The Effect Of Salvia Miltiorrhiza On Proliferation And Apoptosis Of The Vascular Smooth Muscle Cells

Objective Along with the development of basic research, people realize that the vascular smooth muscle cell (vascular smooth muscle cell, VSMC) becomes one of the target cells on cardiovascular drug therapy. The excessive reactive oxygen species (reactive oxygen species, ROS)will produce after the local vascular is injuried, which play an important role on stimulating VSMC proliferation. Therefore, effectively blocking the ROS on the VSMC proliferation and apoptosis is one of the most important measures on the prevention of atherosclerosis (atherosclerosis, AS). In recent years, using VSMC as the cell model to screen out lower toxicity and side effects of the drug that effectively inhibit the proliferation and,and studying of its action mechanism, it is one of the medical research hotspots.The effective composition of Salvia miltiorrhiza is mainly its water-soluble substance. It can inhibit of platelet’s adhesion、aggregation、release, increase the serum level of cAMP, inhibit TXA2formation, promote the formation of PGI2, eliminate oxygen free radicals and fight lipid peroxidation. This topic is about using cell culture technique for the observation of Salvin miltiorrhiza’s influence on rats’VSMC proliferat ion and apoptosis. The rats are induced by hydrogen peroxide (H2O2) and other reactive oxygen species (ROS).The observation provides experimental basis for the protection of against H2O2mediated lipid peroxidation injury.Methods Take180-200g Wistar rats’(Huazhong University of Science and Technology animal center of Tongji Medical College provides) thoracic aorta in membrane with tissue explants adherent method and enzyme digestion to culture VSMC, Observe VSMC characteristic " peak" and" Valley" shape to grow by inverted phase contrast microscope. Take3-5generation cultured cells by antialpha SM actin monoclonal antibody identification, cells whose positive rate arrives at more than85%are chosen for experiment. Add hydrogen peroxide (H2O2) to the culture solution to induce the proliferation of rats’VSMC. Determine VSMC proliferation most notably H2O2concentration for experimental concentration by the experiment.Inject with Danshen (lyophilized), through the experimental test to determine the minimum concentration of experiments and the optimal action time. Finally, establish vitro model for H2O2stimulation of aortic vascular smooth muscle cells proliferation; Measure and analyze the proliferation of SMC by MTT and immunohistochemical method with Salvia miltiorrhiza; and measure the Bcl-2and Bax protein expression to further analyse the correlation between smooth muscle cell proliferation and apoptosis. The experimental groups:1) normal control group:normal cultured VSMC;2) H2O2stimulation in the control group;3) H2O2+Salvia low dose group;4) H2O2+Salvia middle dose group;5) H2O2+Salvia high dose group. Do the experiment for proliferation and apoptosis of VSMC. observe and analyse Salvia miltiorrhiza’s effects on VSMC cell cycle phase and apoptosis of the rats that are induced by oxygen species (H2O2)Results:1.After Wistar rat’s thoracic aorta vascular smooth muscle was successfully cultured through primary and passage generation. Take3-5generation cell in logarithmic phase to identify alpha SM act in monoclonal ant ibody. If more than85%are positive, they can be used in the experiment.2. The concentration of H2O2in the0.02-180μM range can promote VSMC proliferation. When the concentration of H2O2is30μM, the proliferation is the most significant. When the concentration exceeds225μM, and compared to negative controls, P<0.01, then it proves that when the H2O2concentration is more than225μM, the cell has certain toxicity, and it can inhibit the proliferation of VSMC. At the time of effect, After36hours’stimulation, the proliferation of VSMC is the most significant; continuous stimulation of6hours, VSMC began to proliferate. Along with the prolongation of time, proliferation is also enhanced;It reaches the strongest till36hours. Then it is comparatively weakened.3. When Salvia concentration is0.4mg/ml, it can inhibit VSMC, with the increase of concentration, inhibition is strengthened stage by stage. After it arrives at the3.2mg/ml, inhibitory effect tends to be stable. After36hours’ stimulation of Danshen, the VSMC inhibition rate reaches51.5%, Aftet that the inhibitory effect of VSMC converges to a steady state.4. Compared with the normal control group, H2O2stimulation control group of bcl-2’s expression increased significantly (P<0.05), the expression of Bax was reduced, but it isn’t statistically significant. Compared with H2O2stimulation control group, Salvia miltiorrhiza middle dose group and high dose group Bax expression increased significantly (P<0.05), while bcl-2expression decreased (P<0.05), Salvia miltiorrhiza in low dose group, expression of Bax increased, bcl-2expression decreased. However, they were not statistically significant.Conclusions:1.When the vascular smooth muscle cells under in vitro conditions, H2O2can promote its proliferation2. Salvia can inhibit the proliferation of vascular smooth muscle cells that is stimulated by H2O2, and can promote its apoptosis3. Salvia inhibits vascular smooth muscle cell’s prol iferat ion and promote its apoptosis. The mechanism may be as follows, Danshen inhibits bcl-2gene and expression, promotes Bax gene and expression, reduces Bcl-2/Bax ratio, thereby it couses the cell proliferation and apoptosis to achieve a normal balance.