Experimental Study On The Mechanism Of Toll-like Receptor Singal Pathway In A Rat Model Of Virus Hepatitis And The Intervention Effect Of Clearing Heat And Eliminating Dampness

ObjectionThe 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.Methods1. 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 virusGroup 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.Results1. NO 1 experimentThere 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 experimentThe 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 experiment3.1 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).Conclusions1.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.