| Objects:Viral myocarditis（VMC）is a kind of myocardial disease with pathology injuryof myocardium degeneration and necrosis, fiberours and infiltration ininflammatory cells. VMC can be induced by almost any virus, especially acardiotropic variant of coxsackievirus group B（CVB3）. Thus the VMC model inducedby CVB3has been mainly researched.The increasing of incidence of VMC shows serious threat to the health ofchildren and young ages. And studies of treating mechanism of VMC is imperative.The disease name of VMC is not exist in Traditional Chinese Medicine. InTCM VMC can be categorized as febrile disease according to its incidencecharacteristics, or cardiopalmus, deficiency and exhaustion, heartacheaccording to its clinical symptoms.In recent years of clinical researches of VMC, many effective experiences andtreatments had been concluded in Traditional Chinese Medicine that showedobvious efficacy and safety, and the necessity of studying on TCM treating VMCas well.Combined with Traditional Chinese medicine theory and clinical practices,Chinese herbal compound with astragalus, white ginseng, salvia, curcuma,liriope etc. was found out to supplementing Qi and activating blood circulation,which was therapeutic In this study.The indexes of pathogen and myocardial protection chosen in this study revealthe therapeutic mechanism of TCM treating VMC.After modeling the CVB3induced VMC, the expression of cytochrome c oxidasesubunit I, cytochrome coxidase subunit II，ATP synthase F0subunit6，ribosomalprotein L27a in host cell of CVB3and Chinese herbal compound Group were observedby suppression subtractive hybridization (SSH). Exploring the regulationtargets and the mechanism on gene level with the Chinese herbal compound ofsupplementing Qi and activating blood circulation. This study showed that the suppression subtractive hybridization (SSH) isa differential display technique that improved and effective. In Chinese herbalcompound Group, the expression of cytochrome c oxidase subunit I，cytoc hromec oxidase subunit II，ATP synthase F0subunit6，ribosomal protein L27a wasincreased, which suggested that the Chinese herbal compound of supplementingQi and activating blood circulation treated VMC by regulating this gene, andembodied the theoretical thought of Strengthening Healthy Qi to EliminatePathogens in TCM.Methods1.The recovery and culture of Hela cell: The Hela freezing tube was taken outfrom the cryopreserved in liquid nitrogen, and rapidly removed it into a heatedwater box at37℃, shook it in the heated water box constantly to rewarm forabout30seconds until Hela freezing tube dissolved, put the cell suspensioninto sterile centrifuge tube rapidly, added3ml DMEM medium which containing10%fetal bovine serum,1000rpm centrifugated for5min, discarded thesupernatant, add15ml DMEM culture liquid with10%FBS, blowing the depositioninto single cell suspension with pipette repeatedly before the movement intonew culture flask, then incubated it at37degrees centigrade,5%CO2,100%saturated humidity box. Human cervical carcinoma cell was adherent growth cells,growing in DMEM cell culture medium containing10%fetal bovine serum, cellsentered into logarithmic growth phase after24h, the total bottom of the bottlecould be packed with cells about72h, when cells were grown to80%~90%fusion,digested the adherent cells with pancreatin/EDTA, the process required in theultra-clean table, strict aseptic operation.2．Cytotoxicity determination of the Chinese herbal compound of supplementingQi and activating blood circulation: Digested the Hela cells which entered thelogarithmic growth phase by pancreatin/EDTA, trypan blue was used to stainand took the record the number of living cells with counting plate (>95%),regulation of living cells at a concentration of5x105/ml, the recovered Hela cells were inoculated in96culture plates with each hole0.2ml，culturedin incubator with37℃，5%CO2. incubator for24h.when Hela cells all adherentgrowth, to test the maximum nontoxic dosage in the Chinese herbal compound ofsupplementing Qi and activating blood circulation while Hela cells displayedmonolayer growth.Prepared the stock solution of the Chinese herbal compound ofsupplementing Qi and activating blood circulation into11concentrations withDMEM. Each concentration with4multiple wells,0.2ml per well, and4controlwells for normal growth medium. The cell morphology was observed at24h and48hreference to the normal control wells. The maximum nontoxic concentration（TD0）was determined to be the maximum diluted concentration that can not inducecytopathogenic effect.3.Toxicity test of CVB3: After Hela cells displayed monolayer growth in96wellsplates, the growth medium in plates was removed, and the culture wells were washedwith HBSS for2times. The CVB3was diluted from10－1to10－8by decupleconcentration with2%fetal bovine serum maintenance solution, and wasinoculated in96wells plate,0.1ml per well. Each concentration with4multiplewells and4normal control wells, add maintenance solution0.1ml, cultured inincubator with37℃，5%CO2. The cell morphology was observed at24h and48h.4. Inhibition of the Chinese herbal compound of supplementing Qi and activatingblood circulation on CVB3induced cardiac myocyte death(MTT method): Myocardialcells at logarithmic growth phase were collected, each well was added mediantissue infection concentration TCID50CVB30.2ml to myocardial cells adsorptionfor2h.Removed CVB3contained culture solution, washed once tenderly with2%fetal bovine serum maintenance solution, added growth solution with the Chineseherbal compound of the8th concentration from the initial maximum nontoxic dosage,0.2ml per well. Each concentration with4multiple wells and1zero well. After48h,0.18ml DMEM and0.02ML MTT were added each well, cultured in incubator for4h, removed culture solution carefully, then0.15ml DMSO was added, after8minutes oscillation,490nm OD of each well were tested by enzyme linked immunityinstrument. 5．Effects of the Chinese herbal compound of supplementing Qi andactivating blood circulation on gene expression of cytochrome coxidase subunit I，cytochrome c oxidase subunit II，ATP synthase F0subunit6，ribosomal protein L27a in CVB3induced VMC myocardial cells:Isolation of differentially expressed gene in rat myocardial cells of the CVB3infected group and the Chinese herbal compound group by SSH, and confirmthe results above by real-time RT-PCR.Results:3.1Determination of Boosting Qi and Enlivening Blood Formula cytotoxicity: themaximum non-toxic dose for the formula is7.813mg/ml.3.2CVB3virulence titration: the infection rate of50%cultured CVB3virus cellsis1×10-4.5.3.3Boosting qi and enlivening blood formula inhibition of myocardial cell deathfrom CVB3virus(MTT method):use of maximum non-toxic dose boosting qi andenlivening blood formula on post-attack myocardial cells， had the largestabsorption value， and in comparison with other dilutions there was a cleardifference， p<0.001. This shows that the maximum non-toxic dose of boostingqi and enlivening blood formula has a clear inhibitory effect on CVB3inducedmyocardial cell death.3.4Effects of the Chinese herbal compound injection of supplementingQi and activating blood circulation on gene expression of cytochromec oxidase subunit I，cytochrome c oxidase subunit II，ATP synthaseF0subunit6，ribosomal protein L27a in CVB3induced VMC myocardialcells: The result of SSH showed that in the Chinese herbal compound groupthe expression of cytochrome c oxidase subunit I， cytochrome coxidase subunit II，ATP synthase F0subunit6，ribosomal protein L27awas higher than the CVB3infected group, which was confirmed by real-timeRT-PCR. Conclusion:1. The CVB3induced VMC was successfully modeled by cultured primary myocardialcells.2. It was confirmed that the Chinese herbal compound of supplementing Qiand activating blood circulation is multifunctional, multileveled andmulti-targeted for CVB3induced VMC by observing its effects onexpression of cytochrome c oxidase subunit I, cytochrome c oxidasesubunit II，ATP synthase F0subunit6，ribosomal protein L27a.3. This study further verified that supplementing Qi and activating bloodcirculation is a effective way of treating VMC, and the Chinese herbal compoundof supplementing Qi and activating blood circulation is a curative prescription,which suggested its broad application prospect in treating VMC. |
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