Maresin-1对急性胰腺炎小鼠炎症因子及组织病理损伤的影响

Effects of Maresin-1 on inflammatory factors and histopathological injury in mice with acute pancreatitis

目的基于p38丝裂原活化蛋白激酶/核转录因子-κB(p38 MAPK/NF-κB)信号通路,探究Maresin-1对急性胰腺炎(AP)小鼠的影响。方法随机将50只小鼠分为假手术组(sham组)、急性胰腺炎组(AP组)、Maresin-1低剂量组(Mar-1 L组)、Maresin-1高剂量组(Mar-1 H组)和p38 MAPK信号通路抑制剂组(SB203580组),每组各10只。除sham组外,其余各组小鼠均采用腹腔注射雨蛙素联合脂多糖(LPS)建立AP模型;sham组注射等量0.9%氯化钠溶液。Mar-1 L组、Mar-1 H组和SB203580组分别在注射Maresin-1和SB203580雨蛙素3 h后,第1次腹腔注射Maresin-1(25、50 ng/kg)和SB203580溶液(5 mg/kg),12 h后进行第2次腹腔注射;sham组和AP组注射等量0.9%氯化钠溶液。采用苏木精-伊红(HE)染色观察小鼠胰腺组织病理损伤;酶联免疫吸附实验(ELISA)法检测血清淀粉酶、脂肪酶、炎性因子水平;试剂盒测定胰腺组织髓过氧化物酶(MPO)活性;免疫组化法检测胰腺组织中CD68表达;RT-qPCR检测胰腺组织炎性因子及p38 MAPK、NF-κB p65 mRNA的表达;蛋白免疫印迹法(Western-blot)检测胰腺组织p38 MAPK、NF-κB p65蛋白表达。结果sham组小鼠胰腺组织结构完整,无细胞坏死和出血;与sham组比较,AP组可见胰腺组织结构紊乱,腺泡细胞水肿、坏死,胰腺组织病理评分、MPO活性、血清淀粉酶和脂肪酶浓度升高,血清和胰腺组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)水平升高,IL-4、IL-10水平降低,巨噬细胞数增多(P均<0.05);胰腺组织p38 MAPK、NF-κB p65 mRNA和蛋白水平升高(P<0.05)。与AP组比较,Mar-1 L组、Mar-1 H组、SB203580组胰腺损伤明显减轻,仅见腺泡细胞少量坏死与出血,胰腺组织病理评分、MPO活性、血清淀粉酶和脂肪酶浓度降低,血清和胰腺组织TNF-α、IL-6、IL-1β水平降低,IL-4、IL-10水平升高,胰腺组织p38 MAPK、NF-κB p65 mRNA和蛋白水平降低,巨噬细胞数减少(均P<0.05)。结论Maresin-1能够缓解AP小鼠胰腺组织病理损伤,减轻胰腺组织中性粒细胞和巨噬细胞浸润,降低胰腺及全身炎症反应,其可能通过抑制p38 MAPK/NF-κB信号通路发挥作用。

Objective To explore the effect of Maresin-1 on acute pancreatitis(AP)mice by p38 mitogen activated protein kinase/nuclear transcription factor-κB(p38 MAPK/NF-κB)signal pathway.Methods Fifty mice were randomly divided into sham group,AP group,Maresin-1 low dose group(Mar-1 L group),Maresin-1 high dose group(Mar-1 H group)and p38 MAPK signal pathway inhibitor group(SB203580 group),with 10 mice in each group.Except sham group,AP models were established by intraperitoneal injection of cerulein combined with lipopolysaccharide(LPS)in other groups.Sham group was injected with the same amount of 0.9%sodium chloride solution.Maresin-1 and SB203580 solutions were injected intraperitoneally for the first time in Mar-1 L group(25 ng/kg),Mar-1 H group(50 ng/kg)and SB203580 group(5 mg/kg)3 hours after the injection of cerulein,and the second intraperitoneal injection was performed 12 hours later;Sham group and AP group were injected with the same amount of 0.9%sodium chloride solution.HE staining was used to observe the pathological damage of mouse pancreas;ELISA was used to observe the levels of serum amylase,lipase and inflammatory factors;Kit was used to measure myeloperoxidase(MPO)activity of pancreatic tissue;Immunohistochemistry was used to measure the expression of CD68 in pancreatic tissue;RT-qPCR was used to detect the expression of inflammatory factors,p38 MAPK and NF-κB p65 mRNA in pancreatic tissue;Western blot was used to detect the expression of p38 MAPK and NF-κB p65 protein in pancreatic tissue.Results In sham group,the pancreatic tissue structure was intact without cell necrosis and hemorrhage;Compared with sham group,AP group showed the disorder of pancreatic tissue structure,edema and necrosis of acinar cells,and increased pathological score of pancreatic tissue;MPO activity,serum amylase and lipase concentration increased(all P<0.05);The levels of TNF-α,IL-6,IL-1βincreased in serum and pancreatic tissue,but the levels of IL-4 and IL-10 decreased;The number of macrophages increased all(P<0.05);The levels of p38 MAPK,NF-κB p65 mRNA and protein in pancreatic tissue were increased(P<0.05).Compared with AP group,the pancreatic injury in Mar-1 L group,Mar-1 H group and SB203580 group was reduced,only small amount of acinar cell necrosis and bleeding were observed,and the pancreatic histopathological score was reduced;MPO activity,serum amylase and lipase concentration decreased;The levels of TNF-α,IL-6,IL-1βdecreased in serum and pancreatic tissue,but the levels of IL-4 and IL-10 increased;The number of macrophages decreased(P<0.05);The levels of p38 MAPK,NF-κB p65 mRNA and protein in pancreatic tissue were decreased(all P<0.05).Conclusions Maresin-1 can alleviate the pathological injury of pancreatic tissue in AP mice,reduce the infiltration of neutrophils and macrophages in pancreatic tissue,and reduce pancreatic and systemic inflammatory response,which may be related to the inhibition of p38 MAPK/NF-κB signal pathway.