Effects Of Chang’an Ⅱ Recipe In Regulating Visceral Hypersensitivity Of IBS-D By Mast Cell Activation Of AhR-Notchl Pathway

BackgroundIrritable bowel syndrome(IBS)is a functional gastrointestinal disease with abdominal pain or abdominal discomfort as the main symptoms,accompanied by changes in defecation habits or abnormal stool characteristics.Among the subtypes of IBS,diarrhea predominant irritable bowel syndrome(IBS-D)is the most common in China,accounting for 66.3-74.1%.The pathophysiological mechanism of IBS-D is still not completely clear.Visceral hypersensitivity is an important pathogenesis of IBS-D.about 94%of IBS-D patients have visceral hypersensitivity.Low-grade mucosal inflammation is one of the main reasons for inducing visceral hypersensitivity,and the inflammatory infiltration of intestinal mucosa of IBS-D is mainly mast cells.Mast cells may play an important role in activating AhR-notch1 pathway.Notch signal regulates the differentiation,maturation and function of mast cells.The enhancement of Notch signal can increase the number of mast cells in intestinal mucosa,participate in the process of mast cell activation and degranulation,and increase the release of inflammatory factors.AhR plays an important role in maintaining the homeostasis of epithelial cells and immune cells.The loss of AhR can cause the dysfunction of the cells,including mast cells.Recent studies have shown that the decrease of AhR in epithelial cells may lead to the activation of Notch1 signal and the injury of mucosal barrier.However,it is unclear whether the AhR-Notchl pathway is involved in mast cell activation in the colon of IBS-D patients.The overall treatment strategy of IBS-D aims to alleviate the main symptoms,with single drug target and high recurrence rate.Traditional Chinese medicine compound has many advantages and characteristics,such as multi-component,multi-target,multichannel and low economic cost.Therefore,exploring the effective mechanism of traditional Chinese medicine is of great significance to improve the therapeutic effect of IBS-D.Chang’an II Recipe is an empirical Recipe for the management of liver depression and spleen deficiency syndrome of IBS-D.Our previous research found that Chang’an Ⅱ Recipe can inhibit the activation of intestinal mucosal mast cells,reduce the release of bioactive substances,reduce the excitability of intestinal nociceptive sensory neurons,and improve visceral hypersensitivity.Therefore,aiming at the syndrome of liver depression and spleen deficiency of IBS-D,this study takes the activation of mast cells mediated by AhR-Notchl pathway as the starting point to explore its mechanism of participating in IBS-D visceral hypersensitivity and the therapeutic mechanism of Chang’an Ⅱ Recipe.Part one In VivoObjectiveTo establish IBS-D rat model of liver depression and spleen deficiency syndrome combined with disease and syndrome,and observe the curative effect of Chang’an Ⅱformula;Take ahr-notch1 pathway mediated mast cell activation as the starting point to reveal the possible mechanism of Chang’an Ⅱ formula in improving IBS-D visceral hypersensitivity.Method1.IBS-D rat model with liver depression and spleen deficiency syndrome was established by the superposition of three factors:neonatal maternal separation,TNBS enema and mild stimulation of chronic unpredictable stress.The rat model and the effectiveness of Chang’an Ⅱ Recipe were evaluated from the general situation,body weight,food intake,grasp force,Two-Bottle choice ratio,serum D-xylose content,fecal water content,AWR score and pathological tissue sections of rats.2.Observe the effect of Chang’an Ⅱ Recipe on visceral hypersensitivity:The protein expressions of PAR2,TRPV1 were identified by Western blot.3.Observe the effect of Chang’an Ⅱ Recipe on mast cell activation:The protein expressions of MCT were identified by Western blot;Mast cells were counted by toluidine blue staining;The contents of serum MCT,histamine,IL-4 and TNF-α were spotted by ELISA;The distribution and expression of MCT were distinguished by immunohistochemistry.4.Observe the effect of Chang’an Ⅱ Recipe on AhR-Notch pathway:The distribution and expression of AhR,Notchl were distinguished by immunohistochemistry;The protein expressions of AhR,CYP1A1,Notch 1,Hesl were identified by Western blot;The gene expressions of AhR mRNA,CYP1A1 mRNA,Notch1 mRNA and Hesl mRNA were detected by RT-PCR.Result1.Model and efficacy evaluation resultCompared with the normal control group,the body weight,food intake,grasp force and Two-Bottle choice ratio of the model group were significantly lower(P<0.05),and the fecal water content,AWR score and serum D-xylose content were significantly higher(P<0.05).After drug intervention,compared with the model group,Chang’anⅡ Recipe and Kyn can significantly increase the weight,food intake,grasp force and Two-Bottle choice ratio of model rats(P<0.05),and reduce the fecal water content,AWR score and serum D-xylose content(P<0.05).2.Visceral hypersensitivity assessment resultsWB:Compared with the normal control group,the expression of PAR2,TRPV1 protein increased significantly(P<0.05);Compared with the model group,Chang’anⅡ Recipe and Kyn could significantly decrease the expression of PAR2,TRPV1(P<0.05).3.Mast cell activation evaluation resultsIHC:MCT positive staining was mainly distributed in lamina propria and submucosa.Compared with the normal control group,and the MOD values of MCT increased significantly(P<0.05);Compared with the model group,Chang’an Ⅱ Recipe and Kyn could significantly reduce the MOD value of MCT(P<0.05).WB:Compared with the normal control group,the expression of MCT increased significantly(P<0.05);Compared with the model group,Chang’an Ⅱ Recipe and Kyn could decrease the expression MCT(P<0.05).Mast cell count:Compared with the normal control group,the number of mast cells in colonic mucosa of model group increased significantly(P<0.05),and the number of mast cells decreased significantly after treatment with Chang’an Ⅱ Recipe and Kyn(P<0.05).Elisa:Compared with the normal control group,serum MCT,histamine,IL-4 and TNF-α in the model group.The contents of MCT,histamine,IL-4 and TNF-α decreased significantly after treatment with Chang’an II Recipe and Kyn(P<0.05).4.AhR-Notchl pathway resultsIHC:AhR and Notch1 positive staining were widely distributed in the whole mucosal epithelium,submucosa and muscle layer.Compared with the normal control group,the MOD value of AhR in the model group decreased significantly(P<0.05),and the MOD values of Notchl increased significantly(P<0.05);Compared with the model group,Chang’an Ⅱ Recipe and Kyn could significantly increase the MOD value of AhR,significantly reduce the MOD value of Notchl(P<0.05).WB:Compared with the normal control group,the expression of AhR and CYP1A1 protein in the model group decreased significantly(P<0.05),and the expression of Notchl,Hes1 protein increased significantly(P<0.05);Compared with the model group,Chang’an Ⅱ Recipe and Kyn could significantly rise the expression of AhR and CYP1A1 and decrease the expression of Notch1,Hes1(P<0.05).RT-PCR:Compared with the normal control group,the gene expression of AhR mRNA and CYP1A1 mRNA in the model group decreased significantly(P<0.05),and the gene expression of Notch1 mRNA and Hesl mRNA increased significantly(P<0.05);Compared with the model group,Chang’an Ⅱ Recipe and Kyn could significantly increase the gene expression of AhR mRNA and CYP1A1 mRNA(P<0.05),and reduce the gene expression of Notch1 mRNA and Hes1 mRNA(P<0.05).Conclusion1.The compound modeling method of neonatal maternal separation,TNBS enema and mild stimulation of chronic unpredictable stress can successfully establish the IBSD rat model with liver depression and spleen deficiency syndrome.2.Chang’an Ⅱ Recipe can effectively improve the decrease of body weight,food intake and intestinal absorption capacity of IBS-D rats can improve the deterioration of diarrhea,reduce visceral sensitivity and low-grade intestinal inflammation in model rats.3.Chang’an Ⅱ Recipe can elevate the expression of AhR and CYP1A1,inhibit the expression of Notchl,Hes1,MCT and TRPV1,prevent the mast cell activation in rat colon and decrease the the release of MCT,histamine,IL-4 and TNF-α,reduce the expression of intestinal afferent nerve nociceptors PAR2 and TRPV1,which can relieve visceral hypersensitivity and alleviate IBS-D symptoms.Part Two In VitroObjectiveTo observe the effect of AhR-Notchl pathway on the activation of RBL-2H3 cells,and to explore the target of Chang’an II Recipe in regulating the activation of RBL-2H3 cells.Method1.The activation model of RBL-2H3 cells was established by DNP-IgE sensitization and DNP-HSA re-excitation.The best concentration of DNP-IgE and DNP-HSA modeling and the optimal time of DNP-HSA excitation were screened out by β-Hex release rate.2.Observe To observe the molecular mechanism of Chang’an Ⅱ medicated serum on mast cell activation:CCK8 was used to detect the effect of different concentrations of Chang’an II Recipe medicated serum on the viability of RBL-2H3 cells;The supernatant of each group were spotted by ELISA of the β-Hex release rate and the contents of serum MCT,histamine,IL-4 and TNF-α;Western blot was used to detect the expressions of AhR and Notchl protein,and the protein ratios of p-p38/p38,pERK/ERK and p-JNK/JNK;Western blot was used to detect the expressions of AhR and Notchl protein.Result1.Establishment of RBL-2H3 cell activation model □The activation degree of RBL-2H3 cells increased with the increase of DNP-IgE and DNP-HSA concentrations,and both reached the highest value at the concentration of 200ng/mL.Subsequently,the release rate somewhat decreased.At the same time,the activation degree of RBL-2H3 cells increased with the increase of DNP-HSA excitation time,and reached the peak at 1h.2.Molecular mechanism resultsCCK8 test:The cell viability of Chang’an Ⅱ 5%medicated serum was higher than that of 10%,15%and 20%medicated serum.Therefore,5%medicated serum was selected as the administration scheme for 24 hours.β-Hex release rate:Compared with the normal control group,The P-Hex release rate of the model group was increased significantly(P<0.05);Compared with the model group,The β-Hex release rates of of Kyn group and Chang’an Ⅱ Group were decreased significantly(P<0.05);Compared with Kyn group,The β-Hex release rate of Kyn+CH223191 group was increased,but the difference was not statistically significant(P>0.05);Compared with Chang’an Ⅱ group,The β-Hex release rates of Chang’an Ⅱ+CH223191 group was increased significantly(P<0.05).Elisa:Compared with the normal control group,the contents of MCT,histamine,IL-4,TNF-α in the model group increased significantly(P<0.05);Compared with the model group,the contents of MCT,histamine,IL-4,TNF-α in the Kyn group and Chang’an Ⅱ group,decreased significantly(P<0.05);Compared with Kyn group,the contents of MCT,histamine,IL-4 in the Kyn+CH223191 group increased significantly(P<0.05),the content of TNF-α has an increasing trend(P>0.05);Compared with Chang’an Ⅱ group,the contents of IL-4,TNF-α in the Chang’an II+CH223191 group increased significantly(P<0.05),the content of MCT,histamine have an increasing trend(P>0.05)WB(MAPK):Compared with the normal group,the ratios of p-p38/p38,p-JNK/JNK and p-ERK/ERK in the model group were significantly higher(P<0.05);Compared with the model group,the ratios of p-p38/p38,p-JNK/JNK and p-ERK/ERK in Kyn group and Chang’an Ⅱ group decreased significantly(P<0.05);Compared with Kyn group,the ratio of p-p38/p38(P<0.05),p-JNK/JNK(P>0.05),p-ERK/ERK(P<0.05)was increased in Kyn+CH223191 group;Compared with Chang’an Ⅱ group,the ratio of(P<0.05),p-JNK/JNK(P>0.05),p-ERK/ERK(P>0.05)was increased in Chang’an Ⅱ+CH223191 group.WB(AhR、Notchl):Compared with the normal group,the expression level of AhR protein in the model group decreased significantly(P<0.05),and the expression level of Notchl protein increased significantly(P<0.05);Compared with the model group,the expression levels of AhR protein and Notchl protein in Kyn group and Chang’an Ⅱ group were significantly higher(P<0.05);Compared with Kyn group,the expression level of AhR protein in Kyn+CH223191 group decreased significantly and the expression level of Notchl protein increased significantly(P<0.05).Compared with Chang’an Ⅱ group,the expression level of AhR protein in Chang’an II+CH223191 group decreased significantly and the expression level of Notchl protein increased significantly(P<0.05).Conclusion1.Sensitization with final concentration of 200ng/mL DNP-IgE and stimulation with 200ng/mL DNP-HSA for 1h is the best method to establish the activation and degranulation model of RBL-2H3 cells.2.The medicated serum of Chang’an Ⅱ Recipe can promote the expression of AhR protein,thus inhibit the expression of Notchl protein and the phosphorylation levels of p38,ERK1/2 and JNK,reduce the release of MCT,histamine,IL-4 and TNF-α,restrain RBL-2H3 cell activation.