Research Between Pharmacological Effects Of Guang-Pheretima And Proliferation Of Rat Airway Smooth Muscle Cell

ObjectiveGuang-Pheretima is widely used in the treatment of asthma in China,it has been recorded in Shennong's Classic of Materia Medica for its excellent anti-inflammation,anti-asthma,thrombolysis,anticonvulsants and diursis activities.Although Guang-Pheretima exerts prominent anti-inflammatory efficacy in clinical use,its pharmacological effects and underlying molecular mechanism has not been studied in-depth.As a consequence,we explored the effect of Guang-Pheretima decoction intervened to Airway smooth muscle proliferation model,for attempting to systematically reveal the anti-asthmatic activities of Guang-Pheretima in molecular mechanism through cells experiments.Finally,we provide relevant theoretical basis and experimental basis for the development of new therapeutic target and clinical rational application.MethodIn the culture of primary airway smooth muscle cells(ASMC)of rat experiment:In a sterile environment,we isolated trachea of female rats and striped connective tissue and fat,cultured and purified primary airway smooth muscle cells(ASMC)by tissue block method.Finally,we identification airway smooth muscle cells(ASMC)by Cell Immunofluorescence Reaction.In the experiment of rat model of airway smooth muscle cell(ASMC)proliferation:histamine was used to sensitize and chakkenge the airway smooth muscle cells,and used thiazole blue(MTT)to investigate the concentration and time range of histopathic proliferation induced by Hist.In the study of the pharmacological effects of Guang-Pheretima intervened to airwy smooth muscle cells(ASMC):Observation by thiazole blue(MTT)assay on various concentrayions of Guang-Pheretima decoction for the influence of the cell vitality,and choosed the suitable concentrations to continue the following experiments.We selected real-time polymerase chain reaction(Q-PCR)and Western Blotting to determined the concentrations of the ERK1/2 mRNA,Cyclindl mRNA and p-ERK1/2 protein to expore the molecular mechanism of the Guang-Pheretima in anti-asthmatic activities.ResultsIn the culture of primary airway smooth muscle cells(ASMC)of rat experiment:we cultivated a stable state and high yield of ASMC primary cells.After 3?4d,we can observe there are some round shuttle small cell climbed out,the growth rate of the ASMC run into logarithmic growth phase during 5 to 6 days and the cell cycle is 6 or 7 days in the In the 25cm culture flask.By using Cell immunofluorescence reaction,we identified there are more than 95%cell is airway smooth muscle cells of rat,which was used as a research object for the follow-up study.In the experiment of rat model of airway smooth muscle cell(ASMC)proliferation:1.0 mol/1 histamine was used to sensitize and challenge the airway smooth muscle cell(ASMC)of the rat,which was cultured between 12 and 24 hours.We found the number of airway smooth muscle cells was increased obviously and constructed the ASMC proliferation model in vitro successful.In the study of the pharmacological effects of Guang-Pheretima intervened to airwy smooth muscle cells(ASMC):we found the number of ASMCs showed a trend of increasing first and then decreasing with the increase of concentrations of Guang-Pheretima,when the concentrations range of 0,01-0.2g/ml,the number of ASMCs were increase(P<0.05)and the concentrations range of 0.2-2g/ml,the number of ASMCs were decreasing(P<0.05).By using real-time polymerase chain reaction(Q-PCR)and Western Blotting,we evaluated the concentrations range of 0.25-lg/ml of Guang-Pheretima can suppress the cell to express ERK1/2 mRNA and Cyclindl mRNA(P<0.05),when the concentration of Guang-Pheretima was lg/ml,it can inhibited the generation of p-ERKl/2 proteins(P<0.05).ConclusionAfter investigated the anti-asthmatic activities and mechanism of Guang-Pheretima by the means of cell experiments,we found Guang-Pheretima can inhibit the proliferation of rat airway smooth muscle cells(ASMC)effectively.Guang-Pheretima can decrease the expression of ERK1/2 mRNA,Cyclindl mRNA and p-ERK1/2 proteon,and inhibit the activity of ERK1/2 pathway to inhibit the proliferation of ASMC.Therefore,this study serve as ascientific basis of healing asthma and other inflammatory diseases with Guang-Pheretima.