The Research On Matrine's Inhibition Of Apoptosis Of Myocardiocytes Induced By CVB₃ And Its Signal Transduction Pathway

Background and ObjectiveViral myocarditis (VMC) is one of the most common infective one in cardiovascular diseases, while the acute and severe VMC may cause acute myocardial infarction symptom, heart failure, cardiac shock and even sudden death, and someone develops into dilated cardiomyopathy wich survival rate about five years is around 50%. VMC has been threatening children health severely and lack of dependable therapeutic methods, so it is very important to focus on studying its treatment and medicine development.It is an new way to find a medicine to cure VMC in Chinese herbal medicine. It has been proved that Matrine generally consists in pulse family, sophora flavescens Ait., S.alopecuroides L, and S. Subprostrata Chun et T. Chen. It has been report that Matrine has antiviral activity in vitro but also shown therapeutical effects on Balb/c mice infected by Coxsackie virus B₃(CVB₃)in vivo.The objective of study is through simulating VMC model infected by CVB₃ in vitro and vivo to observe the protect effects of Matrine on myocardiocytes and to approach the action mechanism on VMC infected by CVB₃ of Ca²⁺,Ca²⁺-relied calpain-2, cysteine proteinase caspase-12 and (PI3K)/Akt. The therapeutic effects of Matrine have be investigated through apoptosis and apoptosis pathway in order to provide an experimental base to the safe, reasonable and target application in clinic. Methods1 ObservationCells:Construct VMC model in vitro by infecting myocardiocytes of newborn Wistar rat. Groups: negative control (NC), virus group (VK), Ribavirin (RBV), Matrine high does treatment (600mg·L^-1), midst does treatment (300mg·L^-1), lower does treatment (150mg·L^-1), lowest does treatment (75 mg·L^-1). They were also divided into three kinds of style which are simultaneous medicine and virus, medicine before and after virus in vitro. Cell survival rate was measured by MTT, and observe the protection from cytopathic effects (CPE), percentage of beat cells (PBC) and creatine kinase isoenzyme MB (CK-MB).Animals:Similarly, VMC Balb/c also were divided into different groups to detect survival rate, the level of CK-MB in serum, TCID50, histopathology changes.2 Investigation on mechanism:Cells: Further, detecting apoptosis rate by Annexin V/PI through FACS, measuring [Ca²⁺]i by laser confocal microscopy and Fluo-3/AM fluorescent probe in vitro. Having detected protein expression of calpain-2,Phospho-Akt^Ser-473 by immunocytochemical method and Western blotting, and measuring caspase-12 activity on cleavage of specific substrate ATAD-AFC through fluorometer to search into the anti-apoptosis mechanism of Matrine on myocardiocytes infected by CVB₃.Animals: While in vivo, detecting apoptosis with TUNEL kit. Have detected protein expression of calpain-2,Phospho-Akt^Ser-473 by immunohistochemical method and Western blotting, and caspase-12 activity on cleavage of specific substrate through fluorometer to search into the anti-apoptosis mechanism of Matrine on VMC Balb/c.Results1 Results of observationCells:By three kinds of style, simultaneous medicine and virus, medicine before and after virus about 1.5h in vitro, the replication of virus be reduced by Matrine; it can significantly be observed that Matrine could increase PBC, reduce CPE and CK-MB of myocardiocytes.Animals:Survival rate can be significantly increased in Matrine groups with VMC. In the groups of Matrine treatment, TCID50 and histopathology changes in VMC mice myocardium were reduced. Every group of Matrine all can reduce the level of CK-MB in serum, but difference exists by comparing with NC.2 Investigation on mechanism:Apoptosis level of cells:Apoptosis of myocardiocytes can be inhibited by Matrine significantly (the apoptosis rate of VK is 80.3±5.8%, while that of treatment groups is 52.9±5.5% at 36h, P<0.05). Compared with NC, Fluorescence intensity of Ca²⁺ in VK was increased. Matrine can suppress the increase of fluorescence intensity of Ca²⁺ and that result from KCl. Some of protein expression in VK began at 24h, calpain-2 increased remarkably at 36h. The expressed calpain-2 protein could be inhibited by Matrine. Phospho-Akt^Ser-473 is highly expressed in medicine groups, which was higher at 24h than 36h. But PI3K inhibitor could reverse the enhancement action of Matrine. Activity of caspase-12 was increased at 24h, at summit at 36h, and decreased at 48h, compared with NC there was obvious difference(P<0.05). Matrine could suppress the activity of caspase12 compared with VK(P<0.05), but which has statistical significance compared with NC (P<0.05).Apoptosis level of animals: scattered apoptotic cells appeared in the first five days in VK. A large number of apoptotic cells can be observed in the 10th day and the apoptosis lesions are mainly concentrated in the focus of infection and areas surrounded. Matrine can significantly inhibit apoptosis of myocardiocytes of VMC animals. Expression of calpain-2 protein can be detected highly in VK, and while from the 5th day to the first 10 days the expression of calpain-2 went on increasing in VK. Matrine can significantly inhibit calpain-2 expression. Phospho-Akt^Ser-473 highly expressed in drug treatment. In the 5th day a large number of phosphorylation protein of Akt^Ser-473 can be detected, while in the first 10 days it significantly decreased with the increased pathological changes of myocardium. Matrine can promote the expression of Phospho-Akt^Ser-473. In virus group activity of caspase-12 increased in the first 5 days and reached its peak in the first 10 days, in the 15th day the activity of caspase 12 began to drop significantly compared with control group (P<0.05). Matrine can also inhibit caspase-12 activity of myocardium in VMC animals (P<0.05), but higher than CN (P<0.05).Conclusion1. It was proved that matrine could reduce CK-MB of VMC model in vitro and vivo, inhibit cytopathic effect and apoptosis resulted from CVB3, and greatly protect myocardiocytes infected by CVB3.2. The study confirms that calcium overload, calpain-2, caspase-12 participate in the apoptosis process of cardiac muscle cell because of CVB3.3. Matrine can restrain obviously calcium overload, the protein expression of calpain-2 and the activity of caspase-12, but higher than CN.4. Matrine can promote Phospho-Akt^Ser-473 expression, and the PI3K inhibitor can reverse the effects of Matrine which promote the protein expression of Phospho-Akt^Ser-473.According to all above, Matrine inhibit apoptosis of myocardiocytes induced by CVB3 and protect cells through the factors which may be the pathway of endocytoplasmic reticulum about Ca²⁺/Calpain-2/Caspase-12 and PI3K/Akt signal transduction pathway. To our knowledge, this is the first report of such effects of Matrine and caspase-12 on apoptosis in a viral infection model and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.