The Effect And Mechanism Of Melatonin On Irritable Bowel Syndrome Through Inhibiting Mast Cell Activation

Backgroud:Irritable bowel syndrome is a common functional gastrointestinal disorder that significantly affects patients’ quality of life.However,the specific mechanism of its development has not yet been concluded.Visceral hypersensitivity is considered to be one of the main pathophysiological mechanisms of IBS,which may be the direct cause of repeated chronic abdominal pain,abdominal distension,abnormal defecation and other symptoms.As an important intestinal hormone,melatonin can regulate intestinal motility,stimulate mucus secretion,protect intestinal mucosal barrier,and inhibit intestinal inflammation,it plays an important physiological role in the intestine.A number of clinical studies have found that exogenous melatonin can help relieve the visceral hypersensitivity of IBS,but the specific mechanism is rarely reported.Therefore,this study intends to further clarify the influence of melatonin on visceral hypersensitivity of IBS and explore its potential mechanism,providing a scientific basis for finding a new treatment plan for IBS.Methods:1.In vivo:1.1 Newborn male SD rats were separated from their mothers(for 3h every day from the 2nd to 14th day after birth)to establish the animal model of IBS.Intervention with 1mM melatonin in drinking water was given.Set the control group at the same time.01.2 Colorectal distension test(CRD)was used to detect the visceral sensitivity of rats.1.3 Immunohistochemical staining and toluidine blue staining were performed on the distal colon tissues to evaluate the number of mast cells and the activation level.1.4 The whole colon tissue was taken for protein quantitative analysis.2.In vitro:2.1 RBL-2H3 cell model was constructed by transient transfection of Homo-MRGPRX2 gene.2.2 RBL-2H3 cells transfected with Homo-MRGPRX2 gene were incubated with different concentrations of C48/80 for different times to explore the optimal reaction conditions for C48/80-induced mast cell activation.2.3 C48/80 activated mast cells(Homo-MRGPRX2 transfected RBL-2H3 cell line)were treated with different concentrations of melatonin(10 μM and 1 mM),and a blank control group was set.2.3.1 The release rate of β-hexosaminidase was detected by enzyme labeling method to evaluate the degranulation level of mast cells.2.3.2 The expression levels of IL-6 and TNF-α in mast cells were detected by ELISA.Results:1.Visceral hypersensitivity and local mast cell activation in rats resulted from maternal and infant separation.Colorectal dilation experiment showed that the visceral sensitivity of maternal separated rats increased significantly under different balloon pressure levels compared with the control rats without special treatment.Immunohistochemistry of colon tissue showed that the number of mast cell increased,and the expression of tryptase was significantly up-regulated,indicating the activation of mast cells.2.Melatonin intervention affects IBS phenotype and intestinal mast cell activationThe visceral sensitivity of maternal separated rats treated with melatonin was significantly lower than that of rats treated without melatonin,which was close to that of the blank control group without special treatment.Immunohistochemical staining indicated that the expression of tryptase in the colon of the melatonin intervention group was significantly decreased compared with that of the non-melatonin intervention group.Toluidine blue staining suggested that the number of colon mast cells decreased after melatonin intervention.These results suggest that melatonin can effectively reverse visceral hypersensitivity and intestinal mast cell activation in rats induced by maternal separation.3.Low concentration of C48/80 induced Homo-MRGPRX2 gene transfected RBL-2H3 cell activationMast cell stimulator C48/80 showed strong concentration-dependent cytotoxicity,while wild-type RBL-2H3 cells did not respond to safe concentrations of C48/80.By transfecting Homo-MRGPRX2 gene,RBL-2H3 cells were able to respond to low concentration of C48/80.4.Melatonin inhibits C48/80-induced RBL-2H3 cell activationC48/80 activated mast cells(RBL-2H3 cells transfected with Homo-MRGPRX2 gene)were treated with different concentrations of melatonin(10 μM and 1 mM),andβ-hexosaminidase release rate and the levels of TNF-α and IL-6 in cell culture supernatant were detected.The results showed that melatonin significantly inhibited the release of β-hexosaminidase and cytokines.It is suggested that melatonin can effectively inhibit the release of inflammatory mediators synthesized in advance and de novo during mast cell activation.5.The specific action mechanism of melatonin may be related to actin cytoskeleton.By means of quantitative analysis of proteins in the whole colon tissue of maternal separated rats and melatonin intervention rats,it was found that there was significant enrichment of differential proteins in the actin cytoskeleton pathway,and RAP1A,Myh10,Speg and Myh14 in this pathway were central proteins of the protein-interaction network.It suggests that melatonin may play a role through the actin cytoskeletal pathway.Conclusion:1.Melatonin can alleviate visceral hypersensitivity by reducing the activation level of local intestinal mast cells.2.Melatonin inhibits C48/80-mediated RBL-2H3 cell activation in vitro.3.Melatonin may inhibit mast cell activation through the actin cytoskeleton pathway and alleviate IBS visceral hypersensitivity.