目的 研究银翘散及其拆方（君药、臣药、佐药、使药）对流感病毒感染自然杀伤细胞活性及转录组的影响。方法 以小鼠流感病毒PR8（A/Puerto Rico/8/1934，H1N1型）感染ICR小鼠制备肺炎模型，分为对照组、模型组和给药组，每组10只；银翘散、君药（金银花、连翘）、臣药（薄荷、牛蒡子、淡豆豉、荆芥穗）、佐药（桔梗、芦苇根、淡竹叶）和使药（甘草）、阳性药物利巴韦林分别ig模型小鼠5 d；第5天检测小鼠体质量、肺质量，计算肺质量抑制率；固定肺脏，观察病理学变化（HE染色）。以PR8病毒感染C57BL/6J小鼠制备肺炎模型，分为对照组、模型组和给药组，每组3只，上述药物分别ig 5 d，第5天摘取肺脏，制备白细胞悬液，采用流式细胞术检测自然杀伤（nature killer，NK）细胞表面激活性受体Ly-49D和Ly-49H的含量，以及细胞毒性受体NKp46的含量。为了进一步探究药物与病毒感染NK细胞之间的互作关系，建立人流感病毒HK8（A/Hong Kong/8/68，H3N2型）感染人自然杀伤细胞系NK-92MI模型，比较银翘散及臣药对病毒感染NK细胞转录组作用的异同。结果 银翘散及其拆方对PR8病毒感染引起的小鼠肺炎均有一定的抑制作用，抑制作用由强到弱为臣药 > 银翘散 > 使药 > 君药 > 佐药，其中臣药和银翘散组具有统计学差异（P<0.05）。银翘散及其拆方均能增加肺脏NK细胞激活性受体Ly-49D和Ly-49H的含量，其促进作用由强到弱为臣药 > 使药 > 银翘散 > 君药 > 佐药，其中银翘散、君药、臣药和使药组均具有统计学差异（P<0.05、0.01）。结合上述2个指标，臣药表现出与银翘散相似的药效特征，且略优于银翘散。对于PR8病毒感染小鼠肺脏NK细胞毒性受体NKp46的含量，臣药也表现出良好的诱导表达作用。HE染色显示，经臣药治疗的PR8病毒感染小鼠，其肺脏结构完好，肺泡和支气管腔内未出现炎症细胞和红细胞浸润。NK细胞转录组分析显示，臣药对于HK8病毒感染的NK细胞的作用位点，不仅囊括了银翘散的作用位点，而且比银翘散更为广泛。结论 对于流感病毒感染小鼠，银翘散的拆方臣药表现出与银翘散全方相似的药效，且略优于银翘散，这可能与臣药对NK细胞活性的调节作用比银翘散更广泛有关。

Objective To study the effect of Yinqiao San (银翘散, YQS) and its decomposed recipes on the activation and transcriptome of natural killer cells infected with influenza virus. Methods In influenza virus PR8 (A/Puerto Rico/8/1934, H1N1) infected mice (n=10), ribavirin, YQS, Monarch drug (Lonicerae Japonicae Flos and Forsythiae Fructus), Ministerial drug (Menthae Haplocalycis Herba, Arctii Fructus, Sojae Semen Praeparatum, and Schizonepetae Spice), Assistant drug (Platycodonis Radix, reed root, and Lophatheri Herba) and Guide drug (Glycyrrhizae Radix et Rhizoma) was administrated orally for 5 d. The body weight and lung weight of the mice were measured on day 5, and the pulmonary inhibition rate was calculated. The lung was fixed and pathology was analyzed (HE staining). PR8 virus was used to infect C57BL/6J mice (n=3). The above drugs were administered orally for 5 d, and the lungs were extracted on day 5 to prepare the white blood cell suspension. Flow cytometry was used to detect the content of the natural killer (NK) cell surface activating receptors Ly-49D and Ly-49H, as well as the content of the NK cell cytotoxic receptor NKp46. For further exploration of the interaction between the drug and virus-infected NK cells, an in vitro assay was conducted. The NK-92MI cell is a human natural killer cell line. The cells were infected with H3N2 (A/Hong Kong/8/68) human influenza virus and were used to compare the effect of YQS and its decomposed recipes on its transcriptome. Results The results showed that YQS and its decomposed recipes had a certain inhibitory effect on the pneumonia of mice caused by PR8 virus infection. The inhibitory effect from strong to weak was:Ministerial drug > YQS > Guide drug > Monarch drug > Assistant drug; There was a statistical difference in Ministerial drug and YQS group compared with virus infection group. On the influence of the content of the lung NK cell activating receptors Ly-49D and Ly-49H, YQS and its decomposed recipes could both increase the expression of the two receptors, and its promoting effect from strong to weak was:Ministerial drug > Guide drug > YQS > Monarch drug > Assistant drug; there were significant differences in YQS, Monarch drug, Ministerial drug and Guide drug groups compared with virus infection group. In combination with the above two indexes, the efficacy of Ministerial drug was similar to that of YQS, and slightly better than YQS. Moreover, for the content of cytotoxic receptor NKp46 in pulmonary NK cells infected with PR8 virus in mice, Ministerial drug also showed a good induced effect. HE staining of the lung showed that mice infected with PR8 virus treated with Ministerial drug had intact lung structure, and no inflammatory cells or red blood cell infiltration were observed in alveoli or bronchial lumens. Transcriptome analysis showed that the targets of Ministerial drug on virus-infected NK cells not only included the acting sites in YQS but also included the acting sites not involved in YQS. Conclusion For mice infected with influenza virus, Ministerial drug showed a similar or better efficacy than YQS, which may be related to the wider regulatory effect of Ministerial drug on NK cell activity than YQS.

R285

国家自然科学基金资助项目（81460700）