大黄通过NRAV调节miR-509-3p/Rab5c轴抑制呼吸道合胞病毒的效果

Rhubarb inhibits respiratory syncytial virus by regulating the miR-509-3p/Rab5c axis via

目的 通过体外实验评估大黄免煎颗粒对呼吸道合胞病毒(RSV)的抑制效果,探究大黄对RSV感染过程中关键环节的影响和细胞内信号通路的调节等。方法 通过体外培养A549细胞,使用A549细胞设置空白对照组、感染对照组、-2 h组、0 h组、2 h组、4 h组、6 h组、8 h组在感染RSV的不同时间点加入大黄高剂量溶液,使用定量聚合酶链反应检测细胞内病毒基因(RSV-N)的相对量的表达,使用病毒空斑实验法测定上清病毒滴度,感染RSV 72 h后使用cck8法测定细胞活性;使用A549细胞设置感染对照组、利巴韦林组、大黄高剂量组、大黄中剂量组、大黄低剂量组,感染24 h后使用Western blot检测不同处理组RSV携带的绿色荧光蛋白(RSV-GFP)、Rab相关蛋白(Rab5c)蛋白表达水平,qPCR检测不同处理组抗病毒反应的负调负因子(NRAV)、Rab5c、RSV-N基因的相对量的表达。结果 不同浓度大黄培养A549细胞72 h后存活率比较差异有统计学意义(P<0.01),与5μg/mL比较,500μg/mL差异有统计学意义(P<0.05),不同浓度利巴韦林培养A549细胞72 h后细胞存活率比较差异无统计学意义(F=1.89,P=0.17),选取25、50、100μg/mL分别作为大黄低剂量组、大黄中剂量组、大黄高剂量组,利巴韦林处理浓度为500μmol/L;-2 h组细胞存活率较2 h、4 h、6 h、8 h组升高(均P<0.05),-2 h组细胞病毒滴度、RSV-N基因较2 h、4 h、6 h、8 h组降低(均P<0.05),且-2 h组细胞存活率、病毒滴度及RSV-N基因与0 h组比较无差异(均P>0.05);大黄高剂量组、大黄中剂量组、大黄低剂量组较感染对照组细胞的NRAV、RSV-N、Rab5c基因降低(均P<0.05)。结论 大黄可能通过降低A549细胞的NRAV水平促进miR-509-3p对Rab5c mRNA的靶向降解,降低了Rab5c的表达减缓了细胞内囊泡转运,从而发挥抑制RSV增殖的能力。

Objective To evaluate the inhibitory effect of Rhubarb Mianjian Granules on respiratory syncytial virus(RSV)in vitro,and to explore the influence of Rhubarb on the key links in the process of RSV infection and the regulation of intracellular signaling pathways.Methods A549 cells were cultured in vitro,and treated with blank control or RSV infection.At-2h,0h,2h,4h,6h and 8h of RSV infection,A549 cells were induced with high-dose Rhubarb solution,followed by detecting the relative level of RSV-N after 24h by quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).The viral titer of the supernatant was determined by viral plaque assay.After 72h of RSV infection,cell viability was measured by cell counting kit-8(CCK-8)assay.In addition,A549 cells infected with RSV were treated with ribavirin,or high-dose,medium-dose and low-dose Rhubarb.After 24h of RSV infection,protein levels of RSV with Green Fluorescent Protein(RSV-GFP)and Rab5c were detected by Western blot,and relative levels of negative regulator of antiviral response(NRAV),Rab5c and RSV-N were detected by qRT-PCR.Results There was a significant difference in the survival rate of A549 cells induced with Rhubarb for 72h at varied concentrations(P<0.01).A significant difference was detected in cell viability of A549 cells induced with 5μg/ml versus 500μg/ml(P<0.05).There was no significant difference in the survival rate of A549 cells cultured with ribaverin for 72h at varied concentrations of(F=1.89,P=0.17).Rhubarb at 25,50 and 100μg/ml were selected as the low dose,medium dose and high dose,respectively,and the ribaverin treatment was selected at the concentration of 500μmol/L.In comparison to cell survival at-2h(2h prior to RSV infection),the survival at 2h,4h,6h and 8h was significantly elevated(all P<0.05).In comparison to viral titer and expression level of RSV-N at-2h,those at 2h,4h,6h and 8h were significantly reduced(all P<0.05).No significant differences were detected in the survival rate,viral titer and expression level of RSV-N between-2h and 0h(all P>0.05).High-dose,medium-dose and low-dose Rhubarb significantly downregulated NRAV,RSV-N and Rab5c(all P<0.05).Conclusion Rhubarb may promote the targeted degradation of Rab5c mRNA by miR-509-3p via downregulating NRAV in A549 cells,which inhibits the expression of Rab5c and slows down intracellular vesicle transport,thereby inhibiting the proliferation of RSV.