| ObjectiveIrritable Bowel Syndrome-Diarrhea(IBS-D),belonging to the category of TCM diarrhea,melancholia and so on,is a chronic functional intestinal disease with a recurrent pain,bloating,diarrhea as the main symptoms.IBS-D is the result from many factors such as the social environment,psychological,intestinal,genetic.According to TCM,liver stagnation and spleen deficiency diarrhea is the main pathogenesis and the most common type of syndrome is the liver stagnation and spleen deficiency.Many patients are always with anxiety and depression and other mental disorders and antidepressants are always used to treat the IBS-D as a adjuvant therapy.Changji' an formula had certain effect on IBS-D.Our research group successfully established the IBS-D rat model of liver stagnation and spleen deficiency by the stimulation of maternal separation,restraint stress and rectum aceticacid irritation.Changji' an prescription can improve the visceral hypersensitivity,diarrhea,regulating nervous-endocrine-immune system related factors,such as the mast cell particles,CD4+/CD8+ and 5-HT,NPY and GABA.BDNF is a kind of active protein that can promote the survival,growth and differentiation of central and peripheral neurons.It plays an important role in many kinds of mental diseases,such as anxiety and depression.A study reported that BDNF was high expression in the colonic mucosa biopsy specimens of the patients with IBS-D.BDNF and abdominal pain severity have good correlation and some researches suggest that BDNF can through the JAK/STAT pathway regulating the GABAARa 1,but the mechanism of BDNF and related factors in IBS-D mechanism is unclear.According to Changji' an prescription' s function on the nervous system of liver stagnation and spleen deficiency IBS-D rats,the project through mutual authentication of animal and cell experiments,explores Changji' an prescription how to improve the liver depression symptoms mechanism of the liver stagnation and spleen deficiency IBS-D rats.By establishing IBS-D animal model with liver-depression and spleen-deficiency,to explore how Changji' an formula improves the liver depression symptoms in IBS-D rats,to observe the effect of Changji' an prescription on BDNF protein and mRNA expression in IBS-D rat serum,hippocampus,colon tissue and to observe the effect of Changji' an prescription on mRNA expression of IBS-D rats' GABAAR? 1?GABAARa 2?TrkB?CREB in hippocampal.Besides,to observe the the regulatory effect of Changji'an prescription on JAK2/STAT3 signal pathway in hippocampus of IBS-D rats and then the mechanism of Changji' an prescription on the improvement of the liver stagnation for IBS-D rats with liver stagnation and spleen deficiency was explored.To screen the conditions of corticosterone induced injury of PC12 cells by cell experiments.In addition,to reveal the specific molecular mechanism of Changji'an prescription to improve "live stagnation" symptom on IBS-D rats with the liver and spleen deficiency,the intervention effect of Changji'an prescription on corticosterone induced PC12 cell injury model in BDNF/JAK2/STAT3/GABAAR pathway.MethodsPart 1.The study of molecular mechanism of Changji' an prescription to improve"live stagnation" symptom on IBS-D rats.1.IBS-D rat model was established by using separation of breast milk,stimulation of acetic acid and constraint of four limbs.All model rats were randomly divided into the model control group,the Pinaverium Bromide group(0.018g·· kg-1),the high dose of Chang ji' an group(33.48g · kg-1),and the low dose of Changji' an group(16.74g · kg-1),six in each group.All medication lasted for 14 successive days.Normal saline was given to another six normal rats in the normal control group.After medication,to detect the IBS-D rat depressive behavior by the sucrose preference test and open field test and to observe the morphological changes of hippocampal neurons with HE staining.2.After medication,the content of BDNF was detected by ELISA.The gene expression of BDNF in hippocampus and colon were detected by real-time fluorescent quantitative PCR.Besides,the protein expression of BDNF in hippocampus and colon were detected by immune-histochemical method and Western Blot method.3.After medication,the mRNA expression of GABAARa 1,GABAARa 2,TrkB and CREB in hippocampus were detected by real-time fluorescent quantitative PCR.4.After medication,the gene expression of JAK2,STAT3,TNF-a and IL6 in hippocampus were detected by real-time fluorescent quantitative PCR.In situ expression of STAT3 protein was detected by immune-histochemical method.The protein expression of JAK2,STAT3 and pSTAT3 were detected by Western Blot method.Part 2.The regulation effect of Changji'an prescription on corticosterone induced PC12 cell injury model of BDNF/JAK2/STAT3/GABAAR1.PC12 cells were cultured and the effects of different inoculation density,incubation time and corticosterone concentration on the proliferation of PC12 cells were detected by CCK8 method.Also,the morphology of cells was observed by microscope.2.Appropriate drug concentration was selected by CCK8 method and was divided into high(5.Omg/ml),medium(2.5mg/ml)and low dose groups(1.25 mg/ml)to intervene corticosterone(200 ? M)induced PC12 cell injury model.The intracellular free Ca2+ concentration was detected by flow cytometry and the concentration of NO was detected by ELIASA.The gene expression of BDNF,JAK2,STAT3,TrkB,CREB,GABAARa 1 and GABAARa 2 were detected by q-PCR.The protein expression of BDNF,JAK2,STAT3 and pStat3 were detected by Western Blot method.ResultsPart 1.The study of molecular mechanism of Changji' an prescription to improve"live stagnation" symptom on IBS-D rats.1.Compared to the normal group,sugar water percentage preference was significantly lower(P<0.05),open field test scores of horizontal movement and vertical movement and residence time in the central cell was significantly reduced(P<0.05)in rats of model group.Compared to the model group,the sugar water percentage preference,open field test scores of horizontal movement and vertical movement and residence time in the central cell was obviously increased in the high dose of Changji' an group and the low dose of Changji' an group.The sugar water percentage preference,open field test scores of horizontal movement and vertical movement and residence time in the central cell showed tendency to ascend in Pinaverium Bromide group but there was significant difference only in the vertical motion score(P<0.05).In the normal control group,the CA1 cells in the hippocampus of the rats showed a clear hierarchical structure,arranged in neat and compact,with the same size,clear cell structure,and the nucleus was round or oval.In the model group,the levels of CA1 cells in the hippocampus of rats were slightly disordered,and the arrangement was not very regular.Each administration group was improved.There was no significant difference in the morphology of hippocampal DG cells in each group.The cells were arranged in a neat and orderly manner and round or oval in shape.The nucleolus was clear and the chromatin distributed evenly.2.Compared to the normal group,the content of BDNF was obviously decreased(P<0.05)in the model group.After the drug treatment,the content of BDNF of each dose group was significantly higher than that of the model group(P<0.05),and there was no significant difference compared with the control group(P>0.05).Compared to the normol group,the mRNA expression of BDNF in hippocampus and colon of IBS-D rats was significantly increased(P<0.05),while the expression of it in each dose group was significantly lower than those in model group(P<0.05).Compared to the normol group,the protein expression of BDNF in hippocampus and colon of IBS-D rats was significantly increased(P<0.05),while the expression of it was significantly lower in the high dose of Chang ji' an group and the low dose of Chang ji' an group compared with the model group(P<0.05).Compared with the model group,the protein expression of BDNF in colon of rats in pinaverium bromide group decreased significantly(P<0.05),while the expression of it decreased in the hippocampus but there was no significant difference(P>0.05).3.Compared with the normal group,the mRNA expression of GABAARa 1,GABAAR a 2 and CREB in the hippocampus of IBS-D model rats was decreased(P<0.05)and the mRNA expression of TrkB was significantly increased(P<0.05).After drug treatment,the mRNA relative expression of GABAARa 1,GABAARa 2 and CREB in high dose of Changji' an group was higher than that in model group(P<0.05)while the mRNA expression of TrkB decreased compared with the model group(P<0.05).The mRNA relative expression of CREB increased while that of TrkB decreased in the low dose of Changji'an group compared with the model group(P<0.05)and the mRNA relative expression of GABAARa 1 and GABAARa 2 showed tendency to ascend but there was no significant difference(P>0.05).In the Pinaverium bromide group,the mRNA relative expression of GABAARa 1,GABAAR a 2 and CREB was higher than that in model group(P<0.05)while that of TrkB decreased compared with the model group(P<0.05).4.Compared with the normal group,the mRNA expression of JAK2,STAT3 and TNF-? in the hippocampus of IBS-D model rats was significantly increased(P<0.05)while IL6 had an upward trend but there was no significant difference(P>0.05).After drug treatment,the mRNA expression of JAK2,STAT3 and TNF-a in the high dose of Changji' an group was lower than that of model group(P<0.05).The mRNA expression of STAT3 and TNF-? in the low dose of Changji' an group was lower than that of model group(P<0.05)and that expression of JAK2 had a tendency to decrease,but the difference was not statistically significant(P>0.05).The mRNA relative expression of STAT3 in Pinaverium bromide group was lower than that of model group(P<0.05),while the expression of TNF-? and JAK2 had a tendency to decrease,but the difference was not statistically significant(P<0.05).Compared with the normal group,the protein expression of JAK2,STAT3 and pSTAT3 in hippocampus of IBS-D model rats was significantly increased(P<0.05).Compared with the model group,in the high dose of Changji'an group,the protein expression of JAK2,STAT3 and pSTAT3 in rat hippocampal decreased(P<0.05),that of JAK2 and STAT3 decreased and the that of pSTAT3 also decreased but no significant difference in the low dose of Changji'an group(P>0.05).The protein expression of JAK2,STAT3 and pSTAT were decreased in the hippocampus of rats in pinaverium bromide group,but no significant difference(P>0.05).Part 2.The regulation effect of Changji'an prescription on corticosterone induced PC12 cell injury model of BDNF/JAK2/STAT3/GABAAR1.The growth of PC12 cells was normal in 72h and the inhibition rate of 400? M of corticosterone on proliferation of PC12 cells was about 61.61%(P<0.05)while most of the cells contract.The inhibition rate of 300? M of corticosterone on proliferation of PC12 cells was about 48.58%and 250? M was about 49.58%(P<0.05)and there are more cells to shrink floating.The inhibition rate of 200? M of corticosterone on proliferation of PC12 cells was about 36.41(P<0.05)while contraction of floating cells were less,most of the cells were spindle shaped and synapse was obvious.The inhibition rate of 100? M of corticosterone on proliferation of PC12 cells was about 15.33%(P>0.05).Most of the cells were spindle shaped,the synapses were obvious,and the individual cells contracted and floated.2.After the injury of PC12 cells induced by corticosterone,the intracellular free Ca2+ concentration and secretion of NO increased significantly(P<0.05)while the high,medium and low dose of changji'an prescription can reduce the concentration of Ca2+ and the secretion of NO(P<0.05).The mRNA expression of BDNF,TrkB,JAK2 and STAT3 in the model group was increased,the expression of GABAARa 1 and GABAARa 2 was decreased and there was no significant change of the mRNA expression of CREB(P>0.05).Compared with the model group,the mRNA expression of BDNF,TrkB,JAK2 and STAT3 decreased(P<0.05)while the expression of GABAARa 1 and GABA,ARa 2 was increased(P<0.05)in the high dose of changji'an prescription.The mRNA expression of TrkB,JAK2 and STAT3 decreased(P<0.05)while the expression of GABAARa 1 and GABAARa 2 increased(P<0.05)in the middle dose of changji' an prescription.The mRNA expression of TrkB,JAK2 and STAT3 decreased(P<0.05)while the expression of GABAARa 1 increased(P<0.05).Compared with the normal control group,the protein expression of BDNF,JAK2,STAT3 and pSTAT3 in model group increased significantly(P<0.05).Compared with the model group,the protein expression of BDNF,JAK2,STAT3 and pSTAT3 in the high,medium and low dose of Changji'an prescription group was decrease(P<0.05).Conclusion1.It' s proved that the rat model which was established by the method of three factors fits the characteristics of IBS-D of liver-depression and spleen deficiency,obvious anxiety and depression like behaviors.Changji' an prescription has obvious effect on IBS-D of liver stagnation symptoms.The expression of BDNF was abnormal in serum,hippocampus and colon of liver and spleen deficiency IBS-D while Changji' an prescription can increase the expression of BDNF in serum and reduce the expression of BDNF in the hippocampus and the colon.At the same time,Changji' an prescription can increase the mRNA expression of GABAAR? 1,GABAARa 2 and CREB in hippocampal of liver and spleen deficiency IBS-D rats and decrease the mRNA expression of TrkB.In the hippocampus of IBS-D rats,the JAK2/STAT3 pathway is activated and Changji' an prescription can inhibit the pathway activity.Also,Changji'an prescription can decrease the level of BDNF in hippocampus,inhibit the activity of JAK2/STAT3 pathway and decrease the expression of GABAARa 1 and GABAARa 2.This is probably the mechanism of Changji'an prescription to improve the IBS-D of"liver stagnation".2.Changji' an prescription can inhibit the Ca2+ concentration in PC12 cells which induced damage by the corticosterone and inhibit the secretion of NO.It can decrease the expression of BDNF and TrkB in PC12 cells induced by corticosterone,inhibit the JAK2/STAT3 signaling pathway,and increase the expression level of GABAARa 1 and GABAARa 2. |
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