| Diarrhea,as a common clinical symptoms,is often caused by various infectious and non-infective factors.Diarrhea mainly refers to the frequency of increased stool than usual habits,thin feces and increased water content.Diarrhea generally defecates more than 200 g a day,or contains undigested food or pus blood,mucus,which usually accompanied by defecation urgency,anal discomfort,incontinence and other symptoms.At present,western medicine clinical treatment usually adopts the symptomatic and etiological treatment,the rehydration corrects the electrolyte disorder and uses the antibiotic to suppress the intestinal infection.Long-term use of antibiotics is prone to drug resistance,and the abuse of antibiotics may also lead to intestinal flora disorders,causing iatrogenic diarrhea.Traditional Chinese medicine has its unique advantages in the treatment of acute and chronic diarrhea,at the same time,medication safety and curative effect are also better.The Mongolian oak bark,the bark of the oak,neutral in nature and tastes bitter and astringent.There are records in Jilin Chinese Herbal Medicine and National Compilation of Chinese Herbal Medicine: oak bark has the effect of clearing heat and detoxifying,eliminating swelling and dampness,treating bacillary dysentery,acute gastroenteritis,dyspepsia in children,jaundice,acute and chronic bronchitis and other diseases.In this paper,the drug efficacy and pharmacological mechanism of oak bark in the treatment of diarrheal diseases was studied based on two parts: in vivo experiment and cell experiment.Firstly,the rat diarrhea model was established by perfusion of senna leaves,and the effect of oak bark on the treatment of rats and colon motility index and histopat hology were observed,and the colon detected for AQP3 and AQP4 protein expression.Secondly,the cells were stimulated and modeled using the tool drugs acetylcholine and atropine after screening the appropriate drug concentration by in vitro culture of the rat intestine cajal interstitial cells.Thirdly,the protein expression contents of cAMP,PKA and CREB and p CREB in tissues were determined by molecular biology technology using the cAMP-PKA cell signaling pathway.To investigate the regulatory effect of Mongolian oak bark on cAMP-PKA pathway in rat colon and cajal interstitial cells,and to explore whether the pharmacological mechanism of oak bark is related to cAMP-PKA cell signaling pathway and regulation of downstream aquaporins.Part One Study on pharmacodynamics of Mongolian oak bark on antidiarrheal effectObjective: To observer the effect of oak bark decoction on diarrhea index and colonic motor function in rats,and to evaluate the effects of oak bark.Methods: 1.SD rats grouping and modeling: 60 SD rats divided into 6 groups randomly,each group have 10 rats.The six groups are: NC group,model group,PC group,low-dose oak bark group,middle-dose oak bark group and high-dose oak bark group.Except the normal group was fed with saline water,the other five groups were fed with Senna every day to replicate the diarrhea model for one week.2.Pharmacodynamic observation: After the preparation of the model,each treatment group was treated with oral administration of drugs,while the blank group and model group were treated with normal saline.The general behavior and status of rats in the model group and treatment group were observed,and the diarrhea of rats was recorded.Finally,the diarrhea index within 6 hours was used as the evaluation index.3.Data acquisition of colonic pressure in rats:The colonic motility curve of rats in each group was recorded by four-channel physiological recorder,and the colonic motility index in random 5min was calculated and the mean value was taken.4.Make colonic tissue sections :The pathological sections of colonic tissue of rats by HE,and the pathological changes of colonic tissue of rats in each group were observed.Results: 1.Modeling results of Senna leaves: After continuous intragastric administration of Senna for a week,compared with the blank group,the diarrhea rate of the model rats reached 100%,yellow thin stool attachment could be seen around the anus,the coat color was slightly dark,and the rats has no obvious change,it mean the model was successfully prepared.2.Effect of oak bark on diarrhea index of model rats: After treatment,the number of defecation and diarrhea index of model rats decreased compared with those of simple model rats.There was significant difference between the positive control group and the middle and high dose groups(P< 0.01).The effect of high dose was the best,and the decreasing trend of low dose was less than that of other treatment groups.3.Effect of oak bark on colonic motility index of model rats: after analyzing the data,it was found that the colonic index of rats after successful modeling was significantly higher than that of the normal group(P<0.05).After treatment,the colonic motility index of rats in the positive control group,middle and high dose oak bark groups decreased compared with the model group(P<0.05),especially in the high dose group(P<0.01).There was no significant difference in low dose group(P >0.05).4.Effect of oak bark on intestinal pathological changes in model rats:By observing the HE staining sections of colonic tissue in each group under light microscope,it was found that the structure of intestinal mucosa in normal blank group was intact,the cells were arranged neatly and smoothly,but there was mild hyperemia and edema in colonic mucosa of rats after modeling.In the experiment,after the treatment with oak bark decoction,compared with the model group,the colonic tissue hyperemia and edema of rats were improved to some extent,and the mucosal tissue was intact.Conclusion:The diarrhea model of rats was successfully reproduced by senna leaf,and the therapeutic effect of oak bark on diarrhea was verified.Part Two Study on the mechanism of treatment of diarrhea with Mongolian oak barkObjective: The mechanism of anti diarrhea effect of oak bark was explored by detecting the activation of mast cells,the number of ICC cells,the expression of AQP3,AQP4,CREB and p CREB,the level of cAMP and PKA.Methods: 1.The sections of rat colonic tissue were stained with toluidine blue,and the degranulation of colonic mast cells in each group was observed.2.Immunohistochemical and WB techniques were used to text the positive expression and protein expression of AQP3 and AQP4 in colon tissue.ICC cells in the colon were labeled and counted by c-kit antibody.3.Culturing the cells of ICC and Screening the concentration of oak bark by CCK8 method.Divided cells into normal group,low,middle and high dose groups of oak bark,and then treated with atropine and acetylcholine respectively.4.The protein expression levels of CREB and p CREB in cajal interstitial cells were measured by WB method and the gray value was analyzed.5.The contents of cAMP and PKA in cajal interstitial cells were detected by Elisa method.Results: 1.Toluidine blue staining results: After the establishment of the model,the degranulation rate of mast cells increased obviously,and decreased after the intervention of treatment drugs,and the degranulation rate of oak bark in middle and high dose groups decreased significantly(P < 0.01).2.Immunohistochemical results: 2.1 The effect of oak bark on the number of ICC in colon tissue: the number of ICC diarrhea model group was obviously less than that of the NC group.After treatment,the expression of ICC in the positive control group and the middle and high dose groups of oak bark increased significantly,the results were statistically significant(P<0.05),but there was no significant difference between the low dose group and the model group(P > 0.05).2.2 The effect of oak bark on AQP3 expression in colon tissue:the positive expression of AQP3 in the model group was less than that in the blank group.After treatment,the positive expression in the middle and high dose groups of oak bark increased significantly(P < 0.05),but there was no significant difference between the low dose group and the model group(P > 0.05).2.3 The effect of oak bark on AQP4 expression in colon: the positive expression in model group was higher than that in blank group,and the positive expression in each treatment group was lower than that in model group(P < 0.05).The positive expression of oak bark in high dose group was the lowest.3.WB experimental results: 3.1 AQP3 protein expression level in tissues: the protein expression level in the model group was obviously lower than that in the NC group,and the protein expression level in the three doses of treatment group increased(P < 0.05),among which the middle dose of histone was the most.3.2 AQP4 protein expression level in tissues: the protein expression level of AQP4 in tissue: after modeling,compared with the blank group,the expression level of AQP4 protein increased significantly,and the protein expression level of AQP4 in each treatment group decreased,among which the positive control group and the middle and high dose groups of oak bark decreased significantly compared with the model group(P < 0.05).There was no significant difference between the low dose group and the model group(P > 0.05).3.3 The expression level of CREB in cajal interstitial cells: compared with the normal group,the expression level of CREB in the atropine group and the acetylcholine group decreased,and there was no obvious difference between the oak bark group and the normal group(P > 0.05).In the atropine group,with the increase of the dose of oak bark,the expression level decreased,but acetylcholine had no obvious trend.3.4 The expression level of p CREB in ICC group: compared with the normal group,the expression level of atropine group decreased,the acetylcholine group also decreased,and there was no significant difference between the treatment group and the normal group(P < 0.05),the expression level of atropine group also decreased with the increase of the dose of oak bark,and the low and middle dose group was statistically significant.No obvious trend in acetylcholine group(P > 0.05).4.Elisa experimental results:The trend chart of cAMP and PKA expression level is consistent,although it is not obvious according to statistics(P > 0.05),but through the histogram,it was found that the expression level of cAMP and PKA increased with the increase of dose in the oak bark group,while in the atropine group,with the increase of the dose of oak bark,the expression standard of cAMP and PKA decreased,which was opposite to that of the acetylcholine group.Conclusion: The Chinese medicine Mongolian oak bark has a certain therapeutic effect on diarrhea,and its therapeutic mechanism may be related to regulation of downstream CREB phosphorylation and AQP3,AQP4 protein expression through cAMP-PKA signal pathway. |
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