解毒活血方调控NF-κB/NLRP3/Caspase-1促进大鼠胸主动脉损伤血管再内皮化

Jiedu Huoxue Prescription Regulates NF-κB/NLRP3/Caspase-1 to Promote Vascular Reendothelialization in Rat Model of Injured Thoracic Aorta

目的:探讨解毒活血方调节核转录因子(NF)-κB/NOD样受体蛋白3(NLRP3)/胱天蛋白酶(Caspase)-1介导细胞焦亡促进损伤血管再内皮化的可能机制。方法:通过球囊损伤的方法建立大鼠胸主动脉损伤模型,36只大鼠分为假手术组、模型组、解毒活血方低、中、高剂量组、阿托伐他汀钙片组,在术后28 d采集主动脉损伤段,苏木素-伊红(HE)染色观察血管结构形态学变化、伊文思蓝染色观察血管再内皮化情况,酶联免疫吸附测定法(ELISA)检测血清中炎症相关因子肿瘤坏死因子-α(TNF-α)、细胞间黏附分子-1(ICAM-1)、白细胞介素-1β(IL-1β)及内皮相关因子一氧化氮(NO)的变化,蛋白免疫印迹法(Western blot)检测血管组织中内皮型一氧化氮合酶(eNOS)、NF-κB p65、磷酸化(p-)NF-κB p65、NLRP3、Caspase-1的表达。结果:模型组血管内膜增厚,组织血管壁平滑肌细胞增生且排列紊乱,组织可见明显炎症细胞浸润,管腔面积狭窄,解毒活血方各剂量组和阿托伐他汀钙片组胸主动脉血管病理损伤均有不同程度改善。球囊损伤后,模型组内皮覆盖率明显下降,解毒活血方高剂量组和阿托伐他汀钙片组再内皮化率明显升高(P<0.05)。与正常组比较,模型组大鼠血清TNF-α、ICAM-1、IL-1β含量均显著升高(P<0.01),血清中血管活性因子NO含量均显著下降(P<0.01),血管组织中eNOS蛋白表达显著下降(P<0.01),模型组大鼠细胞焦亡相关通路蛋白NLPR3、Caspase-1和NF-κB p65磷酸化的表达明显升高(P<0.05,P<0.01);与模型组比较,给药组大鼠血清TNF-α、ICAM-1、IL-1β明显降低(P<0.05,P<0.01),NO明显升高(P<0.05,P<0.01),血管组织中eNOS表达显著升高(P<0.01),各给药组能降低NLRP3、Caspase-1和NF-κB p65磷酸化蛋白的表达水平(P<0.05,P<0.01)。结论:解毒活血方可以促进球囊损伤血管再内皮化抑制内膜增生,其机制可能与调控NF-κB/NLRP3/Caspase-1抑制细胞焦亡,抑制内皮细胞炎性损伤有关。

Objective:To investigate the mechanism of Jiedu Huoxue prescription in promoting the reendothelialization of injured vessels by regulating the nuclear factor(NF)-κB/NOD-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease(Caspase)-1-mediated pyroptosis.Method:A rat model of injured thoracic aorta was established by balloon injury,and 36 rats were assigned into shame surgery,model,low-,medium-,and high-dose Jiedu Huoxue prescription,and atorvastatin calcium tablet groups.The injured aortic segment was collected 28 days after surgery.Hematoxylin-eosin(HE)staining and Evans blue staining were conducted to reveal the changes of vascular structural morphology and the reendothelialization of blood vessels,respectively.The enzyme-linked immunosorbent assay(ELISA)was employed to determine the levels of tumor necrosis factor-α(TNF-α),intercellular adhesion molecule-1(ICAM-1),interleukin(IL)-1β,and nitric oxide(NO)in the serum.Western blotting was employed to determine the expression of endothelial nitric oxide synthase(eNOS),NF-κB p65,phospho-NF-κB p65(p-NF-κB p65),NLRP3,and Caspase-1 in the vascular tissue.Result:The model group showed thickened endovascular membrane,proliferation and disarrangement of smooth muscle cells of the artery wall,obvious inflammatory cell infiltration,and narrowed luminal area.Jiedu Huoxue prescription and atorvastatin calcium tablets mitigated the pathological changes of the thoracic aorta in different degrees.After balloon injury,the endothelial coverage rate of the model group decreased significantly,while Jiedu Huoxue prescription and atorvastatin calcium tablets increased the reendothelialization rate(P<0.05).Compared with the shame surgery group,the model group showed elevated levels of TNF-α,ICAM-1,and IL-1β(P<0.01)and lowered NO level(P<0.01)in the serum.In addition,the model group presented down-regulated protein level of eNOS(P<0.01)and up-regulated phosphorylation of pyroptosisassociated proteins NLPR3,Caspase-1,and NF-κB p65 in the vascular tissue(P<0.05,P<0.01).Compared with the model group,Jiedu Huoxue prescription and atorvastatin calcium tablets lowered TNF-α,ICAM-1,and IL-1βlevels(P<0.05,P<0.01)and elevated the NO level in the serum(P<0.05,P<0.01).Moreover,the drugs up-regulated the expression of eNOS(P<0.01)and down-regulated the expression of NLRP3,Caspase-1,and NF-κB p65(P<0.05,P<0.01)in the vascular tissue.Conclusion:Jiedu Huoxue prescription can promote the reendothelialization and inhibit the intimal hyperplasia of vessels after balloon injury by regulating the NF-κB/NLRP3/Caspase-1 pathway to inhibit pyroptosis and reduce endothelial inflammatory injury.