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ObjectiveTo characterize the efficacy components of Guizhi Jia Gegentang（GGT） in intervening influenza virus pneumonia by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）. MethodBALB/c mice were randomly divided into normal group and GGT group（36 g·kg-1·d-1） with six mice in each group. GGT group was continuously administered GGT extract for 5 d， while the normal group was administered an equal amount of ultrapure water. Serum and lung tissue were collected after administration， and UPLC-Q-Exactive Orbitrap MS was used to characterize the prototypical and metabolic components of GGT in serum and lung tissue of mice. The components existed simultaneously in the serum and lung tissue of mice from the GGT group were defined as its functional components， and the targets of efficacy components were searched by SwissTargetPrediction database， and GeneCards database was used to query the target of influenza virus pneumonia， and then the intersection was taken to obtain potential targets of GGT for intervening in the disease. Protein-protein interaction（PPI） network analysis of potential targets was performed by STRING database， and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis on potential targets was performed by Metascape. ResultA total of 29 prototypical components and 28 metabolic components of GGT were detected in the drug-containing serum of mice， of which 11 prototypical components and 4 metabolic components were detected in the lung tissue of mice. The main metabolic pathways included reduction， hydroxylation， methylation， glucuronidation and sulfation. The results of PPI network and KEGG analysis showed that these functional components may act through their effects on targets such as albumin（ALB）， epidermal growth factor receptor（EGFR）， steroid receptor coactivator（SRC）， Toll-like receptor 4（TLR4）， nuclear transcription factor（NF）-κB and adhesion junction. ConclusionThe 11 prototypical components and 4 metabolites present simultaneously in the drug-containing serum and lung tissue of mice may be the potential therapeutic components of GGT in interfering with influenza viral pneumonia， and act through interfering with inflammatory metabolic pathways. This study can provide a reference for the mechanism study of GGT in the treatment of influenza viral pneumonia.
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OBJECTIVE To study the toxic mechanism of Mahuang xixin fuzi decoction （MXF） on normal mice. METHODS Totally 48 SPF grade BABL/C mice were randomly divided into blank group， MXF low-dose， medium-dose and high-dose groups， with 12 mice in each group. MXF low-dose， medium-dose and high-dose groups were given drug intragastrically at the dose of 11.262， 33.786， 45.050 g/kg， respectively. Blank group was administered with equal volume of normal saline， once a day， for consecutive 7 d. The body weight， anal temperature and survival rate were recorded， organ index and serum biochemical factors were detected. After the last administration， fecal samples of mice were collected and detected by UHPLC-QE/MS. RESULTS Compared with blank group， the body weight was decreased significantly from the 3rd to the 5th day after administration in MXF medium-dose group， and from the 2nd to the 7th day after administration in MXF high-dose group significantly （P＜0.05）. There was no significant difference in anal temperature among the treatment groups； the average survival rates of MXF medium-dose and high-dose groups were 58.33% and 50.00%， respectively. Compared with blank group， there were significant difference in the indexes of spleen， lung， thymus， adrenal gland and creatine kinase in MXF low-dose， medium-dose and high-dose groups， the testis index in MXF low-dose and high-dose groups， the creatine kinase isoenzyme/creatine kinase ratio in MXF low-dose group， the α-hydroxybutyrate dehydrogenase， lactate dehydrogenase and alkaline phosphatase in MXF medium-dose group， the urine and cystatin C in MXF medium-dose and high-dose groups （P＜0.05）. The fecal metabonomic analysis showed that 19 biomarkers such as phenylpyruvate， L-tyrosine， phosphatidylcholine， glycerol 3-phosphate in MXF low-dose， medium-dose and high-dose groups were significantly different from those in the blank group. CONCLUSIONS When MXF reaches a certain dose， it will have adverse effects on the body weight， multiple organs and serum biochemical indicators of mice， thus showing a certain toxic effect. Its mechanism may be related to disrupting the intestinal flora metabolism， causing inflammatory reaction and immune disorders.
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OBJECTIVE：To study the effects of Mahuang xixin fuzi decoction on Toll-like receptors （TLRs）response and cytochrome C oxidase （Cyt-CO）-mediated apoptosis regulation in mice with influenza disease of kidney-yang deficiency. METHODS：Totally 48 male Balb/c mice were randomly divided into normal group （n＝12）and modeling group （n＝36）. The modeling group was intraperitoneally injected with estradiol benzoate solution （8 mg/kg）and intranasally injected with influenza virus H 1N1（20 μL/mice）to establish the influenza disease compound model of kidney-yang deficiency. After modeling ，the mice were randomly divided into model group ，positive drug group （Oseltamivir phosphate capsules ，0.195 g/kg），Mahuang xixin fuzi decoction group （1.802 g/kg，by crude dru g），with 12 mice in each group. Each group was given relevant medicine intragastrically，normal group and model group were given， corresponding volume of normal saline intragastrically 20 mL/kg，once a day ，for consecutive 6 days. During admi-nistration，body weight and anal temperature of mice were mail：xsy407861520@163.com measured daily ；the percentage of initial body weight was calculated. After last medication ，the organ （spleen，thymus and lung ）indexes were calculated ；the pathological changes of lung tissue were observed. The viral load of influenza A virus H 1N1 in lung tissue was detected （reflected by M gene mRNA expression）；mRNA expressions of TLR3，TLR7，myeloid differentiation factor （MyD88）and Caspase- 3 in cardiac tissue as well as the activity of Cyt-CO and the content of cytochrome C （Cyt-C）were also determined. RESULTS ：Compared with normal group，initial body weight percentage and anal temperature of the model group continued to decrease （P＜0.05）；the spleen and thymus indexes were decreased significantly （P＜0.05），while lung index was increased significantly （P＜0.05）；the lung tissue lesions were serious. Viral load in lung tissue ，mRNA expressions of TLR 3，TLR7，MyD88 and Caspase- 3 in cardiac tissue as well as the content of Cyt-C were increased significantly （P＜0.05 or P＜0.01），while the activity of Cyt-CO in cardiac tissue was significantly decreased （P＜0.01）. Compared with model group ，initial body weight percentage and anal temperature of mice in Mahuang xixin fuzi decoction group showed an increasing trend from the fourth day of administration （P＜0.05 or P＜0.01）. The spleen and thymus indexes were increased significantly （P＜0.05），while the lung index was significantly decreased （P＜0.05）；the pathological injury of lung tissue was significantly improved ；viral load in lung tissue ，mRNA expressions of TLR 3 and Caspase- 3 as well as the content of Cyt-C in cardiac tissue were decreased significantly （P＜0.05 or P＜0.01），while the activity of Cyt-CO was increased significantly in cardiac tissue （P＜0.01）. CONCLUSIONS ：Mahuang xixin fuzi decoction can improve influenza disease of kidney-yang deficiency in mice ，the effect may related to inhibit TLRs response and apoptosis regulation pathway mediated by Cyt-CO.
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ObjectiveComparingfourgroupsofimmunosuppressivemousemodelsestablishedby cyclophosphamide administration in different doses and periods , we used partial least squares ( PLS) analysis to evaluate the immunosuppressive mouse models comprehensively and select the appropriate way to establish this model .Methods In this experimental study, 58 male KM mice were randomly divided into five groups: normal group (10 mice) were given normal saline daily by i.p.injection, model group 1 (12 mice) was given 40 mg/kg CTX daily by i.p.injection for 10 days, model group 2 (12 mice) was given 80 mg/kg CTX daily by i.p.injection for 3 days, model group 3 (12 mice) was given 40 mg/kg CTX daily by i.p.injection twice for a week, model group 4 (12 mice) was given 50 mg/kg CTX daily by i.p.injection for 7 days.After the injection of cyclophosphamide , the daily metabolic activities were detected everyday such as body weight , water intake , and food intake , organ index and immunological indexes such as blood RT of the model mice were measured as well .Using partial least squares ( PLS) analysis to the models and analyzing the final weight , final anal temperature , organ index , and blood routine examination in order to evaluate the immunosuppressive mouse models comprehensively .Results Compared with the normal group , different dosages of CTX reduced the weight and anal temperature of model mice (P<0.05), the food intake and water intake (P<0.01), and the spleen index and thymus index ( P<0.01 ) .Besides , the amount and percentage of basophilic granulocytes decreased ( P <0.05 ) , and the percentage of MCHC , macrophage went up , as well as the absolute value of WBC and lymphocytes were decreased in the model groups 1, 2, and 4 (P<0.05).According to the PLS analysis, there were significant differences among models 1, 2, and 4 when compared with the normal group , especially the most different in the model group 1.Conclusions After the PLS analysis of different indexes , the optimal way to establish immunosuppressive mouse models is the procedure with 40 mg/kg CTX daily injected i .p.for ten days .Our findings provide experimental evidence for the establishment of immunosuppressive mouse models .