丁酸钠对严重烫伤小鼠肠道屏障的作用与相关机制

Effects of sodium butyrate on intestinal barrier of severe scald mice and the related mechanism

目的探讨丁酸钠对严重烫伤小鼠肠道屏障的作用及相关机制。方法将18只雌性8~12周龄C57BL/6小鼠按随机数字表法分为假伤组、单纯烫伤组、烫伤+丁酸钠组,每组6只。将单纯烫伤组、烫伤+丁酸钠组小鼠背部浸入90℃热水中9s,造成30%体表总面积Ⅲ度烫伤;将假伤组小鼠背部浸入37℃温水中9 s模拟致假伤。3组小鼠均于伤后即刻腹腔内注射乳酸林格液1mL。此外,烫伤+丁酸钠组小鼠分别于伤前30 min及伤后即刻经腹腔注射300 mg/kg剂量丁酸钠溶液;假伤组及单纯烫伤组小鼠仅腹腔注射相同体积的无菌磷酸盐缓冲液。伤后24 h,取3组小鼠抽取门静脉血采用异硫氰酸荧光素-葡聚糖荧光探针示踪法检测肠道通透性,取回肠组织采用蛋白质印迹法检测肠黏膜带状闭合蛋白1(ZO-1)、闭合蛋白、密封蛋白1、密封蛋白2、核苷酸结合寡聚化结构域蛋白样受体热蛋白结构域相关蛋白3(NLRP3)、白细胞介素1β(IL-1β)、IL-18蛋白表达,采用间接免疫荧光法检测肠黏膜ZO-1分布。对数据行单因素方差分析、LSD检验及Bonferroni校正。结果(1)伤后24 h,假伤组、单纯烫伤组、烫伤+丁酸钠组小鼠肠道通透性分别为0.88±0.19、2.62±0.48、1.23±0.16。与假伤组比较,单纯烫伤组小鼠肠道通透性明显增强(P<0.01),烫伤+丁酸钠组无明显变化(P>0.05)。与单纯烫伤组比较,烫伤+丁酸钠组小鼠肠道通透性明显减弱(P<0.01)。(2)伤后24 h,与假伤组比较,单纯烫伤组和烫伤+丁酸钠组小鼠肠黏膜紧密连接蛋白ZO-1、闭合蛋白及密封蛋白1表达量均明显降低(P<0.05),密封蛋白2表达量明显增加(P<0.05)。伤后24 h,与单纯烫伤组比较,烫伤+丁酸钠组小鼠肠黏膜ZO-1及闭合蛋白表达量明显增加(P<0.05),密封蛋白2表达量明显减少(P<0.05),密封蛋白1表达量无明显变化(P>0.05)。(3)伤后24 h,与假伤组比较,单纯烫伤组和烫伤+丁酸钠组小鼠肠黏膜NLRP3、IL-1β、IL-18蛋白表达量均明显增加(P<0.05);与单纯烫伤组比较,烫伤+丁酸钠组小鼠肠黏膜NLRP3、IL-1β及IL-18蛋白表达量均明显减少(P<0.05)。(4)伤后24 h,假伤组小鼠肠黏膜ZO-1沿细胞膜均匀分布,光滑连续;单纯烫伤组小鼠肠黏膜ZO-1分布不规则,有断裂;烫伤+丁酸钠组小鼠肠黏膜ZO-1分布改变较单纯烫伤组有所改善。结论丁酸钠能够抑制严重烫伤后小鼠肠黏膜NLRP3炎症小体激活,减少IL-1β和IL-18产生,从而减轻紧密连接蛋白表达和分布异常,保护肠道屏障功能。

Objective To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism.Methods Eighteen C57BL/6 female mice,aged eight to twelve weeks,were divided into sham scald group,pure scald group,and scald+sodium butyrate group according to random number table,with 6 mice in each group.Back of each mouse in pure scald group and scald+sodium butyrate group were immersed into 90℃ water for 9 s,causing full-thickness scald of 30%total body surface area,while back of each mouse in sham scald group were immersed into 37℃water for 9 s,causing sham injury.All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer′s solution immediately after injury.Besides,mice in scald+sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury,while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution.At post injury hour(PIH)24,portal vein of mice in 3 groups was harvested,intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method,then lileal tissue of mice in 3 groups was harvested,protein expressions of zonula occludens l(ZO-1),occludin,claudin-1,claudin-2,nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3(NLRP3),interleukin-1β(IL-1β),and IL-18 were detected by Western blotting,and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence.Data were processed with one-way analysis of variance,least-significant difference test,and Bonferroni correction.Results(1)At PIH 24,the intestinal permeability of mice in sham scald group,pure scald group,and scald+sodium butyrate group was 0.88±0.19,2.62±0.48,1.23±0.16,respectively.Compared with that in sham scald group,the intestinal permeability of mice in pure scald group was significantly elevated(P<0.01),while the intestinal permeability of mice in scald+sodium butyrate group showed no obvious change(P>0.05).Compared with that in pure scald group,the intestinal permeability of mice in scald+sodium butyrate group was significantly decreased(P<0.01).(2)At PIH 24,compared with those in sham scald group,the protein expressions of ZO-1,occludin,and claudin-1 of mice in pure scald group and scald+sodium butyrate group were significantly decreased(P<0.05),while the protein expression of claudin-2 was significantly increased(P<0.05).At PIH 24,compared with those of pure scald group,the protein expressions of ZO-1 and occludin of mice in scald+sodium butyrate group were significantly elevated(P<0.05),while the protein expression of claudin-2 was significantly decreased(P<0.05),the protein expression of claudin-1 showed no significant difference(P>0.05).(3)At PIH 24,compared with those in sham scald group,the protein expressions of NLRP3,IL-1β,and IL-18 of mice in pure scald group and scald+sodium butyrate group were significantly increased(P<0.05).Compared with those of pure scald group,the protein expressions of NLRP3,IL-1β,and IL-18 of mice in scald+sodium butyrate group were significantly decreased(P<0.05).(4)At PIH 24,ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly,continuously and homogeneously along the membrane.ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks.The distribution of ZO-1 in intestinal mucosa of mice in scald+sodium butyrate group was ameliorated compared with that in pure scald group.Conclusions Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice,which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.