| Mycoplasmas, which have a wide host range that includes humans, animals, insects and plants, are known as the smallest free-living and self-replicating microorganisms. They may cause respiratory diseases that have a significant impact on health of humans and animals. The hallmark of these diseases is immunologic suppression, leading to the persistence of mycoplasma infection and chronic respiratory inflammation. Hottuynia Cordata, acrid in taste and slightly cold in nature, with lung-meridian tropism and heat-clearing, detoxifying, carbuncle dissolving and pus discharging effects, is used to treat lung symptoms such as phlegm, heat, dyspnea and cough. According to traditional Chinese medical theories, the onset, development and prognosis are determined by the antagonism of the strength of the vital-qi and pathogenic factors and are closely related to the immune function. Therefore, strengthening the body resistance to eliminate pathogenic factors also called improving immune function in modern immunological theory, is well accepted as the main therapeutic principle in traditional Chinese medicine as the simplicity for modern immunology.ObjectiveTaking Mycoplasma Gallisepticum as subject, this study is about to elucidate the inhibitory effect of Hottuynia Cordata, extracts purified by modern traditional Chinese medical extraction techniques on mycoplasmas, using microbiological and pharmacological methods; to explore the active components that effectively inhibit mycoplasma infection and their immune mechanism; to fully clarify that heat-clearing and detoxifying drugs are of both immune regulatory and pathogenic eradicating actions; to provide scientific foundation for developing safe, effective and quality controllable drugs made from active Herba Houttuyniae extract. Methods1. Three active components including total polysaccharide, total flavone and volatile oil were extracted from Hottuynia Cordata, by water extract-alcohol precipitation, alcohol extraction, Soxhlet’s reflux extraction, microwave extraction and steam distillation, extraction methods are established and optimized, and the content of the main components is determined.2. The active components of Hottuynia Cordata,in vitro extracts that can effectively inhibit Mycoplasma Gallisepticum was determined by flow cytometry based on well selected fluorescent dye and optimized staining conditions, which was further verified by the traditional paper strip method.3. The model of chicken embryo infected with MG infected chick embryo model was established by inoculating gradient diluted mycoplasma growth medium into the allantoic cavity, and was further verified by clinical observation, histological techniques and pathogen examination.4) The expression of CD4/CD8and cytokines including IL-4, IL-6, IL-12and IFN-y in Mycoplasma infected embryo and Herba Houttuyniae extract treated chicken embryo model was detected by histological technique, quantitative RT-PCR and flow cytometery to evaluate the inhibitory effectiveness, immunological mechanism and possible pathway of Hottuynia Cordata,extract against MG infection. Results1. After comparison study on various extraction techniques including ultrasonic-assisted extraction, Soxhlet’s reflux extraction, water extract and ethanol reflux extraction, water extraction was regarded as the best one to extract the active components of Hottuynia Cordata,, including total polysaccharide, total flavones and volatile oil. The extraction conditions were optimized by orthogonal test, which are lh for infusion,1:30for the ratio of material to solvent,90min for extraction time and3times for extracting repetition. The total polysaccharide yield was2.24%, while the total flavone content was2.54%. Using GC-MS,21classes of volatile oil extracted from Hottuynia Cordata, by Steam distillation method were identified including3alkenes,4aldehydes,2esters,2flavonoids,8alcohols and2acids.2. Three kinds of fluorescent dyes were selected for determining the active components of Hottuynia Cordata, extract inhibiting Mycoplasma Gallisepticumin vivo. By optimizing detection conditions including bacteria medium concentration, fluorescent dye,incubation time and instrument operation, two double staining methods, SYBR/PI and CFDA/PI, were selected for flow cytometry to evaluate traditional Chinese drug sensitivity of inhibiting Mycoplasma Gallisepticum. The optimal working conditions for SYBR/PI double staining were1:500000for SYBR working dilution,1μg.mL-1for PI working concentration,7.4for the pH of PBS as working buffer and10min for incubation time, while1μM for CFDA working concentration,1μg.mL-1for PI working concentration,7.4for the pH of PBS as working buffer and10min for incubation time were the optimal working conditions for CFDA/PI double staining. Absolute counting method was established as well. Using these above methods, volatile oil was identified as the active component of Hottuynia Cordata,inhibiting MGin vitro, which was further verified by the traditional paper strip method.3. It was revealed by clinical, histological and pathogenic inspection that the median dose group of chick embryo model infected by MG underwent relatively depressed activity, low mortality (17%), congestion and mild edema in bronchus tissue on pathological sections, and MG mRNA positive detected by RT-PCR. Therefore, median dose was accepted as the optimal infection dosage for MG infecting chicken embryo model, which is inoculating100μL growth medium (bacteria concentration is108ccumL-1) in each chicken embryo via allantoic cavity.4. Herba Houttuyniae extract1) The lung tissues was infected and destructed by Mycoplasma Gallisepticum, characterized by thickened alveolar space, narrowed alveolar cavity, congestion in alveolar wall, edem in alveolar cavity and inflammatory cell infiltration observed by histopathological inspection. Various degrees of improvement in the lung tissues of chicken embryos treated by Hottuynia Cordata,were observed, which were characterized by structure rehabilitation and few Inflammatory cell infiltration.2) Compared with theinfected group, the level of IL-12mRNA was significantly higher (P<0.05), IFN-y mRNA was higher and IL-4mRNA was lower in the volatile oil treated group. The IFN-y mRNA level was significantly higher (P<0.05) in the spleen of total polysaccharide treated embryos and the IL-12mRNA was also higher but not significantly different (P>0.05). The IL-4mRNA level was higher while the IL-6mRNA level was lower in all three extraction treated groups compared with the infected group, but there was no statistical significance (P>0.05) observed, and the level of IFN-y'IL-12mRNA was not obvious changed. The IL-6mRNA level in all three treated groups was decreased but not significantly different. The IL-4and IL-6mRNA levels were decreased and IFN-y、IL-12mRNA were increased in all three extraction treated groups, compared with the infected control group.3) Compared with the uninfected control group, the proportion of CD4+and CD8+T cells of the mycoplasma-infected control group was significantly lower (P<0.01). The difference between the volatile oil treated group and the mycoplasma-infected control group is significant (P<0.05). The proportion of CD4+and CD8+T cells in other groups was relatively higher, compared with the mycoplasma-infected control group, but was of no statistical significance.Conclusions1) Three active components including total polysaccharide, total flavone and volatile oil, were extracted from Hottuynia Cordata,2) Volatile oil was identified as the active component of Herba Houttuyniae inhibiting Mycoplasma Gallisepticumin vitro by flow cytometry established as a quick drug sensitivity screening method.3) The model of chicken embryo infected with MG was established.4) Volatile oil extracted from Hottuynia Cordatamay improve cell immunity by significantly increase the amount of CD4+T cells, increasing Thl cytokine expression such as IFN-y and IL-12, and decreasing Th2cytokine expression such as IL-4to rebalance Thl/Th2ratio. Total flavone extracted from Hottuynia Cordata may improve cell immunity by regulating Th2reaction, such as decreasing the elevated IL-4and IL-6expression. Polysaccharide extracted from Hottuynia Cordatamay regulate Thl reaction by increasing IFN-y and IL-12expression in spleen. |
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