Experimental Study Of Expression Of Periostin In The Mice With Viral Myocarditis And The Therapeutic Efficacy Of [Cordyceps Sinensis (Berk.) Sacc.] And Irbesartan In Myocardial Fibrosis

Viral myocarditis (VMC) is one of the most common infective diseases in cardiovascular diseases. Most of the patients are annoyed with acute VMC, but someone develops into the chronic VMC, dilated cardiomyopathy with survival rate about five years is around 50%. VMC has been threatening children health severely. Myocardial fibrosis refers to the excessive accumulation of collagen fibers in the normal tissues and structures, marked elevation of collagen concentration, or the changes of collagen constituents. This pathological change exists in every stage of VMC. Now, it is believed that this change is associated tightly with cardiac arrhythmia, cardiac dysfunction and dilated cardiomyopathy. The periostin gene was initially cloned from a mouse calvarial cell line (MC3T3-E1) and originally named osteoblast specific factor-2 (OSF-2). Periostin is a member of a growing family of matricellular proteins. Periostin has been shown to play a crucial role during tissue morphogenesis. Periostin is attended as a novel factor responsible for ventricular dilation. Perostin has been shown to be involved in the progress of Myocardial fibrosis after myocardial infarction It is believed that the abnormal expression of periostin may be related with the occurrence and development of VMC.(1) The expression and function of periostin in mice model with viral myocarditisMaterials and methods : In this study we first established VMC models through BALB/c mice infected with coxsackie virus B3. 100 pure bred male Balb/c mice aged 4 weeks and weighted 18±1g, were divided into control group(n=40) and VMC group (n=60) randomly. VMC model was founded by injecting intraperitoneally 0.1ml CVB3 Nancy solution one time a day for three days, while the control group was injected with 0.1ml Engeal's fluid excluding virus. The model mice were sacrificed by extirpating eyeballs on day 7, day 14,day 28, and day 56 respectively, after injecting CVB3 ,0.8-1.0ml blood were extracted by burettes. The serums were separated and stored at -70℃, preparing for the test of Ang II and CK-MB. Then all mice were killed by cutting necks, obtaining hearts and putting them partly into 10% formaldehyde solution for biopsy, the other part was stored at -70℃for RT-PCR. The mice of control group were treated by the same methods. After virus inoculation until day 0,day 7,day14,day28,day56, we examined the contents of periostin in the myocardium by SP and RT-PCR . Meanwhile, we examined the score for myocardial necrosis, the level of CK-MB and AngII in serum, CVF and the contents of TGF-β1 mRNA in the myocardium. Based on the results, we further studied the relationship between periostin and other index. Our study would provide new theoretic base and experimental evidence for prevention and therapy of VMC.Results: We found that the model mice displayed tarnishable furs, reduced movements, been weary spirits and poor appetites ,vaulted backs, showed indifference to stimulation or tended to irritation and been lower temperature since the third day. Death occured on day 4, and reached the top from day 7 to day 14. Among 60 mice in model group, 21 died. The death rate was 35.0%. None of the control mice had any symptom and death. The serum CK-MB concentrations of the VMC group were much higher than that of the control mice at each time spot(P<0.01).The peak is in day 14 , after that day the serum CK-MB concentrations decreased. The other indexs include serum AngII concentrations ,CVF, TGF-β1 increased gradually in the course of disease. The expression of periostin was detected in myocardial tissue at the day0, day7, day14, day28, and day56 after CVB3 infection quantitatively by semiquantitative analysis RT-PCR. The results showed that the expression of periostin increased slightly at the 7 day after infection, then the expression was increased continuously, and the expression of periostin was highest at the 56th day after infection. Linear correlation analysis showed the contents of periostin in the myocardium with the level of AngII in serum, CVF and the contents of TGF-β1 in the myocardium is positive correlation remarkably, but score for myocardial necrosis and level of CK-MB in serum have no correlation with the contents of periostin in the myocardium.Conclusions: It suggested that periostin may be involved in the myocardial fibrosis in myocardium of VMC. Ang II and TGF-β1 maybe increase periostin expression, and eventually contributed to the myocardial fibrosis in VMC mice model. Periostin may be a new target in the therapy of VMC. (2): The therapeutical effect and possible mechanism of [Cordyceps sinensis (Berk.) Sacc.] (CS) and Irbesartan in the VMC mice models in chronic phase.Materials and methods :70 pure bred male Balb/c mice aged 4 weeks and weighted 18±1g, were divided into control group(n=10) and VMC group (n=60) randomly. VMC model was founded by injecting intraperitoneally 0.1ml CVB3 Nancy solution one time a month for three monthes , while the control group was injected with 0.1ml Engeal's fluid excluding virus.Then we successfully established the models of viral myocarditis in chronic phase through BALB/C mice infected with CVB3 .42 models of viral myocarditis in chronic phase were divided into 4 groups. They were VMC-control group (n=12), Irbesartan group (n=10), CS-large group (n=10) and CS-small group(n=10). VMC-control group was treated intragastric with water 1ml/d . Irbesartan group was treated intragastric with Irbesartan at a dose of 10mg/kg.d . CS-large group was treated intragastric with CS at a dose of 7.5g/kg.d CS-small group was treated intragastric with CS at a dose of 2.5g/kg.d .After feeding drugs for 60 days, we weighed the mice, examined the heart function changes, observed Myocardial ultrastructural changes and myocardial collagen fiber distribution,measured CVF and the level of AngII in blood serum. At the same time, the contents of collagen I and collagen III were detected by immunohistochemical techniques. TGF-β1and periostin were detected by reverse transeription polymerase chain reaction Then the therapeutic effect of CS and Irbesartan were discussed.Results:Compared with VMC-control group, the mortality matched Irbesartan group, CS-large group showed significant decrease, while the parameters of heart function were improved (P<0.01), but the parameters is not normal yet (P<0.01). Compared with VMC-control group, the level of Ang II in serum was significantly inhibited by CS (P<0.01). But Irbesartan seemed not act(P>0.05). Compared with VMC-control group, Irbesartan group and CS-large group have lower parameters of CVF(p<0.05). The effect of CS large group is better than the effects of Irbesartan group and CS-smallgroup (p<0.05). Compared with VMC-control group, the expressions of TGF-β1and periostin were attenuated in Irbesartan group and CS-large group (p<0.05). There was no evident difference between CS-small group and VMC-control group(P>0.05). Compared with VMC-control group ,the expression of collagen I was decreased in Irbesartan group, CS-large group and CS-small group (p<0.05). AT the same time, the expression of collagen III was decreased in CS-large group and CS-small group (p<0.05).Conclusions: periostin play an important role in the process of viral myocarditis. CS can offer some protection to myocardial fibrosis in viral myocarditis mice . The inhibition of the expression of periostin may be one of the mechanisms .