柴芩承气汤对急性坏死性胰腺炎肠道平滑肌细胞内CPI-17/MLCP信号通路的影响

Prevention and improvement of gastrointestinal dismotility timely and reduction of inflammation were contributed to the prognosis of severe acute pancreatitis（SAP）,including alleviation of SAP developed into multiple organ failure and prevention of the development of pancreatic or peripancreatic infection in the late stage of SAP. Calcium-dependent pathway is a key link to start gastrointestinal motility, and calcium sensitivity pathway（MLCP signaling pathway） is an important way that causes the changes of calcium sensitivity and the contraction of intestinal smooth muscle. Inflammatory cytokines is an important part of exacerbation gastrointestinal dysmotility. Proteinkinase C-potentiated phosphatase inhibitor of l7 ku(CPI-17) is not the hub of the MLCP pathway, but also regulated by inflammatory cytokines. Our previous studies have confirmed prescription medicine Chaiqingchengqi decoction（CQCQD）can reduce the levels of inflammatory mediators, and can promote calcium store release of calcium and promote colonic smooth muscle contraction by increasing the expression of PhospholipaseC -β1 and protein kinase C –α of the alcium-dependent pathway in rats of severe acute pancreatitis. In the present study, we intend to investigate the effects of the CQCQD on the MLCP signaling pathway of intestinal smooth muscle cells through detection the changes of the expression of mRNA and related proteins, inflammatory cytokines in acute necrotizing pancreatitis rats intragastrically administered. This study is expected to further elucidate the role of Chinese medicine to prevent acute pancreatitis innovative mechanism has been found.

及时恢复胃肠动力，减少炎症反应有助于缓解重症急性胰腺炎向多器官功能衰竭发展，且有利于防止后期胰腺或胰周感染。钙依赖途径是启动胃肠运动的重要环节，钙敏感性途径即MLCP信号通路是引起平滑肌钙敏感性改变和其收缩的关键，而炎症介质是加重胃肠动力障碍的重要因素。蛋白激酶C加强的磷酸酶抑制剂17（CPI-17）是MLCP信号通路的枢纽，同时受到炎症介质的调节。我们前期研究证实中药验方柴芩承气汤可减少急性胰腺炎炎症介质释放，上调大鼠肠道平滑肌细胞钙依赖途径磷脂酶C-β1、蛋白激酶C-α表达，促进钙库释放钙，启动胃肠运动。我们推断柴芩承气汤降低该病炎症介质释放，恢复胃肠动力，还与肠道平滑肌细胞MLCP信号通路有关。本项目检测急性坏死性胰腺炎大鼠肠道平滑肌细胞MLCP信号通路相关蛋白及炎症介质表达的变化，研究其对该病肠道平滑肌细胞MLCP信号通路的影响。为深入阐明中药防治急性胰腺炎的作用机制获得新发现。

及时恢复胃肠动力，减少炎症反应有助于缓解重症急性胰腺炎（SAP）向多器官功能衰竭发展。钙依赖途径是启动胃肠运动的重要环节，钙敏感性途径即MLCP信号通路是引起平滑肌钙敏感性改变和其收缩的关键，而炎症介质是加重胃肠动力障碍的重要因素。蛋白激酶C加强的磷酸酶抑制剂17（CPI-17）为细胞内多种激酶调节MLCP信号通路的枢纽，是引起肠道平滑肌钙敏感性改变和平滑肌收缩的重要分子，同时受到炎症介质的调节。本研究采用L-鸟氨酸制作急性胰腺炎（ANP）大鼠模型，从细胞层面阐述了柴芩承气汤(CQCQD)在急性胰腺炎CPI-17/MLCP信号通路的影响及机制。1）本研究采用避免干扰胃肠动力的药物L-鸟氨酸成功制作ANP大鼠模型，并选择注射该药后第24小时为治疗干预时间点。2）CQCQD及卡巴胆碱（CCh）可降低胰腺及小肠组织病理损害，说明CQCQD可从整体上降低ANP大鼠的严重程度。3）前期研究已发现CQCQD可减少促炎介质TNF-α和IL-6释放，降低血清内毒素水平。本研究亦发现，CQCQD可降低ANP大鼠血清炎症介质TNF-α、IL-1β水平，同时发现CQCQD可降低抑制胃肠运动的胃肠激素血管活性肽（VIP）血清水平，从血清学角度，证明了CQCQD可降低血清炎症介质，改善胃肠激素水平，从而改善胃肠动力功能障碍。4）前期研究已证实CQCQD可上调大鼠肠道平滑肌细胞钙依赖途径磷脂酶C-β1、蛋白激酶C-α的表达，促进钙库释放钙，启动胃肠运动。本研究证实了CQCQD及CCh可显著下调ANP大鼠小肠平滑肌细胞中炎症介质IL-1β表达，一定程度下调TNF-α，显著上调CPI-17 mRNA及蛋白表达，使p-CPI-17蛋白表达上调，同时上调p-MYPT-1、p-LC-20蛋白表达，促进ANP大鼠小肠平滑肌细胞内Ca2+浓度显著升高，增加其平滑肌收缩功能，从而恢复其胃肠运动。本研究证明了SAP状态下，炎症介质TNF-α、IL-1β影响小肠平滑肌细胞内CPI-17/MLCP信号通路，验证了CPI-17/MLCP通路是SAP肠道动力障碍发生的关键靶点；CQCQD可减少炎症介质，使关键蛋白CPI-17磷酸化，增加平滑肌细胞收缩，进一步探明CQCQD促进ANP大鼠胃肠动力的具体作用靶点，为SAP的防治提供新思路及药物治疗新靶点，具有理论和实际意义。