肝郁脾虚外感小鼠模型的建立及评价

Establishment and evaluation of mice model with liver stagnation and spleen deficiency infected by influenza virus

目的:建立肝郁脾虚外感复合病证小鼠模型,并通过柴胡桂枝汤和连花清瘟胶囊进行正治和反证。方法:腹腔注射利血平(0.32mg·kg^-1·d^-1)10d建立肝郁脾虚小鼠模型,在此基础上滴鼻感染甲型H1N1流感病毒建立肝郁脾虚外感小鼠模型;在接种流感病毒后,分为肝郁脾虚外感组、连花清瘟组、柴胡桂枝组;连花清瘟组和柴胡桂枝组分别灌胃连花清瘟胶囊(0.546g·kg^-1·d^-1)和柴胡桂枝汤(6.11g·kg^-1·d^-1),连续6d。每天观察记录小鼠的一般体征,肛温、体质量,饮食量、饮水量,测定小鼠的悬尾不动时间、旷场活动情况;测定小鼠的肝、脾、胸腺、肾上腺、肺指数;测定小鼠血清中淀粉酶(比色法)和胃泌素(ELISA法)含量;qPCR法测定小鼠肺组织中流感病毒H1N1的M基因表达量,并观察小鼠肺组织的病理变化。结果:与正常组比较,肝郁脾虚模型组小鼠体质量、饮食量、饮水量均显著降低(P<0.01,P<0.05),悬尾不动时间显著增加、旷场活动总路程显著减少(P<0.01),肝、脾、胸腺、肾上腺指数均显著降低(P<0.05,P<0.01),血清中淀粉酶和胃泌素含量均显著降低(P<0.01)。与正常组比较,肝郁脾虚外感组小鼠肛温、体质量、饮食量、饮水量均显著降低(P<0.01),肝、胸腺、肾上腺指数显著降低(P<0.01),肺指数显著升高(P<0.01),血清中淀粉酶和胃泌素含量均显著降低(P<0.01),肺组织有较高的流感病毒H1N1的M基因表达并伴有明显的病毒性肺炎症状;与肝郁脾虚外感组比较,柴胡桂枝组小鼠肛温、体质量、饮食量、饮水量均显著升高(P<0.01,P<0.05),肝、胸腺、肾上腺指数显著升高(P<0.01),肺指数显著降低(P<0.01),血清中淀粉酶和胃泌素含量均显著升高(P<0.01,P<0.05),肺组织中流感病毒H1N1的M基因表达量显著降低(P<0.01),病毒性肺炎症状明显缓解。结论:本研究建立的肝郁脾虚外感小鼠模型在一定程度上符合中医肝郁脾虚(少阳病)外感证的特点,柴胡桂枝汤和连花清瘟胶囊分别从正治和反证角度证实此模型成立,为之后的相关药效和机制研究奠定基础。

Objective:To establish a mouse model of exogenous syndrome of liver depression and spleen deficiency infected by influenza virus,and to treat and reverse syndrome by Chaihu Guizhi Decoction and Lianhua Qingwen Capsules.Methods:The mouse model of liver stagnation and spleen deficiency was established by intraperitoneal injection of reserpine(0.32 mg·kg^-1·d^-1)for 10 days,and then the mouse model of liver depression and spleen deficiency was established by intranasal infection with H1 N1 influenza virus A.After inoculation with influenza virus,the mice were divided into three groups:liver stagnation and spleen deficiency exogenous group,Lianhua Qingwen group and Chaihu Guizhi group.The rats in Lianhua Qingwen group and Chaihu Guizhi group were treated with Lianhua Qingwen Capsules(0.546 g·kg^-1·d^-1)and Chaihu Guizhi Decoction(6.11 g·kg^-1·d^-1)respectively for 6 days.The general signs,anal temperature,body mass,food intake and water intake of mice were observed and recorded every day.The tail suspension time and open field activity of mice were measured.The indexes of liver,spleen,thymus,adrenal gland and lung were measured.The contents of amylase(colorimetric method)and gastrin(ELISA method)in serum were determined.The M gene expression of influenza virus H1 N1 in lung tissue was determined by qPCR method,and the pathological changes of lung tissue were observed.Results:Compared with the normal group,the body weight,food intake and water intake of the mice in the liver depression and spleen deficiency model group were significantly decreased(P<0.01,P<0.05),the tail suspension time was significantly increased,the total distance of open field activity was significantly decreased(P<0.01),the indexes of liver,spleen,thymus and adrenal gland were significantly decreased(P<0.05,P<0.01),and the contents of amylase and gastrin in serum were significantly decreased(P<0.01).Compared with the normal group,the anal temperature,body mass,food intake and water intake of the mice with liver depression and spleen deficiency were significantly decreased(P<0.01),the indexes of liver,thymus and adrenal gland were significantly decreased(P<0.01),the lung index was significantly increased(P<0.01),and the contents of amylase and gastrin in serum were significantly lower(P<0.01).The M gene expression of influenza virus H1 N1 in lung tissue was higher and accompanied with obvious symptoms of viral pneumonia.Compared with the liver depression and spleen deficiency external sensation group,the anal temperature,body mass,food intake and water intake in the Chaihu Guizhi group were significantly increased(P<0.01,P<0.05),and the indexes of liver,thymus and adrenal gland were significantly increased(P<0.01).The lung index decreased significantly(P<0.01),and the contents of amylase and gastrin in serum increased significantly(P<0.01,P<0.05).The M gene expression of influenza virus H1 N1 in lung tissue was significantly decreased(P<0.01)and the symptoms of viral pneumonia were significantly relieved.Conclusion:The mouse model of liver depression and spleen deficiency established in this study accords with the characteristics of exogenous syndrome of liver depression and spleen deficiency(Shaoyang disease)to a certain extent.Chaihu Guizhi Decoction and Lianhua Qingwen Capsules confirmed the establishment of this model from the point of view of positive treatment and counter-syndrome to lay a foundation for the study of related pharmacodynamics and mechanism.