敲低RIP140基因的ADSCs通过TLR4/NF-κB信号通路改善心肌肥厚大鼠心功能

ADSCs knockdown RIP140 gene through TLR4/NF-κB signal pathway improves cardiac function in rats with myocardial hypertrophy

目的探讨敲低受体相互作用蛋白140(RIP140)基因的脂肪间充质干细胞(ADSCs)通过TLR4/NF-κB信号通路改善心肌肥厚(MH)大鼠心功能的机制。方法利用慢病毒载体构建敲低RIP140基因的ADSCs。采用连续7 d皮下注射异丙肾上腺素(ISO)(7 mg/kg)建立MH大鼠模型。第8 d时移植ADSCs。具体分组如下:将60只大鼠随机分为sham组(连续7 d注射PBS)、MH组(连续7 d注射ISO)、PBS组(连续7 d注射ISO+第8 d注射PBS)、ADSCs组(连续7 d注射ISO+第8 d注射ADSCs)、LV-shRIP140-ADSCs组(连续7 d注射ISO+第8 d注射LV-shRIP140-ADSCs)和LV-shNC-ADSCs组(连续7 d注射ISO+第8 d注射LV-shNC-ADSCs),每组10只。ADSCs移植28 d后进行指标检测。超声心动图评估大鼠心功能;检测心肌酶水平;HE染色观察心肌组织病理学改变;TUNEL染色检测细胞凋亡;ELISA检测炎症因子的表达;RT-qPCR和Western-blot检测TLR4、NF-κB p65 mRNA和蛋白表达。结果与sham组相比,MH组心肌纤维排列紊乱,心肌细胞肿大、坏死;LVEF、LVFS水平降低,cTnT、CK-MB、TNF-α、IL-1β、IL-6、TLR4、NF-κB p65 mRNA和TLR4、p-NF-κB p65/NF-κB p65蛋白水平升高(均P<0.05);与PBS组相比,ADSCs组心肌纤维、细胞损伤明显改善;LVEF、LVFS水平升高,cTnT、CK-MB、TNF-α、IL-1β、IL-6、TLR4、NF-κB p65 mRNA和TLR4、p-NF-κB p65/NF-κB p65蛋白水平降低(均P<0.05);与LV-shNC-ADSCs组相比,LV-shRIP140-ADSCs组LVEF、LVFS水平升高,cTnT、CK-MB、TNF-α、IL-1β、IL-6、TLR4、p-NF-κB p65/NF-κB p65 mRNA和TLR4、p-NF-κB p65/NF-κB p65蛋白水平降低(均P<0.05)。结论敲低RIP140的ADSCs通过抑制TLR4/NF-κB信号通路减轻心肌肥厚大鼠心肌组织细胞凋亡及炎症反应,促进大鼠心功能恢复。

Objective To explore mechanism of ADSCs knockdown RIP140 gene through TLR4/NF-κB signal pathway improving cardiac function in myocardial hypertrophy(MH)rats.Methods The low RIP140 gene ADSCs was built with slow virus carrie.The rat model of myocardial hypertrophy was established by subcutaneous injection of ISO(7 mg/kg)for 7 days.ADSCs were transplanted on the 8th day.The specific groups were as follows:60 rats were randomly divided into sham group(PBS was injected for 7 consecutive days),MH group(ISO was injected for 7 consecutive days),PBS group(ISO was injected for 7 consecutive days+PBS was injected for 8th day),ADSCs group(ISO was injected for 7 consecutive days+ADSCs was injected for 8th day),LV shRIP140 ADSCs group(ISO was injected for 7 consecutive days+LV shRIP140 ADSCs was injected for 8th day)and LV shNC ADSCs group(ISO was injected for 7 consecutive days+LV shNC ADSCs was injected for 8th day),with 10 rats in each group.ADSCs were detected 28 days after transplantation.Echocardiography was used to evaluate the myocardial function of rats.Myocardial enzyme levels were detected.Myocardial histopathological changes were observed by HE staining.TUNEL staining was used to detect cell apoptosis.The expression of inflammatory factors was detected by ELISA.RT-qPCR and Western-blot test TLR4,NF-kappa B predominate p65 mRNA and protein expression.Results Compared with sham group,MH group had disordered arrangement of myocardial fibers,enlargement and necrosis of myocardial cells;LVEF and LVFS levels decreased,cTnT,CK-MB,TNF-α,IL-1β,IL-6,TLR4,NF-κB p65 mRNA and TLR4,p-NF-κB p65/NF-κB p65 protein levels increased(all P<0.05).Compared with PBS group,The myocardial fiber and cell structure of ADSCs group were improved;LVEF,LVFS levels increased,cTnT,CK-MB,TNF-α,IL-1β,IL-6,TLR4,NF-κB p65 mRNA and TLR4,p-NF-κB p65/NF-κB p65 protein levels were reduced(all P<0.05).Compared with LV-shNC-ADSCs group,LVEF,LVFS levels in LV-shRIP140-ADSCs increased,cTnT,CK-MB,TNF-α,IL-1β,IL-6,TLR4,NF-κB p65 mRNA and pTLR4,p-NF-κB p65/NF-κB p65 rotein levels were reduced(all P<0.05).Conclusion ADSCs that knock down RIP140 gene can reduce the apoptosis and inflammatory reaction of myocardial tissue in rats with myocardial hypertrophy by inhibiting TLR4/NF-κB signal pathway,and promote the recovery of cardiac function in rats.