降钙素原通过影响磷酸化蛋白激酶Cα、闭合蛋白表达损害脓毒症大鼠肠黏膜屏障功能

Procalcitonin impairs intestinal mucosal barrier by decreasing occludin expression via activation of p-PKCαin septic rats

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.