疏风宣肺方和解表清里方干预流感病毒性肺炎小鼠细胞凋亡的研究

Shufengxuanfei and Jiebiaoqingli formulas on cell apoptosis in pneumonia mice infected with influenza virus

目的病毒感染宿主细胞后,细胞凋亡天门冬氨酸特异性半胱氨酸蛋白(caspase)途径激活限制病毒复制,但甲型流感病毒感染引起的细胞凋亡的分子机制尚不完全清楚。探讨流感病毒性肺炎小鼠肺组织细胞凋亡相关的基因及2种不同抗流感病毒方药的治疗作用。方法制备甲型流感病毒性肺炎小鼠模型,随机分为空白组、肺炎模型组、奥司他韦对照组、疏风宣肺方高、中、低剂量组,解表清里方高、中、低剂量组。提取各组小鼠肺组织总RNA,采用基因组芯片筛选出与凋亡密切相关的差异表达基因,并用荧光定量PCR对天门冬氨酸特异性半胱氨酸蛋白基因进行验证。结果肺炎模型组肺组织中差异表达基因casp-3、casp-8、casp-9、Fas、FasL、TNF、TNFrasf1a、Tradd、IL1a、IL1b、IL1r1、IL1r2、casp-7明显上调,与空白组比较差异有统计学意义。疏风宣肺方中剂量组差异表达基因casp-8、casp-7、Fas、FasL、TNF、TNFrasf1a、Tradd、IL1a、IL1b、IL1r1、IL1r2明显下调。疏风宣肺方低剂量组下调上述基因的作用强于解表清里方低剂量组,疏风宣肺方中剂量组治疗效果优于解表清里方中剂量组。疏风宣肺方低剂量组与解表清里方中剂量组对casp-3、-8、-9表达有显著抑制,差异有统计学意义(P<0.01)。不同剂量疏风宣肺方的治疗效果与解表清里方比较如下:疏风宣肺方低剂量组>疏风宣肺方中剂量组>解表清里方中剂量组>解表清里方低剂量组。结论疏风宣肺方对细胞凋亡调控基因的调控作用强于解表清里方,上调casp-3、-8、-9表达,对抗流感病毒感染后的细胞凋亡较强,对甲型流感病毒性肺炎小鼠治疗作用优于解表清里方。

Objective During viral infection, apoptosis by caspase pathway is induced as a consequence of activation of host cellular defense mechanism to limit the virus replication, but the mechanism of apoptosis infected with influenza virus remains to be clari- fied. The aim of this study was to investigate the related genes of apoptosis of pneumonia mice infected with influenza virus and the thera- peutic effects of two herbal anti-virus formulas. Methods The model of pneumonia by nasal dropping influenza virus A was estab- lished in mice, and then divided randomly into nine groups： normal group, pneumonia model group, oseltamivir control group, low-dose, medium-dose and high-dose Shnfengxuanfei groups, low-dose, medium-dose and high-dose Jiebiaoqing3i groups. Total RNA was extracted in each group and gene chips were used to screen the genes involved in apoptosis. These candidate genes were verified by Real-Time PCR. Results Compared with the normal group, casp-8,casp-9, Fas, FasL, TNF, TNFrasfla, Tradd, ILlα, ILlb, ILlrl, ILlr2 and casp-Tgene increased in pneumonia model group. Casp-8, casp-7, Fas, FasL,TNF,TNFrasfla, Tradd, ILlα, ILlb, 1Llrland ILlr2 gene decreased in medium-dose Shufengxuanfei group. The role of downregulation of these above genes in low dose Shnfengxuanfei group was better than in low dose Jiebiaoqingli group. The therapeutic effects of medium dose Shnfengxuanfei was better than in medium dose Jiebi- aoqingli. Compared with the model group, the casp-3, -8,-9 mRNA expression decreased in low-dose Shufengxuanfei group and medium-dose Jiebiaoqingli group （ P 〈 0.01 ）. The therapeutic effects of different doses of Shufengxuanfei and Jiebiaoqingli as follows： low-dose Shufengxuanfei 〉 medium-dose Shufengxuanfei 〉 medium-dose Jiebiao- qingli 〉 low-dose Jiebiaoqingli. Conclusion The regulation effect of apoptosis gene in Shufengxuanfei formula was superior to that of Jiebiao- qingli. Shufengxuanfei formula suppressed casp-3, -8,-9 mRNA expres- sion, and protected against the apoptosis induced by influenza virus.