Study On The Intervention Mechanism Of Chang’an Ⅱ To IBS-D Visceral Hypersensitivity Based On Glu And Its Receptor Regulatory Subunit NR2B

BackgroundIrritable bowel syndrome(IBS)is a common functional bowel disease which is mainly manifested by abdominal pain,abdominal distension and changes in defecation habits.At present,the disease is epidemic worldwide and seriously affects the quality of patients life.Irritable bowel syndrome is divided into four subtypes according to different defecation characters,among which irritable bowel syndrome with predominant(IBS-D)has attracted variants of concern in the research.The pathogenesis of IBS-D has not been fully clarified.With the deepening of research,it is found that low-grade inflammation,intestinal dysbacteriosis,abnormal brain intestinal axis interaction and visceral hypersensitivity plays an important role in the occurrence of the disease,aming which visceral hypersensitivity is considered as the core mechanism of IBS-D.Hyperalgesia and allodynia are the main manifestations of visceral hypersensitivity,which means that subthreshold stimulation can make the painfule of the body more obvious and maintain the painful for a long time.The regulation of neural activity can be effected by many links,and the expression of immunomodulatory receptors in nerve cells and the localization of neuropeptide receptors in immune cells shows that nerve-immune interaction effect plays an important role in regulating body activities to adapt the environment.However,the continuous nerve-immune interaction may cause inappropriate feelings and reactions when external stimuli was disappear.Epidemiological investigation shows that infection is the most important independent risk factor for IBS.Studies related to IBS after infection suggest that abnormal nerve immune interaction effect may be the key link leading to visceral hypersensitivity.As the peripheral nerve with autonomous function,enteric nerve is in direct contact with gastrointestinal tissue and at the front-end of gastrointestinal sensory signal processing.Therefore,the role of enteric nervous system in the occurrence of visceral hypersensitivity should not be underestimated.Glutamate is not only the energy source of some tissues,but also an important neurotransmitter in the body.Studies have confirmed that as the transmitter,glutamate not only exists in the center,but also in the enteric nervous system.Glutamate receptors mainly include metabotropic receptor and ionotropic receptors,among which N-methyl-D-aspartate receptor(NMDAR)has attracted variants of concern in the process of excitatory signal transmission,NMDAR receptor is a complex composed of different subunits,in which N-methyl-D-aspartic acid receptor 2B(NR2B),as a regulatory subunit is involved in regulating the ion channel state of NMDAR receptor.The intracellular Ca2+level will rise up,causing the activation of calcium dependent protein kinase and the corresponding cascade reaction when the ion channel is opened after NMDAR was activited.In addition,as a neuromodulator,glutamate is also regulated by other links,Among them,excitatory amino acid transporter 2(EAAT2)is an important glutamate uptake protein,which is responsible for regulating the concentration of glutamate transmitter in synaptic space.However,whether nervous-immune interaction is related to the activation of glutamate and its ionic receptor NMDAR in the development of visceral hypersensitivity of IBS-D after infection has not been fully clarified.Traditional Chinese medicine classifies IBS-D diseases into the categories of the disease such as "diarrhea","abdominal pain",and attaches importance to regulating the liver and spleen in the treatment.Chang’an No.II formula is an agreed prescription from the Department of Gastroenterology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,which shows the effect of Shugan Jianpi(SGJP),warming middle energizer to stop diarrhea,and it also shows a significant therapeutic effect on IBS-D in clinical,but the specific mechanism is unclear.ObjectiveTo study the role of glutamate and its receptor regulatory subunit NR2B changes in visceral hypersensitivity of IBS-D and the intervention research of Chang’an II formula.Method1.The blood serum of 20 patients with IBS-D liver stagnation and spleen deficiency syndrome and 20 healthy volunteers were collected,the glutamic acid and its main related substances,such as glutamine,arginine,ornithine,proline,histidine,y-aminobutyric acid and polyamine levels in the two groups was detected by High performance chromatography,and NR2B/S-100 β in colon tissues of the two groups were detected by immunofluorescence assays.2.The IBS-D rat model of liver stagnation and spleen deficiency syndrome was established by NMS+ acetic acid enema+unpredictable chronic stimulation.The model was evaluated by comparing the general situation,fecal water content,AWR score,body weight,food intake,sucrose preference test and grasping power of the rats in each group.The rats in the model group were randomly divided into groups and were treated by pinaverium bromide,R025-6981(NR2B Retardant),Chang’an Ⅱformula at high,middle,and low concentrations to intervene and observe the effect of Chang’an Ⅱ formula.3.The protein expressions of NR2B,CaMKII,CREB and BDNF in colon tissue of the rats in each group were detected by Western blot,and the protein expressions of EAAT2,Syn,PSD-95 and c-fos were detected at the same time to clarify the changes of glutamate and glutamine ionotropic receptor NMDAR pathway;meanwhile,the protein expression of NR2B,EAAT2 and SP was detected by immunohistochemistry method,the co-localization of NR2B and NK-1R was detected by immunofluorescence,the changes of enteric nerve synapses in submucosa and muscular layer were observed by transmission electron microscope,and the gene expression of NR2BmRNA,CREBmRNA and BDNFmRNA were detected by RTPCR;4.The levels of blood serum brain-gut peptide SP,BDNF and CGRP were detected by ELISA.The expression level of inflammatory factor IL-1β,TNF-α,IL-4,IL10,NGF,5-HT and NO were detected,the role of nerve-immune interaction in visceral hypersensitivity and the effect of Chang’an Ⅱ formula were observed.Result1.1 The determination of serum glutamate family related substances in IBS-D group and control groupCompared with control group,the level of serum glutamate in IBS-D group was significantly higher(18798.80 ± 4928.62ng/ml vs 13142.38 ± 2898.66ng/ml,P<0.05);The levels of blood serum glutamine,arginine and histidine in IBS-D group were significantly higher than those in control group(94266.75 ± 20119.53ng/ml vs 71284.66±9181.94 ng/ml P<0.05,23209.54±5532.40ng/ml vs 13651.18±3330.26ng/ml P<0.05,17364.39± 3339.06 ng/ml vs 12533.79± 1169.02 ng/ml P<0.05);the concentration of blood serum y-aminobutyric acid in IBS-D group was significantly lower than those in control group(0.87 ± 3.89 ng/ml vs 5.67± 2.78 ng/ml,P<0.05);The blood serum levels of agmatine,putrescine and spermine in IBS-D group were significantly higher than those in control group(0.51 ± 0.21 ng/ml vs 0.29± 0.08 ng/ml,P<0.05;8.00± 3.18 ng/ml vs 5.48 ± 1.53 ng/ml,P<0.05;3.98 ±1.52 ng/ml vs 2.46±0.96 ng/ml,P<0.05).1.2 The immunofluorescence of colonic mucosa in control group and IBS-D groupNR2B/S-100β co-staining was mainly located in submucosa,the expression of NR2B/s-100β in IBS-D group was significantly higher than that in control group;PSD-95/S-100β co-staining was also mainly located in the submucosa,and the expression of PSD-95/s-100β in IBS-D group was lower than that in control group.2.1 The changes after drug interventionThe change of fecal water content:pinaverium bromide group,CHH group and CHM group were significantly lower than those in the model group(P<0.05).Changes of abdominal wall withdrawal reflex:the scores of each medication group were lower than those of the model group when 1.5ml of gas was injected,in which the scores of pinaverium bromide group,Retardant group,CHH group and CHM group were significantly lower than those of in the model group(P<0.05);the scores of each medication group were significantly lower than those of the model group(P<0.05)when 2.0ml of gas was injected.The change of body weight:the body weight of each medication group was higher than that of the model group,and there was significant difference between the pivirium bromide group,CHH group and the model group(P<0.05).Changes of food intake:the food intake of each medication group was higher than that of the model group,and there was significant difference between CHH group,CHM group and the model group(P<0.05).Changes of sugar aqueous preference:the amount of sugar aqueous preference in each-medication group was higher than that in the model group,and there was significant difference between the Retardant group,CHH group and CHM group,as well as the model group(P<0.05).The changes of grasping force:the grasping force of the pivirium bromide group,CHH group was significantly higher than that of the model group(P<0.05).2.2 Histopathological observation and serological results of colonic tissueThere was no significant hyperemia and edema were found in control group,the glands were arranged regularly,the tissue structure of colonic mucosa was complete,the mucosa in the model group was incomplete,and the cell arrangement was disordered.Moreover,compared with the model group,the mucosal structure in the pivirium bromide group,Retardant group and CHH group was recovered completely and the cell arrangement was neat.Blood serum DAO and D-LA:the blood serum DAO and D-LA in the model group were significantly higher than those in control group(P<0.001).The results of each medication group were lower than those in the model group(P<0.05).3.1 Western blot results of the rat colonic tissueCompared with control group,the expression of NR2B,CaMKII,CREB,BDNF and c-fos protein in the model group was increased significantly(P<0.05),and the expression of PSD-P5,Syn and EAAT22 was decreased significantly(P<0.05);Compared with the model group,the expression of NR2B,CaMKII and CREB protein was decreased significantly in each medication group(P<0.05),the expression of BDNF was decreased significantly in other medication groups except CHM group(P<0.05),the expression of c-fos was decreased significantly in other groups except CHL group(P<0.05),the expression of PSD-95 was increased significantly in CHH group(P<0.05),the expression of Syn was increased significantly in Retardant and CHH group(P<0.05),and the expression of EAAT2 was increased significantly in pinaverium bromide group,Retardant group,CHH group and CHL group was increased significantly(P<0.05).3.2 Immunohistochemical results of colonic tissue of the rats in each groupCompared with control group,the expression of PSD-95 in the model group was decreased significantly(P<0.05),and the expression of SP was increased significantly(P<0.05);compared with the model group,PSD-95 was increased significantly in the Retardant group(P<0.05),while SP was increased significantly in the CHH group(P<0.05).3.3 Transmission electron microscopic observation of enteric nerve of the rats in each groupThe enteric nerve in the model group showed swollen,the membrane dissolved,there were many synaptic vesicles,but lack of integrity and arranged disorderly.Compared with the model group,the enteric nerve membrane in the pividium bromide group,Retardant group and CHH group was complete,and the gap was significantly wider than that in the model group.The enteric nerve gap in the CHM group and CHL group was narrowing and arranged less orderly than that in control group.3.4 RT-PCR results of colonic tissue of the rats in each groupNR2BmRNA expression:compared with control group,NR2BmRNA in the model group was significantly higher(P<0.05),and the expression of NR2BmRNA in each medication group was significantly lower than that in the model group(P<0.05).CREBmRNA expression:compared with control group,CREBmRNA was significantly higher in the model group(P<0.05),while significantly lower in pividium bromide group,Retardant group and CHH group as compared with that in the model group(P<0.05)BDNFmRNA expression:compared with control group,the expression of BDNFmRNA in the model group was significantly higher(P<0.05),while the expression of BDNFmRNA in other medication groups was significantly lower than that in the model group,except CHL group(P<0.05).4 The ELISA results of rat blood serum4.1 SP,CGRP and BDNF levels in Blood serumCompared with control group,the levels of blood serum SP,CGRP and BDNF in the model group was increased significantly(P<0.05).Compared with the model group,SP and CGRP in each medication group was decreased significantly(P<0.05),and BDNF was decreased significantly in the Retardant group and CHH group(P<0.05).4.2 The IL-1 β、TNF-α、IL-4,IL-10 levels in blood serumCompared with control group,the level of bood serum IL-1β,TNF-α,IL-4 in the model group was increased significantly(P<0.05),while the level of IL-10 was decreased significantly(P<0.05);compared with the model group,IL-1β was decreased significantly in pivirium bromide group,Retardant group,CHH group and CHLM group(P<0.05),TNF-α was decreased significantly in Retardant group and CHH group(P<0.05),IL-4 was decreased significantly in each medication group,and IL-10 was increased significantly in all the medication group,except CHL group(P<0.05).4.3 The levels of NGF,5-HT and NO in blood serumCompared with control group,the levels of NGF and 5-HT in blood serum in the model group were significantly higher(P<0.05),and the level of NO was significantly lower(P<0.05);compared with the model group,the levels of NGF was decreased significantly,except CHL group,and the level of 5-HT was significantly decreased in each medication group,while the level of NO was significantly increased in each medication group(P<0.05).Conclusion1.IBS-D disease is related to the abnormal metabolism of endogenous glutamate and the increasing expression of NR2B in enteric nerve,which may play an important role in visceral hypersensitivity.2.Chang’an II formula can regulate the level of endogenous glutamate,reduce visceral hypersensitivity,repair mucosal barrier and effectively improve the syndrome of liver depression and spleen deficiency of IBS-D.3.Chang’an II formula can inhibit the reduction of PSD-95 and EAAT2 caused by NR2B overexpression to repair the enteric nerve damage due to infection.Meanwhile,it can also inhibit NR2B-CREB-BDNF pathway to reduce the expression of brain-gut peptide and proinflammatory factors to reduce the visceral hypersensitivity caused by continuous nerve-immune interaction after infection.