| Acute pancreatitis is a common acute abdomen disease of internal medicine,which is urgent and developing fastly,and may be endangerous if there is no treatment on time.The causes of acute pancreatitis are various,such as gallstones and other biliary diseases,alcoholism and overeating,gastroduodenal and other related diseases.The pathologic mechanism of acute pancreatitis has not been elucidated,and there is no specific therapeutic drugs in clinic,especially acute severe pancreatitis which is still symptomatic treatment and the poor prognosis.Various factors such as abnormal activation of the enzyme,inflammatory mediators,oxidative stress,microcirculatory disorders,and nuclear factor kappa B?NF-?B?were all involved in the pathogenesis.Although the pathogenesis of pancreatitis has not been fully elucidated,studies have shown that activating the inflammatory signaling pathway in the pancreatic acinar cells is an important molecular mechanism for acute pancreatitis.The pancreatic lesions and the following activated macrophages and neutrophils release huge an amounts of inflammatory cytokines and inflammation"waterfall cascade"accompanied acute pancreatitis which will result in systemic inflammatory response syndrome?SIRS?and multiple organ dysfunction syndrome?MODS?.Inflammatory cytokines such as TNF-?,IL-6,IL-1?,IL-10 and ICAM-1 and immune cells are important factors affecting the mortality of inflammatory cells.These inflammatory mediators promote the occurrence and development of pancreatitis through complex signaling pathways.The IL-6/IL-6R/STAT3 signaling pathway plays an important role in acute pancreatitis and systemic inflammatory response.The transmembrane protein 16A?TMEM16A?is a calcium-activated chloride channel that can be activated by intracellular calcium,which is demonstrated as by three laboratories at the same time in 2008.The function of TMEM16A may be associated with the cell division cycle and cell proliferation.TMEM16A expreses in a variety of cells and participates in the salivary glands,sweat gland,respiratory tract,intestinal epithelial cell secretion,smooth muscle contraction,control of gastrointestinal pacemaker and other physiological processes.In addition,TMEM16A refers to the pathophysiological processes of tumors,hypertension and pain.TMEM16A was involved in the pathogenesis of respiratory inflammation such as asthma.TMEM16A is expressed in pancreatic duct cells,islets and acinar cells,and is involved in the occurrence of ductal carcinoma and insulin secretion and the acidic regulation of pancreatic cells.However,the role and mechanism of TMEM16A in acute pancreatitis is not reported.So we carry out the following research:Part?TMEM16A was up-regulated in acute pancreatitisObjective:To observe the change of TMEM16A channel protein at different time points of acute pancreatitis caused by caerulein in vivo and in vitro.Methods:In vivo,all mice were randomly divided into control group,caerulein 6h group,caerulein 12h groupe and caerulein 24h group and 6 mice per group.Mice were raised adaptively for 3 days after purchase and the mice were breeded under the condition of free water and fasting 12 h before implementation night.The experimental mice were produced by intraperitoneal injection caerulein of saline solution,with dosage of 50?g/kg,hourly for seven times,and the control mice with intraperitoneal injection of saline.The blood was collected from the medial canthus of the mice,and the blood was removed and the serum was preserved after the last injection to 6 hours,12 hours and 24 hours.After the death of cervical dislocation of mice,the pancreatic tissue was extracted,and western blot was used to detect TMEM16A channel protein in pancreatic tissue.Another was fixed in4%formaldehyde solution for the method of HE staining verified pathomorphism detectionand immunohistochemical method to detect TMEM16A channel protein expressionin the pancretitis.In vitro,the pancreas cells were primary cultured with Waymouths media from mice and dealed with 10-8M caerulein when the cells grew to 90 percent of the culture dish.And the cells were collected after different time.Cell culture supernatant were collected.Cell culture was carried out in AR42J cells of rat pancreatic acinar cell line,and the cells were collected after different time points after dealing with 10-8M caerulein,and the cell culture was retained.The expression of TMEM16A channel protein was detected by western blot and Immunofluorescence.Results:The mice model of acute pancreatitis was successfully induced by caerulein.The expression of TMEM16A channel protein was up-regulated in acute pancreatitis.The expression of TMEM16A channel protein was increased in the pancreatic acinar cells of primary culture.TMEM16A channel protein was discovered in normal AR42J cell,and TMEM16A channel protein was over-expressed in inflammatory AR42J cells.Conclusion:TMEM16A channel protein was expressed in pancreatic acinar cell;And TMEM16A channel protein expression was up-regulated in the acute pancreatitis model induced by caerulein.Part?Up-regulation of TMEM16A was caused by increasing IL-6 and its activation to IL-6/IL-6R/STAT3 signaling pathway in acute pancreatitisObjective:To observe IL-6 affect on IL-6/IL-6R/STAT3 signaling pathway in acute pancreatitis and its regulation effect on TMEM16A protein.Methods:ELISA was used to detect IL-6 in the pancreatic tissue and serum of acute pancreatitis mice besides of the cultured supernatant of inflammatory AR42J cells and pancreatic acinar primary cells in caerulein.The expression of IL-6/IL-6R/STAT3signaling pathway protein and TMEME16A were investigated after the treatment with caerulein to AR42J cells.The dose-dependent of TMEM16A expression of AR42J cells treated by IL-6 were screened.The expression of IL-6/IL-6R/STAT3 signaling pathway and TMEM16A were detected after IL-6 treatment of AR42J cells;The expression of IL-6/IL-6R/STAT3 signaling pathway and TMEM16A protein were reseached after dealing with IL-6 and IL-6R neutralizing antibody simultaneously on AR42J cells.The expression of IL-6/IL-6R/STAT3 signaling pathway and TMEM16A protein were tested after IL-6and STAT3 inhibitors Cucurbitacin?treated simultaneously to AR42J cells.Results:IL-6 in the pancreas tissue and serum of acute pancreatitis mice was increased as well as that in the cultured supernatant of the isolated pancreatic acinar cells and AR42J cells.IL-6 was enhanced in AR42J cells treated by caerulein,which activated IL-6/IL-6R/STAT3 signaling pathway to promote the expression of TMEM16A.The expression of TMEM16A was promoted in AR42J cells by IL-6 with optimal concentration of 0.25?g/ml for 48 hours;TMEM16A protein was increased by IL-6 which interacts to IL-6R in AR42J cells;IL-6 enforced the expression of TMEM16A by promoting the phosphorylation of STAT3 in AR42J cells.Conclusion:IL-6 promotes The expression of TMEM16A in AR42J cells was promoted by IL-6 through IL-6/IL-6R/STAT3;The high expression of TMEM16A was caused by the increasing IL-6 through IL-6/IL-6R/STAT3 signaling pathway in acute pancreatitis induced by caerulein.Part?The up-regulation of TMEM16A promotes the developent of acute pancreatitisObjective:To observe the effects of TMEM16A protein on the inflammatory pathogenesis of acute pancreatitis,IL-6 content and nuclear transcription factor NF-kappa B.Methods:The experimental mice were randomly assigned by weight to control group,model group,the T16Ainh-A01 pretreat group and T16Ainh-A01 posttreat group.The acute pancretitis was produced intraperitoneally by caerulein of saline solution with dosage of 50?g/kg hourly for seven times while the control mice with equal saline.TMEM16A specific inhibitor T16Ainh-A01 was injected intraperitoneally 30 minutes before the first caerulein called the T16Ainh-A01?1mg/kg?pretreat group and 30 minutes after the first injection as the T16Ainh-A01 posttreat group.The blood was collected from the medial canthus of the mice,the serum was taken out and the centrifuge at 12 hours of the last injection.The pancreatic tissue was extracted and the content of IL-6 in serum and pancreatic tissue homogenate was detected by ELISA after cervical dislocation of mice.Western blot was used to detect the expression of TMEM16A protein and NF-?B/p65 in pancreatic tissue.Another part pancreatic tissue fixed in 4%formaldehyde solution to HE staining method detecting the pathological morphology and to immunohistochemical method for detection of pancreatic tissue TMEM16A channel protein expression.By plasmid transformation and amplification,plasmid DNA extraction,pGFP-TMEM16A plasmid was transfected into AR42J cells with the lipofection 2000 transfection reagent.After G418 screening and extracting cell cytoplasm protein and nucleoprotein respectively,the NF-?B/p65 was assessed in AR42J cytoplasm and nucleus by immunofluorescence method.Results:TMEM16A channel protein expression was increased after 12 hours with caerulein in AR42J,and p65 in cytoplasm decreased while the nucleus of p65 protein expression increased,which implicated p65 protein transferring into nuclear in pancreatitis of AR42J.After treated with T16Ainh-A01,TMEM16A channel protein was decreased,and p65 protein in cytoplasm was increased while decreased in the nucleus,which inhibited p65 protein into nucleus.The content of IL-6 was increased significantly in serum and pancreatic tissue after 12 hours caerulein while it was decreased after given TMEM16A specific inhibitors T16Ainh-A01 vs the caerulein mice.Acute inflammation of the pancreas was lobular gap widened,edema,inflammatory cells exudation and acinar cell swelling and edema after caerulein.And the pancreatitis was alleviated after intraperitoneal injection of TMEM16A specific inhibitor T16Ainh-A01 vs the caerulein group.TMEM16A protein was detected after transfected and screened by G418 and the transfection efficiency was about 50%.After over-expression of TMEM16A,the p65protein in the cytoplasm decreased but increased in the nucleus,and the ratio of p65 in nuclear/plasma was increased significantly and the nuclear transfer occurred in p65.The cytoplasm p65 protein was increased while decreased in the nucleus when inducing acute inflammation model by caerulein in knockout TMEM16A cells,at the same time the content of IL-6 in cell supernatant was decreased.Conclusion:The up-expression of TMEM16A protein in acute pancreatitis promoted the NF-?B nuclear transfer of pancreatic inflammatory cells,and expedited the synthesis and release of IL-6 and accelerated the pathological process of acute pancreatitis. |
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