Up-regulation Of ASIC1 By MiR-485 Mediates The Spinal Cord Mechanism Of Chronic Colonic Visceral Pain Pain In The Offspring Rats Following Prenatal Maternal Stress

Aims:Irritable bowel syndrome(IBS)is a common chronic functional gastrointestinal disease,which is characterized by abdominal pain,bloating,and changes in bowel habits.In recent years,studies on IBS have been becoming more and more in-depth,while its essential pathogenesis is still unclear.Adverse stress occurred in early life may affect the gastrointestinal structure and function of the offspring.Previous studies in our laboratory have shown that the acid-sensitive ion channel 1(ASIC 1)is involved in gastric hyperalgesia in adult offspring rats subjected with prenatal maternal stress.However,whether it is involved in the pathogenesis of IBS and its mechanism are still unclear.And miR-485 is able to target the expression of ASIC 1.Thus,whether miR-485 participates in the occurrence of IBS by up-regulating the expression of ASIC1 has become the main research object of our study.Methods:1.Pregnant female SD rats were randomly divided into control(CON)group and experimental(PMS)group.PMS rats were subjected with heterogeneous intermittent stimulation(HIS)from the 7th day of pregnancy until delivery,and CON rats received no treatments.When the offspring grew to 6 weeks old,the visceral pain response was evaluated by detecting the rat’s colorectal distension threshold(DT).2.Western Blotting and RT-PCR were perfomed to detect the expressing of ASICs and related microRNAs.3.Intrathecal injection of ASICs antagonist amiloride or miR-485 Agomir was used to detect visceral pain response in PMS offspring rats.4.Spinal patch clamp was employed to record synaptic transmission characteristics of spinal dorsal horn neurons in CON/PMS offspring rats,and changes of synaptic transmission characteristics after drug administration on PMS offspring rats.5.Immunofluorescence and Fish were used to detect the localization of ASICl in spinal dorsal horn and the co-localization of miR-485 and ASIC 1.Results:1.At the age of 6 weeks and 8 weeks,PMS offspring rats showed obvious visceral pain.The ASIC1 protein expression in the spinal cord of T13-L2 segments increased significantly in 6 weeks of age,while the mRNA level did not change.In addition,ASIC1 was found to express mainly in the neurons of the spinal dorsal shallow horn.2.Intrathecal injection of ASICs antagonist amiloride could significantly alleviate visceral pain of PMS offspring rats.3.Compared with CON offspring rats,synaptic transmission in the spinal dorsal horn of PMS offspring rats was robustly enhanced,and intrathecal ASICs antagonist amiloride could largely reverse the increase of synaptic transmission in the spinal dorsal horn of PMS offspring rats.4.In the spinal dorsal horn of PMS offspring rats,miR-485 was significantly decreased.Intrathecal injection of miR-485 Agomir significantly attenuated visceral pain of PMS offspring rats.In addition,miR-485 administration largely reversed the increase in synaptic transmission in the spinal dorsal horn of PMS offspring rats.5.ASICl and miR-485 were co-expressed on neurons in the spinal dorsal horn.In addition,following intrathecal injection of miR-485 Agomir into PMS offspring rats,ASIC1 protein levels were significantly decreased.Conclusions:miR-485 participates in chronic visceral pain in PMS offspring rats by up-regulating the expression of ASIC1.