Spinal Mechanisms Of ASIC1 Regulating Expression Of NKCC1 In Rats With Chronic Colorectal Pain

Aims:Irritable bowel syndrome(IBS)is a common functional gastrointestinal disorder that is widely distributed around the world.Symptoms include abdominal pain,bloating and changes in bowel habits.The pathogenesis of IBS is complex and has not yet been fully understood.Early life stress affects social behavior and emotional development.We simulated this early life stress by neonatal maternal deprivation(NMD)to induce chronic visceral pain sensitivity in SD rats.Acid-sensing ion channel 1(ASIC1)plays an important role in pain regulation.Na+-K+-C1--C1--1(NKCC1)is a cationic chloride co-transporter that regulates intracellular and extracellular Cl-concentration and plays an important role in the regulation of pain.Whether ASIC 1 and NKCC1 play a regulatory role in chronic visceral pain and the relationship between the two are not fully understood.Therefore,this project mainly studies the regulatory mechanism of ASIC1 and NKCC1 in chronic visceral pain sensitivity,and then seeks new therapeutic targets for the treatment of chronic visceral pain.Methods:1.NMD modeling:from the second day,newborn SD rats were separated from their mothers for 3 hours every day for 14 consecutive days.The control group received no treatment.After the pups had grown to 6 weeks,these rats were evaluated for visceral pain sensitivity through the colorectal distention(CRD)2.Patch clamp recording was used to detect excitability and changes in synaptic transmission of spinal dorsal horn neurons in CON and NMD rats at T13-L2 segment.3.Expression changes of ASICs,NKCC1 and KCC2 were detected by Western Blotting.4.Intrathecal injection of ASICs antagonist Amiloride or NKCC1 inhibitor Bumetanide(BMT)was used to detect visceral pain sensitivity in NMD rats.5.Patch clamp was used to detect changes in synaptic transmission before and after incubation with Amiloride,an antagonist of ASICs,or BMT,an NKCC1 inhibitor.6.The distribution of ASIC1 in spinal dorsal horn was detected by immunofluorescence assay.Results:1.NMD rats showed significant visceral pain sensitivity at the age of 6 weeks,and ASIC1 protein expression was significantly up-regulated in the spinal dorsal horn of T13-L2 segment.Immunofluorescence showed that ASIC1 was mainly distributed in the superficial neurons in the dorsal horn of the spinal cord.2.Compared with CON rats,the excitability of spinal dorsal horn neurons in the T13-L2 segment of NMD rats was significantly enhanced,and the excitatory synaptic transmission was significantly enhanced,but the inhibitory synaptic transmission was significantly decreased3.Intrathecal injection of Amiloride,an ASICs antagonist,significantly attenuated chronic visceral pain sensitivity in NMD rats.4.The expression of NKCC1 was significantly up-regulated,and intrathecal injection of BMT,an inhibitor of NKCC1,could significantly reverse the chronic visceral pain of NMD rats.5.Incubation of Amiloride significantly reduced the amplitude of mEPSCs in the spinal dorsal horn of NMD rats without affecting the frequency of mEPSCs,indicating that ASIC1 enhanced excitatory synaptic transmission through the postsynaptic mechanism.Incubation of BMT significantly increased the amplitude of mIPSCs in the spinal dorsal horn of NMD rats without affecting the frequency of mIPSCs,suggesting that NKCC1 reduced inhibitory synaptic transmission through the postsynaptic mechanism.6.Intrathecal injection of Amiloride,an ASICs antagonist,significantly suppressed the expression of NKCC1 in the spinal dorsal horn of NMD rats.Conclusions:NMD enhanced the spinal cord ASIC1 expression level,which was likely mediated by up-regulating the NKCC1 expression,thus enhancing excitatory synaptic transmission and suppressing inhibitory synaptic transmission at the spinal cord level,and eventually contributing to the chronic visceral pain.