The Study Of Cytokines's Expression In Murine Myocardium Of Viral Myocarditis And Influence To It When Treated With Astragalus Membranaceus

1. To study the time course of the expression change of TNF-amRNA in murine myocardium of Viral Myocarditis(VMC);At the same time the influence to the expression of TNF-amRNA when treated with Astragalus Membranaceus(AM) was also studied.2. To study the influence to the expression of Th 1-associated cytokine IL-2mRNA and Th 2-associated cytokine IL-10mRNA in murine myocardium of VMC when treated with AM.Methods:4-week-old balb/c mice were inoculated intraperitoneally with 0.2ml Coxsackievirus B3m(CVB3m) which was diluted in hanks solution to concentration of 10?The day of virus inoculation was regarded as the day 0,and the murine activities eating condition somatotype and change of hair color were observed daily after inoculation.4 surviving mice were killed randomly by cervical dislocation on days 1 3 7 9 11and 21 after inoculation.Their removed hearts were stored at -70 refrigerator and prepared for semiquantitative reverse-transcription polymerase chain reaction(RT-PCR).The therapy with AM which started after virus inoculation for 0.5h was perfusing AM to murine stomach in a dose of 0.2ml for twice a day consecutively to day 10.All surviving mice were killed by cervical dislocation on day 11 .Their removed hearts were stored at -70癈 refrigerator and prepared for semiquantitative RT-PCR.Total RNA was isolated by Trizol isolationprocedure from each 5omg heart sample.Total RNA was used for DNA synthesis by reverse transcription.Then DNA was amplified by polymerase chain reaction(PCR) when added a sense primer and an antisense primer of TNF- IL-2 IL-10and -actin.So the gene sequenses of TNF- IL-2 IL-10 and referring gene -actin were amplified respectively for each DNA sample.The PCR products were judged whether the same as we designed when compared with PCR marker by ethidium bromide(EB)-stained 1% agarose gel electrophoresis.Then the density of PCR products were analyzed by Kodak image analyzing system and the relative value of TNF-amRNA IL-2mRNA and IL-10mRNA were caculated. Results: 1. The expression of TNF-amRNA in murine myocardium of CVB VMC andthe influence to it when treated with AM.(1)The weak expression of TNF-amRNA in murine myocardium was seen on day 3 after virus inoculation,then a gradual expression increase was seen and the peak expression was on day 9 after inoculation, from then on the expression decreased,but still could be seen obviously on day 21 after inoculation;(2) When compared with the viral control group that was not received AM therapy the expression of TNF-amRNA in murine myocardium of CVB VMC decreased(P<0.05) when treated with AM. 2. The influence to the expression of Th associated cytokine andTh-associated cytokine in murine myocardium of VMC when treated withAM.When compared with the viral control group that was not received AMtherapy the expression of Th -associated cytokine IL-2mRNAdecreased(P<0.05) and Th -asspciated cytokine IL-10 mRNA increased(P<0.05)at the same time when treated with AM.Conclusions:1. TNF-a expressed by myocardial cells(consist of cardiac myocytes fibroblasts and endothelial cells) at the early time of VMC,and the peak expression occurred when massive cell infiltration appeared in the heart ,this suggested TNF-a was expressed maily by infiltrating cells at this time.2.AM exhibited its immunoregulatory effect partly through reducing the expression of TNF-amRNA in murine myocardium.3. AM could restrain Th reaction and accelerate Th reaction in the early phase of VMC,so it exhibited its beneficial immunoregulatory effect through ameliorating the interaction of Th cells and Th cells.