The Role Of ASIC1 And PICK1 In Esophageal Visceral Hypersensitivity Of Rats With Gastroesophageal Reflux Disease

ObjectiveGastroesophageal reflux disease（GERD）is one of the most common digestive system diseases,the reason of which is complicated.It has been widely accepted that direct chemical damage of gastric contents,local inflammatory response and visceral hypersensitivity of esophagus are involved in the occurrence and development of GERD.Increasing evidence indicated that GERD patients,especially those with NERD,have an increased sensitivity to chemical or mechanical stimulations,suggesting that visceral hypersensitivity play an important role in generation and duration of symptoms of GERD.Acid-sensing ion channels（ASICs）is a kind of ligand-gated cation channels that are activated when extracellular pH decreases or proton increases,thus generating a Na⁺inward current,relating to conducting chemical and mechanical signals.It is clear that ASICs are associated with nociception and central sensitization,and play an important role in regulation and conduction of pain.Protein interacting with C-kinase 1（PICK1）is a highly conserved peripheral membrane protein,containing PDZ（PSD-95/Dlg/ZO-1）and BAR（Bin-Amphiphysin-Rvs）domains and making it possible to bind to a variety of proteins,relating to protein transport and synaptic transmission.It is confirmed that PICK1regulates expression and function of ASIC1 by binding to C-terminal acid domain of ASIC1.Currently,the mechanism of visceral hypersensitivity in GERD patients is still unknown.Whether ASIC1 or PICK1 is involved in the occurrence and development of GERD has not been studied clearly.This study intended to clarify mechanism of visceral hypersensitivity and possible role of ASIC1 and PICK1 in the progression of GERD.MethodSprague-Dawley rats were performed pylorus restriction with different diameter（Group A:put a special 18Fr Nelaton catheter in the pylorus;group B:limit pylorus diameter to 4.17mm;group C:limit pylorus diameter to 3.42mm;D group:limit pylorus diameter to 2.98mm）combined with ligation of the junction between forestomach and corpus,in order to establish the gastroesophageal reflux disease rat models.Group S is the control.Postoperative weights of rats were measured.Histological examination of esophagus was carried out and stained with haematoxylin and eosin（H&E）.The esophagus was observed and the hematoxylin and eosin（HE）staining was used to analyze the incidence of GERD of each procedure.The rat models of GERD were established by ligation of junction between forestomach and corpus combined with pylorus restriction limiting pylorus 3.42mm in diameter.The mechanical paw withdraw threshold and thermal paw withdraw latency were measured on 1 day before operation and the 3^th,7^th,11^th and15^th day after operation.The esophageal specific dorsal root ganglions（DRG）were observed by retrograde neural tracing with DiI tracer injected in esophageal wall,whole call patch clamp was performed on the 15^th day postoperatively.The expression of ASIC1and PICK1 in esophageal mucosa and T3^T5 DRG was detected by Western Blot and RT-PCR.SPSS18.0 software was used to statistical analysis,P<0.05 considered statistically significant.Results1.Comparison of rat models of gastroesophageal reflux disease with different diameter of pylorusThe wights of rats in group S and group B were increased after operation,there was no significant differences in weight of rats between those two groups（296+19.408g vs278.50+19.156g,P=0.077）.There was a significant difference in weight loss of rats in group A,C,D compared with that in group S（168.75±13.823g,177.50±15.501g,152.00±5.701g,P<0.001）.The survival rates of group A,B,C and D were 40%,85%,60%and 25%respectively.15 days after surgery,esophagus of rats in group S presented an intact and thin epithelial layer with few inflammatory cells in submucosal layer,while esophagus of the remaining groups exhibited marked increase thickness of epithelium,obvious hyperplasia of basal cells,heavy infiltration of inflammatory cells in submucosal,apparent elongation of papilla to more than 2/3 of the epithelium and so on.The occurrence ratios of esophagitis in group A,B,C,D were 100%,41.18%,91.67%and100%respectively.We conclude that can successfully prepare gastroesophageal reflux disease rat models could be established successfully by pylorus restriction combining with gastric fundus ligation,and 3.42mm may be the most fitable diameter of pylorus restriction.2.Changes in mechanical paw withdraw reflex threshold,thermal paw withdraw latency and sensitivity of DRG neurons of GERD ratsThere was no significant differences between the two groups before operation and on the third day after operation（49.14±15.44g vs 54.63±8.80g,P=0.511;9.64±6.19g vs6.33±4.42g,P=0.307）.Mechanical paw withdraw threshold significantly decreased in GERD rats,compared with that in control rats at the 7^th,11^th and 15^th day postoperatively（7.98±5.66g vs 24.28±17.02g,P<0.05;12.08±13.78g vs 36.12±16.60g,P<0.01;16.16±17.71g vs 39.40±14.97g,P<0.05）.There was no significant differences in thermal paw withdraw latency between the two groups before and on the3^th,7^th,11^th and 15^th day after operation(18.34±1.14s vs18.31±0.86s,P=0.948;（9.10±0.91s vs 8.43±0.80s,P=0.139;9.95±1.51s vs 9.06±1.35s,P=0.232;13.26±1.61s vs 11.70±1.84s,P=0.092;13.48±1.63s vs 12.70±1.46s,P=0.335）.Those results indicated that there were somatosensory changes to mechanical stimulations in GERD rats,but no significant changes to thermal stimulation.The whole cell patch clamp showed that the resting membrane potential of esophageal DRG neurons in GERD rats were significantly lower than that in control rats（-45.27±0.56m V vs-51.91±0.34mV,P<0.001）.The basal intensity of esophageal DRG neurons in GERD rats were significantly lower than that of control rats（25.91±5.98pA vs83.64±3.88pA,P<0.001）,and the frequency of action potential activitied by 2,3times of basal intensity in GERD rats were significantly increased,compared with that in control rats（6.27±0.76 vs 3.36±0.41,P<0.001;10.91±0.77 vs 5.64±0.49,P<0.001）.All result above indicated that the sensitivity of DRG neurons increased significantly in GERD rats.3.The expression of ASIC1 was down-regulated in esophageal mucosa of GERD rats,while PICK1 was up-regulated in esophageal mucosa of GERD ratWestern blot showed that the expression of ASIC1 was decreased in esophageal mucosa of GERD rats（1.1594±0.4678 vs 0.7190±0.2284,P<0.05）.There was no significant differences in expression of ASIC1 protein in DRG between GERD rats and control rats（1.4948±0.2657 vs 1.5180±0.6213,P=0.925）.Compared with control rats,the expression of PICK1 was up-regulated in esophageal mucosa of GERD rats（0.5109±0.2484 vs 0.9141±o.3924,P<0.05）.There was no significant differences in the expression of PICK1 protein in DRG between GERD rats and control rats（0.4963±0.4560 vs 0.3183±0.2476,P=0.375）.TheRT-PCR results showed that the level of ASIC1 m RNA in esophageal mucosa were significantly lower in GERD rats than that in control rats（1.0207±0.2126 vs 0.6559±0.1193,P<0.01）.The expression of ASIC1 mRNA was decreased in DRG of GERD rats and control rats（0.9204±0.2190 vs 1.1055±0.2811,P=0.232）.Compared with control rats,the expression of PICK1 mRNA in esophageal mucosa of GERD rats were significantly different（1.3428±0.1621 vs 1.0111±0.1626,P<0.01）.There was no significant differences in the level of PICK1 mRNA in DRG between GERD rats and control rats（1.0295±0.2249 vs 1.0176±0.1978,P=0.924）.Conclusion1.Pylorus restriction in 3.42mm diameter combined with ligation of the junction between forestomach and corpus may be a reliable method to establish of GERD rat models;2.The sensitivity of DRG neurons in GERD rats significantly increased,the somatic sensitivity to mechanical stimuli increased,suggesting that esophageal hypersensitivity affects the formation of somatic sensations.3.The expression of ASIC1 was decreased,while the expression of PICK1 was increased in esophageal mucosa of GERD rats,suggesting that ASIC1,PICK1 participate in the formation of esophageal hypersensitivity in GERD rats.