The Role And Mechanism Of Muscarinic Acetylcholine Receptor 3 In Pancreatitis

Background and objective:Pancreatitis,both acute and chronic forms,is an inflammatory disease of the pancreas which is painful and often lethal in severe cases.So far there is no proven pharmacologic entity to treat acute pancreatitis（AP）.Supportive care with intravenous fluids,bowel rest and pain management are the mainstays of therapy.Investigation into the pathogenesis and treatment of pancreatitis in humans faces many obstacles because of its deep anatomical location in the human body.Most of our knowledge on pancreatitis is based on research conducted using experimental animal models.There are difficulties in translating animal research into clinical practice.Alternative clinically relevant animal models need to be established for the investigation into the mechanisms of pancreatitis pathogenesis and the development of effective preventive and therapeutic interventions.Currently,the most widely used method for research is pancreatitis induced by hyperstimulation with cerulein,a cholecystokinin（CCK）analogue,in rats and mice.However,we and others have previously shown there are no functional CCK receptors on human pancreatic acinar cells,calling into question the relevance of cerulein-induced pancreatitis models to the human disease.Muscarinic receptor 3（M3R）is highly expressed on human pancreatic acinar cells.Both CCKAR and M3R belong to Gq-protein coupled receptors and activation of these receptors causes similar biological effects on isolated pancreatic acinar cells,including calcium and PKC signaling pathways,amylase secretion,morphological changes,and LDH release.The most direct evidence suggests that in vivo exposure to potent cholinergic agonists,as seen with scorpion bites or organophosphate intoxication,is linked to the development human acute pancreatitis.Atropine,a cholinergic receptor antagonist,ameliorated pancreatitis induced by the combination of alcohol and cerulein,suggesting that cholinergic pathways are involved pancreatitis in vivo.Because both acetylcholine-activated nicotinic and muscarinic receptors are widely expressed in the central nervous system,ganglionic neurons,muscles,mucosa,etc,it is difficult in conventional pharmacology to study the specific roles of acinar M3R in the pathogenesis of pancreatitis.In order to specifically activate the M3R in mouse pancreatic acinar cells,we decided to use a new genetic mouse model with conditional expression of a modified human M3 receptor by chemical-genetic approach to control the activation of receptors,the Designer receptors exclusively activated by designer drugs（DREADD）.The DREADD mutant M3R（h M3R）losses responsiveness to the endogenous ligand acetylcholine,but can be activated by exogenous CNO（clozapine-N-oxide）.Therefore,measures to activate the M3R specifically on pancreatic acinar cells shall be developed to explore its role and mechanism of pancreatitis.Materials and methods:（1）To compare the expression and function of different M receptors and CCK receptors in human pancreatic acinar cells1.RNA sequencing and RT-PCR were used to detect the m RNA expression of different subtypes of M receptors and CCK receptors in pancreatic acinar cells from healthy human.2.To observe the function of human pancreatic acini cells response to the M receptor agonists carbachol and CCK receptor agonists CCK-8,including the release of amylase and morphology.（2）Construction of a mouse model expressing with pancreatic-specific mutant M3 receptors to investigate the role of M3 receptors in pancreatic injury.1.For conditional pancreatic acinar specific expression of h M3R,LSL-h M3R（Loxp-Stop-Loxp-h M3R）mice were crossed with BAC-Ela-Cre^ERT（BAC）mice which express Cre^ERT driven by the pancreatic acinar-specific elastase-I promoter.Expression of h M3R alleles was induced by administration of tamoxifen.The protein expression of h M3R was verified by Western Blot.2.We studied the effects of CCK,carbachol and CNO stimulation in isolated pancreatic acinar cells from control BAC and h M3/BAC mice,including the release of amylase and morphology.3.We explore different doses of intraperitoneal injection of CNO（0,1,2,4,6,8,10,20mg/kg for 5 times（1/h）（n=3））,and induce the occurrence of AP through activation of h M3 receptor.Based on the trend changes of above results,the effective dose of CNO was selected to construct the AP model in h M3/BAC mice（n=3）stimulated with 6mg/kg dose of CNO for different times.4.The AP model was constructed by injecting CNO,and detected the degree of pancreatic edema,serum amylase level and pancreatic pathological injury for h M3/BAC mice compared with BAC and h M4/BAC mice.Immunohistochemical staining of neutrophils（CD11b）and macrophages（F4-80）was used to observe inflammatory cells.5.To observe the typical changes of impaired autophagy in the AP model of h M3/BAC mice.6.Pdx1 promoter was selected to mediate the expression of Cre enzyme,and the degree of pancreatic edema,serum amylase level,inflammatory cells and pancreatic pathological injury induced by CNO in h M3/Pdx1 mice were observed.7.Comparing the M3 receptor models of CNO-stimulated with AP induced by cerulein,BAC mice were selected to be given an intraperitoneal injection of 100cerulein for 8 times（1/h）,and BAC,h M3/BAC and h M3/Pdx1 mice were given a single CNO（6mg/kg）.Pancreatic tissue edema,serum amylase level,pancreatic histopathology,immunohistochemical staining（cleaved Caspase-3（C-caspase-3）and necrosis（HMGB1））were compared.（3）To investigate the activation of NF-κB pathway involved in the process of M3 receptor-induced AP and the effect of IKKβby regulating NF-κB pathway on the prognosis of AP.1.To observe the activation of the NF-κB signaling pathway in the AP model induced by stimulation of the M3 receptor in pancreas compare to the cerulein-induced AP and normal saline groups,and to detect the levels of IκBαand phosphorylated p65（Ser 536）and the localization of p65 proteins in the pancreatic tissuses by IHC.2.After activating M3 receptors,we observe changes in m RNA levels of many inflammatory factors such as MCP1,COX-2,TNF-α,IL-1β,and IL-6 which are the downstream of NF-κB signaling pathway.3.To further explore the relationship between the NF-κB pathway and the prognosis of pancreatitis,the effect of IKKβ,a key protein upstream of the NF-κB pathway,on the activation of M3 receptor-induced pancreatitis was observed.Conditional IKKβknockout transgenic mice were induced by tamoxifen after cross-breeding with pancreas-specific Cre mice.The h M3/BAC,IKKβ^f/+/h M3/BAC and IKKβ^f/f/h M3/BAC mice were treated with CNO or normal saline as a control to observe the indexes of AP severity.4.To observe the effects of specific IKKβinhibitor,TPCA-1 for pancreatic damage and inflammation of the AP model induced by cerulein and h M3/BAC induced by CNO.（4）Characteristics of chronic pancreatitis induced by repeated stimulation of M3 receptors.1.The m RNA levels of TGF-βandα-SMA were significantly up-regulated in the early stages of AP,especially in CNO-stimulated h M3/BAC mice,suggesting a possible progression to chronic inflammatory processes.,The progression and recovery of pancreatic lesions and the chronic inflammatory process were observed within one week in the AP model induced by cerulein or stimulation of the M3receptor.2.As the activation of M3 receptors in the pancreas shows more severe characteristics of pancreatic damage and slower recovery process than the cerulein-induced AP model.It is speculated that pancreatitis induced by h M3 receptor mice is more likely to become chronic pancreatitis.To test this hypothesis,recurrent pancreatitis was induced in BAC mice by multiple injections of cerulein or CNO for h M3/BAC and h M3/Pdx1 mice.Pancreatic damage was assessed after 28 days.3.To observe the typical characteristics of CP in mice activated by cerulein or M3 receptor by Sirius red staining,Ki-67 staining,and macrophage marker（F4-80）staining.（5）Pharmacological antagonist M3 receptor used to intervene the acute pancreatitis induced by cerulein.1.To observe the effect of pre-treatment specific M3 receptor inhibitor,darifenacin hydrobromide on pancreatic damage and inflammation in AP models of wild type（WT）mice and Try/BAC mice induced by cerulein.Results:（1）Human pancreatic acinar cells express M3 receptors but not CCKA receptors1.Total RNA was obtained from pancreatic acinar cells isolated from healthy donors.By RNA sequencing,it was found that the expression of CCKA receptor was hardly expressed,and the M3 receptor was significantly expressed in the results.RT-PCR was used to verify the results of RNA sequencing.Only M3 receptors were found in human-derived pancreatic cells compared to other M receptors and CCK receptors.2.Different doses of CCK-8 did not respond to human pancreatic acinar cells,and releasing amylase maintained a low concentration.The release of amylase increased significantly with the increase of the concentration of carbachol,and the release of amylase in supernatant reached the peak at the concentration of 10^-6M.Morphological changes of human acinar cells were observed to be garlands induced by carbachol stimulation.（B）Acute pancreatitis was induced by activation of M3 receptor in mice1.The h M3/BAC mice with specific expression of mutant M3 receptor in pancreatic acinar cells were constructed.The expression of human M3 receptor in h M3/BAC mice was observed at the protein level,indicating the presence of gene recombination and the expression of human mutant M3 receptor in pancreatic acinar cells.2.In pancreatic acinar cells from the control BAC mice,CCK and carbachol induced membrane blebbing and acinar membrane damage as indicated by ethidium bromide staining and biphasic amylase secretion.No response to CNO was observed in the control pancreatic acinar cells.In contrast,pancreatic acinar cells from h M3R mice responded to CCK,carbachol,as well as CNO in similar patterns.These data indicate that CNO and carbachol activation of M receptors caused pancreatic acinar cell damage.3.By exploring different doses and times of CNO administration to stimulate the occurrence of M3 receptor-induced AP in mice,it was found that the AP model could be constructed in h M3/BAC mice stimulated with a single dose of CNO of 6mg/kg.4.Compared with the control group and h M4 receptor mice,the degree of pancreatic edema and serum amylase level were increased in the CNO-treated h M3receptor mice.There were also obvious edema,inflammatory cells and pancreatic acinar cell necrosis in the pancreatic histopathology of h M3/BAC mice.5.After CNO activated the pancreatic M3 receptor in h M3/BAC mice,the increase of p62 protein and LC3I to LC3II in the pancreatic tissue were observed in the early stage.Impaired autophagy was observed in an AP model induced by activation of the pancreatic M3 receptor.6.Pdx1 promoter was used to mediate the expression of Cre enzyme.Similar to h M3/BAC mice,CNO induced h M3/Pdx1 mice can also cause significant pancreatic edema,serum amylase release,and pancreatic injury including infiltration of inflammatory cells.7.Compared with cerulein-induced AP,CNO stimulating M3 receptors of h M3/BAC and h M3/Pdx1 mice can cause more pancreatic tissue edema,serum amylase level,severe pancreatic damage,apoptosis and necrosis.（3）Stimulation of M3 receptors in mice leads to activation of the NF-κB pathway and the reduction of IKKβin the regulation of the NF-κB pathway improves the prognosis of AP.1.Compared with the control group,the expression of IκBαwas significantly reduced and the expression of p-P65（Ser 536）was significantly increased in the pancreatic M3 receptor of h M3/BAC mice activated by CNO.By IHC localization analysis,a large number of nuclear staining of P65 protein were observed in pancreatic tissue of h M3/BAC mice.It is suggested that the activation of pancreatic M3 receptor can induce the activation of NF-κB pathway.2.Compared with the control group and the AP group induced by cerulein,the m RNA levels of MCP1,TNF-α,IL-1β,and IL-6 in pancreatic tissue of h M3/BAC mice stimulated by CNO significantly increased,suggesting higher levels of inflammation.3.By specifically knockout IKKβthat was upstream of the NF-κB pathway on pancreatic acinar cells,it was observed that the level of IKKβin the pancreatic tissue of mice was significantly reduced.Pancreatic IKKβknockdown in AP models significantly reduced serum amylase levels,pancreatic pathological damage,recruitment of inflammatory cells,and apoptosis.Comparing h M3/BAC group treated with CNO,IKKβ^f/f/h M3/BAC mice were significantly reduced in pancreatic damage,inflammation and apoptosis.Therefore,knockout IKKβexpression in pancreas can alleviate M3receptor-induced pancreatitis.4.In the AP model induced by cerulein and h M3/BAC mice with CNO,treatment with the selective IKKβinhibitor,TPCA-1 reduced pancreatic pathological damage and inflammatory cells.（4）Chronic pancreatitis can be developed by repeated stimulation of the M3receptor in mice.1.In the early stage of acute pancreatitis,m RNA levels of TGF-βandα-SMA were significantly increased especially in the CNO-stimulated h M3/BAC mice,suggesting that they may progress to the chronic inflammatory process.By observing the progress and recovery of pancreatic lesions within a week after AP induction,h M3/BAC mice recovered approximately one week after a single CNO injection,but a little chronic inflammatory infiltration was also observed.In contrast,after a single CNO-induced acute pancreatitis in h M3/Pdx1 mice,the pancreatic weight became significantly smaller.The results suggest that activation of the M3 receptor can cause delayed recovery of acute pancreatitis.2.The pancreas of mice treated with cerulein for three days returned to normal after4 weeks,while the pancreatic pathology of most h M3/BAC mice and all h M3/Pdx1 mice showed more severe and wide-spread with massive chronic inflammation,fibrosis,adipose tissue infiltration,and pancreatic atrophy.Even h M3/Pdx1 mice showed acinar-ductal metaplasia（ADM）.Therefore,repeated stimulation of pancreatic M3receptors can develop chronic pancreatitis.3.Compared with the cerulein stimulation,fibrosis（Sirus red staining）,reactive cell proliferations（Ki-67）,and chronic inflammatory cell infiltrations were more prominent in the M3 receptor mice,especially in the h M3/Pdx1 mice（5）M3 receptor antagonist alleviate cerulein-induced pancreatitis.1.In the AP model of wild-type mice and Try/BAC mice induced by cerulein,pretreatment with selective M3 receptor antagonist darifenacin hydrobromide can significantly improve pancreatic damage.Conclusions1.The expression and function of M3 receptor in human pancreatic acinar cells were confirmed,but there was no CCKA receptor.2.Isolated pancreatic acinar cells of transgenic mice with mutant M3 receptor,CNO stimulation of mutant M3 receptor can cause cell damage.Activation of the mutant M3receptor in mice（h M3/BAC and h M3/Pdx1）induced the occurrence of AP,which established a more severe AP model with clinical relevance compared with the model of cerulein.3.Activation of NF-κB signaling pathway and increased inflammation can be observed in the AP model induced by M3 receptors.Knockout of IKKβin mice can alleviate pancreatitis induced by activation of M3 receptors,and it was observed that the specific IKKβantagonist TPCA-1 can also alleviate pancreatitis.4.Recovery of M3 receptor-induced pancreatitis was delayed,and chronic pancreatitis could be developed by repeated stimulation of M3 receptors in mice（h M3/BAC and h M3/Pdx1）.5.The specific antagonist of M3 receptor,darifenacin hydrobromide,can alleviate AP injury induced by cerulein.Targeting the M3 receptor may provide a new approach to the prevention and treatment strategies of human pancreatitis.