Effect Of Sanguinarine On Cell Viability And Apoptosis Of Rat Intestinal Smooth Muscle Cells

Sanguinarine (Sanguinarine, SA), belongs to benzylisoquinoline alkaloids, derived from the root of Sanguinaria canadendid and some poppy fumaria species, which has been shown to possess antimicrobial, anti-inflammatory, anticancer and antitumor pharmacological effects. The studies on the anticancer mechanisms of SA were gradually developed, it is generally accepted that its anticancer effects were caused by the induction of apoptosis in different pathways. Based on this, we selected the rat intestinal smooth muscle cells (ISMCs) in vitro as a cell model, and discussed the effect on cell viability and apoptosis, providing reference for further study on SA.1. Take the SD fetal rats as sources of tissue, we isolated and purified the rat intestinal smooth muscle cells (ISMCs) with type Ⅱ collagenase digestion method, and identified the ISMCs in morphology and immunohistochemistry methods. The results demonstrated that, the ISMCs was successfully cultured by the enzymic digestion method and growed in the "peak and valley" mode under microscope, with the big cell nucleus located in the central of the cells; Immunohistochemistry Assay announced strong positive expression of a-actin, cell purity reached more than90%, and there had no abnormal changes in cell morphological features.2. We drawned growth curve of ISMCs by means of MTT colorimetry; then treated ISMCs with0.1,0.25,0.5,1,2,5,10,20,40,80,100μmol/L final concentrations of sanguinarine in12h,24h and48h, and detected the effects on proliferation of ISMCs. The results showed that growth curve of ISMCs is similar with general cells, shaped like a "S", and in the third day after seeding ISMCs entered into the logarithmic growth phase; meanwhile, in the cell proliferation assay, SA treatment was found to result in a dose-dependent decrease in the viability of ISMCs albeit at different levels because saguinarine-mediated loss of viability occurred at5μmol/L dose and was much more pronounced in48h.3. To determinate the oxidative damage, we measured ROS and MDA content after treating with SA; and also explored the possibility of the induction of apotosis by SA combining with the DNA Ladder assay and flow cytometry. The results showed that SA treatment induced a dose-dependent increase in intracellular ROS and MDA content, and increase of ROS could be inhibited by antioxidants GSH and NAC; DNA Ladder assay demonstrated that compared to vehicle-treated control, SA treatment did not result in the formation of DNA Ladder, but Flow cytometry showed that the high concentration of SA could resulted in apoptosis.