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Cα(p-protein kinase Cα,p-PKCα)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120~150g 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)。(1)正常组:不做任何处理;(2)脓毒症组:盲肠结扎穿孔法(cecal ligation and puncture,CLP)造模;(3)脓毒症+PCT组:大鼠CLP造模后,尾静脉注射PCT;(4)脓毒症+PCT+Go6983组:大鼠CLP造模后,先后经尾静脉注射PCT及Go6983(PKCα磷酸化抑制剂);(5)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(TdT-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[(0.39±0.14)mmol/L比(5.15±0.66)mmol/L,(12.17±3.34)U/L比(31.43±3.94)U/L,P均<0.001];肠道组织中肠黏膜细胞p-PKCα表达增加[(58.85±11.43)×10^(3)IOD比(111.25±14.96)×10^(3)IOD],闭合蛋白表达量减少[(317.41±23.46)×10^(3)IOD比(192.98±15.97)×10^(3)IOD],肠黏膜细胞凋亡增加[(15.93±0.87)×10^(3)IOD比(20.53±1.87)×10^(3)IOD],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[(5.15±0.66)mmol/L比(7.55±0.76)mmol/L,(31.43±3.94)U/L比(58.71±7.54)U/L,P均<0.001],肠道组织中肠黏膜细胞p-PKCα表达增加[(111.25±14.96)×10^(3)IOD比(220.51±24.04)×10^(3)IOD],闭合蛋白表达量减少[(192.98±15.97)×10^(3)IOD比(91.28±9.7)×10^(3)IOD],肠黏膜细胞凋亡增加[(20.53±1.87)×10^(3)IOD比(28.05±1.48)×10^(3)IOD],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[(7.55±0.76)mmol/L比(5.55±0.73)mmol/L,(58.71±7.54)U/L比(42.68±2.78)U/L,P均<0.001],肠道组织中肠黏膜细胞p-PKCα表达减少[(220.51±24.04)×10^(3)IOD比(157.13±12.79)×10^(3)IOD,P=0.002],闭合蛋白表达量增多[(91.28±9.7)×10^(3)IOD比(131.23±11.58)×10^(3)IOD,P<0.001],肠黏膜细胞凋亡减少[(28.05±1.48)×10^(3)IOD比(22.66±0.72)×10^(3)IOD,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+Go6983组大鼠血清D-lac、DAO水平下降[(5.55±0.73)mmol/L比(2.82±0.49)mmol/L,(42.68±2.78)U/L比(23.28±3.86)U/L,P<0.001],肠道组织中肠黏膜细胞p-PKCα表达减少[(157.13±12.79)×10^(3)IOD比(81.84±11.53)×10^(3)IOD],闭合蛋白表达量增加[(131.23±11.58)×10^(3)IOD比(260.37±11.44)×10^(3)IOD],肠黏膜细胞凋亡减少[(22.66±0.72)×10^(3)IOD比(19.14±1.05)×10^(3)IOD],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-PKCα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。
Objective To investigate whether procalcitonin(PCT)damages the intestinal mucosal barrier in sepsis rats by increasing the expression of intestinal protein kinase Cα(p-PKCα)and reducing the expression of occludin.Method Twenty male SD rats with SPF grade(120g~150g)were divided into five groups with random number table method:including①normal group:no treatment;②sepsis group:cecal ligation(CLP)model;③sepsis+PCT group:PCT was injected into the caudal vein after CLP modeling;④sepsis+PCT+Go 6983 group:PCT and Go 6983(p-PKCαinhibitor)were injected into the tail vein after CLP modeling;and⑤sepsis+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 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[(0.39±0.14)mmol/L vs(5.15±0.66)mmol/L,P<0.001],serum DAO level[(12.17±3.34)U/L vs(31.43±3.94)U/L,P<0.001]and the expression of p-PKCα[(58.85±11.43)×10^(3) vs(111.25±14.96)×10^(3),IOD,P<0.001],and a decrease in the expression of occludin[(317.41±23.46)×10^(3) vs(192.98±15.97)×10^(3),IOD,P<0.001].Intestinal mucosal cell apoptosis also increased[(15.93±0.87)×10^(3) vs(20.53±1.87)×10^(3),IOD,P<0.001],along with 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[(0.39±0.14)mmol/L vs(5.15±0.66)mmol/L,P<0.001],serum DAO level[(12.17±3.34)U/L vs(31.43±3.94)U/L,P<0.001]and the expression of p-PKCα[(58.85±11.43)×10^(3) vs(111.25±14.96)×10^(3),IOD,P<0.001],and a decrease in the expression of occludin[(317.41±23.46)×10^(3) vs(192.98±15.97)×10^(3),IOD,P<0.001].Intestinal mucosal cell apoptosis also increased[(15.93±0.87)×10^(3) vs(20.53±1.87)×10^(3),IOD,P<0.001],along with an increase in intestinal tissue pathological injury.Compared to the sepsis+PCT group,the sepsis+PCT+Go 6983 group showed a decrease in the increase level of serum D-lac[(7.55±0.76)mmol/L vs(5.55±0.73)mmol/L,P<0.001],serum DAO level[(58.71±7.54)U/L vs(42.68±2.78)U/L,P<0.001],and the expression of p-PKCα[(220.51±24.04)×10^(3) vs(157.13±12.79)×10^(3),IOD,P=0.002],and an increase in occludin expression[(91.28±9.7)×10^(3) vs(131.23±11.58)×10^(3),IOD,P<0.001],a decrease in intestinal mucosal cell apoptosis[(28.05±1.48)×10^(3) vs(22.66±0.72)×10^(3),IOD,P<0.001],and alleviated pathological damage of intestinal tissue.Compared to the sepsis+PCT+Go 6983 group,the sepsis+Go 6983 group showed a decrease in serum D-lac level[(5.55±0.73)mmol/L vs(2.82±0.49)mmol/L,P<0.001],serum DAO level[(42.68±2.78)U/L vs(23.28±3.86)U/L,P<0.001],and the expression of p-PKCα[(157.13±12.79)×10^(3) vs(81.84±11.53)×10^(3),IOD,P<0.001],along with an increase in the expression of occludin[(131.23±11.58)×10^(3) vs(260.37±11.44)×10^(3),IOD,P<0.001],a decrease in intestinal mucosal cell apoptosis[(22.66±0.72)×10^(3) vs(19.14±1.05)×10^(3),IOD,P<0.001],and alleviated pathological damage of intestinal tissue.Conclusion PCT was found to increase the expression of p-PKCαwhile decreasing the expression of occludin in intestinal mucosal cells,which is associated with the exacerbation of intestinal barrier function injury in septic rats.
作者
刘慧恒
王隽笙
林锦洲
郭梦雨
胡婷
Liu Huiheng;Wang Junsheng;Lin Jinzhou;Guo Mengyu;Hu Ting(Department of Emergency,Zhongshan Hospital,Xiamen University,Xiamen 361001,Fujian,China)
出处
《创伤与急诊电子杂志》
2023年第1期1-9,共9页
Journal of Trauma and Emergency(Electronic Version)
基金
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)。