Study On The Mechanisms Underlying Regulation Of Colonic Neuropathy By 5-HT In Diabetic Rats

Background: Diabetic peripheral neuropathy(DPN)is one of the most common complications in patients with diabetes,there is rarely effective treatment for DPN.5-hydroxytryptamine(5-HT)released from enterochromaffin cells regulates physiological functions of gastrointestinal(GI)tracts,participates in the inflammation in GI tracts and protects enteric nervous system from apoptosis.Some evidence indicates that 5-HT is involved in diabetes and its complications,however,little is known that the effect of 5-HT on colonic neuropathy in diabetes.Thus,the role of 5-HT in colonic neuropathy in diabetes was investigated and the mechamisms underlying the effects induced by 5-HT were explored.Method and Results: Type 1 diabetes was established by intraperitoneal injection of streptozotocin(STZ,50mg/kg)in Sprague-Dawly(SD)rats.The expression of panneuronal marker protein gene product(PGP)9.5 and 5-HT in colon was examined by immunofluorescent(IF)staining and the level of 5-HT in serum was detected by HPLCMS in control(same volume of vehicle)and diabetic rats at week 1,2,3,4 after induction of diabetes.We found that the number of PGP9.5-immunoreactive nerve fibers was gradually decreaed from week 1 to week 4,showing statistical significance at week 3 and 4.The level of serum 5-HT was also significantly lowered at week 3 and 4.The number of 5-HT-immunoreactive cells was reduced at week 1 and 2 after induction of diabetes.At week 2 after induction of diabetes,chronic i.p.injection of 5-HT(10 mg/kg,every other day for two weeks)were performed.The day after completing 5-HT injection,the expression of PGP9.5 was examined by IF staining.We found that 5-HT attenuated STZ-induced loss of PGP9.5-immunoreactive nerve fibers in colon.In addition,5-HT improved STZ-induced colonic dyskinesis by assessing colonic transmit time and 5-HT4 receptor agonist showed the similar effect.To explore the mechanisms underling the protection induced by 5-HT,the expression of molecules associated to cell injury and death was examined by western blot(WB).At week 4 after induction of diabetes,the expression of receptor-interacting protein(RIP)3 and mixed-lineage kinase domain-like protein(MLKL)was increasd.5-HT inhibited the upregulation of RIP3 and MLKL induced by STZ.However,there is no difference in expression of caspase-9 in non-diabetic,diabetic-control and diabetic-5-HT treated rats.At last,the expression of myelin basic protein(MBP)associated with the induction and maintenance of myelin in L6-S1 dorsal root ganglion(DRG)was detected by WB.We found that the expression of MBP in L6-S1 DRG was significantly decreased at week 4 after induction of diabetes and 5-HT reversed the above change in MBP expression.Conclusion: Diabetes results in loss of nerve fibers in colon,dyskinesis of colon and decreaed level of peripheral 5-HT.Exogenous 5-HT significantly delays the loss of nerve fibers,improves colonic dyskinesis,inhibits the upregulation of RIP3/MLKL in colon and downregulation of MBP in L6-S1 DRG.These results suggest exogenous 5-HT improves colonic neuropathy,which might be due to the inhibition of necroptosis signaling pathways that lead to the dysmyelination of glial cells.This will provide scientific rationale for treating diabetic neuropathy in GI tract via targeting 5-HT receptors and necroptosis signaling pathways,eventually provides new strategy for clinical treatments.