褪黑素对胰腺腺泡细胞钙超载的调节及其作用机制

The regulation and the mechanism of melatonin in calcium overload of pancreatic acinar cell

目的探讨褪黑素（MT）对急性坏死性胰腺炎（ANP）胰腺腺泡细胞钙超载的调节作用及其机制。方法54只SD大鼠均分为假手术组（SO组）、急性坏死性胰腺炎组（ANP组，胰胆管逆行注射牛磺胆酸钠法造模）和MT组（腹腔注射MT后30minANP造模）。术后1、4、8h分批处死大鼠并进行胰腺病理检查。用荧光控测法测定胰腺组织内游离钙离子浓度，实时荧光定量PCR及免疫印迹法分别检测胰腺细胞中钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）mRNA和蛋白表达水平。结果ANP组随时间延长胰腺病理损伤进行性加重，MT组胰腺病理损伤较ANP组明显减轻（1、4、8h时病理评分比较的t值分别为-7．95、-9．72、-7．69，P值均-0．00）。同时间点比较，ANP组胰腺组织内游离钙离子浓度均较SO组明显增高（1、4、8h时的t值分别-13．09、18．58、16．56，P值均=0．00），MT组较SO组略高，但较ANP组明显降低（1、4、8h时的t值分别：-10．03、-11．75、-11．02，P值均=0．00）。与S0组相比，ANP组CaMKIImRNA及蛋白表达水平均明显升高，MT对其表达有明显的抑制作用。结论MT干预可抑制CaMKⅡ表达，从而减轻胰腺腺泡内钙超载，起到保护胰腺组织的作用。

Objective To investigate the regulation and the mechanism of Melatonin in calcium overload of pancreatic acinar cell in acute necrotizing pancreatitis （ANP）. Methods Fifty-four Sprague-Dawley （SD） rats were equally divided into three groups： sham-operation group （SO group）, ANP group （created with retrograde cholangiopancreatography injection of sodium taurocholate） and MT group （ANP model made after intra-peritoneal injection MT for 30 rains）. Rats were sacrificed at 1, 4 and 8hours after operation and pancreas tissues were underwent pathological examination. The free calcium concentration of pancreas tissues was determined by fluorescence minitoring method and the expression of CaMK Ⅱ in pancreas tissues at mRNA and protein level was tested by real-time PCR and Western Blot. Results Pancreatic pathological injury in ANP groups was progressively severe as time extended, which was obviously ameliorated in MT group compared with ANP group （the t value of compared pathological score at 1, 4 and 8 hour was： -7.95, -9.72 and -7.69, all P =0.00）. Compared at same time point, the free Ca2＋ concentration of pancreas tissues in ANP group was significantly higher than that of SO group （the t value of 1, 4 and 8 hour was 13. 09, 18. 58 and 16.56, all P=0.00）. It was a little bit higher in MT group compared with that of SO group, howeverwas significantly lower than that of ANP group （the t value of 1, 4 and 8 hour was --10.03, -11.75 and -11.02, all P〈0.00）. Compared with SO group, the expression of CaMK Ⅱ at mRNA and protein level significantly increased in ANP group~ MT significantly inhibited its expression. Conclusions The expression of CaMK Ⅱ may be inhibited by MT interfere, and then lower the calcium overload in pancreatic acinar cell, which play a role in pancreas tissue protection.