Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome / 中国实验方剂学杂志

Objective:To determine the therapeutic effect of <italic>in vitro</italic> cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method:BALB/c mice were randomly divided into six groups according to their weight grade： normal group， HCoV-229E infection group， cold and damp group， a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome， and high and low dose group of <italic>in vitro</italic> cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. <italic>In vitro</italic> cultivation of bezoar （0.128，0.064 g·kg^-1） was administrated by gavage for 3 days from the day of infection. The observation indexes included： general state observation of mice， inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the viral load in the lung tissues of mice. Serum levels of motilin（MTL）， gastrin （GAS）， and cytokines interleukin（IL）-10，IL-6， tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）and interferon-<italic>γ</italic>（IFN-<italic>γ</italic>） in lung tissue of mice were determined by enzyme-linked immunosorbent assay（ELISA）. The percentages of CD4⁺ T lymphocytes，CD8⁺ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of <italic>in vitro</italic> cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group， model group mice lung index increased significantly （<italic>P</italic><0.01）， nucleic acids significantly increased expression of lung tissue in mice （<italic>P</italic><0.01）， significantly higher serum MTL content in mice， GAS content significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， lung tissue cells in the immune factor TNF-<italic>α</italic>， IL-10 and IL-6 were significantly increased （<italic>P</italic><0.01）， peripheral blood lymphocyte CD4⁺ T cells in mice， The percentages of CD8⁺ T cells and B cells were significantly decreased （<italic>P</italic><0.01）. Compared with model group， <italic>in vitro</italic> cultivation bezoar mice lung index of high and low dose group were significantly lower （<italic>P</italic><0.01）， the lung tissue of mice express nucleic acid decreased significantly （<italic>P</italic><0.01）， MTL content decreased significantly （<italic>P</italic><0.01）， the lung tissue of mice in the IL-6， IL-10， the TNF-<italic>α</italic>， IFN-<italic>γ</italic> levels were significantly lower （<italic>P</italic><0.01）， <italic>in vitro</italic> cultivation bezoar high dose group can significantly increase the CD4⁺ T cell percentage （<italic>P</italic><0.05）， <italic>in vitro</italic> cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation， pulmonary interstitial edema， as well as blood stasis symptoms. Conclusion:<italic>In vitro</italic> cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice， improving the pathological damage of the lung tissue， adjusting the immune effective and inhibiting the clearing of inflammatory factors， and to provide a laboratory basis for clinical medication.

Study on therapeutic effect of Chaiyin Particles on combining disease with syndrome of human coronavirus pneumonia with pestilence attacking lung syndrome based on regulation of immune function / 中国中药杂志

According to the classification of traditional Chinese medicine syndromes of coronavirus disease 2019 by the national competent authority, this study determined that human coronavirus 229 E(HCoV-229 E) was infected in a mouse model of cold and dampness syndrome, so as to build the human coronavirus pneumonia with pestilence attacking lung syndrome model. The model can simulate the traditional Chinese medicine treatment of common disease syndromes in Coronavirus Disease 2019 Diagnosis and Treatment Program(the sixth edition for trial). Specific steps were as follows. ABALB/c mouse model of cold and dampness syndrome was established, based on which, HCoV-229 E virus was infected; then the experiment was divided into normal control group, infection control group, cold-dampness control group, cold-dampness infection group(the model group), high-dose Chaiyin Particles group(8.8 g·kg~(-1)·d~(-1)), and low-dose Chaiyin Particles group(4.4 g·kg~(-1)·d~(-1)). On the day of infection, Chaiyin Particles was given for three consecutive days. Lung tissues were collected the day after the last dose, and the lung index and inhibition rate were calculated. The nucleic acid of lung tissue was extracted, and the HCoV-229 E virus load was detected by Real-time fluorescent quantitative RT-PCR. Blood leukocytes were separated, and the percentage of T and B lymphocytes was detected by flow cytometry. Lung tissue protein was extracted, and IL-6, IL-10, TNF-α and IFN-γ contents were detected by ELISA. High and low-dose Chaiyin Particles significantly reduced the lung index(P<0.01) of mice of human coronavirus pneumonia with pestilence attacking the lung syndrome, and the inhibition rates were 61.02% and 55.45%, respectively. Compared with the model control group, high and low-dose Chaiyin Particles significantly increased cross blood CD4~+ T lymphocytes, CD8~+T lymphocytes and total B lymphocyte percentage(P<0.05, P<0.01), and reduced IL-10, TNF-α and IFN-γ levels in lungs(P<0.01). In vitro results showed that TC_(50), TC_0, IC_(50) and TI of Chaiyin Particles were 4.46 mg·mL~(-1), 3.13 mg·mL~(-1), 1.12 mg·mL~(-1) and 4. The control group of in vitro culture cells had no HCoV-229 E virus nucleic acid expression. The expression of HCoV-229 E virus nucleic acid in the virus control group was 1.48×10~7 copies/mL, and Chaiyin Particles significantly reduced HCoV-229 E expression at doses of 3.13 and 1.56 mg·mL~(-1), and the expression of HCoV-229 E nucleic acid was 9.47×10~5 and 9.47×10~6 copies/mL, respectively. Chaiyin Particles has a better effect on the mouse model with human coronavirus pneumonia with pestilence attacking the lung syndrome, and could play a role by enhancing immunity, and reducing inflammatory factor expression.

Effect of matrine sodium chloride injection on a mouse model combining disease with syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking the lung / 药学学报

"TCM syndrome of plague attack lung" is a classification of traditional Chinese medicine syndromes of the novel coronavirus pneumonia by the Beijing Municipal Administration of Traditional Chinese Medicine. In this study, a mouse model combining disease with syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking the lung was established for the first time, and the therapeutic effect of matrine sodium chloride injection was evaluated based on immune regulation and inflammatory damage. Lung index, lung index inhibition rate and HE stain were used to evaluate the therapeutic effect of matrine sodium chloride injection on the model mice; the viral load in lung tissue was measured by RT-PCR to evaluate its antiviral effect; the percentage of CD4+ T cells, CD8+ T cells and B cells were detected by flow cytometry to evaluate its immunomodulatory effect; the production of interleukin 6 (IL-6), IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured by ELISA to evaluate its anti-inflammatory effect. All interventions and operations in the experiment were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) and Beijing Experimental Animal Ethics Committee. The results showed that intraperitoneal injection of the high-dose (36.67 mL·kg-1·d-1) and low-dose (18.33 mL·kg-1·d-1) of matrine sodium chloride injection significantly improved the pathological damage of lung tissue and reduced lung index. The lung index inhibition rates were 86.86% and 76.53%, respectively. The production of IL-6, IL-10, TNF-α, IFN-γ, as well as the viral load in lung tissue were reduced significantly compared to the model; the percentage of CD4+ T cells, CD8+ T cells and B cells in peripheral blood were increased compared to the model. These results indicated that the matrine sodium chloride injection has an evident therapeutic effect on the model, and its mechanism was related to the inhibition virus replication, regulation of immunity function and inhibition of inflammatory factor release. This study provided laboratory data support for matrine sodium chloride injection which was used to treat the novel coronavirus pneumonia in clinical in Hubei province. These results indicated that the matrine sodium chloride injection has a good prospect for prevention and treatment of the novel coronavirus pneumonia.

Intervening Effect of Promoting Lung and Resolving Phlegm Method on Mouse Model Combining Disease and Syndrome of Human Coronavirus Pneumonia with Cold-dampness Pestilence Attacking Lung / 中国实验方剂学杂志

Objective:To observe the therapeutic effect of Kesuting syrups and Keqing capsules, which have the function of promoting lung and resolving phlegm, on a mouse model combining disease and syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking lung. Method:The therapeutic effects of Kesuting syrups (the doses of 22, 11 mL·kg-1) and Keqing capsules (the doses of 1.155, 0.577 5 g·kg-1) on this model were evaluated by the inflammatory changes of lung tissue, the expression of viral nucleic acid, the contents of inflammatory factors [interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α and interferon-γ (IFN-γ)], and the percentages of immune cells in peripheral blood (CD4+ T cells, CD8+ T cells and B cells). Result:Compared with the model group, high- and low-dose groups of Keqing capsules and Kesuting syrups could significantly reduce the inflammatory damage in the lung tissues of mice, Keqing capsules could significantly increase the percentages of CD4+ T cells, CD8+ T cells and B cells in peripheral blood, Keqing capsules and Kesuting syrups could reduce the expression levels of IL-6, IL-10, TNF-α and IFN-γ, inhibit the viral load in lung tissue, as well as improve the pathogenic manifestations of lung tissue. Conclusion:As the first-line drugs for novel coronavirus pneumonia, Keqing capsules and Kesuting syrups have significant therapeutic effect on the mouse model combining disease and syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking lung, and the mechanism may be related to regulating immune function and reducing cytokine storm.

Efficacy Evaluation of Jinchai Kangbingdu Capsule Based on Coronaryvirus Pneumonia with 'Hanshi Yidu Xifei' syndrome Model / 中国实验方剂学杂志

Objective:To study the therapeutic effect of Jinchai Kangbingdu capsule based on human coronavirus pneumonia with 'Hanshi Yidu Xifei' syndrome model, in order to provide experimental basis for evaluating its effect in preventing and treating coronavirus infection. Method:The 48 Balb/c mice were randomly divided into normal group, virus infection group, cold-dampness group, cold-dampness epidemic toxin lung syndrome model group, and high and low-dose Jinchai Kangbingdu capsule groups (1.76, 0.88 g·kg-1·d-1). A cold-dampness stimulation combined with human coronavirus 229E infection was used to imitate human coronavirus pneumonia with 'Hanshi Yidu Xifei' syndrome model. Behavioral characteristics, lung index, viral load, and lung tissue pathological changes in Balb/c mice were observed to evaluate the therapeutic effect of Jinchai Kangbingdu capsules. The contents of interleukin-6（IL-6），IL-10，tumor necrosis factor-α（TNF-α），interferon-γ（IFN-γ） in lung tissue and motilin（MTL），gastrin（GAS） in serum were detected by enzyme-linked immunosorbent assay(ELISA), and the contents of CD4+ T cells, CD8 + T cells, and B cells in peripheral blood were detected by flow cytometry. Result:Compared with the cold-dampness epidemic toxin lung syndrome model group, Jinchai Kangbingdu capsule can increase the activity and response ability of 'Hanshi Yidu Xifei' syndrome model mice, and change the skin and stool status of mice. High and low-dose Jinchai Kangbingdu capsule groups can significantly reduce the lung index (P<0.01), while significantly increased the content of CD4+ T cells, CD8+ T cells, and B cells (P<0.05, P<0.01). Low-dose Jinchai Kangbingdu capsule group could significantly decrease the MTL content in serum and the levels of IL-6, IL-10, TNF-α, IFN-γ in lung tissue (P<0.01), whereas alleviate the pathological damage of lung tissue. Conclusion:Jinchai Kangbingdu capsule showed a therapeutic effect on human coronavirus pneumonia with 'Hanshi Yidu Xifei' syndrome model, and can improve the behavioral characterization and gastrointestinal index level of cold-dampness syndrome, while reduce lung index and viral load in lung tissue. The mechanism may be related to the decrease of the content of inflammatory factors and the increase of the number of lymphocytes.