Biological Activity Evaluation Of The Famous Classical Formulae Qing-Jin-Hua-Tan-Decoction

Objective1.Study the anti-inflammatory effect of QJHTT for acute lung injury model rats,and provide experimental evidence for its clinical application;2.Establish a quality control method based on the biological activity of QJHTT,to provide an effective supplement to the quality control.BackgroundIn recent years,because of the changes in the climatic environment,the global respiratory disease has a high incidence.QJHTT was contained in Yixuetonghzi,written by Ye Wenling,a physician in the Ming Dynasty,and is a classical prescription for the treatment of lung disease,this recipe is included in the "Catalogue of Ancient Classical Prescriptions（First Batch）" released by the State Administration of Traditional Chinese Medicine in 2018.QJHTT was composed of eleven medicinal materials.In ancient times,QJHTT was often used in the treatment of lung diseases caused by phlegm heat in the lungs.In modern clinical practice,it is widely used in the AECOPD,CAP,acute and chronic bronchitis,bronchiectasis,and so on.In the treatment of infectious pulmonary diseases,due to its comprehensive effects in the treatment of diseases,such as anti-inflammatory,relaxing tracheal smooth muscle,immunoregulation,and reducing drug resistance,reports on its mechanism and clinical applications have gradually increased in recent years.However,researches about the quality of the original formula and the basic research on the effective material are rare,and due to the difference between the modern preparation process,the raw material medicine and the ancient times,especially the raw material medicine is susceptible to the influence of the natural environment and human factors,which makes the quality consistency of QJHTT is difficult to control,so it is difficult to ensure the consistency of clinical efficacy.Therefore,the research and formulation of scientific and comprehensive quality standards are the main problems in the research and development of QJHTT.In recent years,the biological activity assay developed,and has the advantages of strong specificity,close correlation with the effectiveness,clear dose-effect relationship,simple and fast advantages.Many studies have reported its application in the evaluation of biological effects of traditional Chinese medicine.Therefore,this experiment combines the pharmacological effects and clinical application status of QJHTT,explores the pharmacodynamic effects of QJHTT on acute lung injury model rats;Based on the pharmacological effects of QJHTT on anti-inflammation and immune regulation,established a RAW264.7 macrophage inflammation and phagocytosis model,the effects of QJHTT on the IL-6 content of RAW264.7 secretion by LPS induced and the phagocytic index of neutral red were investigated,to find a biological limits for quality control.Research content1.Anti-inflammatory effect of QJHTT for acute lung injury modelFifty-five male SD rats were divided into 6 groups,control group,model group,QJHTT low,medium,high dose groups,and positive drug group.The doses of each g roup were 9.25 g·kg^-1·d^-1,18.50 g·kg^-1.d^-1,and 37.00 g·kg^-1·d^-1.The positive drug gro up was given dexamethasone methosone sodium phosphate injection（intraperitoneal i njection）,the dosage is 2 mg·kg^-1.After 3 days of adaptive feeding,the rats were injec ted with 1%sodium pentobarbital at a dose of 50 mg·kg^-1,and LPS was instilled into t he trachea of 200μg per mouse（200μg dissolved in 100μL of normal saline）to establi sh an acute lung injury model rat.In the control group,the same amount of normal sal ine was instilled in the trachea by the same method,and the administration was started after the model was established 48h.The dexamethasone group was given a one-time intraperitoneal injection,and the QJHTT group was given by intragastric administrati on for 7 days.The same amount of saline for control group.After 7 days,the rats were anesthetized with 1%sodium pentobarbital at the dose of 50 mg·kg^-1,the pathologica 1 changes of lung tissues were observed by HE staining;the levels of IL-1β,TNF-α in the lung tissue supernatant were detected by ELISA essays;the changes in the copy n umber of MtDNA in the platelets of rats were detected by RT-qPCR essays.2.Biological Evaluation for Quality Control of Water Soluble Extract of QJHTT Based on the phagocytic and secretory functions of macrophagesThe RAW264.7 macrophage phagocytic model and inflammation model were established,and the inhibitory rate of LPS-induced IL-6 secretion and the phagocytic index of neutral red of RAW264.7 macrophages by QJHTT were acted as indicators to investigate the biological activity of QJHTT,fined a limits for quality control.Results1.Anti-inflammatory effect of QJHTT for acute lung injury modelPathological observation of rat lung tissue showed that the lung tissue of the control group was normal,the cell structure was complete,the alveolar cavity was clearly visible,the lung septum was not thickened or inflammatory cell infiltration,and the lung interstitium had no edema.The lung tissue of the model group had obvious pathological damage,pulmonary blood vessels and alveolar cavity,the alveolar septum was obviously thickened,and the pulmonary interstitium was congested and edema.Compared with the model group,the lung tissue pathological damage of the QJHTT is significantly reduced.ELISA test results showed that,the levels of IL-1β and TNF-α in the lung tissues of the model group increased significantly,after administration,the content of IL-1β decreased significantly at the low and high dose of QJHTT compared with the model group,and the dexamethasone group were also significantly reduced;The RT-qPCR experiment results showed that,compared with the control group,the copy number of MtDNA in the platelets of the model group increased significantly,compared with the model group,the number of the MtDNA of the QJHTT middle-dose group,high-dose group and dexamethasone group is significantly reduced.2.Biological Evaluation for Quality Control of Water Soluble Extract of QJHTT Based on the phagocytic and secretory functions of macrophagesThe experiment results showed that the optimal incubation density of macrophages is 3×10⁴ per well,and incubated with the QJHTT for 24h,LPS concentration is 1μg·mL^-1.When the final concentration of QJHTT is 62.5～250μg·mL^-1,it has a significant effect on the proliferation of macrophages.But when the concentration is 500μg·mL^-1,neither toxic effect nor obvious proliferation effect,and at this concentration,QJHTT has a significant inhibitory effect on the IL-6 secretion of RAW264.7 macrophages induced by LPS,the inhibition rate is relatively stable,so 500μg·mL^-1 is selected as the limit concentration,and at this concentration,the phagocytic index of macrophages to neutral red was not less than 113%,the inhibition rate of LPS-induced IL-6 secretion of RAW264.7 macrophages was not less than 45%,and both have good repeatability.Conclusion1.This study confirmed that QJHTT has a significant therapeutic effect on LPS-induced acute lung injury,and can significantly improve lung inflammation.2.Combined with the anti-inflammatory and immunomodulatory pharmacological activities of QJHTT,this study established a biological method for evaluating the quality of QJHTT based on the phagocytic and secretory functions of macrophages,and 500μg·mL^-1 was confirmed as the limit concentration,the phagocytosis index of macrophage is improved,and has significant inhibition rate of IL-6 secretion（P<0.05）,that can be judged qualified.