The Influence Of Chai Shi Antipyretic Granule On Japanese Encephalitis Virus Infected BHK Cells In Vitro And Study On The Law Of TCM Syndrome Differentiation Of Japanese Encephalitis

Part1.Objectives:Based on traditional Chinese medicine theory, in accordance with theprincipleofevidence-based medicine, the clinical studyoflarge sample, multi center, randomized, TCM syndrome elements and prognosis out come of different syndrome type carding on J-E, summarizesits pathogenesis, transmission and prognosis ofTCM, verification plan scientifically and accurately, to improve the Chinese plan (2011). Application of different TCMtreatmentof JE patients, the clinical efficacy was analyzed, summarizedthe treatmentofTCMandChinesemedicine screening effective JE. Thetheory of TCM and clinicalunderstanding and deepen JE, to providescientific and technological support to establish the standard of Chinese medicine for the treatment of virus infectious diseasesystem.Methods:From June to October in2012, to inspect the diagnosed284cases of patientswithepidemicencephalitisB were prospectiveclinicalstudy. On2009Chinesemedicine industryspecial"Chinese med-icine in prevention and treatment of epidemic encephalitis Clin ical Regularity and treatment ofreference"(JE projects) Chines-e plan research group established in the acuteperiod, allcasesweredivided intomild (DUYun lung stomach), ordinarytype (toxinhurtingbraincollaterals), heavy (virus syndromeofinvasionofpericardium), very heavy (yinandYang failuresyndrome), fillinthe "JE cases questionnaire"(CRF), recorded demographicdata, symptoms, signsthe severity, frequency, time, disappear (or reduce) thetime, TCMsyndrome and thetongueandpulse information, TCMsyndromedifferentiation, hospitalization, medicalrecords, ending judgment, follow-uprecord. TCM syndrome elements of the CRF tablerecords were co-llected and sortedaccording to different syndromes.Accordingto the "method of grouping implementation JE project" clinicalprograms, light, ordinarytypepatients randomized group, heavy, ve-ry heavy inthe cohort. Through the computers of tware(producedby the center for clinical evaluation China Academy of traditional Chinese Medicine) to generate random numbers, the central randomization system, block and dynamic random combination metho-d, using the SAS9.1.3statistical software PROC PLAN program were random lyassigned, the subjects receiveed treatment with mach-ine arrangement,divided into treatment group and control group.Western medicine control group with routine western medical treatment. Traditional Chinese medicine in the treatment groupwere treated with traditional Chinese medicine:①TCM prescription，②Chai Shi antipyretic granule，③Oral anti-viral④Xiyangping Injection.To alleviate the influence on prognosis of efficacy, clinical efficacy, comparedoftwo treatment methods of fever, coma, convulsions. In particular, with fever as the breakthroughpoint, to observe the clinical curative effect of TCM therapy eff ective drugs.Quantitative data description will calculate the mean, standard deviation, median, minimumvalue,maximum value. Qualitative data to describe the number and percentage of cases.Statisticalanalysis by SPSS16.0software, two groups of comparability anal-ysis, qualitative data using chi square test/Fisher exact probability/Wilcoxon rank sum test. Quantitative data with normal distribution by the t test (t ’variance incomplete inspection), does not meet the normaldistribution using the Wilcoxon rank sumtest, differences among groups by single factor analysis of variance (one-way, ANOVA) between the two two groups with LSD method. P<0.05is considered to be statistically significant difference between the test, P<0.01means significant difference.Results:1.Case information:In2012,the study observed a total of284JE cases, syndrome differentiation in acute period: Du Yun lungand stomach type in61cases, accounting for21.48%of the totalcases; toxin damaging brain collaterals type in114cases,40.14%of the total number of cases; virus invasion of pericardium type in98cases,34.51%of the total number of cases; Yin and Yang failure type in11cases, accounting for3.87%of the total cases. The minimum age for March, a maximum of15years old, the average age is5years old in July; the male to female ratio was1.63:1.2.The transfer and prognosis of lung and stomach syndrome: poison accumulate in the Wei, and Wei Qi is not smooth, easy to cu re; toxin damaging brain collaterals is very hot gas, evil in middle Jiao, awind of trillion, most clinical cure.3.Toxic syndromeofinvasionofpericardium for heatcellulitesYingfen, consuming Qi and body fluidand blood disease, or camp,air consumption of blood, or both, and damp phlegm endogenous,abo-ut30%patients with poor prognosis; Yin and Yang and the failu-re to see to fry the Qi and Yin,into the inner closed outer de, Yin Yang Exhaustion like, in critical condition, to save lives.And, JEcondition change more quickly and more critical than thegeneral epidemic febrile disease.4.Both of the five kinds of TCM syndrome type of TCM syndrome elements and2011plan type elements comparison, found thattwo different parts:①fever with tightness in the chest, poor appetite, loose stools, moss thin white or whitethick, suggestingthat the heat dampness pathogen.The part of evil gas camp or the campblood disease, longer duration of illness, stress, during the recovery period of18%cases occurred in various neurological impairment in cash, were observed at the end there are still11.6%cases of disability, showing the phlegm, meridian dystrophysyndrome.5.the mass effect：The end of the observation, analysis of Chinese and Western medicine groupandthecontrolgroup ofWesternmedicine treatment, results show, random groups: the treatment group cure rate was90.6%, improvement rate was7.69%, effective rate1.71%; control group, the cure rate was74.14%, improvement rate was18.97%, effective rate6.7%. Effect of queue groups: treatment group, the cure rate was68.49%, improvement rate was23.29%, effective rate8.22%; control group, the cure rate was46.8 8%, improvement rate was40.62%, effecttive rate1.25%. Study group two, significantly better than the control group curative effect in the treatment group, there are significant differences(P<0.05).6.Clinical analysis of three main symotoms:Random group, pyretolysis median3days,4days in control group; cohort, pyretolysis median4days,6days in control group; through comparisonof survival analysis, two research group combined with TCM trea-tment group antifebrile trend faster than the control group, the difference was significant (P<0.05).Random group, the treatment group consciousness recovery time was72hours,120hours in the control group; cohort, pyretolysis a median of85hours,132hours in the control group; through comparison of production analysis, two research group combinedwith TCM treatment groupconsciousness recovery normal trend faster than the control group, the difference was significant(P<0.05).Screening of remission CRF tic each type method, were recorded, and compared with baseline(watchthefirst day), resultsshowed: treatmentof third random day group, two groups wererelieved, and the treatment group was better than that of the control group relief; cohort for fifth days, the two group compared with the base line was relieved, and the treatment remission groupthan in the control group; the difference was significant (P<0.05).7.284patients with epidemic encephalitis B patients,194cases in the treatment group were treated with traditional Chinese medicine traditionnal Chinese medicine according to the execution of the project, the use rate of100%, the total cure rate was83.6%, 8.Other tranditonal Chinese msdicine: Potassium Sodium Deh-ydroandroandrographolide Succinate Injection, Reduning injection, Tanreqing injection, Xingnaojing Injection9.Chai Shi antipyretic granule in the treatment of epidemicencephalitis B in44cases, the mean feveronset time was10.23±6.24h, average completely antifebrile time was68.92±18.54h; the control group treatment for JE in31cases, the mean fever onset time was12.53±7.31h, average completely antifebrile time was106.55±24.83h respectively; the statistical analysis, the difference was significant (P<0.05).10. Chai Shi antipyretic granule in treatment of JE, the queue group cure rate of the patients more advantages than randomgroup, in the fever at the same time, can effectively alleviatethe coma,convulsions, shorten the course of treatment, reducethe incidence of sequelae. Compared with the control group, thedifference was significant (P<0.05).11. Safety inspection:no damage to liver and kidney functi-on, serious in two groups. Adverse eventsand drug free, veryfew cases of gastrointestinal reaction, disappeared after symptomatic treatment.Conclusion:This study shows that,epidemic encephalitis B in the sum-er, more common in preschool and school aged children. Je TCM pathogenesis and transmission rules accord with weiqiyingxuetransmission characteristiccs, even JE transmission changesrapidly, theillness is generally more severe febrile disease. By2012theclinical TCM syndrome elements analysis, and compared with the tr-aditional Chinese medicine syndrome differentiation of JE plan contents, prove JE projectChinese plans for the JE differential treatment accorded with clinical practice, it is of higher scientificity and accuracy. At the same time, found the2012JE pathogenic for summerheatdampness, treatment principlein the "detoxification" basis, pay attention to heat and removing, andlight penetration dampness herbs. And in2011the Chinese plan to optimize the recovery period,increase the liver kidney yin deficiency, phlegm and blood stasis blocking collaterals.We demonstrate that the Chinese medicine infection prominent role in prevention and treatment ofinfectious disease epidemicencephalitis B virus with a large number of clinical data and modernscientific and technical methods, in relieving clinical symptoms, improve the cure rate hasobviousadvantages, reduce sequela etc.. Screeningofeffective traditional Chinese medicine such asXiyanping injection, Xingnaojing injection, Chai Shi antipyretic granule, Antiviral Oral-Liquid, Angong Niuhuang Pill and so on. Among them, Chai Shi antipyretic granule clinical treatment effect is mainly manifested in the JE fever fast onset time, fever has strong effect, effective control of seizures andreduce the disturbance of consciousness, shorten the course of treatment, at the same time, can reduce severe encephalitis sequela occurred. Advantages of treating severe cases obviously. Objectives:In vitro cell culture technology, anti Japanese encephalitis virus on Chai Shi antipyretic granule at the cellular level (JEV) explores the role function and target, for traditional Chinese medicine toprovide objective experimental basis for the Anti Japanese encephalitis virus, thereby expandingthe Chai Shi antipyreticgranule antiviral spectrum, at the same time, to provide ascientific basis for the next step of molecular studies on the gene level and whole animal test.Methods:Using the established model of Japanese encephalitis virus infection of cells in BHK-21cell line, withthe activity of the virus detection experiments, using BHK cells to detect drug toxicity, drug intervention cells to differentconcentrations, by different administration, the use of synchronous infection assay and plaque reduction was determined by counting the number of virus titer,respectively study drugs on inhibition of JEV infection cells, direct killing effect on the virus, anddrugs on JEV adsorption, invasive cell inhibitory effect of blocking proliferation and intracellular.The other set of ribavirin drug control group and virus control group.The measurement data are mean+standard deviation,, differences among groups by singlefactor analysis of variance (one-way, ANOVA) between the two two groups with LSD method; count datausing X2test statistics, all statistics calculated by using statistical software SPSS16.0, P<0.05was statistically significant, significant P<0.01. Results:1. Estabilshment of cell model in vitro JEV infection: thisexperiment uses JEV is virulent, after the activation of inoculated in BHK-21cells, the cell membrane in the small48-72becameround, cell chromatin deepen, cell shedding, apoptosis and pathological changes, the virus in the cell lesiontime tends to be stable after3times.2.Drug toxicity experiment：ribavirin with increasing drug concentration, showing the effect of inhibition of cellexpansion, at concentration of1600ug/ml can be three times the cell growth slowed, but did not induce apoptosis; and Chai Shi antipyretic Granule on the cell toxicity in the killing of cells, when theconcentration reaches320ug/ml, the majority of BHK-21cells showed necrosis. Maximum non-toxicconcentration Chai Shi antipyreticgranule liquid (TD0) for160ug/ml, ribavirin10ug/ml can achievethe inhibition of Japanese encephalitis virus.3. The suppression of viral replication：Chai Shi comparedto40,80160group ug/ml, Leigh Bhave Lin antipyretic granule group and virus control group, there were significant differences (P<0.01); compared with Chai Shi antipyretic granule160ug/ml group and Chai Shi antipyretic granule40ug/ml group, there was statistically significant differentce (P<0.05); compared withthe high concentration of traditional Chinese medicine group andthe Leigh Bhave Lin group, the difference was not statisticallysignificant.4.Direct killing effect of Chai Shi on JEV: Chai Shi Chai Shi antipyretic granule160ug/mlgroup compared with the virus control group, virus titer decreased by about53333.3PFU/ml, but n osignificant difference by statistical analysis.5.Study on the effect of target inhibition of JEV replication Chai Shi antipyretic granule： To reduce the rate of virus replication pretreatmentofcells160ug/ml after ChaiShi antipyretic granule group, controlgroupand virus, therewasstatisticallysignificantdifference (P<0.05); ChaiShi antipyretic granule80160ug/ml group was administered in virus infected cells, to a certain extent blocked the JEV intrusion, compared with virus control group, there was statistically significant difference (P<0.05) effect on the virus, there is no difference between the twoconcentrations; Chai Shi antipyretic granule group160ug/ml replication link within the cell to the virus, its effect on viral replication is the virus control group, there was statistically significant difference (P<0.05).Conclusion:In this experiment,the successful use of BHK-21cell wasestablished in vitro cell model of JE virus infection, to observe the effect of antipyretic Granule on Chai Shi boycott of Japanese encephalitis virus infection. The results showed, Chai Shi antipyretic granule in vitro cell model can better inhibition of Japanese encephalitis virus infection, and the inhibition associated with traditional Chinese medicine concentration, high concentration group effect and ribavirin. The mechanisms of drug resistance performance of JEV cell entry blocking inhibition proliferation andintracellular drug on JEV adsorption. At the same time, the Chai Shi antipyretic granule hasprotective effects on the hostcell.