Experimental Research Of Cyclovirobuxine D On Rat Ileum In Vivo And Its Mechanisms Exploration

Objective:To explore the effects of cyclovirobuxine D(CVB-D) on rats ileum in vivo.To investigate the effective mechanisms from the level of cells.Methods:1.To observe the effects of CVB-D on the contractive amplitude and frequency of ileum with BL-410 biological function experimental system,various concentration of CVB-D were intraduodenally given.Then nifedipine(calcium channel blocker) was given to explore its mechanism primely.2.Rats ileal myocytes were isolated and cultured using mixed enzyme method (collagenase,pronase,bovine serum albumin),growing curve of primary culture cells was drew.Immunocytochemical stain with specific smooth muscleα-actin antibody was applied to identify the primary culture cells.3.To sthdy the effects of CVB-D on proliferation of ileal myocytes with MTT method.To observe the toxic effects of CVB-D on ileal myocytes with LDH method.4.Using calcium sensitive fluorescence indicator Fura-2,the calcium concentration in isolated ileal myocytes was determined to investigate the effective mechanisms from the level of cells.Results:1.After 110 minutes,the contractive amplitude of rats ileum in administration groups increased,but after pretreatment with calcium channel blocker-nifedipine, the above effects could be antagonized(P＜0.05).But the effects of CVB-D on contractive frequency of ileum were not marked(P＞0.05).2.Freshly isolated cells mostly were spindle-shaped with different lengths or ellipse -shaped.In primary culture ileal myocytes attached to the culture vessels after 48 hours,proliferated after 3-5 days and reached confluence after 10 days with a 'hill-and-valley' pattern.Ileal myocytes could be passaged when cells grew to single layer compactly.The positive effect by immunocytochemical stain was irregular shape with brown plasm and blue nuclei.It turned out that almost all cells had those characters.3.Using MTT method,the low and middle dosage CVB-D significantly promoted cells proliferation(P＜0.01),while the ligh group had suppressing effeet(P＜0.05).Using LDH method,compared with control group,the low and middle dosage CVB-D significantly decreased LDH level which indicated it could protect cells.(P＜0.01).The high dosage had high LDH level which indicated that over high dosage CVB-D may injur cells or promot apoptosis.4.Resting[Ca²⁺]_i in ileal myocytes under extracellular calcium concentrations 1.5mmol/L could be concentration-dependently increased by CVB-D,and verapamil could inhibit the[Ca²⁺]_i elevation induced by CVB-D,inhibition ratio was 17.9%.Under extracellular calcium concentrations Oretool/L,CVB-D also could induce[Ca²⁺]_i increasing,inhibition ratio of Procaine was 29.2%.Conclusion:CVB-D can strengthen contractive amplitude of rats ileum in vivo,and the mechanisms may be related to increasing[Ca²⁺]_i of ileal myocytes,which mainly come from influx of extracellular calcium by voltage-dependent calcium channel and mobilization of ryanodine-sensitive intracellular stored calcium.