The Mechanisms Of Probiotic VSL#3 In A Rat Model Of Visceral Hypersensitivity Involves The Mast Cell-PAR2-TRPV1 Pathway

Objective: Visceral hypersensitivity,which is a change in visceral perception of pain and characterized by hyperalgesia and allodynia,is the important clinical manifestation in patients with diarrhea predominant irritable bowel syndrome.The pathogenesis of visceral hypersensitivity may be closely related to increased sensitivity of peripheral receptors and abnormal signal transduction.Further understanding of the peripheral mechanism of visceral hypersensitivity is helpful to reveal the pathogenesis of IBS and discover effective treatment measures.Animal and clinical experiments show that intestinal flora imbalance can lead to gastrointestinal motility disorder,intestinal mucosal barrier and immunity dysfunction,and affect brain-gut axis interaction,which can lead to visceral hypersensitivity.With the recognition of the importance of intestinal flora in IBS,attention has been focused on the manipulation of intestinal flora,which may be an effective treatment for IBS.Some therapeutic interventions for intestinal microflora are being studied,including probiotics,which is the focus of current research.Probiotics can alleviate the visceral hypersensitivity of IBS,which has been proved to be effective,but its mechanism is still unclear.In vivo,intestinal sensory information is transferred through the intestinal sensory afferent nerve fibers to the central nervous system（CNS）.The cell body of intestinal sensory afferent nerve fibers is located in the dorsal root ganglion（DRG）.Lumbosacral neurons are located in the L6-S1 dorsal root ganglion segment of the spinal cord,which is closely related to the transmission of intestinal pain signals.PAR2 and TRPV1 on DRG neurons play an important role in the transmission of visceral pain signals.Tryptase released by mast cells can specifically shear PAR2 receptors in spinal afferent neurons,and then PAR2 activates TRPV1 on sensitized nociceptive neurons.Sensitized receptors in nociceptive neurons cause the release of peptides such as SP and CGRP in sensory nerve endings,which are closely related to pain sensation.Therefore,detection of PAR2-TRPV1 pathway activation in neurons of L6-S1 DRGs is one effective method to evaluate postsynaptic neuron activation caused by visceral pain.The increase of mast cell number or degranulation may be related to visceral hypersensitivity in IBS.Although the application of mast cell stabilizer can alleviate visceral hypersensitivity and IBS symptoms,the specific mechanism is still not clear.Previous studies revealed that stress could induce visceral hypersensitivity in rats,and much more activated mast cells in intestinal mucosa,wherea the intervention of probiotic VSL#3 reduced visceral sensitivity significantly.The changes of mast cells and TRPV1 pathway in this model were observed in the preliminary experiment.It was speculated that the mast cell-PAR2-TRPV1 pathway is key to the formation of visceral hypersensitivity,which maybe involved in the mechanism of probiotic VSL#3.Based on these background,the aim of our study was to evaluate:（1）establishment of visceral hypersensitivity rat model;（2）the changes of intestinal flora and mast cells in visceral hypersensitivity rats（3）the involvement of the mast cell-PAR2-TRPV1 pathway in the formation of VH;（4）whether probiotic VSL#3 treatment can regulate VH;and the effect of probiotic VSL#3 treatment on the mast cell-PAR2-TRPV1 pathway.Methods: Experiment 1.To establish visceral hypersensitivity rat model and research on its intestinal flora alteration 1.The visceral hypersensitivity model was induced by 40 ml/L acetic acid enema combined with restriction of partial limb movement.The control group was free to take water and food without any disposal.The experimental animals were divided into four groups: Con group,VH group,VH+VSL#3 group and VH+ketotifen group.2.Stool samples were collected from each group（n=3）for a further analysis using 16 Sr RNA pyrosequencing analysis.3.Colon tissues of rats were obtained from each group.Mast cells were detected by toluidine blue staining,and the degranulation of mast cells was assessed by transmission electron microscopy.Experiment 2.Mast cell-PAR2-TRPV1 pathway in visceral hypersensitivity 1.Visceral hypersensitivity rat model was induced by acetic acid enema combined with partial limb restraint method.2.The experimental animals were divided into Con group,VH group,VH+Ketotifen group,VH+FSLLRY-NH2 group and VH+SB366791 group.3.The fecal water content of rats in each group was measured.The time of the first excretion of black stool was measured.The rats were sacrificed after abdominal muscle contraction reflex（AWR）was observed.Colon tissue and L6S1 dorsal root ganglion of rats in each group were retained.HE staining was used to observe the morphology of colonic mucosa and the infiltration of inflammatory cells.Mast cells were detected by toluidine blue staining.The expression of PAR2 and TRPV1 protein in DRG neurons was detected by immunohistochemistry and Western blot.Experiment 3.The mechanism of probiotics VSL#3 in visceral hypersensitivity rat model involves mast cell-PAR2-TRPV1 pathway 1.Visceral hypersensitivity rat model was induced by acetic acid enema combined with partial limb restraint stress 2.The experimental animals were divided into Con group,VH group,VH+VSL#3 group,VH+VSL#3+SLIGRL-NH2 group and VH+VSL#3+Capsaicin group.3.The fecal water content of rats in each group was measured.The time of the first excretion of black stool was detected.The rats were sacrificed after abdominal muscle contraction reflex（AWR）was detected.Colon tissue and L6S1 dorsal root ganglion of rats in each group were retained.HE staining was used to observe the morphology of colonic mucosa and the infiltration of inflammatory cells.Mast cells were detected by toluidine blue staining.The expression of PAR2 and TRPV1 protein in DRG neurons was detected by immunohistochemistry and Western blot.Results: 1.Visceral hypersensitivity rat model could successfully be induced by acetic acid enema combined with partial limb restraint method.Compared with rats in Con group,AWR score,number of mast cells,and degranulaiton of mast cells increased in VH rats.Administration of ketotifen or VSL#3 could reduce AWR score,mast cell numbers in the colon,and degranulation of mast cells in VH rats.2.No difference of the species and diversity of intestinal flora were found between VH group and Con group.The abundance of Clostridium_sensu_strictio₁ was increased in VH group compared to Con group,which could be restored by application of probiotic VSL#3.Clostridium_sensu_strictio₁ might be an important factor in visceral hypersensitivity.3.Ketotifen decreasd the expression of PAR2 and TRPV1 in（L6-S1）DRG in visceral hypersensitivity rats.4.PAR2 antagonist caused reduction of AWR score and downregulaiton of PAR2 and TRPV1 expression in（L6-S1）DRG in visceral hypersensitivity rats.TRPV1 antagonist could reduce AWR score and TRPV1 protein expression in（L6-S1）DRG in visceral hypersensitivity rats.5.Compared with VH group,the expression of PAR2 and TRPV1 in（L6-S1）DRG in VH+VSL#3 group decreased.Compared with VH+VSL#3 group,AWR score and expression of PAR2 and TRPV1 protein in（L6-S1）DRG increased in VH+ VSL#3+PAR2 agonist group.The AWR score and the expression of TRPV1 protein in（L6-S1）DRG in VH+VSL#3+TRPV1 agonist group increased compared with that in VH+VSL#3 group.The results indicated that PAR2 agonist and TRPV1 agonist could block the effect of probiotic VSL#3.Conclusion: The probiotic VSL#3 decreases visceral sensitivity in a rat model of VH.The mechanism may be related with the mast cell-PAR2-TRPV1 signaling pathway.