Upregulated Expression Of IGF-IIF In The Myocardium Of Mice With Viral Myocarditis

0bjective Establish a murine viral myocarditis(VMC) model and then investigate the dynamic expression of insulin-like growth factor II receptor(IGF-IIR) protein and m RNA in the myocardium of mice with VMC at different phases.Combined with cardiac pathology integration, we study the relationship between IGF-IIR with myocardium injury and further more explore the possible mechanisms of IGF-IIR involved in VMC. This may provide new therapeutic targets for further prevention of VMC.Methods The 120 4-week-old male BALB/c mice were randomly divided into two groups.There were 80 in the experimental group(VMC group) and 40 in the control group. To establish VMC models, the mice of VMC group were inoculated intraperitoneally with DMEM solution containing coxsackie virus B3(CVB3). The control group received the same amount of virus-free DMEM solution. Ten mice of each group were sacrificed on days 3,7,14 and 28 after virus inoculation,then their heart tissues were cut along the longitudinal section and collected for analysis. Part of the tissues were fixed with formalin, dehydrated, embedded in paraffin and sliced.Myocardial histopathology was observed by hematoxylin and eosin staining(HE) and scored.The levels of myocardial IGF-IIR protein was detected by immunohistochemistry(IHC).The other part of the tissues were stored in the sterile vials without enzyme, throwed into the liguid nitrogen tank quickly and thansfered to the freezer with 80 degrees below zero. The myocardial IGF-IIR m RNA relative expression was detected by semi-quantitative RT-PCR, Analyzing the dynamic expression levels of IGF-IIR and its correlation with the degree of myocardial lesions.Results 1、The VMC group of mice infected with CVB3 were listless, lack of exercise, hair removal and so on gradually. The mortality rate is 23.75%.We can see the whole heart was edematous and there some necrosis in the outer membrane of the murine cardiac anatomy. We can see the myocardial cells swelling, degeneration, necrosis, fracture, nuclear condensation and focal inflammatory cell infiltration by HE staining. In the other hand the mice of the normal control group showed a normal spirit,diet, activity and there is no death. The myocardial fibers were arranged in neat rows and did not show any pathological changes by HE staining. The score of the cardiac pathology integral of VMC group mice in 3,7,14,28 days after viral inoculation were respectively(1.10 ± 0.568),(2.60 ± 0.699),(1.40 ± 0.516),(0.60 ± 0.516), and the differences were statistically significant compared to the control group(P <0.001);2、The amount of IGF-IIR protein was observed under a microscope by the IHC method. We can see very small amount of brown particle deposition in the cytoplasm and membrane of the control group mice. But the positive brown particles was increased significantly and colored deeper at each time point of the VMC myocardial after virus inoculation. The semi-quantitative by measuring the average optical density showed that the expression of myocardial IGF-IIR protein in VMC group began to increase gradually after the first three days, reach the peak after seven days, then began to decline and remained at a relatively high level all the time. The expression of the IGF-IIR protein of VMC group mice in 3,7,14,28 days after viral inoculation were respectively(0.257±0.0191),(0.396±0.0225),(0.292±0.0132),(0.288±0.0136), and the differences were statistically significant compared to the control group(P <0.001);3、The RT-PCR of IGF-IIR m RNA showed that the corresponding lane of the internal control and the target gene can see the DNA fragment clearly. We got the semi-quantitalive analysis results by the ratio of the gray value between the target gene and the internal control gene. The results showed that the expression of myocardial IGF-IIR gene in VMC group began to increase gradually after the first three days of getting viral inoculation, reach the peak after seven days, then began to decline and remained at a relatively high level all the time. The expression level of the IGF-IIR gene of VMC group mice in 3,7,14,28 days after viral inoculation were respectively(0.584±0.0738),(0.754±0.1090),(0.683±0.0995),(0.636±0.0925), and the differences were statistically significant compared to the control group(P <0.001);4、Linear correlation analysis showed that the amount of the IGF-IIR protein levels at different phases measured by IHC was positively correlated with cardiac pathology points(r=0.628,P =0.000)The amount of the IGF-IIR m RNA gene expression levels at different phases measured by RT-PCR was positively correlated with cardiac pathology points(r=0.413,P =0.008). The amount of the IGF-IIR protein levels measured by IHC was positively correlated with the amount of the IGF-IIR m RNA gene expression levels measured by RT-PCR(r=0.465,P =0.003).Conclusion The upregulated expression of IGF-IIR may play a role in the myocardium injury of VMC.