The Impact Of Calmodulin-Dependent Protein Kinase Ⅱ Promote Rats Vascular Remodeling By Magnesium Lithospermate

Objective:To inhibit Ca MKⅡ expression level, using magnesium lithospermate intervene in primary cultured rat aortic smooth muscle cells, observation of smooth muscle cells of proliferation and apoptosis, and to explore the relationship of specific Ca MKII and mi RNAs(mi RNA-21, mi RNA-221, mi RNA-223, mi RNA–143 and mi RNA- 145).Methods: The research object is primary cultured aortic smooth muscle cells of SD rat, according to the different drug concentrations, the cells can be divided into four groups: normal group, the high concentration group(100 μmol), the medium concentration group(50μmol) and the low concentration group(25μmol).CCK-8 method is applied to detect the change of the cells proliferation activity. Flow cytometry instrument is used to test the change of cells apoptosis. Using RT-PCR detect Ca MKII and mi RNAs expression change.Results:(1) The RT-PCR test showed that, after magnesium lithospermate intervened in rat aortic smooth muscle cells 72 hours, compared with the normal cells, the expression level of Ca MKII significantly decreased, with significantly difference.( P<0.01)(2)CCK-8 experimental results showed that, after 100 umol magnesium lithospermate intervened in smooth muscle cells 72 h,compared with the normal growth of cells in the group, the cells proliferation activity was significantly inhibited, and with significant difference(P<0.01); the medium and the low concentration groups had different degree trend of inhibition, but with no significant difference(P > 0.05).After 100 umol magnesium lithospermate intervened in smooth muscle cells 96 h, the cells proliferation activity was significantly inhibited, with significant difference(P<0.05);the medium and the low concentration groups have different degree trend of inhibition.After 100 umol magnesium lithospermate intervened in smooth muscle cells 120 h, compared with the normal growth of cells in the group, the cells proliferation activity was significantly inhibited, with significant difference(P<0.05);after 50 umol magnesium lithospermate intervene in smooth muscle cells 120 h, the cells proliferation activity was significantly inhibited, with significant difference(P<0.01);the low concentration group had a tendency to inhibit, but with no significant difference(P > 0.05).(3)The cell counting of Transwell inserts under the microscope were 112.0±2.1(cell),29.0±1.5(high concentration group), the experimental group compared to control group,the migration force significantly decreased(P < 0.05).(4) Flow cytometry experimental result showed that each group’s apoptosis rate was 7.9%(normal group), 11.5%(low concentration group), 13.5%(medium concentration group), 30.8%(high concentration); after magnesium lithospermate intervened smooth muscle cells,the apoptosis rate increased significantly, and with concentration dependence(7.9%vs11.5%;7.9%vs13.5%;7.9%vs30.8%).(5)The RT-PCR test showed that, after inhibiting Ca MKII, mi RNA-21 expression was significantly decreased(P < 0.05); the mi RNA-221 and the mi RNA-223 expression had a downward trend, but compared with the control group, there was no significant difference(P>0.05); the mi RNA-143, the mi RNA-145 expression was significantly increased(P<0.01).Conclusion: The experimental results, Magnesium lithospermate can reduce intracellular Ca MKII expression level, inhibit cells proliferation, and promote apoptosis.mi RNAs may be based on Ca MKII gene regulation, realize its role to promote vascular remodeling. We confirmed in the formation of vascular remodeling, Ca MKII is the one of important target genes of specific mi RNAs.