| Object Based on the effect of PKA/cAMP in DRG neurons on the regulation of colonic smooth muscle Ca2+gating channels,the possible mechanism of Shugan Jianpi Recipe in treating irritable bowel syndrome was discussed.Methods?1?According to the randomized controlled study design method,Thirty-six SPF male Sprague-Dawley rats were randomly divided into6 groups according to body weight,6 in each group,ie normal control group;medium,low,medium and high dose Chinese medicine group?western medicine group and irritable bowel syndrome model group.After 2 weeks of restraint-stress-capsaicin simulation,the Chinese medicine group was given low,medium and high doses of the stomach,and the western medicine group was given pivalvonium orally,and the model group was given equal dose of normal saline.All rats were sacrificed at the weekend,and the colonic smooth muscle tension was measured by removing the descending colon about 2 cm and fixing it to a tension transducer.?2?According to the serum pharmacology method,the serum containing serum of Shugan Jianpi Recipe was prepared.The rat colonic smooth muscle cells were isolated and cultured.The effects of different concentrations of Shugan Jianpi Recipe on colonic smooth muscle cells were detected by MTT method.Flow cytometry and Fluo-3AM fluorescent probe were used to detect changes in Ca2+content in rat colonic smooth muscle cells.?3?The rat DRG?Dorsal root ganglion,DRG?cells were isolated and primary cultured,and the cells were identified by immunofluorescence.The DRG cells were grouped by capsaicin?CAP?and then subgrouped?ie low,medium and high dose groups?.The serum and serum were used to detect the expression of PKA/cAMP in rat DRG neurons by RT-PCR and Western-blot.Results?1?The rat model of IBS-D was made by restraint stress-pyrumol model,and the rat colon smooth muscle strips were prepared.There was no significant difference in the muscle tension of colon smooth muscle between the groups.After the addition of acetylcholine,the muscle strip contraction frequency increased,the peak increased,the contraction increased,and there was no statistical difference.After adding 1mg/ml,3 mg/ml,10mg/ml,20 mg/ml Shugan Jianpi,the frequency of muscle strip contraction decreased and the peak value increased.There was a statistical difference between before and after treatment?P<0.05=?.?2?The rat colonic smooth muscle cells were successfully isolated.The morphological cells of colon smooth muscle cells were elliptical in shape and bundled,and the dense and sparse areas were interlaced into a"peak-to-valley"shape.The MTT assay selected 1.56%,0.78%,and 0.39%as the optimal concentration for calcium ion detection.The quantitative results showed no significant difference between the groups?P>0.05?,while flow cytometry showed that the rat colon smooth muscle cells Ca2+The concentration was significantly lower than that of the low,medium and high dose groups.With the increase of drug concentration,the intracellular calcium concentration in the low,medium and high dose groups gradually decreased,and the difference was statistically significant?P<0.05?.?3?The rat DRG cells were successfully isolated and confirmed by cell identification.The DRG cells were successfully isolated.The DRG cells were round in shape,with small cell bodies and long processes.The expression of PKA mRNA and protein in DRG neurons of each model group were low.In the normal control group,and with the increase of concentration,the difference was statistically significant?P<0.05?.The mRNA and protein expression of cAMP in the high dose group was lower than that in the normal group?P<0.05?.There was no difference between the low and middle dose groups?P>0.05?.Conclusion Shugan Jianpi Recipe can down-regulate the expression of cAMP,decrease the activation of PKA,inhibit the influx of Ca2+,and inhibit the contraction of colonic smooth muscle,thereby exerting the effect of preventing irritable bowel syndrome. |
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