| Object(1)The model of diarrhea-predominant Irritable Bowel Syndrome(IBS-D)was induced by chronic restraint stress with a single factor,and was evaluated by stool condition,visceral sensitivity and weight change.The dynamic observation of excitability neurons in the proximal and distal colon longitudinal muscle plexus(longitudinal muscle myenteric plexus,LMMP)at days 14,21,28 after the onset of the IBS-D rat model was conducted to investigate the changes in the number,morphology,and expression of excitatory neurotransmitter-related proteins in the IBS-D rat model induced by chronic restraint stress.(2)To observe the effect of patchouli alcohol(patchouli alcohol,PA)on excitatory neurons LMMP proximal and distal colon in IBS-D rats.To explore the effect of PA on the number and morphology of excitatory neurons in colon and PA on the expression of excitatory neurotransmitter related proteins in rats.The effects of PA on the expression of colon LMMP Myosin VA was observed.(3)The primary rat colon neurons were isolated and cultured to observe the colocalization of Myosin Va and excitatory neurotransmitter related proteins on the neurons,and the colon primary neurons were cultured for Myosin Va knockdown,and the effect of knockdown was verified.To explore the effect of PA administration on Myosin Va regulation related proteins.Methods(1)The study of IBS-D rat model replication and LMMP excitatory neuron:42 SPF male Sprague-Dawley(SD)rats were divided into control group and model group.Model group was restrained at the same time for 2h every day for 14 days.To observe and record the changes of body weight and the fecal area of 4h of rats.In D14,D21 and D28 measurement of abdominal wall retraction reflex(abdominal withdrawal reflex,AWR)Score,As a model evaluation index.In D14、D21、D28,6 control and 6 model rats,Complete lamina formation LMMP distal and proximal colon,The proportion of positive ChAT and SP neurons detected by immunofluorescence,And LMMP total protein extraction,Immunoprotein imprinting(Western Blot,WB)Detection of excitatory neurotransmitter related protein expression.(Real-time quantitative polymerase chain reaction,qPCR)to determine the expression level ChAT、SP mRNA colon LMMP muscle strips.(2)PA effects on visceral sensitivity and changes in excitability neurons LMMP colon in IBS-D rats:30 rats were divided into blank control group,model group,low PA(5mg/kg),medium(10mg/kg)and high(20mg/kg)dose group,Each group has 6.After 14 days of administration,All-layers were prepared from the distal and proximal colon LMMP each group,The proportion of ChAT positive neurons and SP positive neurons and the location of Myosin Va were detected by immunofluorescence,And LMMP total protein extraction,Immune protein printing method to detect the expression of excitatory.Myosin VA localization was detected by WB method.The expression level of ChAT SP mRNA in colon LMMP muscle was determined by qPCR method.(3)The regulatory effect of PA on Myosin VA in rat colonic neurons:The rat colon was dissected,the longitudinal muscle intermuscular plexus was separated,the muscle strips were digested,the cell suspension was prepared and planted on the cell petri dish.The colocalization of Myosin Va and excitatory neurotransmitter related proteins on neurons was detected by immunofluorescence.The RNA,detection Myosin Va expression level of excitatory neurotransmitter related proteins were extracted and the effects of PA administration(20 uM)on Myosin Va regulation related proteins were investigated.Results(1)Replication and evaluation of IBS-D animal model:AWR score of rats in D14 D28 model group was higher than that in the control group;Compared with the control group,the fecal area in D21 D28 was significantly higher than that in the control group Weight of the rats,compared with control group,model group in D7 D14 D28 decreased obviously.This indicates that the visceral sensitivity and defecation frequency of IBS-D rats increased,which accords with the IBS-D clinical characteristics.(2)Results of IBS-D rat colon LMMP excitatory neurons:compared with control group,D14、D21、D28 area of the proximal colon LMMP SP positive neurons in the model group was significantly higher than that in the control group(P<0.05),The area of LMMP SP positive neurons in the distal colon of rats in D14、D28 model group was significantly higher than that in the control group(P<0.05).Detection of expression of colon LMMP SP in rats by Western blotting,D28 protein expression was upregulated in both distal and proximal colon SP,significantly higher than the control group(P<0.05).At D21、D28,Compared with the control group,The proportion of distal and proximal colon LMMP ChAT positive neurons in the model group was significantly higher than that in the control group(P<0.05).Expression of protein in LMMP ChAT colon of rats,D14、D28 protein expression LMMP ChAT the distal colon,It was found that the model group compared with the control group,significantly upregulated(P<0.05).Effects of PA on LMMP excitatory neurons in IBS-D rats:Compared with the model group,PA area of positive neurons of proximal and distal colon LMMP SP decreased significantly in each dose group(P<0.01(P<0.01),return to normal level;WB test showed that the expression of LMMP SP protein in the proximal and distal colons decreased after PA administration.qPCR showed that the mRNA expression of LMMP ChAT in the distal colon of each group was significantly down-regulated after PA administration(P=0.01,P<0.01 P<0.01),the mRNA expression of PA in the proximal colon of low,medium and high administration groups was significantly down-regulated(P<0.05,P<0.05)PA protein expression of proximal colon LMMP ChAT in rats was significantly down-regulated(P<0.01)compared with that in model group,and the protein expression of distal colon LMMP ChAT in rats was significantly down-regulated(P<0.01).PA expression of distal colon LMMP ChAT mRNA was significantly downregulated in low,medium and high administration groups(P=0.01,P<0.01,and P<0.01).PA expression of distal colon LMMP ChAT mRNA was significantly downregulated in low,medium and high administration groups(P=0.01,P<0.01,P<0.01).(3)Effect of PA on the expression of LMMP Myosin VA in colon of IBS-D rats:immunoblotting test PA 14d after administration,LMMP,of proximal colon in rats Compared to the model group,High protein expression in proximal colon LMMP Myosin Va,A significant difference(P=0.012,P=0.003,P<0.001).PA the middle dose and high dose group compared with the model group,Myosin Va protein expression was significantly upregulated,A significant difference(P=0.003,P=0.016).(4)Effects of PA on the regulation of Myosin VA in colonic neurons of rats:After isolating the rat intestinal neurons,Transfection of Myosin Va,using siRNA reagent For gene knockdown,The expression of Myosin Va and excitatory neurotransmitter related proteins in each group was detected by qPCR method,The results showed that compared with the control group,The relative expression levels of Myosin Va and SP mRNA in the model group decreased,had significant difference(P<0.05).Compared to the model group,The relative Myosin Va mRNA expression levels increased after PA incubation,A significant difference(P=0.01);Compared to the model group,The relative Myosin Va SP expression levels increased after PA incubation,A significant difference was found(P=0.013).ChAT mRNA relative expression data of each group were not statistically different.ConclusionThe IBS-D rat model induced by chronic binding stress was consistent with the characteristics of the change of defecation habit and increased visceral sensitivity of the disease.The neuropathological changes of colon appeared after PA administration,the morphological number of LMMP excitatory neurons in colon of IBS-D rats was changed.PA can up-regulate Myosin VA gene transcription and protein expression levels,and promote Myosin VA transport ChAT and SP neurovesicles.After Myosin VA gene knockdown in rat intestinal neurons,PA administration can increase the expression on Myosin VA gene level,suggesting that PA may increase ChAT and SP nerve vesicle transport through Myosin Va. |
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