疏风宣肺解毒方对流感病毒性肺炎小鼠炎性细胞因子的影响

Effects of Shufeng Xuanfei Jiedu formula for the inflammation-related cytokines in pneumonia mice infected with influenza virus

目的:通过基因芯片技术分析疏风宣肺解毒方对流感病毒性肺炎小鼠炎性细胞因子差异表达基因的影响。方法:按随机数字表法将雄性ICR小鼠分为正常组(N组)、流感病毒性肺炎模型组(M组)、奥司他韦对照组(C组)及疏风宣肺解毒方高、中、低剂量组(SH、SM、SL组),每组10只。通过流感病毒亚甲型鼠肺适应株FM1(0.05 mL)滴鼻建立小鼠流感病毒性肺炎模型;N组以0.05 mL生理盐水滴鼻。SH、SM、SL组于滴鼻感染后2 h灌胃疏风宣肺解毒方,含药浓度分别为临床用药的2倍量、等量、1/2量(约为3.8、1.9、1.0 kg/L),每日1次,每次0.2 mL,连用4 d;C组灌服奥司他韦2.5 kg/L;N组和M组灌服蒸馏水。第5天取小鼠全肺,提取肺组织总RNA,与小鼠全基因芯片杂交并检测,筛选参与炎症相关通路中的靶基因。扫描杂交芯片,计算芯片探针信号在各组与M组的强度表达比值,筛选差异表达基因,其中P<0.05且log2比值>1为上调基因,P<0.05且log2比值<-1为下调基因。采用反转录-聚合酶链反应(RT-PCR)检测白细胞介素(IL-1、IL-8)、细胞间黏附分子-1(ICAM-1)的mRNA表达。结果:与N组相比,M组差异基因表达IL-1、IL-8、ICAM-1明显上调(log2N组/M组分别为2.62、2.07、1.41,均P<0.05)。与M组相比,SH、SM、SL、C组差异基因表达IL-1、IL-8、ICAM-1均明显下调(log2SH组/M组分别为-1.91、-1.85、-0.88,log2SM组/M组分别为-3.10、-1.74、-1.84,log2SL组/M组分别为-1.89、-1.39、-0.53,log2C组/M组分别为-2.46、-1.52、-1.44,均P<0.05)。RT-PCR检测结果显示,M组IL-1、IL-8、ICAM-1的mRNA表达水平均较N组显著升高〔IL-1(2^-ΔΔCT):4.63±0.24比1.01±0.13,IL-8(2^-ΔΔCT):6.28±0.13比1.02±0.09,ICAM-1(2^-ΔΔCT):2.90±0.18比1.02±0.12,均P<0.05〕。SH、SM、SL和C组IL-1、IL-8、ICAM-1的mRNA表达水平均较M组组显著降低〔IL-1(2^-ΔΔCT):2.12±0.32、1.71±0.07、2.05±0.16、1.66±0.13比4.63±0.24,IL-8(2^-ΔΔCT):3.89±0.13、2.08±0.19、2.98±0.20、2.02±0.12比6.28±0.13,ICAM-1(2^-ΔΔCT):1.72±0.93、1.34±0.14、1.53±0.25、1.17±0.12比2.90±0.18,均P<0.05〕,但SH、SM、SL和C组间差异无统计学意义。结论:疏风宣肺解毒方通过下调IL-1、IL-8、ICAM-1炎性细胞因子相关基因的表达,抑制流感病毒感染后小鼠的炎症损伤。

Objective To analyze the differential gene expression of Shufeng Xuanfei Jiedu formula on whole expression profiles of the inflammation-related cytokines in mice infected with influenza virus by the gene chip technology.Methods Male ICR mice were divided into normal group(N group),influenza virus pneumonia model group(M group),oseltamivir control group(C group)and Shufeng Xuanfei Jiedu formula high,medium and low dose groups(SH,SM,SL groups)according to the random number table method,with 10 mice in each group.A mouse model of influenza virus pneumonia was established by nasal drip of influenza virus strain FM1(0.05 mL);in group N,0.05 mL normal saline was used.In SH,SM and SL groups,Shufeng Xuanfei Jiedu formula was prescribed after 2 hours of intranasal infection(drug concentration approximately 3.8,1.9 and 1.0 kg/L),0.2 mL once a day for 4 days;in group C,the dosage of oseltamivir was 2.5 kg/L;in group N and group M,distilled water was given.On the 5th day,the whole lung of mice was harvested,and the total RNA of lung tissue was extracted and detected after hybridization with mice whole gene expression spectrum chip.Differential expressed genes of cytokines involved in inflammatory pathways were selected.The intensity expression ratio of the chip probe signal in each group vs.M group was calculated,and P<0.05 and log2 ratio>1 were defined as up-regulated genes,while P<0.05 and log2 ratio<-1 were down-regulated genes.The mRNA expressions of interleukin(IL-1,IL-8)and intercellular adhesion molecule-1(ICAM-1)were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with group N,the differential gene expressions of IL-1,IL-8 and ICAM-1 in group M were significantly up-regulated[log2(N/M)were 2.62,2.07,1.41,respectively,all P<0.05].Compared with group M,the gene expressions of IL-1,IL-8,ICAM-1 were significantly down-regulated in SH,SM,SL and C groups[log2(SH/M)were-1.91,-1.85,-0.88;log2(SM/M)were-3.10,-1.74,-1.84;log2(SL/M)were-1.89,-1.39,-0.53;log2(C/M)were-2.46,-1.52,-1.44,respectively,all P<0.05].RT-PCR showed that the mRNA expressions of IL-1,IL-8 and ICAM-1 in group M were significantly higher than those in group N[IL-1(2-ΔΔCT):4.63±0.24 vs.1.01±0.13,IL-8(2-ΔΔCT):6.28±0.13 vs.1.02±0.09,ICAM-1(2-ΔΔCT):2.90±0.18 vs.1.02±0.12,all P<0.05].The mRNA expressions of IL-1,IL-8,ICAM-1 in SH,SM,SL and C groups were lower than those in group M[IL-1(2-ΔΔCT):2.12±0.32,1.71±0.07,2.05±0.16,1.66±0.13 vs.4.63±0.24;IL-8(2-ΔΔCT):3.89±0.13,2.08±0.19,2.98±0.20,2.02±0.12 vs.6.28±0.13;ICAM-1(2-ΔΔCT):1.72±0.93,1.34±0.14,1.53±0.25,1.17±0.12 vs.2.90±0.18,all P<0.05].There was no significant difference among the SH,SM,SL and C groups.Conclusion Shufeng Xuanfei Jiedu formula inhibits inflammatory damage in mice after influenza virus infection by down-regulating the expressions of IL-1,IL-8,and ICAM-1 inflammatory cytokine-related genes.