植物乳杆菌WCFS1对急性坏死性胰腺炎小鼠胰腺及回肠损伤的影响

Effect of Lactobacillus plantarum WCFS1 on pancreatic and ileal injury in mice with acute necrotizing pancreatitis

目的探讨益生菌植物乳杆菌WCFS1(LP)灌胃对急性坏死性胰腺炎(ANP)小鼠胰腺及回肠损伤的影响。方法24只健康雄性小鼠应用广谱抗生素持续灌胃3周以建立肠道无菌鼠,然后随机分为正常对照组(CON组)、ANP模型组(ANP组)、LP灌胃组(LP组)和LP灌胃后再诱导ANP组(LP+ANP组),每组6只。LP组与LP+ANP组给予1×10^(9)CFU/ml、0.2 ml/d的LP灌胃1周,CON组与ANP组以0.2 ml/d无菌磷酸盐缓冲液灌胃1周。采用雨蛙肽(100μg/kg)腹腔注射10次,每次间隔1 h,第10次注射时腹腔加注脂多糖5 mg/kg的方法构建ANP小鼠模型,2 h后处死小鼠。采用荧光定量PCR法检测小鼠粪便及回肠黏膜中LP数量;取胰腺和回肠组织行病理学检查,观察组织的炎症程度,并行病理学评分;采用酶动力化学法检测血清淀粉酶水平,ELISA法检测血清炎症因子水平及肠通透性指标;荧光定量PCR法检测胰腺与回肠组织炎症因子的表达;免疫荧光组织化学法检测回肠紧密连接蛋白occludin、claudin-1、ZO-1表达量。结果LP灌胃后小鼠粪便及回肠黏膜LP水平显著升高,差异有统计学意义(913.30±39.12比2.39±1.39,23.11±0.50比1.38±0.28,P值均<0.05)。CON组、LP组、ANP组、LP+ANP组胰腺病理评分分别为(0.26±0.41)、(0.17±0.26)、(8.55±0.46)、(6.30±0.45)分;血清淀粉酶水平分别为(219.70±19.73)、(217.60±11.30)、(2896.24±98.32)、(1837.13±131.60)U/L,IL-1β水平分别为(0.87±0.28)、(1.4±0.85)、(67.41±6.45)、(36.33±5.65)pg/ml,IL-6分别为(0.74±0.27)、(0.16±0.16)、(280586.12±39163.92)、(107912.75±31283.47)pg/ml,IL-10分别为(35.52±5.27)、(50.99±15.34)、(2008.45±184.83)、(3070.35±403.71)pg/ml;胰腺组织IL-1βmRNA表达量分别为1.42±0.39、0.95±25、20.53±0.50、10.69±1.01,IL-6 mRNA分别为1.31±0.44、0.93±0.02、21.97±1.71、11.54±1.75,IL-10 mRNA分别为0.93±0.14、0.75±0.15、0.99±0.21、1.76±0.19。LP组与CON组差异均无统计学意义,LP+ANP组胰腺病理评分,血清淀粉酶、IL-1β、IL-6水平,胰腺组织IL-1β、IL-6 mRNA表达量均较ANP组显著下降,而血清IL-10水平及胰腺组织IL-10 mRNA表达量较ANP组显著升高,差异有统计学意义(P值均<0.05)。CON组、LP组、ANP组、LP+ANP组回肠病理评分分别为0、0、(3.17±0.41)、(1.67±0.52)分;血清DAO水平分别为(0.03±0.03)、(0.02±0.02)、(0.50±0.05)、(0.49±0.06)ng/ml,LPS水平分别为(2.75±0.35)、(3.74±0.28)、(7.19±0.92)、(5.88±0.38)ng/ml;回肠组织IL-1βmRNA表达量分别为1.21±0.20、1.17±0.09、1.81±0.25、1.63±0.21,IL-6 mRNA分别为1.01±0.29、2.83±0.42、54.45±8.50、16.87±4.42,IL-10 mRNA分别为1.12±0.41、6.09±2.51、11.65±1.47、29.86±2.93。LP组回肠组织IL-10 mRNA表达量较CON组显著升高,其他指标两组差异均无统计学意义。LP+ANP组回肠病理评分、血清LPS水平、回肠组织IL-6 mRNA表达量均较ANP组显著下降,而IL-10 mRNA水平较ANP组显著升高;回肠紧密连接蛋白occludin、claudin-1、ZO-1表达量较ANP组显著增加,差异均有统计学意义(P值均<0.05)。结论LP灌胃可减轻雨蛙肽诱导的ANP小鼠的胰腺及回肠屏障的损伤程度。

Objective To explore the effect of probiotics Lactiplantibacillus plantarum(LP)WCFS1 by gavage on acute necrotizing pancreatitis(ANP)and associated ileum injury in mice.Methods Twenty-four healthy male mice were gavaged with broad-spectrum antibiotics for 3 weeks to establish microbiota-depleted mice,and then randomly divided into control group(CON),ANP model group(ANP),LP gavage group(LP)and LP gavage and ANP induced group(LP+ANP),with 6 mice in each group.Mice in LP and LP+ANP group were treated by gavage of LP(1×109 CFU/ml,0.2 ml/day per mouse)for 1 week,while CON and ANP were gavaged with sterile phosphate buffered saline for 1 week instead.The ANP model was induced by intraperitoneal injection with caerulein(100μg/kg)for 10 times with 1-hour interval between two injections and the 10th injection with lipopolysaccharide(LPS)5 mg/kg intraperitoneally,and the mice were sacrificed 2 h later.Levels of LP in stool and ileal mucosa were detected by real-time PCR;the pancreas and ileum were collected for pathological examination to observe the extent of tissue inflammation and to score the pathology.Serum amylase activities were determined by enzymatic kinetic chemistry;serum inflammators levels and intestinal permeability were detected by ELISA;levels of inflammators in pancreatic and ileal tissues were detected by real-time PCR;ileal tight-junction proteins(occludin,claudin-1 and ZO-1)were measured by immunofluorescence staining.Results LP levels in the stool and ileal mucosa of mice were significantly increased after LP gavage,and the differences were statistically significant(913.30±39.12 vs 2.39±1.39,23.11±0.50 vs 1.38±0.28,all P value<0.05).The pathological scores of pancreatic tissue of CON,LP,ANP and LP+ANP group were(0.26±0.41),(0.17±0.26),(8.55±0.46)and(6.30±0.45);the serum amylase activities were(219.70±19.73),(217.60±11.30),(2896.24±98.32)and(1837.13±131.60)U/L,IL-1βwere(0.87±0.28),(1.4±0.85),(67.41±6.45)and(36.33±5.65)pg/ml,IL-6 were(0.74±0.27),(0.16±0.16),(280586.12±39163.92)and(107912.75±31283.47)pg/ml,IL-10 were(35.52±5.27),(50.99±15.34),(2008.45±184.83)and(3070.35±403.71)pg/ml;the expression level of pancreatic IL-1βmRNA was 1.42±0.39,0.95±25,20.53±0.50 and 10.69±1.01,IL-6 mRNA was 1.31±0.44,0.93±0.023,21.97±1.71 and 11.54±1.75,IL-10 mRNA was 0.93±0.14,0.75±0.15,0.99±0.21 and 1.76±0.19;there was no significant difference between LP and CON group,and pancreatic pathological scores,serum amylase、IL-1βand IL-6 levels,and the expression level of pancreatic IL-1βand IL-6 mRNA were significantly decreased in LP+ANP group compared with those in ANP group,while serum IL-10 levels and the expression level of pancreatic IL-10 mRNA were significantly increased compared with ANP group,and all the differences were statistically significant(all P values<0.05).The pathological scores of ileal tissue of CON,LP,ANP and LP+ANP group were 0,0,(3.17±0.41)and(1.67±0.52);the levels of serum DAO of CON,LP,ANP and LP+ANP group were(0.03±0.03),(0.02±0.02),(0.50±0.05)and(0.49±0.06)ng/ml;LPS levels were(2.75±0.35),(3.74±0.28),(7.19±0.92)and(5.88±0.38)ng/ml;the expression level of ileal IL-1βmRNA was 1.21±0.20,1.17±0.09,1.81±0.25 and 1.63±0.21;IL-6 mRNA was 1.01±0.29,2.83±0.42,54.45±8.50 and 16.87±4.42;IL-10 mRNA was 1.12±0.41,6.09±2.51,11.65±1.47 and 29.86±2.93.There was no significant difference between LP and CON group,except that the ileal IL-10 mRNA expression was significantly higher than that of CON group.Ileal pathological scores,serum LPS levels and the expression level of ileal IL-6 mRNA were significantly lower in LP+ANP group than those in ANP group,while the expression level of ileal IL-10 mRNA was significantly higher than that of ANP group;the expression of ileal tight junction proteins(ocludin,claudin-1,ZO-1)was significantly higher than those in ANP group,and all the differences were statistically significant(all P values<0.05).Conclusions LP WCFS1 gavage could ameliorate the injury of pancreatic and ileal barrier in caerulein-induced ANP mice.