NOD1基因在Hp感染小鼠炎性反应及免疫功能的作用

Effect of NOD1 gene on the inflammatory response and immune function in the Hp infected mice

目的探讨核苷酸结合寡聚化结构域1(NOD1)基因对幽门螺杆菌(Hp)感染小鼠炎性反应及免疫功能的作用。方法将80只C57BL/6小鼠随机分为对照组和感染组,分别给予磷酸盐缓冲液(PBS)和Hp灌胃,间隔48 h灌胃1次,共5次;末次灌胃后24 h,快速尿素酶试验鉴定是否感染成功,不同时间点处死小鼠,无菌获取胃组织、脾脏组织及血液,苏木素-伊红(HE)染色法鉴定感染小鼠胃组织炎症程度的变化;PCR检测小鼠胃组织中NOD1、受体相互作用蛋白2(RIP2)mRNA的表达;酶联免疫吸附试验(ELISA)检测血清干扰素诱导蛋白10(IP-10)、干扰素β(IFN-β)水平;Western blot检测胃组织细胞核中核因子κB(NF-κB)p65表达水平;流式细胞技术分析CD4+、CD8^(+)T淋巴细胞百分比。结果感染组小鼠均Hp感染阳性。与对照组比较,感染组小鼠胃组织腺体结构破坏、减少、萎缩,肌层间质出现炎性细胞;胃组织中NOD1、RIP2 mRNA表达水平,细胞核中NF-κB p65表达水平在Hp感染48、72、120 h后明显升高(P<0.05),NOD1 mRNA表达水平在Hp感染48 h后达到峰值,RIP2 mRNA表达水平在Hp感染120 h后达到峰值,细胞核中NF-κB p65表达水平在Hp感染72 h后达到峰值;血清IP-10、IFN-β水平在感染8、12、16周后明显升高(P<0.05),Hp感染16周后达到峰值;小鼠脾脏组织CD4^(+)T淋巴细胞百分比在感染12、16周后明显降低(P<0.05),Hp感染12周后最低,CD8^(+)T淋巴细胞百分比在感染8、12、16周后明显升高(P<0.05),Hp感染12周后达到峰值。结论Hp感染可以诱导小鼠产生炎性反应,降低其免疫功能,可能与NOD1和NF-κB激活有关。

Objective To investigate the effect of nucleotide-binding oligomerization domain1(NOD1)gene on the inflammatory response and immune function of the Helicobacter pylori(Hp)infected mice.Methods A total of 80 C57BL/6 mice were randomly divided into the control group and the infection group,and were given phosphate-buffered saline(PBS)and Hp gavage respectively,once every 48 hours,for five times totally.The rapid urease test was used to determine whether the infection was successful at 24 hours after the last intragastric administration.The mice were sacrificed at different time points,and their gastric tissues,spleen tissues and blood specimen were obtained aseptically.Hematoxylin-eosin(HE)staining was used to identify the changes in the degree of inflammation in the gastric tissue of infected mice.Polymerase chain reaction(PCR)was used to detect the expression of NOD1 gene and receptor-interacting protein 2(RIP2)mRNA in the mice gastric tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum interferon-inducible protein 10(IP-10)and interferonβ(IFN-β).Western blot was used to detect the nuclear factor kappa-B(NF-κB)p65 expression in the nucleus of gastric tissue.Flow cytometry was used to analyze the percentage of CD4+and CD8^(+)T lymphocytes.Results Mice in the infected group were all positive for Hp infection.Compared with the control group,the glandular structures of gastric tissues were destroyed,reduced and atrophied,and inflammatory cells appeared in the muscularis interstitium of the mice in the infected group.The expression levels of NOD1 and RIP2 mRNA in gastric tissue and the expression level of NF-κB p65in the nucleus were significantly increased at 48,72 and 120 hours after Hp infection(P<0.05).The expression level of NOD1 mRNA reached the peak at 48 hours after Hp infection,the expression level of RIP2 mRNA reached the peak at 120 hours after Hp infection,and the expression level of NF-κB p65 in the nucleus reached the peak at 72 hours after Hp infection.The levels of serum IP-10 and IFN-βwere significantly increased at 8,12 and 16 weeks after infection,and peaked at 16 weeks after Hp infection.The percentage of CD4^(+)T lymphocytes in the spleen of mice was significantly decreased after 12 and 16 weeks of infection(P<0.05),and reached the lowest level after 12 weeks of Hp infection.The percentage of CD8^(+)T lymphocytes was significantly increased after 8,12,and 16 weeks of infection(P<0.05),and reached the peak at 12 weeks after Hp infection.Conclusion Hp infection can induce inflammatory response in mice and reduce their immune function,which may be related to the activation of NOD1 and NF-κB.