腐殖酸钠对肠产毒素性大肠杆菌K88感染小鼠肠道损伤的保护作用

Protective Effects of Sodium Humate on Intestinal Damage in Mice Infected with Enterotoxigenic Escherichia coli K88

本研究旨在探究腐殖酸钠(HNa)对肠产毒素性大肠杆菌K88(ETEC K88)感染小鼠肠道损伤的保护作用。选取健康的无特定病原体(SPF)级雌性昆明小鼠30只,随机分为3组,对照组、模型组(ETEC组)、HNa干预组(ETEC+HNa组),每组10只。对照组和ETEC组小鼠每天灌胃0.2 mL生理盐水,ETEC+HNa组小鼠每天灌胃0.2 mL 5%HNa;试验第8天,ETEC和ETEC+HNa组小鼠灌胃0.2 mL 5×10^(10)CFU/mL ETEC K88。试验期10 d。采用苏木精-伊红(HE)染色法观察空肠病理变化,采用酶联免疫吸附测定(ELISA)法检测血清炎症因子含量,采用实时荧光定量PCR(qPCR)法检测肠道屏障相关蛋白的mRNA表达,采用蛋白免疫印迹(Western blot)法检测空肠紧密连接、黏附连接、肠道组织炎性小体和细胞凋亡相关蛋白的表达。结果表明:1)与对照组和ETEC+HNa组,ETEC组小鼠体重及空肠绒毛高度、绒毛高度/隐窝深度显著降低(P<0.05)。2)与对照组和ETEC+HNa组相比,ETEC组小鼠血液白细胞、单核细胞、粒细胞数量及血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、二胺氧化酶(DAO)、D-乳酸(D-Lac)含量显著升高(P<0.05),血清白细胞介素-4(IL-4)和白细胞介素-10(IL-10)含量显著降低(P<0.05)。3)与对照组和ETEC+HNa组相比,ETEC组空肠闭合蛋白(Occludin)、封闭蛋白-1(Claudin-1)、上皮型钙黏蛋白(E-cadherin)和黏蛋白-2(MUC-2)的mRNA相对表达量显著降低(P<0.05),空肠Claudin-1、ZO-1、E-cadherin和MUC-2的蛋白表达量也显著降低(P<0.05)。4)与对照组和ETEC+HNa组相比,ETEC组空肠核因子-κB(NF-κB)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱天冬蛋白酶-1(Caspase-1)、白细胞介素-1β(IL-1β)和B细胞淋巴瘤/白血病-2相关X蛋白(Bax)的蛋白表达量显著升高(P<0.05),空肠B细胞淋巴瘤/白血病-2(Bcl-2)的蛋白表达量显著降低(P<0.05)。5)与对照组相比,ETEC组粪便大肠杆菌数量显著增加(P<0.05),粪便乳酸菌数量则显著降低(P<0.05)。与ETEC组相比,ETEC+HNa组粪便大肠杆菌显著降低(P<0.05)。由此可见,HNa可以通过抑制肠道组织炎性小体激活、肠道上皮细胞凋亡以及上调肠道屏障相关蛋白的表达缓解ETEC K88感染引起的肠道损伤。

This experiment was conducted to investigate the protective effects of sodium humate(HNa)on intestinal damage in mice infected with enterotoxigenic Escherichia coli K88(ETEC K88). Thirty healthy specific pathogen free(SPF)Kunming mice were randomly allocated to 3 groups,control group,model group(ETEC group)and HNa intervention group(ETEC+HNa group). Mice in control group and ETEC group were gavaged with 0.2 mL saline every day,and mice in ETEC+HNa group were gavaged with 0.2 mL 5%HNa;on day 8 of the experiment,the mice in ETEC group and ETEC+HNa were gavaged with 0.2 mL 5×10^(10)CFU/mL ETEC K88. The experiment lasted for 10 days. The pathological changes of jejunum were observed by Hematoxylin-eosin(HE)staining method,the serum inflammatory cytokine contents were determined by enzyme linked immunosorbent assay(ELISA)method,the mRNA expression of intestinal barrier related protein was detected by real time fluorescence quantitative PCR(qPCR)method,the protein expression of tight junction,adhesion connection,intestinal tissue inflammasome and cells apoptosis related protein in jejunum was detected by Western blot method. The results showed as follows:1)compared with control group and ETEC+ HNa group,the body weight and villus height,villus height/crypt depth in jejunum of ETEC group were significantly decreased(P<0.05). 2)Compared with control group and ETEC+HNa group,the numbers leukocytes,monocytes,granulocytes in blood and contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),diamine oxidase(DAO),D-lactic acid(D-Lac)in serum of ETEC group were significantly increased(P <0.05),and the contents of interleukin-4(IL-4)and interleukin-10(IL-10)in serum were significantly decreased(P<0.05). 3)Compared with control group and ETEC+HNa group,the mRNA relative expressions of Occludin,Claudin-1,epithelial cadherin(E-cadherin)and mucin-2(MUC-2)in jejunum of ETEC group were significantly decreased(P<0.05),and the protein expressions of Claudin-1,ZO-1,E-cadherin and MUC-2 in jejunum were significantly decreased(P<0.05). 4)Compared with control group and ETEC+HNa group,the protein expressions of nuclear factor kappa B(NF-κB),nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),cysteinyl aspartate specific proteinase-1(Caspase-1),interleukin-1β(IL-1β)and B-cell lymphoma/leukaemia-2-associated X protein(Bax)of ETEC group were significantly increased(P<0.05),and the jejunal B-cell lymphoma/leukaemia-2(Bcl-2)protein expression level was significantly decreased(P<0.05). 5)Compared with the control group,the fecal Escherichia coli number of ETEC group was significantly increased(P<0.05),and the fecal Lactobacillus number was significantly decreased(P<0.05). Compared with the ETEC group,the fecal Escherichia coli number of ETEC+HNa group was significantly decreased(P<0.05). In conclusion,HNa can alleviate ETEC K88 induced intestinal damage by inhibiting the activation of intestinal tissue inflammasome,apoptosis of intestinal epithelial cells and regulating the expression of intestinal barrier related proteins.