Objective To investigate the regulation of two herbal anti-virus formulas on gene expression profile associated with natural killer cell (NK cell) mediated cytotoxicity in pneumonia mice infected with influenza virus.Methods According to random number table,90 ICR mice were divided into nine groups with 10 mice in each group:normal group (N),model group (M),osehamivir group (control group,C),low-dose,medium-dose and high-dose Shufeng Xuanfei formula groups (SL,SM,SH groups),and low-dose,medium-dose and high-dose Jiebiao Qingli formula groups (JL,JM,JH groups).The model of pneumonia was reproduced by nasal dropping influenza virus A (FM1) in mice.N group was given isotonic saline 0.05 ml in nasal drops.After 2 hours of model-building,C group was received 11.375 mg· kg-1· d-1 osehamivir phosphate.Shufeng Xuanfei formula (mainly honeysuckle,forsythia and radix isatidis,etc.) with 3.76,1.88 and 0.94 g ·kg-1 ·d-1 were administrated to SH,SM and SL groups by gastric irrigation respectively.Jiebiao Qingli formula (mainly ephedra,gypsum,glycyrrhiza glabra,etc.) with 4.36,2.18 and 1.09 g ·kg-1 ·d-1 were administrated to JH,JM and JL groups by gastric irrigation respectively.In N and M groups,normal saline was administrated with gastric perfusion.Each group was in equal dose of 0.2 ml daily over a 4-day period.Total RNA in lung tissue of mice were extracted in each group,thengene chips were used to screen these RNA samples.Some genes involved NK cell mediated cytotoxicity were select,with "I" representing of signal intensity.These candidate genes were verified by real-time fluorescent quantitation polymerase chain reaction (PCR) and Western blotting.Results In the pathway of NK cell mediated cytotoxicity,M group up-regulated 43 genes expression,and 36,29,22,21,20 and 10 genes showed down-regulation in SM,JM,SL,JH,SH and JL groups,respectively.Apart from gene co-expression network in SH,SL,JH,JM and JL,SM also expressed other differential genes which SH,SL,JH,JM and JL did not.So medium-does Shufeng Xuanfei formula had the most significant regulation in gene expression of NK cell mediated cytotoxicity.By real-time PCR and Western blotting experiments showed that compared with the M group,mRNA and protein expression of tumor necrosis factor-α (TNF-α) in these two formula groups were significantly down-regulated,especially prominent in SM group and JM group (TNF-α mRNA:1.07 ±0.19,1.19 ±0.14 vs.3.20 ± 0.56,both P＜0.01).Conclusions Influenza viral replication in host cell,which means influenza antigens exposure in infected cells as target cells.NK cells recognize and exert cell mediated cytotoxic function against influenza antigens.Genes associated with NK cell mediated cytotoxicity in influenza infection were up-regulated.Shufeng Xuanfei and Jiebiao Qingli formulas could down-regulate these genes.The mechanism of down-regulated genes is that the number of influenza infected cells and NK cells activation decreases in treatment with two formulas.%目的 观察疏风宣肺方和解表清里方对流感病毒性肺炎小鼠自然杀伤细胞(NK细胞)毒性相关信号转导通路差异基因表达的调控作用.方法 将90只ICR小鼠按随机数字表法分为对照(N)组、模型(M)组、奥司他韦(C)组以及中药疏风宣肺方高、中、低剂量(SH、SM、SL)组和解表清里方高、中、低剂量(JH、JM、JL)组,每组10只.采用流感病毒亚甲型鼠肺适应株FM1滴鼻感染方法制备流感病毒性肺炎模型；N组以0.05 ml等渗盐水滴鼻.于制模后2h,C组给予奥司他韦11.375 mg· kg-1·d-1灌胃；SH、SM、SL组给予疏风宣肺方(主要药物金银花、连翘、板蓝根等)3.76、1.88、0.94 g· kg-1·d-1灌胃；JH、JM、JL组给予解表清里方(主要药物麻黄、石膏、生甘草等)4.36、2.18、1.09 g·kg-1·d-1灌胃；N组及M组则灌胃等量生理盐水；各组均0.2 ml/d,连用4d.提取肺组织总RNA,采用基因芯片筛选出NK细胞毒性信号转导通路相关的差异表达基因,以I表示信号强度；并采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹试验(Western blotting)对部分基因进行验证.结果 在NK细胞毒性相关信号转导通路中,M组较N组上调差异基因43个.用药治疗后,SM组下调差异表达基因36个,JM组下调差异表达基因29个,SL组下调差异表达基因22个,JH组下调差异表达基因21个,SH组下调差异表达基因20个,JL组下调差异表达基因10个；SH 、SL、JH、JM、JL组表达下调的基因均包括在SM组的表达谱中,表明中剂量疏风宣肺方调控NK细胞毒性作用最显著.荧光定量PCR和Western blotting测定结果显示,与M组比较,疏风宣肺方与解表清里方各剂量组对肿瘤坏死因子-α (TNF-α)的mRNA及蛋白表达均有显著抑制作用,疏风宣肺方和解表清里方均以中剂量组最低(TNF-α mRNA:1.07±0.19、1.19±0.14比3.20±0.56,均P＜0.01).结论 流感病毒感染后在宿主细胞内增殖即成为带有病毒抗原的靶细胞,并激活NK细胞毒性信号转导通路的相关基因表达增加,被NK细胞识别并杀伤；两种方药可下调NK细胞中表达上调的差异基因,下调机制与其能明确减少流感病毒性肺炎体内靶抗原、减弱NK细胞识别活化作用有关.
展开▼
