| Purpose:Viral myocarditis refers to infection caused by a variety of in order to myocardial cell degeneration and necrosis,interstitial inflammatory cell infiltration and fibrous exudation for cardiac disease,major changes occur. Its pathogenicity to Coxsackie virus group B virus(CVB) is the main,and in the six CVB serotypes in,CVB3 Anopheles strong mind,therefore,the current principal for the establishment of viral infection in CVB3 myocarditis model to carry out the research,the subject of viral myocarditis to choose research subjects as a main reason is that this disease has a high self-harm,such as children and adults of all ages can be involved stage disease,acute period, and into a dilated the possibility of cardiomyopathy at the same time,can also be caused by small-scale outbreaks,and viral myocarditis in recent years the incidence of the rise is,therefore,against CVB3 viral myocarditis treatment mechanism appears imminent.Subject to the 2-3d newborn rats in vitro neonatal rat myocardial CVB3 infection in cell culture to establish the VMC model;Hoechst33258 fluorescence detection through cardiomyocyte apoptosis;immunohistochemical detection of myocardial cell connexin Cx43,cytoskeleton - actin(actin),cardiac troponin I(cTnl) expression;TEM cells infected with CVB3 myocarditis model of cardiac cell function and morphological changes.Test the transform of Ca2+ and the communication function between cell gap junction in myocardialcell dynamic by SLCM in dynamic state.Yiqihuoxue attempt to verify the compound traditional Chinese medicine to relieve myocardial CVB3 viral myocarditis cell immune injury: inhibition of cardiomyocyte apoptosis occurred;to adjust Ca2+ and to improve the inter-cell gap junctional intercellular communication;repair myocardial ultrastructure of the cytoskeleton and the role of,and further confirmed traditional Chinese medicine compound Yiqihuoxue is the treatment of viral myocarditis in CVB3 valid prescription,Yiqihuoxue treatment of this disease is an effective way. Material and method:①Yiqihuoxue cytotoxicity determination of compound Chinese medicine:Recovery from Hela cells by passage culture bottles after inoculation with 0.2ml of each hole to 96 hole cell culture plate,the home 37℃,5%carbon dioxide incubator to cultivate,when Hela cells were adherent monolayer growth,the measurement Yiqihuoxue largest non-toxic dose of Chinese medicine compound.DMEM culture medium with the traditional Chinese medicine compound Yiqihuoxue injection started from the stock solution concentration of the preparation 11.Each hole 0.2ml,each concentration for 4-hole complex,plus an additional four control normal growth and pore fluid.In 24h,48h observation of cell growth state,a reference hole normal cells to the cytopathic effect is not caused by the largest concentration of drug dilution largest concentration of non-toxic(TD0).②CVB3 virus virulence titration:In 96-well plate in the preparation of single-layer growth of Hela cells,jumped to the growth plate in the liquid to wash the culture fluid HBSS hole 2.Containing 2%fetal bovine serum with the maintenance of fluid,the CVB3 virus to 10-fold dilution concentrations from 10-1 to 10-8,each hole 0.1ml,inoculated in 96-well plate,each concentration for 4-hole complex,another hole four normal controls,the increase in the maintenance of liquid 0.1ml,at 37℃,5%carbon dioxide incubator to cultivate, respectively 24h,48h observation cell lesions.③Yiqihuoxue Chinese herbal compound on CVB3-induced myocardial cell death inhibition(MTT method):Logarithmic phase from myocardial cells,0.2ml of each hole by adding half the volume of the concentration of tissue infections myocytes CVB3 virus adsorption 2h.Suction disposable culture medium containing the virus, containing 2%fetal bovine serum to the maintenance of gently washing liquid on it,adding the largest non-toxic drug dosage from the initial dilution of the concentration of 8 Yiqihuoxue Chinese herbal compound containing the growth solution,0.2 per hole ml,each concentration for 4-hole complex,a separate zero-hole.In 48h,each hole by adding 0.18ml DMEM culture medium,then add 0.02ml MTT solution into incubator to continue to foster 4h,carefully suction hole disposable culture medium,each hole by adding 0.15ml DMSO,the oscillation oscillator home 8min,in enzyme-linked immunosorbent assay detector 490 nm of the hole measured absorbance(OD).④Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis the apoptosis of myocardial cells:Isolation and culture of normal neonatal rat myocardial cells,inoculated in 24-hole cell culture plate so climbing film,48h after the adherent monolayer of Health check up to 70-80% of the normal group,the virus group,the intervention group of Chinese medicine myocytes glass to PBS wash 2,each hole by adding glacial acetic acid methanol fixed 10min,PBS washed 2 times,each hole Stain Hoechst33258 adding 1ml (5μg/ml),light response 15min,PBS washed 3 times,each time 3min,dropping anti-0.05ml fluorescence quenching letters do mount tablet treatment,observed under the fluorescence microscope.⑤Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis myocardial cells cTnI,Cx43,actin expression:Using SABC immunohistochemical method was used to detect cTnI,Cx43,actin expression: incubation with 3%H2O2 cells long slide 10min,PBS wash.Dropwise closed normal goat serum solution,incubated at room temperature for 15 minutes to dump,do not wash.Dropwise a 1:100 dilution of first antibody,37℃and incubated 2h, PBS wash,dropwise biotinylated second antibody,37℃incubated 30min.PBS washed 3 times,each time 5min.SABC complex dropwise,37℃wet box incubated 30min.PBS washed 3 times,each time 5min,DAB color,tap water rinse,hematoxylin staining,dehydration,transparent processing,resin mount,were observed.⑥Yiqihuoxue TEM testing of Chinese medicine compound of CVB3 viral myocarditis ultrastructure of myocardial cells:Cultured myocardial cells to normal 72h after washing with normal HBSS solution,the virus group,the Chinese Medicines Board of myocardial cells,0.25%trypsin digestion of the 3-5min,the collection of cells to centrifuge tube 1.5mlEP in,1500rpm centrifugal 10min,the supernatant discarded in order to 2.5%glutaraldehyde fixed power microscope.⑦FRAP testing CVB3-induced myocardial cell communication function cell gap junction:viral myocarditis cardiac myocyte check up to 70-80%add to 10μg/ml 6—CFDA: application liquor washed by Ca2+,Mg2+ PBS,37℃incubated 5%carbon dioxide 30min.After that washed by Ca2+,Mg2+ PBS again,then test it by FRAP.⑧Yiqihuoxue Chinese herbal compound on CVB3 myocardial cells Ca2+:put culture of normal neonatal rat myocardial cells,at 37℃,incubator to cultivate box under Fluo-3 AM Ca2+ by fluorescent probe,after hank srinsing put it on SLCM to test.Results:①Yiqihuoxue cytotoxicity determination of compound Chinese medicine: Yiqihuoxue Chinese medicine to determine the maximum non-toxic dose of compound 7.813mg/ml。②CVB3 virus virulence titration:CVB3 virus,50%of cultured cells infected with 1×10-4.5。③Yiqihuoxue Chinese herbal compound on CVB3-induced myocardial cell death inhibition(MTT method):Yiqihuoxue largest non-toxic dose of Chinese medicine compound CVB3 intervention of myocardial cells after the attack,the absorbance values the most,with other dilution group,a significant difference,P＜0.001,confirmed that the largest Chinese herbal compound Yiqihuoxue with non-toxic dose inhibited CVB3-induced myocardial cell death role.④Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis the apoptosis of myocardial cells:Normal myocardial nuclei were round,oval,no apoptotic nuclei,and 100×were observed myocardial cell virus group,showing a blue light into tablets,400×microscope observation shows that incomplete nuclear morphology,was debris-like distribution,but also lobulated nucleus can be seen.Cardiac nuclear medicine mostly round,no obvious nuclear morphology of apoptosis,suggesting that traditional Chinese medicine compound Yiqihuoxue CVB3 infected cells can inhibit apoptosis,myocardial protective role.⑤Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis cTnI expression of cell:Immunohistochemical detection of cardiac muscle Ca I(cTnI) expression showed that the normal group there was no apparent cTnI positive brown staining. CTnI-infected group showed strong positive(+++) expression of more positive signals tan color and depth,with the normal group the average optical density of positive goal difference significant,P＜0.05.CTnI Chinese intervention group were weakly positive(+) expression levels were significantly decreased compared with the virus,a significant difference,P＜0.01.⑥Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis cardiac myocyte expression of Cx43: Immunohistochemical detection of Connexin 43 protein found in normal myocardial cells were strongly positive(+++) expression of Cx43 and the virus was very weakly positive(±) expression,and the normal group the average optical density of positive goals,the difference is extremely significant,P＜0.001.Chinese medicine treatment group Cx43-positive(++) expression,and virus group,a significant difference,P＜0.05.⑦Yiqihuoxue Chinese herbal compound on CVB3 viral myocarditis cardiac myocyte expression of actin:Immunohistochemical detection of actin(actin) showed that normal myocardial cells were strongly positive(+++) actin expression.Actin virus was weakly positive(+) expression, compared with the normal group,significant difference,P＜0.05.Chinese medicine treatment group actin-positive(++) expression,and virus group,P＜0.01.⑧Transmission electron microscopy detection of Chinese herbal compound Yiqihuoxue CVB3 viral myocarditis ultrastructure of myocardial cells:CVB3 infection of myocardial cells with normal compared to cell injury,indicating that CVB3 viral myocarditis model the success of the intervention group and the Chinese form of myocardial cells and cell structure close to the normal group, compared with the model group differences,indicating that Chinese Yiqihuoxue and defense compound can reduce the myocardial damage caused by CVB3,repair damage with the role of myocardial ultrastructure.⑨FRAP testing CVB3-induced myocardial cell communication function cell gap junction:normal group's cell gap junction communication was better than model group.P＜0.05.Chinese Yiqihuoxue was also better than model groupP＜0.01.⑩Yiqihuoxue Chinese herbal compound on CVB3 myocardial cells Ca2+:The effect of Yiqihuoxue Chinese herbal compound on Ca2+ in normal neonatal rat myocardial cells was not obviously,P＞0.05,but it can improve the model group,P＜0.05.Conclusion:1.To primary neonatal rat cardiac myocytes in vitro methods,the establishment of the myocardial cells attack CVB3 infection model. 2.Immune from this experiment,molecular,protein and cell levels,such as the level of ultrastructure revealed Yiqihuoxue control of Chinese medicine composed of viral myocarditis myocardial injury mechanisms,reflects the traditional Chinese medicine focus on the "overall" idea of the Diagnosis and Treatment.3.Through apoptosis,CVB3-induced myocardial cell communication function and cell gap junction Cx43,actin,cTnI and myocardial cell ultrastructure such as functional testing and morphological observation,confirmed the drug mainly in the composition of Yiqihuoxue Chinese medicine compound has a multi-role, multi-level,multi-target effect.4.The experiments further confirmed Yiqihuoxue VMC is an effective treatment of the law,and medicine Yiqihuoxue VMC compound is an effective prescription treatment,suggesting that this treatment in the VMC has broad application prospects. |
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