Study On The Effect And Apoptosis Mechanism Of Tibet Artemisiae Capillaries And Its Complex Prescription To Damp-heat Syndrome Of MHV-A59 Virus Hepatitis

BackgroundGuangdong province has always been charactered by the high temperature and rainy. Because of its special climate and the habit of eating cold and oily food, people in Guangdong has formed the body with damp-heat syndrome. The high temperature and rainy climate, besides the special body of people in Guangdong, profit to bacterial and viral propagation. Therefore, Guangdong province has been the dangerous and important region for the infection diseases would happen and spread easily and quickly. Many virus such as Influenza viruses,Corona viruses and Duo viruses have been existed for a long time and were found in Guangdong. For example, the first case of explosive SARS was happened in Guangdong. The Mouse Hepatitis virus(MHV) belongs to the Corona virus genus. It mainly cause the viral hepatitis when infected. Because human can not be caused by it, MHV has been used as the succedaneum when studying the mechanism of human Corona viruses. It was significant to study damp-heat syndrome caused by Corona viruses in Guangdong province. Nowadays, people prevention of Corona viruses mainly through vaccine and antiviral drugs. However, vaccine production is hard to play the role of variation of the virus. Anti-viral agents must be use an early stage if you want to achieve the desired result,. Chinese medicine in the treatment of such diseases has unique advantages. Tibet Artemisiae Capillaries is one of the most particular and effective Tibet medicines for hepatobiliary diseases. It has the functions of heat-clearing and detoxicating and hepatobiliary-protecting. So it has been using for many kinds of hepatobiliary diseases, especially the viral hepatitis, many scholars and experts have been thinking highly of it. Therefore, this experiment was sign to explore the role of Tibet Artemisiae Capillaries of antagonism effects, and look for the possible signal pathway. The experiment is significant to the ground research.ObjectivesTo establish the animal model of damp-heat syndrome of virus hepatitis infected by MHV-A59 according to clinical symptom. To evaluate the role of different modeling condition and to explore correlated pathogenic mechanism of the model. To observe the effect after treating the model by complex prescription of Tibet Artemisiae Capillaries in order to prove the exist of the pathogenic mechanism. To offer the ground theory for the further research and therapy.MethodsThe experiments consisted of two parts. The first part:Establish the animal model of damp-heat syndrome virus hepatitis infected by MHV-A59.12 SPF mouse were randomly divided into two groups:control group and model group,6 mouse for each group. From the 1d to 9d, the control group were fed with normal diet, the model group were fed with high fat diet (80%normal diet+8%honey+12% 1ard preparation; prepared by the Guangdong Laboratory Animal Medicine Center).At the 10d, the mouse in model group were infected by MHV-A59,the control group were given equivalent sterile liquid LB medium. After 24h, put the model group on the high temperature environment:temperature(32±0.5)℃, humidity(55～60)% for 3h; once every two days, and totally the model group was heat treat 3 times. The whole time of modeling was 15 days. Measured the body weight of mice in each group and detected their anus temperature at the Id,9d,14d of 9:30 AM in the physical environment (temp.25℃and RH60%).After the model was finished, we got the blood by excising the eyeball, then maintained the liver by 4%paraformaldehyde. The level of blood lipid in serum of mice was detected by glycerol oxidation. The concentrations of TNF-a in serum of mice were detected by ELISA. The Pathological changes of the liver were observed through the light microscope after the HE staining. RT-PCR was used to detect the quantity of virus in mice liver tissue. The anus temperature,weight,blood-fat,the concentrations of TNF-αand the levels of liver pathological changes of mice in each group were compared. The second part:Research on the effect and mechanism of Tibet Artemisiae Capillaries and its complex prescription to Damp-heat syndrome of virus hepatitis infected by MHV-A59.64 SPF mouse were randomly divided into 8 groups:control group, damp-heat group,virus group,model group,Virazole group,Hao Qin Qing Dan decoction group,Tibet Artemisiae Capillaries group(TAC group) and TAC complex prescription group,8 mouse for each group. To establish the mice model reference to the first part of the experiment. After the virus infect, the medicine was given to the medicine groups,the Chinese medicine twice a day, while the Virazole once a day. After the model was established and the blood serum,liver homogenate were prepared, the serum contents of ALT,AST were detected by Lai's method and the Pathological changes of the liver were observed through the light microscope after the HE staining. the liver homogenate contents of MDA and the activity of SOD were detected by TBA method. The activity of caspase3 was detected by the Microplate Reader. The contents of Fas and caspase3mRNA were detected by Quantitative PCR. To compared the value of active caspase3 absorbance,contents of ALT,AST and contents of MDA and the activity of SOD in different condition groups. To compared Fas and caspase3mRNA and so on between model and the prescription groups.ResultsThe first part:Establish the animal model of damp-heat syndrome virus hepatitis infected by MHV-A59.1,The general condition of miceCompared with the control group, the model mouse were slow-moving, the bodies were emaciated, expressed languid, the activity reduced and showed shortness of breath. The tetraplegia like the Gecko chinensis, the fur looked fluffiness and fell off. While the mouse decreased the food and water intake, the weight reduced obviously. The condition of sedes was sticking. The size of liver of the model mouse became small, with a rough appearance which full of bleeding point. The texture was fragile and the color was black purple.2,The anus temperature and weight of mice(1) At the 1d of modeling, the anus temperature of two groups separately were (34.37±0.38)℃and (34.63±0.36)℃; At the 9d they were (34.35±0.16)℃and (34.87±0.28)℃; At the 15d they were (34.30±0.44)℃and (34.30±0.44)℃.The changes between two groups had the statistical significance (According to the Mauchly's Test of Sphericity,W=0.616, P=0.113, hypothesis cannot be rejected, so F=70.167,P=0.000)(2) At the 1d of modeling, the weight of two groups separately were(9.17±0.38)g and (9.44±0.39) g.At the 9d they were (15.83±0.57)g and (18.05±0.51)g; At the 15d they were (18.02±0.19)g and (16.21±0.30)g。The model group were fed to the 9d, the weight gained much, but decreased to the 14d(after virus and high temperature).The change between two groups had the statistical significance (According to the Mauchly's Test of Sphericity,W=0.906, P=0.641, hypothesis cannot be rejected, so F=5.509,P=0.041)3,Changes in blood lipids of miceThe data of TG between model group and the control group were(1.84±0.56) mmol/l and (4.70±0.95) mmol/l, the data of CHOL were (4.02±0.28) mmol/l and (5.33±1.01) mmol/l. The blood lipids of model group(TG,CHOL) were higher than the control one(t=6.346,P=0.000;t=3.042,P=0.024).4,The Pathological changes of the liverCompared with the control group, the pathological section of model one showed obvious damage:the circumsciption of acini hepatic disappeared, the frame collaped, the liver cell around the central veins was disord and swell like the ballon and acidophilia. The kytoplasm became raritas, and the cell nucleus had the different shape and size.5,Changes of TNF-αin blood serum of miceThe contents of TNF-αin blood serum of both the two groups were (147.16±4.76)pg/ml and (188.10±10.09) pg/ml. The data showed that model group was much higher than the control one (t=8.987,P=0.000) The second part:Research on the effect and mechanism of Tibet Artemisiae Capillaries and its complex prescription to Damp-heat syndrome of virus hepatitis infected by MHV-A59.1,The contents of MHV-A59 in the liverThe corresponding copy number of MHV-A59 in the liver separately were virus group(22.92±3.55),model group(25.43±5.56),Virazole group(21.26±6.21),Hao Qin Qing Dan decoction group(20.75±5.37),Tibet Artemisiae Capillaries group(TAC group) (20.10±5.66)and TAC complex prescription group(18.16±4.96).All kinds of the prescription could reduce the contents of MHV-A59 in different levels, but only the TAC complex prescription had the most obvious effect, the difference had the statistical significance (P=0.017)2,The Pathological changes of the liverDifferent condition groups had the damages in different levels, which the model group was the most serious one. As all the liver pathological sections shown, the TAC group and its complex prescription group had the most effective to reduce the damage of the liver, and the complex prescription group was better than TAC one. Although the liver cell still swelled like the ballon, the integrity of cell and acini hepatic could be seen. The acidoid in the kytoplasm showed reduced. The improvement in the Hao Qin Qing Dan decoction group was not obvious.3,Changes of ALT,AST in blood serum of miceThe contents of ALT,AST in different condition groups were control group(10.28±2.22) U/L and (29.97±8.49) U/L, damp-heat group(13.03±2.51) U/L and (34.80±7.03) U/L,virus group(194.70±24.49) U/L and (163.16±18.77) U/L,model group(227.35±10.22) U/L and(180.82±17.34) U/L. The virus group and model group were much higher than control one (P=0.001,0.000).The difference between control and damp-heat group didn't have the statistically significance (P=0.794,0.607). Model group was higher than virus group, The difference also didn't have the statistically significance (P=0.372,0.067).The contents of ALT,AST in different prescription groups were Virazole group(169.20±20.30) U/L and (137.931±12.197) U/L,Hao Qin Qing Dan decoction group(202.43±9.86) U/L and (175.146±14.781)U/L,Tibet Artemisiae Capillaries group(TAC group) (178.97±16.45) U/L and (139.824±13.153) U/L,TAC complex prescription group(182.73±18.11) U/L and (145.080±20.423) U/L. All kinds of the prescription could reduce the contents of ALT,AST differently. And the Tibet Artemisiae Capillaries group and its complex prescription had the obvious statistical significance (P=0.016,0.000;P=0.040,0.001).There was no statistical significance between TAC complex prescription group and TAC,Hao Qin one (P=0.1000, P=0.577; 0.632)4,Changes of MDA,SOD in liver homogenate of miceThe changes of MDA,SOD in different condition groups were control group(7.18±1.48) nmol/mg and (129.26±7.21) U/mg, damp-heat group(10.48±1.41) nmol/mg and (122.85±7.91)U/mg,virus group(24.05±4.48) nmol/mg and(99.22±2.88) U/mg,model group(39.03±7.76) nmol/mg and (94.82±11.13) U/mg. MDA:The virus and model group were higher than the control one (P-0.000).Damp-heat group was higher than control one but with no statistical significance (P=0.011).SOD:The virus and model group were lower than the control one (P=0.000). Damp-heat group was lower than control one but with no statistical significance (P=0.880).The changes of MDA,SOD in different prescription groups were Virazole group(21.30±3.44) nmol/mg and (120.01±9.02) U/mg,Hao Qin Qing Dan decoction group(29.07±3.94) nmol/mg and (106.00±5.12) U/mg,Tibet Artemisiae Capillaries group(TAC group) (26.14±5.65) nmol/mg and (114.07±8.90) U/mg,TAC complex prescription group(23.61±5.56) nmol/mg and (118.11±12.17) U/mg. Compared with the model group, contents of MDA of the Tibet Artemisiae Capillaries group and its complex prescription were lower (P=0.013,0.049),while the activity of SOD were higher (P=0.031,0.045).There was no statistical significance between them (P=1.000), so as the Hao Qin Qing Dan decoction group (P=0.537,0.377).5,The values of active caspase3 absorbance in different groupsThe values of active caspase3 absorbance in different condition groups separately were control group (0.148±0.028), damp-heat group(0.215±0.037),virus group(0.293±0.033),model group(0.371±0.051).Compared with the control group, other three groups were higher in different levels, and the changes had the statistical significance (P=0.011,0.000,0.000).The model group was the highest one. The value of active caspase3 absorbance in different prescription groups were Virazole group (0.261±0.051),Hao Qin Qing Dan decoction group(0.304±0.067),TAC group (0.202±0.029) and TAC complex prescription group (0.239±0.035). Compared with the model group, the TAC group reduced the most, followed by its complex prescription one, and the changes had the statistical significance (P-0.000).There were with no statistical significance between TAC complex prescription group and TAC one,Hao Qin Qing Dan decoction one(P=0.145,0.037).The Hao Qin Qing Dan decoction group reduced the least in three prescription groups, but it had the statistical significance with the model one(P=0.000).6,Changes of the expression of Fas-mRNA and Caspase3-mRNA in the liver of miceIn each condition group, the corresponding copy number of Fas- mRNA and caspase3mRNA in the liver were control group(1.105±0.166) and (0.317±0.046),damp-heat group(1.657±0.069) and (0.547±0.043). virus group(1.548±0.050) and (0.615±0.027),model group(1.930±0.080) and (0.867±0.102).Compared with the control group, other three condition ones had higher expressions of FasmRNA and Caspase3mRNA in different levels.There were with statistical significance (P=0.000).In all the prescription groups the corresponding copy number were Virazole group(1.620±0.061) and (0.622±0.110), Hao Qin Qing Dan decoction group (1.602±0.044) and (0.583±0.050),Tibet Artemisiae Capillaries group(TAC group) (1.673±0.047) and (0.518±0.103), TAC complex prescription group (1.478±0.05)å'Œ(0.435±0.084).Compared with the control group, the expressions of FasmRNA and caspase3mRNA of model one were much higher with obvious statistical significance (P=0.000). Compared with the model group, other prescription ones could reduce the expressions of Fas- mRNA and caspase3-mRNA in different levels with obvious statistical significance (P=0.000).TAC complex prescription group had the most effective function in the four prescriptions. Compared with the TAC complex prescription group, changes of the expression of two genes of Hao Qin Qing Dan decoction had statistical significance(P=0.024,0.002),while only the change of Fas gene in TAC group had statistical significance(P=0.001).Conclusions:1,In this experiment, the first time use MHV-A59 as a biological factor, with "hot and humid environmental+fat food+biological infection factor" to replicate damp-heat syndrome of virus hepatitis disease. The incidence of the conditions, signs of model are consistent with the symptoms of Damp-heat. So it can be used as one of the damp-heat syndrome models. This study expand the research of the content of the previous model for its innovative; in addition, model as a prerequisite for future research.2,The MHV-A59 infection leads to the increase of ALT,AST in the blood serum, but the damp-heat can aggravate the changes. TAC complex prescription can reduce the content of virus,improve the liver cytothesis and protect the integrity of liver cell, in order to reduce the damages of liver tissue and contents of ALT,AST which were induced by the cell disrupting. So it play a part in liver-protecting.3,The virus and the damp-heat can aggravate the lipid peroxidation and strengthen effect of key enzyme which induce the apoptosis happening in the liver through the synergistic action. TAC complex prescription can reduce the content of MDA and the value of active SOD, so as the expressions of Fas and caspae3-mRNA. So it can be made a conclusion that the apoptosis signaling pathway to which led by Fas maybe one of the pathogenic mechanism of Damp-heat syndrome of virus hepatitis infected by MHV-A59.4,TAC and its complex prescription have more effective functions than Hao Qin Qing Dan decoction no matter the lipid peroxidation or the apoptosis signaling pathway, it consistent with the theory of determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs. Compared with TAC, its complex prescription is more effective for the model. Not only do it prove the existence of apoptosis signaling pathway in the model, but form the ground theory for the therapy of TAC complex prescription. Furthermore, it can provide the standardize and experiment evidences for complex prescription of Chinese medicine.