Objective:To invastigate the targets and signaling pathways of Gubiao Fanggan Modified Formula in regulating the defense against influenza A virus in immunosuppressed mice by network pharmacology,and the key targets were verified by immuno-suppressive mice model.Methods:TCMSP database was used to search the active ingredients of Gubiao Fanggan Modified Formula,and GeneCard,OMIM,PharmGkb databases were used to obtain the potential targets of the active ingredients to prevent influenza,and take their intersection targets were taken;STRING11.5 database was used to make protein-protein interaction network analyzed and finded the core targets;Cytoscape3.8.1 was used to build a traditional Chinese medicine-ingredient-disease target network,and GO enrichment analysis and KEGG enrichment analysis were performed.Intraperitoneal injection of cyclophosphamide was used to construct a mouse model of immune function suppression,normal group,model control group,Gubiao Fanggan Modified Formula group and oseltamivir group were set up,followed by prophylactic administration,and influenza virus intervention was performed on the fourth day.After 7 days of intragastric administration,the key targets were verified by mouse spleen index,HE staining,RT-qPCR and immunohistochemistry.Results:There were 82 active ingredients in five traditional Chinese medicines in Gubiao Fanggan Modi-fied Formula,and 72 common targets of drugs and diseases such as IL-6,TNF-α,IL-2,etc,mainly involving IL-17,TNF and AGE-RAGE signaling pathway.Gubiao Fanggan Modified Formula could increase spleen index and significantly reduce mRNA and protein expressions of IL-6 and TNF-α in spleen tissue of mice(P<0.05 or P<0.01).Conclusion:Gubiao Fanggan Modified Formula may regulate body's immune function through targets such as IL-6 and TNF-α,thereby preventing influenza virus infection.

Objective To compare the anti-inflammatory,antitumor and anti-bacterial effects of the single extract(in granules)and the prepared drug in pieces of Forsythia Suspense(Lianqiao,a traditional Chinese herbal medicine).Methods In zebrafish embryo models of CuSO4 exposure,tail transection and LPS microinjection-induced inflammation,the anti-inflammatory effects of 10 μg/mL DEX,single extract of Forsythia Suspense,and the water extract of the prepared drug(400,600,and 800 μg/mL)were evaluated by observing neutrophil counts,RT-qPCR,HE staining and survival analysis.Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining.The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs.Results In the zebrafish embryo models of inflammation,both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation,reduced the mRNA expressions of TNF-α,IL-6,P38,Jnk,Erk and P65,and increased the survival rate of zebrafish.They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells(HCT116),pancreas adenocarcinoma cells(PANC-1),lung cancer cells(A549),liver cancer cells(Hep3B)and cervical carcinoma cells(Hela)in zebrafish embryos,and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL.Conclusion The two dosage forms of Forsythia Suspense have similar anti-inflammatory,antitumor and antibacterial effects,but their effects for inhibiting IL-6,P65,and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

Objective To compare the anti-inflammatory,antitumor and anti-bacterial effects of the single extract(in granules)and the prepared drug in pieces of Forsythia Suspense(Lianqiao,a traditional Chinese herbal medicine).Methods In zebrafish embryo models of CuSO4 exposure,tail transection and LPS microinjection-induced inflammation,the anti-inflammatory effects of 10 μg/mL DEX,single extract of Forsythia Suspense,and the water extract of the prepared drug(400,600,and 800 μg/mL)were evaluated by observing neutrophil counts,RT-qPCR,HE staining and survival analysis.Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining.The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs.Results In the zebrafish embryo models of inflammation,both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation,reduced the mRNA expressions of TNF-α,IL-6,P38,Jnk,Erk and P65,and increased the survival rate of zebrafish.They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells(HCT116),pancreas adenocarcinoma cells(PANC-1),lung cancer cells(A549),liver cancer cells(Hep3B)and cervical carcinoma cells(Hela)in zebrafish embryos,and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL.Conclusion The two dosage forms of Forsythia Suspense have similar anti-inflammatory,antitumor and antibacterial effects,but their effects for inhibiting IL-6,P65,and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

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.
Animals ; Mice ; Ferroptosis ; Network Pharmacology ; Reactive Oxygen Species ; Vascular Endothelial Growth Factor A ; Influenza A virus ; Iron ; Hypoxia

OBJECTIVE To sort out the chemical drugs for children in China's over-the-counter(OTC) drugs catalogue and analyze the drug categories and the specification of the instructions, so as to find out the key points that lead to the risk of drug use, and put forward suggestions for improvement. METHODS According to the sample of OTC drugs instructions published on the website of the National Medical Products Administration, as well as the results of clinical expert investigation and literature, the OTC drugs catalog of children's chemical drugs was determined and classified, and the investigate the standardization and consistency of the instructions. RESULTS There were 267 kinds of OTC drugs for children sorted out, involving 18 kinds of diseases. A considerable proportion of the instructions were incomplete and nonstandard, and there were few kinds of drugs for respiratory and digestive diseases. CONCLUSION The preparation and publicity of children OTC drugs instructions should be standard, the research and development of children's OTC drugs should be strengthened, the training of pharmacy employees should be strengthened, and ensure the rational and safe use of children's OTC drugs.

OBJECTIVE：To standardize the use of children’s over-the-counter（OTC）drugs in China，control the risk of children’s self medication and ensure the safety of children’s medication. METHODS：The questionnaire was randomly distributed to pharmacy practitioners in retail pharmacies in 13/parents in 16 provinces and municipalities directly under the central government. The results of questionnaire survey were analyzed descriptively. The corresponding suggestions were put forward for the problems existing in the use of OTC drugs for children among pharmacy practitioners in retail pharmacies and parents.RESULTS：Totally 5 367 people filled the questionnaire at pharmacies，and 5 353 questionnaires were filled validly with effective rate of 99.74％；the number of consumers（parents）who filled the questionnaires was 6 111，and 5 094 questionnaires were filled validly with effective rate of 83.36％. The results of survey showed that 93.67％ of retail pharmacies were equipped with 1-2licensed pharmacists；most of the employees in pharmacies had college degree（46.59％），and their professional knowledge was relatively weak，and only 30.00％ of them were very familiar with pharmaceutical professional knowledge. Pharmacy practitioners could provide basic pharmaceutical care，but they were not sensitive to the information of children’s age and weight；only 44.11％of the practitioners would approve the dosage according to the age and weight of the children；98.09％ of pharmacies would regularly conduct OTC drugs knowledge training，but they did not pay attention to the knowledge of children related to drug use. About half of the pharmacy practitioners were very clear about the indications，usage and dosage，precautions of OTC drugs forchildren. About 70％ of parents did not fully understand the difference between prescription drugs，green OTC drugs and red OTC drugs，and did not know the risk of home OTC treatment；63.06％ of parents chose OTC drugs according to their previous experience， and 23.05％ of parents relied on drug advertisements to choose OTC drugs； 92.64％ of parents would read the drug instruction carefully before using OTC drugs， but they had blind spots in understanding many contents of the drug instruction. In the process of OTC drug use，46.21％ of parents had used adult OTC drugs for their children，and 41.54％ of parents did not convert the amount of children；52.89％ of parents said that their children suffered from adverse reactions after taking OTC drugs. CONCLUSIONS：The pharmacy practitioners have low education on the whole，professional level needs to be improved，their understanding of children’s OTC drugs is not comprehensive enough，and there is a lack oftraining on children’s medication knowledge. Parents have a poor awareness of children’s OTC drugs，and there are many problems in the process of using children’s OTC drugs，such as inaccurate dosage，using adult’s drugs. It is suggested that we should improve the access standard of pharmacy practitioners and increase the number of licensed pharmacists；organize special training on safe drug use for children to improve the pharmaceutical service ability of pharmacies；strengthen the supervision of retail pharmacies and formulate pharmaceutical care standards for the use of OTC drugs for children；strengthen the publicity and education on rational drug use for children and popularize the knowledge of safe drug use of OTC drugs for children；give full play to the guiding role of pharmacists and pay attention to the medication guidance of parents.

Chloroquine and hydroxychloroquine were primarily used to treat malaria. With continuous research on their mechanism of action, their application range has gradually expanded to treat serious infectious diseases, rheumatic immune diseases, tumors, etc. Chloroquine and hydroxychloroquine are the two most attractive drugs developed in the past 50 years, and their efficacy in the treatment of various non-malaria diseases has been increasingly recognized by researchers. The results of in-depth research show that chloroquine and hydroxychloroquine have antiviral effects against coronavirus, influenza virus and Ebola virus, have significant clinical efficacy in the treatment of rheumatoid diseases such as systemic lupus erythematosus and rheumatoid arthritis, and can enhance the drug sensitivity of tumor cells by combining with classic anticancer drugs. The main adverse reaction of hydroxychloroquine is eye damage, especially retinopathy. Therefore, patients treated with this drug should undergo regular eye examinations. In this review paper, the chemical structure, pharmacokinetics, application status and pharmaceutical care of chloroquine and hydroxychloroquine were summarized. This paper looks forward to providing help for the rational use of these two drugs in the future.

Objective To construct the Hut78 cell line with EZH2 gene knocked into by CRISPR/Cas9 system. Methods The EZH2 expression vector pMD-18T-EZH2 with homologous arm and the sgRNA expression vector pSpCas9 (BB)-2A-Puro-sgRNA, which could cut the double stranded genomic DNA, were constructed, and the two vectors were co-transfected into Hut78 cells. Then the expression of EZH2 mRNA was detected by qPCR, and the expressions of EZH2 and H3K27me3 proteins were detected by Western blot assay. Results The pMD-18T-EZH2 and pSpCas9(BB)-2A-Puro-sgRNA recombinant vectors were confirmed by DNA sequencing. When Hut78 cells were transfected with the two recombinant plasmid, qPCR results showed that the expression of EZH2 mRNA was significantly increased, and Western blot analysis showed that the expressions of EZH2 and H3K27me3 proteins were significantly increased. Conclusion EZH2 gene is successfully knocked into Hut78 cells by CRISPR/Cas9 system.

Objective To evaluate the tonic and health effects of Xuebijing injection,a traditional Chinese medicine injection,by means of corn low-nutritional mice model combined with benefit-damage index-general score (BDI-GS) evaluation system under a physiological dosage.Further,to investigate the scientific and practical benefits of the evaluation system in efficacy and safety re-evaluation of clinical traditional Chinese medicine injections.Methods Healthy ICR mice during growth period were randomly divided into blank control group and low,middle and high dose groups.All the mice were fed with corn diets,and treated by intraperitoneal injection once a day.The mice in the blank control group were injected with 0.4 ml 0.9％ sodium chloride injection,and the mice in the low,medium and high dose groups were injected with 0.2,0.4 and 0.6 ml Xuebijing injection,respectively,for seven days.On the 8th day,the mice were killed,dissected,and took nine kinds of organs including heart,lung,thymus,spleen,pancreas,kidney,gonad and femur.The weight BDI,index BDI and the corresponding weight BDI general score (GSw) and index BDI general score (GSI) were calculated,and the serum biochemical parameters were measured.Results Under the physiological dose,Xuebijing injection shows good safety of continuous infusion,tonic and health effects,and slight damages on the rest of organs.More specifically,the values of weight BDI and index BDI were all higher than 9.0,and values of GSw and GSI were all higher than the basic value of 9.0.Furthermore,these parameters increased in a dose-dependent manner.In addition,the total protein level of (47.6 ± 4.30) g/L in the high dose groups was significantly higher than (43.18 ± 2.60) g/L in the blank control group (P＜0.05).Conclusions The Xuebijing injection has a lower risk for conventional usage.This injection is safe under the physiological dosage and much safer in higher dosage designed in this study.The low-nutritional model combined with BDI-GS evaluation system can be used for re-evaluating the function and safety of clinical traditional Chinese medicine injections.

Objective To study the pharmacokinetic status of Cyclophosphamide (CYC) in children with lupus nephritis (LN),as well as the relationship between the pharmacokinetic results and clinical indicators,adverse reactions,curative effect evaluation.Methods Thirty patients hospitalized at Tianjin Children's Hospital from January 2014 to December 2016 were treated with glucocorticoid (GC) combined with CYC.Blood samples were collected in 6 point-in-time after intravenous CYC,a high performance liquid chromatography (HPLC) method was used to detect the blood drug concentration,and the pharmacokinetic results were calculated.All patients were followed up for 24 weeks,and the levels of serum albumin (ALB),24 hours urinary protein,serum creatinine (Scr),complement 3 (C3),therapeutic effect and systemic lupus erythematosus disease activity index scores were evaluated at the same time.Results Pharmacokinetic curve showed the second chamber model,the area under the curve (AUC) of time-effect relationship was (143.55 ±42.43) g/(L · h),peak concentration (Cmax) was (20.02 ± 3.55) g/L,and half-time period (T1/2) was(4.21 ± 0.96) h.Age and gender had no influence on AUC,Cmax and T1/2,and statistically significant correlation was found between serum ALB and T1/2,which was positively correlated (r =0.517,P ＜ 0.05).After 16 weeks of treatment,7 patients were partially responsive,22 cases had a complete remission,and the invalid case was only one.Followed up for 24 weeks,6 patients were partially responsive,24 cases had a complete remission.After 16 weeks of treatment,there were statistical differences in AUC among patients with complete response,partial response or no remission.And after 24 weeks,the patients between complete remission and partial remission had no statistically significant difference in the AUC.After 16 weeks and 24 weeks,T1/2 and Cmax had no statistical differences among those groups of patients.Adverse reactions included leukopenia (3 cases,10.00％),gastrointestinal symptoms (4 cases,13.33％) and respiratory tract infection (6 cases,20.00％).There was no statistical relationship between pharmacokinetic parameters and the occurrence of adverse reactions.Single factor analysis showed that there was a statistical significance between ALB,urine protein quantity and treatment effect,but multi-factor analysis showed that the relation between factors and therapeutic effect had no statistical significance.Conclusions HPLC method can be used for the detection of cyclophosphamide blood drug concentration.The cyclophosphamide pharmacokinetic status of children and adult is similar.ALB and T1/2 show a linear positive correlation.Single factor analysis shows a correlation between ALB and treatment effect.Therefore,the increasing level of plasma propagated before treatment can improve therapeutic effect possibly.Cyclophosphamide pharmacokinetic status has nothing to do with the occurrence of adverse drug reactions in the study.