| Betelnut is a traditional Chinese medicine, used for expelling intestinal parasites,antibacterial and curing digestive system diseases. Arecoline hydrobromide is a kind of M-receptor agonist extracted from betelnut. Arecoline hydrobromide combines with the receptor on cell membrane, and increases the concentration of calcium ion by opening the ion channel, to promote the motion of small intestines. The unusual regulation of ion channel will lead to malfunction of gastrointestinal motility. So, it's very important to study the regulation of Arecoline hydrobromide to ion channel of smooth muscle. This experiment will study the change of ion channel mRNA at the level of gene, it will analyze the effect of Arecoline hydrobromide to the smooth muscle cells in molecular further.ObjectiveThis topic uses the method of molecular biology and studys the regulation of Arecoline hydrobromide to the rat smooth mucle cell. According to the differences of molecular level and the expression of mRNA, it discusses the drug effects on the cell in molecular biology mechanism. It proves the promotion of the drug to the colon in molecular biology level, and it provides new method to explain information translation in cells and the regulation of gastrointestinal motility, so it has great meaning in clinical use.Methods1. Intragastric administration in rats:thirty healthy female Vista rats were randomly divided into four groups:control group (n=6) was fed with normal diet and intragastric administration with physiological saline; experimental groups were divided to three groups(n=8 each),given Arecoline hydrobromide with high,middle,low concentration for three weeks.All rats were free to drink,eat and feed in coops in animal experimental lab,where temperature was 20～25℃, light and dark were equally 12 hours.2.RT-PCR:extract mRNA of colon muscle, wash with physiological saline. Reverse Transcription and Polymerase Chain Reaction(RT-PCR),then electrophoresis in buffer solution.3.Image Acquisition and Analysis Software analyze the result of different groups.Results1.Calcium-activated K channel:Control group has no obvious difference with the experimental groups (P>0.05), the differences between experimental groups exhibited no difference (P>0.05)2.Voltage-gated Na channel:Control group has no obvious difference with the experimental groups (P>0.05), the differences between experimental groups exhibited no difference (P>0.05)3.L-type Ca channel:electrophoretic band in experimental groups were lighter than control group, and it increases obviously, control group has obvious difference with the experimental groups (P<0.01);control group has unconspicuous differences with the low consistency group (P>0.05), high consistency and middle consistency experimental groups exhibited difference with control group and low consistency group (P<0.05), but middle group has unconspicuous differences with the high consistency group (P>0.05)Conclusion1. Intragastric administration in rats is an easy way to increase the concentration of the drug,it has little hurt and stimulation to rats, and there's no obvious untoward effect. 2.Detection of mRNA is a way to study the expression the gene at the level of molecular biology. RT-PCR(Reverse Transcription and Polymerase Chain Reaction) has great sensibility,specificity and it's easy to carry out,so it can be used to detect mRNA.3. High and middle consistency Arecoline hydrobromide can increase the expression of mRNA of L-type Ca channel, but different consistency have no obvious action to potassium and sodium channel.4. L-type Ca channel has close relation with cell contraction,according to the PCR result, so we speculate the promotion of the Arecoline hydrobromide to the colon has related to the L-type Ca channel. |
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