Roles Of PKMζ In Spinal Central Sensitization Of Chronic Visceral Pain In Rats With Irritable Bowel Syndrome

Objective In this study, the effect of spinal PKMζ on visceral pain sensitivity was examined in rats with irritable Bowel Syndrome (IBS) in order to deeply understand the pathogenesis of chronic visceral pain on IBS, which could supply a new thought and target point for the pharmacotherapy of visceral pain on IBS.Methods Neonatal male Sprague-Dawley (SD) rats were used as study subject. Chronic visceral pain model rats (IBS) were received neonatal maternal separation for3hours daily during post neonatal days3~21; the control rats were received the same processes except for neonatal maternal separation.①The amplitude of EMG was tested to assess the visceral pain sensitivity of rats when they were adult.②Expression of spinal PKMζ and phosphorylated PKMζ (p-PKMζ) were detected by western blot in control and IBS-like rats.③ZIP (a PKMζ selective antagonist) was given to the IBS-like rats by intrathecal application and the effect of ZIP (different doses,1～10μg) on response to chronic visceral pain was observed both in IBS-like rats and control rats.④Finally the highest dose of ZIP(10μg) was given by intrathecal application both in IBS-like rats and control rats to detect the influence on motor function by open field test.Results1. The visceral pain sensitivity of IBS-like rats significantly increasedThe sensitivity of visceral pain was assessed by recording amplitude of EMG to CRD pressure. Compared with control rats, the visceral pain sensitivity of IBS-like rats significantly increased from (12.35±2.77)%to (115.25±33.72)%under20mmHg CRD, from (203.17±26.68)%to (528.15±67.13)%under40mmHg CRD, from (321.45±21.77)%to (815.10±60.29)%under60mmHg CRD, and from (393.84±28.47)%to (900.41±94.41)%under80mmHg CRD (p <0.05), indicating the visceral pain sensitivity of IBS-like rats significantly increased. 2. The expression of p-PKMζ in spinal thoracolumbar (TL) segments of the IBS-like rats with chronic visceral pain notably enhancedThe expression of TL PKMζ and p-PKMζ was detected by western blot in control and IBS-like rats, and the results shown that the expression of TL p-PKMζ in IBS-like rats was increased significantly when compared with that in control rats (p <0.05). There was no significant difference in TL PKMζ between the control rats and IBS-like rats.3. The expression of p-PKMζ in spinal lumbosacral (LS) segments of the IBS-like rats with chronic visceral pain increased significantlyWestern blot data indicated that expression of LS p-PKMζ significantly enhanced in IBS-like rats when compared with control rats (p <0.05). There was no significant difference on TL PKMζ between the control rats and IBS-like rats.4. Intrathecal injection of ZIP could dose-dependently attenuated the visceral pain hypersensitivity in IBS-like ratsEMG data revealed that intrathecal injection of ZIP dose-dependently attenuated the visceral pain hypersensitivity in IBS-like rats. The attenuated effect of the highest doses of ZIP (10μg) given by intrathecal application could continue until180min post-injection (p <0.05)，and the best reaction time was about30min～90min.5. ZIP by intrathecal application had no influence on motor function either in IBS-like rats or in control rats.Open field test shown that neither the highest doses of ZIP (10μg) nor the control saline had any statistical significance on total walking distance and average speed in IBS-like rats and control rats (p <0.05), assessed at60min post-injection, indicating that there was no effect on the locomotor function in rats after intrathecal injection of10μg ZIP.ConclusionPKMζ may be involved in the spinal central sensitization of chronic visceral pain on IBS after activated by phosphorylation, which suggests PKMζ maybe a new target for clinical drug therapy of chronic visceral pain on IBS.