1、1创伤与急诊电子杂志 2023年3月 第11卷 第1期 J TRAUMA EMERG，Mar.2023，Vol.11，No.1降钙素原通过影响磷酸化蛋白激酶C、闭合蛋白表达损害脓毒症大鼠肠黏膜屏障功能刘慧恒，王隽笙，林锦洲，郭梦雨，胡婷（厦门大学附属中山医院急诊部，福建厦门 361001）摘要 目的 研究降钙素原（procalcitonin，PCT）是否通过增加肠黏膜磷酸化蛋白激酶 C（p-protein kinase C，p-PKC）表达，减少闭合蛋白表达，从而损害脓毒症大鼠肠黏膜屏障。方法 120150g SPF级SD雄性大鼠20只，使用随机数表法随机分5组（每组4只）。正常组：不做任何处
2、理；脓毒症组：盲肠结扎穿孔法（cecal ligation and puncture，CLP）造模；脓毒症+PCT 组：大鼠 CLP 造模后，尾静脉注射 PCT；脓毒症+PCT+Go6983组：大鼠 CLP 造模后，先后经尾静脉注射 PCT 及 Go6983（PKC 磷酸化抑制剂）；脓毒症+Go6983 组：大鼠 CLP 造模后，尾静脉注射 Go6983。术后 24h 于眼静脉取血 1ml行血清 D-乳酸（D-lactic acid，D-lac）、二胺氧化酶（diamine oxidase，DAO）浓度测定，取血后处死大鼠，留取回肠末端肠道组织行组织学、免疫组化、免疫荧光、原位末端标记法（Td
3、T-mediated dUTP nick end labeling，TUNEL）检查。结果 与正常组比较，脓毒症组大鼠血清 D-lac、DAO 水平水平明显升高（0.390.14）mmol/L 比（5.150.66）mmol/L，（12.173.34）U/L 比（31.433.94）U/L，P 均 0.001；肠 道 组 织 中 肠 黏 膜 细 胞p-PKC 表 达 增 加（58.8511.43）103IOD 比（111.2514.96）103IOD，闭 合 蛋 白表 达 量 减 少（317.4123.46）103IOD 比（192.9815.97）103IOD，肠 黏 膜 细 胞凋 亡 增
4、加（15.930.87）103IOD 比（20.531.87）103IOD，P 均 0.001，肠 道组织病理损伤加重。与脓毒症组比较，脓毒症+PCT 组大鼠血清 D-lac、DAO 水平升高（5.150.66）mmol/L 比（7.550.76）mmol/L，（31.433.94）U/L 比（58.717.54）U/L，P 均 0.001，肠道组织中肠黏膜细胞 p-PKC 表达增加（111.2514.96）103IOD比（220.5124.04）103IOD，闭 合 蛋 白 表 达 量 减 少（192.9815.97）103IOD比（91.289.7）103IOD，肠 黏 膜 细 胞 凋 亡
5、 增 加（20.531.87）103IOD 比（28.051.48）103IOD，P 均 0.001，肠道组织病理损伤加重。与脓毒症+PCT 组相比，脓毒症+PCT+Go 6983 组大鼠血清 D-lac、DAO 水平升高程度减少（7.550.76）mmol/L比（5.550.73）mmol/L，（58.717.54）U/L 比（42.682.78）U/L，P 均 0.001，肠道组织中肠黏膜细胞 p-PKC 表达减少（220.5124.04）103IOD 比（157.1312.79）103IOD，P=0.002，闭合蛋白表达量增多（91.289.7）103IOD 比（131.2311.58）
6、103IOD，P 0.001，肠黏膜细胞凋亡减少（28.051.48）103IOD 比（22.660.72）103IOD，P 0.001，肠道组织病理损伤减轻。与脓毒症+PCT+Go 6983 组相比，脓毒症+Go 6983 组大鼠血清 D-lac、DAO 水平下降（5.550.73）mmol/L 比（2.820.49）mmol/L，（42.682.78）U/L 比（23.283.86）U/L，P 0.001，肠 道 组 织 中 肠 黏 膜 细 胞 p-PKC表 达 减 少（157.1312.79）103IOD 比（81.8411.53）103IOD，闭 合 蛋 白 表 达 量脓毒症基金项目：
7、福建省卫生健康科技计划项目厦门市医疗卫生科技计划项目（3502Z20194019）通信作者：王隽笙，Email：2创伤与急诊电子杂志 2023年3月 第11卷 第1期 J TRAUMA EMERG，Mar.2023，Vol.11，No.1增 加（131.2311.58）103IOD 比（260.3711.44）103IOD，肠 黏 膜 细 胞 凋 亡 减少（22.660.72）103IOD 比（19.141.05）103IOD，P 均 0.001，肠道组织病理损伤减轻。结论 PCT 使脓毒症大鼠肠黏膜细胞 p-PKC 表达增加，闭合蛋白表达减少，加重脓毒症大鼠肠屏障功能损害。关键词 降钙素原；
8、脓毒症；肠道屏障功能；磷酸化蛋白激酶 C；闭合蛋白DOI：10.16746/ki.11-9332/r.2023.01.001Procalcitonin impairs intestinal mucosal barrier by decreasing occludin expression via activation of p-PKC in septic ratsLiu Huiheng,Wang Junsheng,Lin Jinzhou,Guo Mengyu,Hu Ting(Department of Emergency,Zhongshan Hospital,Xiamen University
9、,Xiamen 361001,Fujian,China)Abstract Objective To investigate whether procalcitonin(PCT)damages the intestinal mucosal barrier in sepsis rats by increasing the expression of intestinal protein kinase C(p-PKC)and reducing the expression of occludin.Method Twenty male SD rats with SPF grade(120g150g)w
10、ere divided into five groups with random number table method:including normal group:no treatment;sepsis group:cecal ligation(CLP)model;sepsis+PCT group:PCT was injected into the caudal vein after CLP modeling;sepsis+PCT+Go 6983 group:PCT and Go 6983(p-PKC inhibitor)were injected into the tail vein a
11、fter CLP modeling;and sepsis+Go 6983:Go 6983 was injected into the caudal vein after CLP modeling.Serum D-lactic acid(D-lac)and diamine oxidase(DAO)concentrations were measured at 1 ml of ophthalmic vein blood after 24 hours.The rats were killed after blood collection,and the distal ileum intestinal
12、 tissues were retained for histological,immunohistochemical,immunofluorescence and TdT-mediated dUTP nick end labeling(TUNEL)examinations.Result Compared to the normal group,the sepsis group showed a significant increase in serum D-lac level(0.390.14)mmol/L vs(5.150.66)mmol/L,P0.001,serum DAO level(
13、12.173.34)U/L vs(31.433.94)U/L,P0.001 and the expression of p-PKC(58.8511.43)103 vs(111.2514.96)103,IOD,P0.001,and a decrease in the expression of occludin(317.4123.46)103 vs(192.9815.97)103,IOD,P0.001.Intestinal mucosal cell apoptosis also increased(15.930.87)103 vs(20.531.87)103,IOD,P0.001,along w
14、ith an increase in intestinal tissue pathological injury.Compared to the sepsis group,the sepsis+PCT group showed a significant increase in serum D-lac level(0.390.14)mmol/L vs(5.150.66)mmol/L,P0.001,serum DAO level(12.173.34)U/L vs(31.433.94)U/L,P0.001 and the expression of p-PKC(58.8511.43)103 vs(
15、111.2514.96)103,IOD,P0.001,and a decrease in the expression of occludin(317.4123.46)103 vs(192.9815.97)103,IOD,P0.001.Intestinal mucosal cell apoptosis also increased(15.930.87)103 vs(20.531.87)103,IOD,P0.001,along with an increase in intestinal tissue pathological injury.Compared to the sepsis+PCT
16、group,the sepsis+PCT+Go 6983 group showed a decrease in the increase level of serum D-lac(7.550.76)mmol/L vs(5.550.73)mmol/L,P0.001,serum DAO level(58.717.54)U/L vs(42.682.78)U/L,P 0.001,and the expression of p-PKC(220.5124.04)103 vs(157.1312.79)103,IOD,P=0.002,and an increase in occludin expression(91.289.7)103 vs(131.2311.58)103,IOD,P0.001,a decrease in intestinal mucosal cell apoptosis(28.051.48)103 vs(22.660.72)103,IOD,P0.001,and alleviated pathological damage of intestinal tissue.Compared t