黄连素结合M3受体调节小鼠胰腺腺泡细胞钙信号的机制研究

Acute pancreatitis (AP) is a life-threatening serious inflammatory disease. Since the reasons for its pathogenesis are still unclear, the effective treatment of AP is still lack. Recent studies have shown that calcium overload in pancreatic acinar cells(PAC) is an important factor that triggers the onset of AP, which provides a novel therapeutic strategy for treatment of AP through the prevention of cell calcium overload.Emerging evidence shows that berberine can improve the pathological changes in AP model mice, but the cellular mechanism of improvement calcium overload in pancreatic acinar cells are still unclear. During a Chinese-American Ph.D. Co-training Program, the applicant investigated the berberine can significantly inhibit acetylcholine-induced PAC calcium oscillations. Further experiments confirmed that berberine can bind to the mycotoxin M3 receptor subtype (M3R) of PAC. Therefore, we hypothesized that berberine inhibits calcium signal in PAC through the M3 receptor, thereby reducing intracellular calcium overload and preventing intracellular protease activation and PAC necrosis.Based on our preliminary studies, this proposal will use patch clamp and Ca2+ imaging techniques to further study the effects of berberine on PAC intracellular Ca2+ signals. The main research aims include: (1) the pharmacological mechanism of berberine-induced inhibition in Ca2+ signal of PAC, (2) the specific target of berberine effect, and (3) the protective effect of berberine on the pathological injury during acute pancreatitis. This proposal will provide theoretical and experimental evidence to elucidate the cellular mechanisms and targets of berberine in the treatment of AP.

急性胰腺炎（AP）是一种严重的炎症性疾病，其发病机制不明，且尚无有效的治疗手段。胰腺腺泡细胞(PAC)钙过载是触发AP发病的一个重要因素。防止PAC钙过载可为靶向治疗AP提供新思路。研究表明黄连素可改善小鼠AP的病理改变，但其机制不清楚。申请人在美国博士联合培养期间研究发现黄连素可明显抑制乙酰胆碱（ACh）诱发的PAC内钙振荡，进一步实验表明黄连素可与PAC的毒菌碱M3受体亚型（M3R）结合。因此我们推测“黄连素可通过抑制M3R减弱PAC内钙信号，从而减轻PAC内钙过载，防止胞内蛋白酶激活和PAC坏死”。本研究将在前期工作的基础上，利用膜片钳和钙成像技术进一步探讨黄连素调节PAC内钙信号的机制。主要内容：①黄连素抑制PAC内钙信号的药理机制；②黄连素作用的特异性靶点；③黄连素对AP模型小鼠的保护作用。本研究为阐明黄连素治疗AP的细胞机制和靶点提供重要的理论依据和实验基础。

背景与目的：黄连素是一种天然异喹啉生物碱，具有多种药理作用，但其药理靶点和作用机制尚不明确。我们报告了一项新发现，即黄连素抑制乙酰胆碱(ACh)诱导的Ca2+振荡，这可能是L-精氨酸诱导的急性胰腺炎(AP)小鼠模型的发病机制的基础。实验方法：将膜片钳记录和共聚焦Ca2+成像应用于从CD1小鼠制备的急性解离胰腺腺泡细胞，以检查黄连素对ACh诱导的Ca2+振荡的影响。L-精氨酸诱导的急性胰腺炎小鼠模型用于评估黄连素对病理变化的保护作用。主要结果：全细胞膜片钳记录表明，黄连素（0.1至10µM）以浓度依赖性方式减少ACh诱导Ca2+振荡，并且这种抑制作用还取决于ACh浓度。黄连素的抑制作用既不发生在细胞内靶点，也不发生在细胞外胆囊收缩素(CCK)受体、氯离子(Cl-)通道和钙库操纵的Ca2+通道中。总之，结果表明黄连素直接抑制M3毒蕈碱受体，这进一步通过腺泡细胞中黄连素与M3受体之间相互作用得到证实。在L-精氨酸AP模型中，黄连素消除ACh诱导的Ca2+振荡、胰淀粉酶和肺髓过氧化物酶的升高，并改善腺泡细胞病理损伤。结论和意义：我们提供了新的证据表明黄连素抑制M3受体，进而消除ACh诱导的Ca2+振荡和L-精氨酸增强的Ca2+信号传导，这是黄连素对胰腺腺泡细胞抵抗急性胰腺炎模型病理变化的保护作用的基础。