Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-induced ferroptosis / 中国天然药物

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide. Influenza A virus (IAV) has been found to activate multiple programmed cell death pathways, including ferroptosis. Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation, leading to cell death. However, little is known about how influenza viruses induce ferroptosis in the host cells. In this study, based on network pharmacology, we predicted the mechanism of action of Maxing Shigan decoction (MXSGD) in IAV-induced ferroptosis, and found that this process was related to biological processes, cellular components, molecular function and multiple signaling pathways, where the hypoxia inducible factor-1(HIF-1) signaling pathway plays a significant role. Subsequently, we constructed the mouse lung epithelial (MLE-12) cell model by IAV-infected in vitro cell experiments, and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage, increased reactive oxygen species (ROS) release, increased total iron and iron ion contents, decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4 (GPX4), increased expression of acyl-CoA synthetase long chain family member 4 (ACSL4), and enhanced activation of hypoxia inducible factor-1α (HIF-1α), induced nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in the HIF-1 signaling pathway. Treatment with MXSGD effectively reduced intracellular viral load, while reducing ROS, total iron and ferrous ion contents, repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway. Finally, based on animal experiments, it was found that MXSGD effectively alleviated pulmonary congestion, edema and inflammation in IAV-infected mice, and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

Connotation of Xiao Chaihu Decoction combined with Maxing Shigan Decoction based on severe cases and modern pathophysiological mechanism and application for severe pulmonary infection and acute exacerbation of chronic obstructive pulmonary disease in critical care medicine / 中国中药杂志

Xiao Chaihu Decoction combined with Maxing Shigan Decoction is a classic herbal formula. All of them are derived from Treatise on Cold Damage(Shang Han Lun) by ZHANG Zhong-jing. This combination has the effects of harmonizing lesser yang, relieving exterior syndrome, clearing lung heat, and relieving panting. It is mainly used for treating the disease involving the triple-Yang combination of diseases and accumulation of pathogenic heat in the lung. Xiao Chaihu Decoction combined with Maxing Shigan Decoction is a classic combination for the treatment of exogenous diseases involving the triple-Yang combination. They are commonly used in exogenous diseases, especially in the north of China. This combination is also the main treatment strategy for coronavirus disease 2019(COVID-19) accompanied by fever and cough. Maxing Shigan Decoction is a classical herbal formula for treating the syndrome of phlegm-heat obstructing the lung. "Dyspnea after sweating" suggests the accumulation of pathogenic heat in the lung. Patients with mild symptoms may develop cough and asthma along with forehead sweating, and those in critical severe may develop whole-body sweating, especially the front chest. Modern medicine believes that the above situation is related to lung infection. "Mild fever" refers to syndromes rather than pathogenesis. It does not mean that the heat syndrome is not heavy, instead, it suggests that severe heat and inflammation have occurred. The indications of Xiao Chaihu Decoction combined with Maxing Shigan Decoction are as follows.(1) In terms of diseases, it is suitable for the treatment of viral pneumonia, bronchopneumonia, lobar pneumonia, mycoplasma pneumonia, COVID-19 infection, measles with pneumonia, severe acute respiratory syndrome(SARS), avian influenza, H1N1 influenza, chronic obstructive pulmonary disease with acute exacerbation, pertussis, and other influenza and pneumonia.(2) In terms of syndromes, it can be used for the syndromes of bitter mouth, dry pharynx, vertigo, loss of appetite, vexation, vomiting, and fullness and discomfort in the chest and hypochondrium. It can also be used to treat alternate attacks of chill and fever and different degrees of fever, as well as chest tightness, cough, asthma, expectoration, dry mouth, wanting cold drinks, feeling agitated, sweating, yellow urine, dry stool, red tongue, yellow or white fur, and floating, smooth, and powerful pulse, especially the right wrist pulse.

Separation, characterization, and antiviral activity of colloidal phase state of Maxing Shigan Decoction / 中国中药杂志

This study focused on the separation, characterization, content determination, and antiviral efficacy research on colloidal particles with different sizes in Maxing Shigan Decoction(MXSG). The mixed colloidal phase of MXSG was initially separated into small colloidal particle segment(S), medium colloidal particle segment(M), and big colloidal particle segment(B) using ultrafiltration. Further fine separation was performed using size-exclusion chromatography. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were employed to characterize the size and morphology of the separated colloidal particles. UPLC-MS/MS was used to determine the content of ephedrine, amygdalin, glycyrrhizic acid, and the EDTA complexometric titration was used to measure the calcium(Ca~(2+)) content in different colloidal phases. Finally, a respiratory syncytial virus(RSV) infection mouse model was established using intranasal administration. The experimental groups included a blank group, a model group, a ribavirin group, an MXSG group, an S group, an M group, and a B group. Oral administration was given for treatment, and pathological changes in mouse lung tissue and organ indices were evaluated. The results of the study showed that the distribution of ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) content was not uniform among different colloidal segments. Among them, the B segment had the highest proportions of the three components, except for Ca~(2+), accounting for 46.35%, 53.72%, and 92.36%, respectively. Size-exclusion chromatography separated colloidal particles with uniform morphology in the size range of 100-500 nm. Compared to the S and M segments, the B segment showed an increased lung index inhibition rate(38.31%), spleen index, and thymus index in RSV-infected mice, and it improved the infiltration of inflammatory cells and lung injury in the lung tissue of mice. The complex components in MXSG form colloidal particles of various sizes and morphologies through heating, and small-molecule active components such as ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) participate in the assembly to varying degrees. The main material basis for the antiviral effect of MXSG is the colloidal particles with certain particle sizes formed by the assembly of active components during the heating process.

Systematic review of randomized controlled trial of Maxing Shigan Decoction in treatment of community acquired pneumonia / 中国中药杂志

To systemically evaluate the efficacy and safety of Maxing Shigan Decoction in the treatment of community acquired pneumonia(CAP) and provide a reference for the treatment of CAP. Databases of CNKI, Wanfang, VIP, SinoMed, EMbase, Cochrane Library, Web of Science and PubMed were searched(from inception to May 30, 2020) to screen the randomized controlled trials(RCTs) of Maxing Shigan Decoction in treating CAP. Two authors independently screened and selected relevant literature and extracted data based on the inclusion and exclusion criteria. Any disagreement or differences was resolved through discussion. The bias risk assessment tool recommended by Cochrane handbook was used to evaluate the quality of the included studies, and RevMan 5.3 software was used for data analysis. Seventeen RCTs were finally included, involving 1 309 patients. Meta-analysis showed that Maxing Shigan Decoction combined with conventional Western medicine treatment could improve clinical efficacy in patients with CAP more effectively as compared with conventional Western medicine treatment alone, mainly in terms of anti-inflammatory effects, a decrease in C-reactive protein(CRP) content(MD=-6.01, 95%CI[-10.95,-1.06], P=0.02)and white blood cell(WBC) count, a decrease in procalcitonin(PCT) level(MD=-0.74, 95%CI[-0.77,-0.71], P<0.000 1), and shortened recovery time of cough and fever. Maxing Shigan Decoction has certain curative effect on CAP, but there are problems in the methodology of included studies. High-quality stu-dies are still needed for further verification.

Effect of type A influenza virus on autophagy of lung macrophages and intervention of serum of Maxing Shigan decoction / 中国药理学通报

Aim: To study the effect of influenza A virus on autophagy in lung macrophages and the intervention effect of Maxing Shigan decoction. Methods: Influenza A virus infected RAW264. 7 cells as the research object, the experiment set up Maxing Shigan decoction containing serum low, medium and high doses groups, oseltamivir group, 3-MA + influenza virus group, influenza virus group, 3-MA group, rapamycin group and control group. After 12 hours of intervention, the treated cells were examined as follows; (1) the autophagy of each group was detected by laser confocal microscopy; (2) the autophagy and distribution of virus particles were detected by transmission electron microscopy; (3) the expressions of autophagy marker proteins LC3-1 and LC3-II were detected by Western blot. Results: The serum containing Maxing Shigan decoction could inhibit the membrane aggregation of autophagy marker protein LC3 induced by influenza virus in varying degrees, the increase of autophagosome and autophagy lysosome induced by influenza virus, the expression of LC3-II and the ratio of LC3-U to LC3-1 to some extent, and showed a dose-effect relationship (positive correlation). Conclusion Maxing Shigan decoction may regulate the pathogenesis of influenza A virus by inhibiting autophagy induced by influenza A virus.

Effects of Maxing-Shigan decoction on airway remodeling and expression of MMP-9 and TIMP-1 in lung tissues of asthmatic mice / 中国病理生理杂志

AIM:To investigate the effects of Maxing-Shigan decoction on airway remodeling and expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissues of asthmatic mice, and to explore its possible mechanism in treatment of asthma.METHODS:The BALB/c mice were divided into blank control group, model group, low-dose Maxing-Shigan decoction group, middle-dose Maxing-Shigan decoction group, high-dose Maxing-Shigan decoction group and positive control group.The mice were sensitized and challenged with ovalbumin to establish asthma model.The mice in blank control group and model group were given saline by oral administration before 30 min of suscitation.The mice in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups were given Maxing-Shigan decoction at 5.0 g/kg, 10.0 g/kg and 20.0 g/kg, respectively, by oral administration before 30 min of suscitation.The mice in positive control group was given dexamethasone at 0.005 g/kg by oral administration before 30 min of suscitation.After consecutive administration for 7 d, the variations of airway responsiveness, the percentage of the goblet cells, the collagen deposition, and the eosinophil (EOS) counts in bronchoalveolar lavage fluid (BALF) of each group were observed.The protein levels of MMP-9 and TIMP-1 in the lung tissues were determined by ELISA and Western blot.The mRNA expression of MMP-9 and TIMP-1 was detected by RT-qPCR.RESULTS:Compared with blank control group, the airway responsiveness, the goblet cell percentage, the collagen deposition, the EOS counts in BALF, the protein levels of MMP-9 and TIMP-1, and the mRNA expression of MMP-9 and TIMP-1 were significantly increased in model group (P<0.01).Compared with model group, all of the indexes were reversed in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups and positive control group (P<0.05 or P<0.01).CONCLUSION:Maxing-Shigan decoction improves airway remodeling in asthma model mice by down-regulating the expression of MMP-9 and TIMP-1.

Effect of Maxing Shigan Decoction against type A influenza virus infection in mice induced by viral lung injury based on TLR4-MyD88-TRAF6 signal pathways / 中草药

Objective: To explore the effect of TLR4 on type A influenza virus induced lung tissue injury, and to further explore the intervention effect of Maxing Shigan Decoction (MXSGD). Methods: The WT influenza virus model in mice infected with influenza A virus was established. The mice were devided into low, medium, and high dose MXSGD groups, oseltamivir group, IAV group, and Sham group. After 7 d, the treated animals were treated with corresponding clinical equivalent dose of drug. Detection in mouse body weight, lung index, spleen index, thymus index, pathological changes of lung, and ELISA method for the detection of alveolar irrigation lotion in inflammatory cytokines (TNF-α, IL-1β, and IL-6) content, real-time PCR and Western blotting detection of lung tissue TLR4, MyD88, TRAF6 mRNA, and protein expression. Results: MXSGD could up-regulate the weight, spleen index, and thymus index of type A influenza virus infection in WT mice, improve the pathological injury of lung tissue, decrease alveolar lavage lotion proinflammatory cytokines content, and down regulate the expression of MyD88 and TRAF6 mRNA and protein in lung index and lung tissue and there is a certain dose effect relationship. The effect of high dose MXSGD group was close to oseltamivir. Conclusion: MXSGD as effective anti influenza virus of traditional Chinese medicine compound can effectively reduce lung inflammation, protect immune organs, and regulate cytokine balance. The possible mechanism is alleviating the lung injury caused by type A influenza virus infection in mice through inhibition TLR4-MyD88-TRAF6 signaling pathway of activation.

The New Explanation of“Without High Fever”in ShangHanLun / 浙江中医药大学学报

Objective]To clarify the meaning of the phase“without high fever”in ShangHan Lun. [Method]To sort out the explanation of “without high fever”of interpreters from past to nowadays,then to analyse the scriptures of ShangHanLun and Sypnosis of Golden Chamber that involve“without high fever”,to re-define the exact meaning of the phase. [Result] Two main opinions are found about the explanation of“without high fever”in the past literature:one is “heat is in interior not exterior”,while the other is“exterior syndrome is passed”,Both are not accorded with Zhong Jing’s word expression and the real clinical situation. If “high fever”is explained as“the fever characteristics of Yang-ming disease”,the scripture involved and the meaning of“without high fever”can be thoroughly understood.[Conclusion] “Without high fever”is the phase Zhang-zhong-jing used to differentiate the disease in scripture from Yang-ming visceral substantial syndrome .Clarifying the phase“without high fever”helps us to better understand the meaning of related scriptures.

Different Effects of Mahuang Decoction and Maxing Shigan Decoction on Animal Temperature Tropism and Correlation to Differences of Cold and Hot Nature of Chinese Materia Medica / 中草药·英文版

Objective To establish an objective method for evaluating the intrinsic characteristics between cold and hot nature of Chinese materia medica(CMM)through the different effects of Mahuang decoction(MHD)and Maxing Shigan decoction(MSD)on animal temperature tropism.Methods The equipment with cold/hot pads was used to investigate the variety ofthe temperature tropism between two groups of mice treated by MHD and MSD,respectively.Meanwhile,the activities ofadenosine triphosphatase(ATPase),superoxide dismutase,succinate dehydrogenase,and malondialdehyde were measured.Results After treated by MHD,the macroscopic behavioral index of remaining rate on warm pad(40 ℃)of mice decreasedsignificantly(P < 0.05),suggesting the enhancement of cold tropism,meanwhile,the internal indices of ATPase activity and oxygen consuming volume increased significantly(P < 0.05),suggesting the enhancement of energy metabolism.On theother hand,the above-mentioned indices in MSD group changed on the inverse way.Conclusion The relative drug natureof MHD and MSD revealed in this study is consistent with the theoretical prognostication or definition.It indicates that theinternal cold and hot nature of CMM could be reflected in ethological way on the changes of animal temperature tropismwhich might be internally regulated by body energy metabolism.

Effect of Maxing Shigan Decoction on degranulation of mast cell / 中成药

AIM:To investigate the mechanism of Maxing Shigan Decoction(Herba ephedrae,Semen armeniacae amarum,Radix er Rhizoma glycyrrhizae,Gypsum fibrosum) against type Ⅰ hypersenstivity reaction. METHODS: The experiment of passive cutaneous anaphylaxis(PCA) was used to determine the effect of Maxing Shigan Decoction on degranulation of mast cells in vivo.For in vitro study,the drug serum of Maxing Shigan Decoction was added to the culture medium of sensitized RBL-2H3 cells.The antigen-induced release of degranulation was measured by enzymatic assay,histamine by enzyme immunoassay(EIA) and cytokines by enzyme-linked immunosorbent assay(ELISA). RESULTS: The results showed that treatment with drug serum of Maxing Shigan Decoction was followed by a decrease in PCA of rats and in degranulation,histamine,TNF-? and IL-4 from RBL-2H3 cells induced by antigen. CONCLUSION: Maxing Shigan Decoction may suppress the type Ⅰ hypersensitivity reaction by inhibiting the action of mast cells.