基于TLR4/NF-κB/NLRP3通路的羟基红花黄色素A抑制血管紧张素II诱导的血管外膜成纤维细胞迁移研究

Inhibitory effect of hydroxylsafflower yellow A on migration of vascular adventitial fibroblasts induced by angiotensin II based on TLR4/NF-κB/NLRP3 pathway

目的基于通过Toll样受体4(Toll-like receptor 4,TLR4)/核因子-κB(nuclear factor-κB,NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路探讨羟基红花黄色素A(hydroxysafflor yellow A,HSYA)抑制血管紧张素II(angiotensin II,ANG II)诱导的血管外膜成纤维细胞(vascular adventitial fibroblasts,VAFs)迁移作用。方法组织贴块法培养大鼠胸主动脉VAFs,免疫荧光实验检测细胞中Vimentin和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)蛋白表达,鉴定VAFs。设置对照组、ANG II组、HSYA组、脂多糖(lipopolysaccharide,LPS)组和LPS+HSYA组,对照组VAFs给予培养基正常培养,其余各组给予1×10^(-7)mol/L ANG II处理24 h,然后更换为含100 nmol/L LPS或40μmol/L HSYA的培养基,继续培养24 h,划痕实验检测VAFs的迁移能力;CCK-8法检测细胞活力;Western blotting检测TLR4、NF-κB和p-NF-κB蛋白表达;免疫荧光实验检测NLRP3炎性小体相关蛋白NLRP3、半胱氨酸天冬氨酸蛋白酶-1(cystein-asparate protease-1,Caspase-1)及凋亡斑点样蛋白(apoptotic spot-like protein,ASC)共表达情况。结果免疫荧光结果证实VAFs培养成功。划痕实验结果显示,ANG II能够显著增加细胞的迁移率(P<0.01),LPS刺激进一步提高VAFs的迁移率(P<0.01),HSYA能够抑制ANG II及LPS对VAFs迁移的促进作用(P<0.01)。Western blotting结果显示,ANG II显著提高细胞内TLR4和p-NF-κB的表达(P<0.05、0.001),并促进NF-κB入核(P<0.001);HSYA显著抑制ANG II诱导的细胞内TLR4和p-NF-κB的表达(P<0.05、0.01、0.001),并抑制NF-κB入核(P<0.05、0.01、0.001)。免疫荧光实验结果表明,ANG II及LPS促进NLRP3炎症小体相关蛋白NLRP3、Caspase-1及ASC共表达,HSYA可减少ANG II及LPS诱导NLRP3、Caspase-1及ASC蛋白共表达,抑制NLRP3炎症小体的组装。结论HSYA通过抑制TLR4/NF-κB/NLRP3通路减少ANG II诱导的VAFs迁移。

Objective To investigate the effect of hydroxysafflor yellow A(HSYA)on migration of advangiolar fibroblasts(VAFs)induced by angiotensinⅡ(ANGⅡ)based on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway.Methods VAFs were cultured from rat thoracic aorta by tissue patch method,protein expressions of Vimentin andα-smooth muscle actin(α-SMA)was detected by laser confocal assay for identification of VAFs.Control group,ANG II group,HSYA group,lipopolysaccharide(LPS)group and LPS+HSYA group were set up.VAFs in control group were given normal culture medium,and the other groups were given 1×10^(-7)mol/L ANG II for 24 h,and then changed to contain 100 nmol/L LPS or 40μmol/L HSYA medium,continue to culture for 24 h,and migration ability of VAFs was tested by scratch test;CCK-8 method was used to detect cell viability;Western blotting was used to detect TLR4,NF-κB and p-NF-κB protein expression;The co-expression of NLRP3 inflammatory corpuscle-associated protein NLRP3,cystein-aspartate protease-1(Caspase-1)and apoptotic spot-like protein(ASC)were detected by immunofluorescence assay.Results The results of immunofluorescence confirmed that VAFs were successfully cultured.The scratch test results showed that ANG II could significantly increase the cell migration rate(P<0.01),LPS stimulation can further increase the migration of VAFs(P<0.01),HSYA can inhibit the promotion of ANG II and LPS on VAFs migration(P<0.01).Western blotting results showed that ANG II significantly increased intracellular TLR4 and p-NF-κB expressions(P<0.05,0.001),and promoted NF-κB entry(P<0.001);HSYA significantly inhibited the intracellular TLR4 and p-NF-κB expressions induced by ANG II(P<0.05,0.01,0.001),and inhibited NF-κB entered the nucleus(P<0.05,0.01,0.001).The results of immunofluorescence test showed that ANG II and LPS promoted the co-expression of NLRP3,Caspase-1 and ASC proteins associated with NLRP3 inflammatory bodies,and HSYA could reduce the co-expression of NLRP3,Caspase-1 and ASC proteins induced by ANG II and LPS,and inhibited the assembly of NLRP3 inflammatory bodies.Conclusion HSYA can inhibit TLR4/NF-κB/NLRP3 pathway to reduce the migration of VAFs induced by ANG II.