摘要
研究目的
采用复合因素(肥甘饮食+外湿热环境+MHV-A59病毒感染)建立小鼠病毒性肝炎温病湿热证模型,探索新的生物感染因子和动物的温病湿热证模型；通过研究TLR2、TLR4、NF-κBP65、IL-1β、IFN-γ的表达变化,探讨温病湿热证与Toll样受体通路的相关性；运用方证相应理论,使用清热化湿类方药(蒿芩清胆汤、甘露消毒丹)干预上述靶点,分析清热祛湿法干预温病湿热证的药效机制及可能作用机理。
研究方法
1.4周龄SPF级BALB／c雄性小鼠72只随机分为9组,每组8只。正常组予普通饲料,同步饲养；其余8组小鼠予高脂饲料+湿热环境10天(d),第11 d不同浓度小鼠肝炎病毒(MHV-A59)0.5ml／只腹腔注射；攻毒后再连续观察8d。记录全部小鼠体重变化、皮毛、饮食及死亡情况,根据LD50变化,确定最佳攻毒剂量。
2.在实验1基础上,确定最佳攻毒剂量后。4周龄SPF级BALB／c雄性小鼠40只,随机分为以下4组,每组10只。
A组：正常对照组：正常饮食,同步饲养；
B组：单纯湿热组：肥甘饮食+湿热环境；
C组：单纯病毒组：正常饮食+小鼠肝炎病毒；
D组：模型组：肥甘饮食+湿热环境+小鼠肝炎病毒；
模型复制过程中,观察模型动物的症状(纳呆,便溏或便秘,倦怠),饮水量,饮食量,体重,肛温。
采用全自动生化分析仪检测肝功能；HE染色观察肝组织病理变化：组织芯片及荧光定量PCR法检测肝组织TLR2、TLR4、NF-κBP65、IL-1β、IFN-γ的含量。
3.4周龄SPF级BALB/c雄性小鼠80只,随机分为以下8组,每组10只。
A组：正常对照组,造模方法同实验2；
B组：单纯病毒组,造模方法同实验2；
C组：模型组,造模方法同实验2；
D组：单纯病毒蒿芩清胆汤治疗组,造模方法同单纯病毒组,但在病毒感染前一天起予以蒿芩清胆汤灌胃,每日1次给药,连续4d(药物均按传统煎法,剂量参考原方剂量并按人鼠体表面积换算)。
E组：单纯病毒西药治疗组,造模方法同单纯病毒组,但在病毒感染前一天起予以易善复灌胃,每日1次给药,连续4d(药物剂量按人鼠体表面积换算)。
F组：蒿芩清胆汤治疗组：造模方法同模型组,但在病毒感染前一天起予以蒿芩清胆汤灌胃,每日1次给药,连续4d(药物均按传统煎法,剂量参考原方剂量并按人鼠体表面积换算)。
G组：甘露消毒丹治疗组：造模方法同模型组,但在病毒感染前一天起予以甘露消毒丹灌胃,每日1次给药,连续4d(药物均按传统煎法,剂量参考原方剂量并按人鼠体表面积换算)。
H组：西药组：造模方法同模型组,但在病毒感染前一天起予以易善复灌胃,每日1次给药,连续4d(药物剂量按人鼠体表面积换算)。
模型复制过程中,观察模型动物的症状(纳呆,便溏或便秘,倦怠),饮水量,饮食量,体重。
采用全自动生化仪检测各组小鼠的肝功能,HE染色观察各组小鼠肝组织病理变化,免疫组化法检测各组小鼠肝脏TLR2、TLR4、NF-κBP65、IL-1β、IFN-γ阳性表达,荧光定量PCR检测各组小鼠肝组织中TLR2、TLR4、NF-κBP65的mRNA含量。
研究结果
1.实验一
正常组小鼠活动、饮食、大便及精神状态均正常；各模型组小鼠可见耸毛现象,食量减少,活动减少,喜倦缩,大便溏稀符合湿热证改变特点。各模型组小鼠予以不同浓度MHV-A59病毒0.5ml／只腹腔注射后,自第4天起,开始出现小鼠死亡现象,第5天达到高峰,出现暴发型死亡；而未予攻毒的正常组无一例死亡,确定最佳攻毒剂量为10-2,即1：102的浓度腹腔注射感染小鼠,每只0.5ml。最佳观察时间为攻毒后3天内。
2.实验二
温病湿热证的本质中,“湿”性致病因子包括肥甘厚味和外湿环境；“热”性致病因子主要和复合因素造成的内毒素血症有关,TLR2、TLR4以及Toll-NF-κB信号通路激活,以及下游炎性细胞因子如IL-1β等大量分泌为特征。
3.实验三
3.1正常组小鼠体重呈自然增长趋势,大便正常,皮毛有光泽,精神状况好；其余各组小鼠体重亦增长趋势,大便偏稀,肛门污秽。经重复测量方差分析,各组小鼠体重变化无差异(P>0.05)；各组小鼠肝脏指数无统计学差异(P>0.05)。
模型组及治疗组血清ALT、AST存在不同程度的升高,表明小鼠肝细胞存在不同程度坏死现象。G组血清ALT、AST与模型组(B、C组)及治疗组(D、E、F、H组)比较,有统计学意义(P<0.05)。
模型组及治疗组小鼠肝脏TLR2、TLR4阳性细胞分布面积呈上升趋势,与A组比较,有统计学意义(P<0.05)。F组和G组与B组、C组、D组、E组、H组比较有统计学意义(P<0.05),经予清热化湿法治疗后,TLR2、TLR4阳性细胞表达呈下降趋势。
3.2模型组及治疗各组NF-κBP 65mRNA及其蛋白表达之间比较,无统计学意义；与A组比较,有统计学意义(P<0.05)。
3.3模型组及治疗各组IL-1β、IFN-γ的表达均呈上升趋势,以IL-1β变化明显,与A组比较,有统计学意义(P<0.05)；而IFN-γ变化与A组比较,无统计学意义(P>0.05)。
研究结论
1、在本研究模型构建中,病毒感染是造成模型小鼠病理变化的主要因素,饮食因素+气候环境因素+MHV-A59病毒感染的综合因素实验方法造模,所复制的模型较为理想,无论从发病条件,病变脏腑,还是主要症状体征均近似于中医温病湿热证型,且具有操作简单,重复性好的特点。
2、本实验研究证明,小鼠病毒性肝炎湿热证以TLR2、TLR4以及Toll-NF-κB信号通路激活,NF-κBP65及下游炎性细胞因子如IL-1β等大量分泌为特征。TLR-2、TLR-4、IL-1β可以作为温病湿热证辨证的客观指标之一,是病证结合模型的深入探索。
3.研究表明,清热化湿法对于小鼠病毒性肝炎湿热证有一定治疗效应,均能降低湿热证小鼠体温,增加小鼠耗食量、饮水量,改善肝功能,对内毒素受体TLR-2、TLR-4有抑制作用。清热化湿法可能通过影响TLR-2、TLR-4表达发挥改善温病湿热证的效应。
4.清热化湿法对小鼠病毒性肝炎湿热证的NF-κBP65、IL-1β及IFN-γ的肝脏表达无显著改善作用。这表明蒿芩清胆汤或甘露消毒丹可能不是通过影响NF-κBP65、IL-1β及IFN-γ的表达而发挥对温病湿热证小鼠的治疗效应。
Objection
The mechanism of epidemic febrile disease syndrome of damp and heat which caused by combined factors including high- fat diet and hot and humid environment and MHV-A59 virus was discussed in order to evaluate the essence of febrile disease of dampness-heat syndrome and explain the influence of the method of clearing away damp and heat treating on epidemic febrile disease syndrome of damp and heat.
Methods
1. Seventy-two BALB/c rats were randomly divided into nine groups.Each group has eight rats.Normal group was feeding of common diet.the other groups were feeding of high-fat diet and hot and humid environment for ten days. Meanwhile, except normal group, the other groups were injected intraperitoneally the different concertration of MHV-A59 virus for three days.After intraperitoneal injection of MHV-A59 virus, all rats were observed for five days continuely.The changes of rats'weight and diet and death status were surveyed.The optimal dosage of intraperitoneal injection of MHV-A59 virus was according to the levels of LD 50.
2. Based on first experiment, we calculate the optimal dosage of intraperitoneal injection of MHV-A59 virus.Forty BALB/c rats were randomly divided into 4 groups; each group has ten rats.such as:
Group A:normal group:common diet.
Group B:only damp-heat group:high-fat diet and hot and humid environment.
Group C:only virus group:common diet+MHV-A59 virus
Group D:experimental model group:high-fat diet and hot and humid environmentt+MHV-A59 virus.
During the course of animal model replication of epidemic febrile disease syndrome of damp and heat, we survey the syndrome and drinking amount as well as the amount of food and the rectal temperature.
The changes of liver morphology and hepatic function as well as Toll like recept signal pathway and the levels of IL-1βand IFN-γand NF-κBP65 was assayed by Automatic Biochemistry Analyzer、HE statins、tissue microarray and Real-time PCR respectively.
3. Eighty BALB/c rats were randomly divided into 8 groups; each group has ten rats.such as:
Group A:normal group:common diet.
Group B:only virus group:common diet+MHV-A59 virus.
Group C:experimental model group:high-fat diet and hot and humid environmentt+MHV-A59 virus.
Group D:only intraperitoneal injection of MHV-A59 virus which was treated with Hao Qing Qin Dan decoction group.
Group E:only intraperitoneal injection of MHV-A59 virus which was treated with polyene phosphatidylcholine capsules group.
Group F:Hao Qing Qin Dan decoction group. The preparation of animal model like group C which was treated by Hao Qing Qin Dan decoction.
Group G:Gan Lu Xiao Du Dan decoction group. The preparation of animal model like group C which was treated by Gan Lu Xiao Du Dan decoction.
Group H:Control group. The preparation of animal model like group C which was treated by polyene phosphatidylcholine capsules.
During the course of animal model replication of epidemic febrile disease syndrome of damp and heat, we survey the syndrome and drinking amount as well as the amount of food and the rectal temperature.
The changes of liver morphology and hepatic function as well as Toll like recept signal pathway and the levels of IL-1βand IFN-γand NF-κBP65 was assayed by automatic Biochemistry Analyzer、HE statins、tissue microarray and Real-time PCR respectively.
Results
1. NO 1 experiment
There are a cluster of signs in the experimental rats such as loss of appetite、less activity、somnolence、loose stool and so on.Such syndromes conform to the diagnosis of epidemic febrile disease syndrome of damp and heat. MHV-A59 virus was injected intraperitoneal after three days, there is a sign hat the death rate of rats are increasingly.The death summit is discovered in the fifth day.We calculate the optimal dosage of intraperitoneal injection of MHV-A59 virus is 10-2. The optimal observation time interval is three days.
2. NO 2 experiment
The essence of epidemic febrile disease syndrome of damp and heat include two pathogenic factors. "Damp factor" include high-fat diet and damp and heat enviroment. "Heat factor" include Endotoxaemia which is caused by intraperitoneal injection of MHV-A59 virus.
3. NO 3 experiment
3.1 After repetitive measurement and analysis of variance, there is no significant in different group in the changes of weight and liver index (P>0.05)
The changes of ALT and AST were on the trend of increasing in blank group and different intervention group. This means that there is a sign of necrosis in rats liver cell.Compared to G group, these changes in B group C group D group, E group、F group、H group is obvious (P<0.05).
Compared to A group,there is a significant difference in the positive expression of TLR2、TLR4 in B-G group.Meanwhile,there is a significant difference in the changes of TLR2、TLR4 between F group、G group and B group、C group、D group、E group、H group(P<0.05).After treated with clearing away damp and heat decoction,the expression of TLR2、TLR4 is on the decrease (P<0.05).
3.2 There is no significant difference in the protein and mRNA of NF-κBP 65 in B-G group (P>0.05).However; compared with A group, there is a significant in the protein and mRNA of NF-κBP 65. (P<0.05)
3.3 The expression of IL-1βIFN-y is on the increase,especially in the expression of IL-1β.There is a significant difference between A Group and the others in the expression of IL-1β(P<0.05).However; there is no significant difference in the expression of IFN-y in different groups (P>0.05).
Conclusions
1.The epidemic febrile disease syndrome of damp and heat model was replicated by combined factors which include diet factor and environment factor and mice hepatitis virus infection.This model is the similar to human syndrome of damp and heat.The changes of TLR-2、TLR-4、IL-1βcan be as the objective index of febrile disease syndrome of damp and heat.
2. The clearing away damp and heat method has an exact effect on the epidemic febrile disease syndrome of damp and heat,which can decrease the temperature of rats and increase the diet amount of rats as well as can improve hepatic function and inhibit the expression of TLR-2、TLR-4.
3. The clearing away damp and heat method maybe exert an effct on epidemic febrile disease syndrome of damp and heat through influened on the expression of TLR-2、TLR-4.
4. The clearing away damp and heat method has no effect on the expression of NF-κBP 65、IL-1β及IFN-γ.
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