Effects Of Berberine And GW405833 On Pancreatic Acinar Cell Ca²⁺ Signaling And Acute Pancreatitis

Background and objectiveAcute pancreatitis（AP）is an inflammatory disease in which premature activation of trypsin leads to self-digestion of the pancreas.At present,there is still no effective medicine for AP.It has been reported that the initial mechanism of AP is triggered by Ca²⁺overload in pancreatic acinar cells.Recent evidences have shown that berberine and the selective cannabinoid type 2 receptor（CB2R）agonist GW405833 inhibited calcium signaling in pancreatic acinar cells.Other studies have demonstrated that berberine and the selective CB2R receptor agonists have protective effects on experimental acute pancreatitis.However,studies have focused on early（within 1 hour after modeling）or preventive administration of berberine or cannabinoid receptors agonists,while few studies have been conducted on the administration of late acute pancreatitis,and it is unknown whether there is a synergistic effect between berberine and the selective CB2R agonists.Therefore,the purpose of this research is to investigate the effects of berberine combined with the selective CB2R agonist GW on intracellular calcium signaling in pancreatic acinus cells and the effects of different drug doses,different administration methods and combined administration on AP at the later stage of experimental AP.Methods1.Isolation of pancreatic acinar cells of mice:5-month-old or older CD1 and C57male mice were used,and sacrificed via cervical dislocation after anesthesia.Pancreas tissues were quickly isolated,then digested with collagenase solution to obtain acute dissociated acinar cell.2.Single-cell patch clamp recording:The acetylcholine（ACh）-induced Ca²⁺oscillations were recorded using patch-clamp whole-cell recording,and the influence of different concentrations of berberine,GW and combined drug on it was detected.3.Animal experimental groups:（1）AP group（L-arginine（L-Arg）,4 g/kg i.p.）;（2）Berberine（50 mg/kg）combined with sodium decanate（50 mg/kg）i.g.treatment group;（3）Berberine i.p.treatment group（10,5 mg/kg）;（4）GW treatment group（5 mg/kg,i.p.）;（5）Combined treatment group（5 mg/kg（BBR+GW）,i.p.）;（6）Control group.4.Establishment and detection of AP model in mice:3-month-old and older CD1mice were used.Animal model of AP was induced by L-Arg i.p.,2 times,1 hour interval.Medication was given at 8h,32h,56h of AP genesis.At 72h after the second injection of L-Arg,mice were sacrificed,then the blood was collected to measure serum amylase（AMS）activity,and the pancreas were taken for pathological examination.5.Data analysis and processing:Paired T-test was used for self-comparison,and One-Way ANOVA or Kruskal-Wallis test was used for comparison of multiple experimental data.Results1.The results of single-cell patch clamp:Berberine and GW both reduced ACh-induced Ca²⁺oscillations.Compared with the baseline（before berberine and GW combined perfusion）,the combined perfusion of 100 n M berberine and 100 n M GW reduced the normalized net charge of Ca²⁺oscillations to 30%±7.9%（P<0.001,N=7）.Q=2.1 was calculated using the formula of Webb fraction product method,indicating synergistic effect of the combined use of the two drugs at this dose.2.The results of animal experiment:At 8 h after AP genesis,berberine（50 mg/kg）combined with sodium decanoate（50 mg/kg）by gavage could not significantly inhibit serum AMS activity,but could significantly alleviated the pathological injury of pancreas caused by AP.Berberine（10,5 mg/kg）or GW（5 mg/kg）by Intraperitoneal injection could not significantly reduce serum AMS activity and pathological injury of pancreas caused by AP.Combined intraperitoneal injection of berberine（5 mg/kg）and GW（5 mg/kg）could not significantly relieve serum AMS activity and pathological injury of pancreas caused by AP.ConclusionWe first demonstrated that 100 n M berberine combined with 100 n M GW had a coordinate repression on ACh-induced Ca²⁺oscillations.In the later stage of experimental AP,berberine combined with sodium decanoate by gavage had a protective effect on AP,while berberine and GW had no significant protective effect on AP,either alone or in combination by intraperitoneal injection,suggesting that the treatment of AP may be time-dependent.