,IL^(-1)βand TNF-αlevels in cells,the protein expressions of calpain-1,MMP-9,TGF-β1 and phosphorylated NF-κB P65 in cells were increased significantly in the hypoxia model group(P<0.05).Compared with the hypoxia model group,the viability of cells,IL^(-1)β,TNF-αlevels in cells,the protein expressions of calpain-1,MMP-9,TGF-β1 and phosphorylated NF-κB P65 in cells were decreased significantly in the model+APS 0.6 g·L^(-1) group,hypoxia+BAY11-708210μmol·L^(-1) group and hypoxia+MDL-2817010μmol·L^(-1) group(P<0.05).CONCLUSION APS has a protective effect on mice with hypoxia-induced PAH and can inhibit pulmonary inflammation and fibrosis by down-regulating calpain-1/NF-κB signaling pathway.