Role Of TRPA1 In The Initiation And Maintenance Of Post-inflammatory Visceral Hypersensitivity

BackgroundVisceral hypersensitivity refers to the exaggerated sensory reflex of visceral organs to various kinds of noxious stimuli and normal physiological stimuli,it is considered to be an important pathophysiological mechanism of recurrent chronic visceral pain in patients with irritable bowel syndrome(IBS).Approximately 10-31%of IBS patients are reported to be post infectious irritable bowel syndrome(PI-IBS),and it is defined as the acute onset of IBS symptoms(abdominal pain,abdominal bloating,bowel habit and/or stool properties change)following an initial episode of acute gastrointestinal infection in individuals who have had no evidence of previous gastrointestinal infection or inflammation.However,the initiation and maintenance mechanisms of visceral hypersensitivity remain unclear due to the undetectable structural and biochemical abnormalities.Transient receptor potential(TRP)ion channels are a broad group nonselective cation channels,which include many members and detect the pain induced by temperature,mechanical as well as chemical stimuli.Previous studies have shown that TRP vanilloid-1(TRPV1)is wildly involved in the visceral hypersensitivity induced by colorectal stimuli.TRP ankyrin-1(TRPA1),a member of the TRP receptor superfamily,has become a research hotspot due to the co-expression with TRPV1 in sensory neurons.It is mainly distributed in small diameter sensory neurons and can be activated by noxious cold(<17?)as well as a variety of exogenous or endogenous chemical stimulus,including menthol,tetrahydrocannabinol,allylisothiocyanate(AITC),and cinnamaldehyde.Although a few previous studies have found that the TRPA1 receptor could participate in the regulation of visceral hypersensitivity,they only involved in the peripheral nervous system.Here we used 2,4,6-Trinitrobenzene sulfonic acid(TNBS)-induced rat model of post-inflammatory visceral hypersensitivity to extensively explore the role of TRPA1 in the initiation and maintenance of persistent post-inflammatory visceral hypersensitivity from both the peripheral and central nervous systems,which will provide a guidance basis for the treatment of visceral hypersensitivity from the symptomatic treatment to the treatment based on the mechanism in the future.Objective1.To investigate the expression of TRPA1 in the colon by using Western Blot and immunohistochemistry.2.To investigate the expression of TRPA1 in the spinal cord,especially in spinal dorsal horn(SDH)by using Western Blot and immunohistochemistry.3.To explore the role of TRPA1 in the initiation and maintenance of persistent post-inflammatory visceral hypersensitivity.MethodsA total of 24 female Wistar rats(180-200g)were randomly allocated equally into two groups and each one of 12 rats.They received the same volume TNBS(TNBS-treated group)or normal saline(control group)respectively.Six weeks after the administration,the visceral sensitivity to colorectal distension(CRD)was measured by the abdominal withdrawal reflex(AWR)scores.Then rats were killed by decapitation,the colon and spinal cord were dissected immediately.The colonic inflammation state of TNBS-treated and control rats were evaluate by H&E stain;The level of TRPA1 protein expression were measured by Western Blot;The number of TRPA1-immunopositive neurons and integrated optical density values(IOD)of TRPA1 were measured by immunohistochemistry.Results1.At the distension pressure of 15mmHg,30mmHg and 45mmHg,TNBS-treated rats exhibited stronger behavioral response and higher AWR scores.2.Six weeks after the administration,there is no microscopic evidence of inflammation was observed in TNBS-treated and control rats colonic tissues.3.Densitometric analysis of the bands revealed that in both colon and spinal cord,the expression of TRPA1 were significantly greater in the TNBS-treated group than in controls.4.Quantitative immunohistochemical analysis showed that the number of TRPA1-immunopositive neurons and IOD values of TRPA1 in the SDH from TNBS-treated rats were significantly higher than that from control rats.Moreover,TRPA1-immunopositive neurons were mainly localized in the superficial layers of the SDH,especially in the substantia gelatinosa(SG;lamina ? of Rexed).Conclusion1.The expression level of TRPA1 in colon tissue of TNBS-treated group was significantly elevated.2.The expression level of TRPA1 in spinal cord(especially in the SDH)of TNBS-treated group was significantly elevated.3.Up-regulation of TRPA1 in the TNBS-treated rat colon and spinal cord(especially in the SDH)contribute to the initiation and maintenance of persistent post-inflammatory visceral hypersensitivity.