Experimental Research Of The Effect Of The Therapeutic Principle Clearing Heat And Dampness To Relieve Superficies In Treating With Fat Mice Infected By Influenza A (H₁N₁) Virus Under Intermittent Thermal Stress

ObjectiveIn recent years, herbal treatments show particular advantage in treating viral diseases. Especially to say, complex prescriptions have good effects in multi-direction and regulation of immunity, with the aim to getting rid of pathogen without disturbing the body's normal function. Respiratory viral infection is the most common diseases in South of china. Influenza and parainfluenza viruses are detected among adult cases. Researches of TCM have showed that respiratory viral infection in southern china is of heat and dampness character. The therapeutic principles are made according to the pathogen and syndrome, among which clearing heat and dampness, clearing heat and toxin, building up and tonifying immunity are very popular. Some researches showed that the effects of these principles are inhibiting virus cloning, regulating the immunity, or reducing inflammation etc. But there are many unsettled problems in this field.Hao Qin Qing Dan decotion(HQQD) ,which represents clearing heat and dampness, relieving superficies therapeutic principle, is wildly used in treatments on respiratory viral diseases and Febrile diseases in Southern China. To clarify the connotation and epitaxy of heat-dampness syndrome in respiratory viral infection, and explore the mechanism of clearing heat and dampness, relieving superficies therapeutic principle, we use a influenza virus infected mouse model with composite factors in our experiments to imitate the syndrome of respiratory infection in Southern China. Comparing with the other two therapeutic principles, we want to study the mechanism of the clearing heat and dampness, relieving superficies therapeutic principle in this model and give more certifications to use this principle in treating respiratory viral diseases.Method: According to the normal heat-dampness animal model, on the basis of fat animal feeds and heat-dampness conditions, mice are infected by influenza virus (H₁N₁) by dropping. Lung indexes are used in our research to evaluate the anti-virus effect of experimental decoctions. Different lymphocyte groups of model mice are detected by FCM. IFN- y and IL-4 level in the blood are tested by ELISA to estimate Thl/Th2 and immunity balance. NF-KB and aquaporin 4 are detected by immunohistochemical method in lung. We want to explore the main direction of the therapeutic principle by comparing it with Ying Qiao San decotion(HQS, represent relieving superficies by acrid-cold herbs) and Yu Ping Fen San decotion(YPFS, represent building up and tonifying immunity). In our clinical research, 101 adult cases of upper respiratory infection are colleted, syndrome and prescription character of these cases are analyzed and summarized.Results and Analysis:About clinical study: 101 adult cases of acute upper respiratory infection from June to August in 2007 are collected and analyzed. As a result: 1. onset of the disease is closely related to the solar term. Attack rate is highest during Grain in Ear. The main diagnosis is anemogenous febrile disease (wind-heat with dampness, syndrome type); 2.Fever, headache, limbers with aching pain, sore throat are most common symptoms, and the most common simultaneous phenomenon are anorexia, dry mouth, red tongue with thick float;3. Clearing heat and toxin, relieving superficies by acrid-cold herbs and opening the inhibited lung-qi to dissipate dampness are very popular in the prescriptions according to differentiation of symptoms and signs. Sang Ju Yin(SJY),Yin Qiao San(YQS),which represent the main principle in Febrile Disease, are wildly used in Southern China. Pharmaco-experiments have proved that isatis root, dyers woad leaf, forsythia suspense are effective in inhibiting viruses in vitro and vivo. These herbs be useful in antibiosis, relieving fever and analgesia, too. 4. The following local herbs are very popular in prescriptions, such as Operculate cleistocalyx(function: clear summer heat and dampness), snakegourd fruit(function: lower adverse-rising qi and disspate phlegm), roughhaired holly root(function:clear toxin and relieving sore throat ),musslinite etc.About the animal model: 1. the symptoms and pathogenic changes are closely related to influenza virus infection. Fat feeds and intmt heat conditions, not act as the main factors, influence the onset and pathogenic changes of disease; 2. The the balance of T cell groups is broken after virus infection, percent of CD₄⁺T increases and CD₄⁺T decreases, the ratio of CD₄⁺ and CD₈⁺ inverts. Virus infection inhibits the immunity system of mice. Meanwhile, the level of IFN-γincreased and IL-4 keep still. Th1/Th2 balance is directed to Th1. These changes are same between model mice and virus-infected mice; 3. Expression of NF-κB and AQP4 in lung like enhanced in model mice, to show the inflammation and edema in local lung tissues. The slight difference between model mice and virus infected mice is of no significance; 4. To sum up, living causative agent (influenza virus infected successfully) is the key factor to set up the animal model. With heat and dampness factors, virus-infected mice's cell/humoral immunity system is interrupted, some inflammatory factors,such as IFN-γ,NF-κB etc, are over expressed and water metabolism of alveolar wall cells is irregular, which leads to pathogenic injury.About the therapeutic principle: 1. HQQD(clear heat and dampness, relieve superficies therapeutic principle) is effective in inhibiting the pathogenic injury of model mice, which is similar with YQS(relieve superficies by acrid-cold herbs) and YPFS(build up and tonify the immunity); 2.HQQD can increase the percent of CD₄⁺T and decrease the percent of CD₈⁺T, advance cell immunity and keep the immunity balcance. This part is similar with YPFS which represent building up and tonify immunity therapeutic principle. And we presume that accelerating cell immunity maybe one of mechanism that anti-virus function of the principle; 3. HQQD can inhibit the increase of IFN-γand direct the Th1/Th2 to Th2, this function is more significant than YPFS. YQS is not significant in this aspect. We presume that regulating cell/humoral immunity, reducing inflammatory factors expression maybe one of mechanism that anti-virus function of the principle; 4. HQQD(clear heat and dampness to relieve superficies) reduce the positive expression of NF-κB obviously. And there is no difference between HQQD,YQS and YPFS. Furthermore, three decotions(three different therapeutic principle) can reduce the expression of AQP4 on the alveolar wall cells, but YQS and YPFS are more effective.To sum up, three different therapeutic principles are effective in reducing lung index, reliving pathogenesis. HQQD and YPFS are more effective in strengthen T cell immunity; HQQD, which turns Th1/Th2 to Th2, is more effective than YPFS in regulate Th1/Th2 balance. YQS's function may exist in inhibiting and reducing inflammation and edema of lung. As a result, three therapeutic principles(HQQD,YQS,YPFS) have multiple function targets in treating influenza A viral pneumia. HQQD(clear heat and dampness, relieve superficies) has advantage in strengthening cell immunity, reducing inflammatory factors, and regulating Th1/Th2 balance.ConclusionOn the whole, acute upper respiratory infection in Southern China belongs to anemogenous febrile disease, and the main syndrome type is wind-heat with dampness. Different therapeutic principle has its advantage in treating respiratory vrial infection according to differentiation of symptoms and signs in different area. As far as heat-dampness animal model in our research are concerned, immunity system derangement after virus infection is key pathogenesis in heat-dampness mouse influenza pneumia model comparing with fat feeds and hot condition. The state of model mouse(immunity derangement, over expression of inflammatory factors, disharmony of water metabolism ) may be the target of heat and dampness. HQQD(clearing heat and dampness, relieving superficies therapeutic principle) can effectively reduce the lung index, relieve the pathogenic changes. HQQD can regulate T cell immunity and Th1/Th2 balance significantly, comparing with YQS and YPFS. Our experimental result hints that HQQD probably not the same role with the other two principles. Our research provides more evidences to respiratory viral infection treatment by using heat and dampness theory.