The Role Of Transient Receptor Potential Channel A1 In Colonic Motility Recovery After Pelvic Nerve Injury Mice

Background:The incidence of colorectal cancer has been ranked third in common tumors.Most of rectal cancer patients undergo anal sphincter surgery.25%to 90%of patients were suffering from bowel habits changes after surgery,which seriously affects the patients’quality of life,known as anterior resection syndrome.Several studies have shown that an important factor in anterior resection syndrome is the damage of the pelvic nerve plexus.A small number of patients have improved over time.The mechanism of this compensatory recovery is still unclear,and this adaptive recovery takes a long time,and the degree is limited.If the mechanism of adaptive recovery can be clarified,it is of great clinical significance to implement the corresponding interventions to accelerate the recovery process.Ridolfi TJ et al found that the pelvic nerve denervation（PND）rats can recover from colonic function in about one week.Our previous study found that:after pelvic nerve denervation（PND）in rats,the expression of transient receptor potential ankyrin1（TRPA1）in the colonic mucosa of rats was firstly down-regulated and recovered,and the trend ofthe colonic motility recoveryis consistent,suggesting that the colonic motility recovery after rat pelvic denervation is related to TRPA1.TRPA 1 is a separate subtype of transient receptor potential channels（TRPs）that play an important role in pain sensation,nociceptive cold stimulation,and mechanical perception.TRPA1 is expressed in enterochromaffincells（ECC）.As a sensory molecule of EC cells,TRPA1 senses the stimulation of physicochemical factors in the intestinal lumen,mediates the release of 5-HT from EC cells,and regulates the intestinal tract.In the gastrointestinal track,the 5-HT₃receptor is involved in intestinal motility regulation.To further clarify the role of TRPA1 in colonicadaptive recovery after pelvic denervation,this study established a PND model mouse to observe TRPA1 gene deletion,and changes in colonic motility by TRPA1 agonist and inhibitor intervention,and the expression of 5-HT₃ receptor were observed to explore the possible mechanism of TRPA1colonic motility recovery in mice after pelvic nerve denervation.Objective:1.To verify the adaptive recovery of colonic motility in PND mice.2.To observe the effect of TRPAl agonists and antagonist on colonic function in PND mice.3.To detect the expression of TRPA1,and 5-HT₃ receptor in the colon of PND mice,and to explore its role in colonic motility recovery.4.Using TRPA1 knockout mice to study the changes of colonic motility and the possible mechanism of TRPA1 in colonic motility recovery.Methods:1.Experimental subjects and groups1.1 216 healthy and clean male C57 mice were randomly divided into two parts:108for colonic transit test and 108 for visceral sensitivity test.The colonic transit test group was further divided into two groups:pelvic nerve denervation（PND）model group（n=54）and sham group（n=54）.The two groups were divided into:no drug-treated group;TRP1agonist curcumintreated group;TRPA1 antagonist HC-030031 treated group,18 in each group.The visceral sensitivity testgroup is divided into the same groups as the colonic transit test group.1.2 60 healthy and clean male TRPA1 knockout micewere randomly divided into two groups:colonic transit test group（n=30）and visceral sensitivity test group（n=30）.The components of the colonic transmission function test were:15 in the PND model group and15 in the sham operation group.The visceral sensitivity test group was grouped in the same way.2.Model preparation2.1.PND model group:after the laparotomy,the bilateral pelvic nerves were found and separated by microscopic instruments.2.2.Sham-operated group:after the mice were opened,the abdomen was placed in the abdominal cavity for 20 minutes.2.3 colonic transit test:in the mouse cecum pre-embedded silicone tube and fixed.2.4 visceral sensitivity test:platinum electrode was placed in the extra-abdominal oblique muscle of mice.3.Detectionindex3.1 Detection of colonic transittest:at the 1st,3rd,and 7th day after surgery,0.1ml of methylene blue mixture was slowly injected into the colon from the preset silicone tube,and then 0.1mL of air was injected to make the methylene blue all enter into the colon.After 20 minutes,the mice were sacrificed,the entire colon was taken out,and the length of the methylene blue staining portion was measured with a ruler,and the length was calculated as the percentage of the colon,which is the ratio of colonic transit.3.2Visceral sensitivity test:at the first,third,and seventh days after surgery,the BL-420S biosignal acquisition system was connected from the indwelling electrode,and the rectal sensitivity was measured by colorectal dilatation（CRD）-visceral motion reflex（VMR）.3.3 Immunohistochemical detection of TRPA1 and 5-HT₃ receptor expression:the distal colon specimens of mice were taken at the 1st,3rd,and 7th postoperative day,fixed in 4%paraformaldehyde,and embedded in paraffin.TRPA1 immunohistochemical staining was performed using a hypersensitive 3-step method.3.4 Western Blot test:the distal colon specimens of mice were taken at the 1st,3rd,and 7th postoperative day,and the TRPA1,5-HT₃ receptor were detected according to the Western Blot procedure.Results:1.Colonic adaptive recovery after mouse PND model:The colonic transit test showed that compared with the sham operation group,the colonic transit of the PND model group was significantly decreased on the first day after operation（P<0.001）,and the colonic colonic transit on the third day after surgery had a recovery trend but still decreased（P=0.040）,by day 7,the difference between two groups have no significant（P=0.073）.The visceral sensitivity test showed that compared with the sham operation group,the DND model group had significantly lower visceral sensitivity in the first day after surgery（P<0.001）,and the visceral sensitivity on the third day after surgery had a recovery trend but still decreased（P=0.001）,at POD7,there was no difference in visceral sensitivity between the two groups（P=0.309）,and there was an adaptive recovery trend.2.After treated with TRPA1 agonist:Compared with the normal control group and the sham operation group,the colonic transit of the PND model group was significantly increased on the 1st,3rd,and 7th day after surgery,and was higher than that of the control group（P=0.001,P<0.001）.In contrast,after TRPA1 antagonist intervention:compared with the normal control group,the colonic transmission function of the mice on the 1st,3rd,and7th day after surgery was significantly decreased（P<0.05）,and there is no recovery trend in colonic transit test.3.The results observed in TRPA1 knockout mice are consistent with the results of the application of TRPA1 antagonists.4.Immunohistochemistry showed that TRPA1 was expressed in the colonic mucosa of mice,and after the pelvic nerve innervation,the TRPA1 appeared to gradually decrease and then gradually increased,as well as 5-HT₃ receptor.5.The expression of TRPA1 and 5-HT₃ receptor in mouse colon suggests that the expression of TRPA1 and 5-HT₃ receptor in the colon of PND group was lower than that in the sham operation group（P=0.006,P=0.004）.There was also a decrease in 3 days（P=0.043,P=0.012）.At POD7,there was no significant difference between the 7th day and the sham operation group（P>0.05）,which showed a recovery trend.Compared with wild-type mice,the expression of 5-HT₃ receptor was significantly decreased in TRPA1knockout mice（P<0.05）.After depilatory innervation,5-HT₃ receptor expression and sham operation group were not statistically significantlydifference（P>0.05）.Conclusions:1.After pelvic nerve denervation,the mouse’s intestinal motility also showed an adaptive recovery phenomenon.2.TRPA1 agonist curcumin can significantly promote colonic motility recovery in pelvic denervation mice,the adaptive recovery trend disappeared after the application of TRPA1 antagonist and TRPA1 gene knockout.3.TRPA1 is an irreplaceable role in the mechanism of colonicadaptive recovery in mice after pelvic denervation.4.TRPA1 may regulate colonic motility recovery through the 5-HT₃ receptor.