Study On The Therapeutic Effect And Mechanism Of Qingxing Granules For Dengue Fever Based On In Vitro And In Vivo Models

ObjectiveDengue fever(DF)is a common mosquito-borne viral disease that has become a major problem of international public health in recent years.This experiment mainly established DF models in vivo and in vitro to study the therapeutic effect and mechanism of Qingxing Granules(QX).The PERK/ATF6 signaling pathway in the Unfolded Protein Reaction(UPR)was used to explore the therapeutic effect of QX.All these provide a basis for further research on the PERK/ATF6 signaling pathway in UPR,and an evidence and direction for drug development of DF treatment.Methods1.Antipyretic and analgesic experiments: Animal experiments are conducted to investigate the pharmacological effects of QX on dry yeast induced fever in rats,endotoxin induced fever in rabbits,hot plate induced pain response in mice,and acetic acid induced pain response in mice,providing pharmacological basis for the later treatment of DF.2.In vitro experiment: By culturing C6/36 cells to a good growth state,the CCK8 method is used to detect the effect of different concentrations of QX on the growth of C6/36 cells in the 96 well plate.Also,the half inhibitory concentration(IC50)of QX is calculated to determine its maximum non-toxic concentration.Then,calculating cell survival rate using MTT method and detecting the m RNA expression of DENV-2 using Q-PCR is to verify whether QX has a certain therapeutic effect on DF.3.In vivo experiments:3.1 Grouping and administration: 70 SPF female ICR mice are randomly divided into seven groups:(1)normal group(CON);(2)model group(MOD);(3)ibuprofen group(IBP 4.51ml/kg);(4)mannan disinfectant group(GLXD 4.51g/kg);(5)low-dose QX group(QX-L 7.21g/kg);(6)medium-dose QX group(QX-M 14.42g/kg);(7)high-dose QX group(QX-H 28.84g/kg).Then,TCID50104(100μl DENV-2 virus)is injected into the abdominal cavity once a day for modeling for 3 days.Starting from the first day of modeling,observing the basic condition of the mice and recording their body temperature and weight during the period.Besides,blood from the jaw is collected on the 3rd,5th and7 th days for administration.3.2.Sampling and indicator measurement: Starting from the afternoon of the end of administration,the samples are taken and blood is collected through eyeball extraction.All ICR mice are euthanized by cervical dislocation method,and brain tissue,lungs,liver,spleen,small intestine and kidneys are stripped and washed with PBS for the surface.According to the required volume of the experimental design,each group is placed in a4% paraformaldehyde tissue pathology bottle and fixed at room temperature for preservation for pathological HE staining experiments.Then,cutting 20 mg of liver tissue in each group and placing it in a container containing 200μl Trizol cracking solution.50 mg from the container is packed into 1.5ml EP tube and stored in a refrigerator at-80 ℃.The rest is packaged separately with data label in a 5ml EP tube in the same way.ICR mouse serum for Q-PCR is to detected the DENV-2 m RNA expression and for ELISA detection of AST,ALT,IFN-γ cell factors and so on.Liver tissue is used for QPCR detecting AST,ALT,IFN-γ,IL-6,IL-10,TNF-α,GRP78,ATF6,PERK target genes and so on.The main non-structural protein NS1 in DENV is tested by using Western blot and others,and the expression of the related proteins in the PERK/ATF6 signaling pathway in UPR.Results1.The antipyretic and analgesic experiments show that QX can reduce the body temperature of rats after 6 hours of modeling in the experiment on the effect of dry yeast induced fever in rats(P<0.05).In the experiment on the effect of endotoxins on fever in rabbits,the body temperature of rabbits after modeling is reduced(P<0.05).Among them,QX-H could significantly reduce the body temperature of rabbits from 5 to 6 hours after modeling(P<0.05).Also,QX could reduce IL-1βand TNF-α content in rabbits after 3hours of modeling(P<0.05).Further,in the experiment on the effect of hot plate induced pain response in mice,QX significantly prolong the pain threshold(s)(P<0.05)at 60 minutes of administration,and QX-M significantly prolong the pain threshold(s)(P<0.05)at 90 minutes.In the experiment on the effect of acetic acid induced pain response in mice,QX-M and QX-H significantly prolong the latency period(P<0.05)and reduce the number of twists in mice(P<0.05).2.In vitro experiments have shown that different concentrations of QX have different effects on the survival rate of C6/36 cells.As the QX dilution ratio decreases,the survival rate shows a gradient increase.The IC50 is calculated to be 2.256mg/ml.The maximum non-toxic concentration is determined to be 1 mg/ml when the cell survival rate reaches 80% or above.The MTT assay shows that the cell survival rates of QX at1mg/ml,0.5mg/ml and 0.25mg/ml are higher than those of the virus group(P<0.05).3.In in vivo experiments,pathological HE staining results of liver,small intestine and brain tissues suggest that QX can reduce inflammation caused by DENV compared to the MOD group.A significant therapeutic effect of QX on small intestinal tissue lesions is obtained from them.ELISA detection of QX can reduce the content of AST,ALT and IFN-γ(P<0.05).Compared with MOD,Q-PCR and Western blot results show that QX can reduce the content of AST,ALT,IFN-γ,IL-6 and TNF-α,and the m RNA expression level of PERK.QX can also reduce the expression levels of NS1,ATF6 and PERK proteins(P<0.05).Conclusion1.QX has antipyretic and analgesic effects and can reduce the expression level of inflammatory factors,which provide a pharmacological basis for the later treatment of DF.2.QX can effectively inhibit DENV replication in C6/36 cells and has therapeutic effects on C6/36 mosquito cells infected with DENV-2.3.QX exert therapeutic effects on DF through the PERK/ATF6 pathway in UPR.