未甲基化寡聚脱氧核苷酸对链脲佐菌素诱导糖尿病大鼠Toll样受体9和核转录因子B的影响

CpG-ODN activation of TLR9 signaling pathways and NF-κB in STZ-induced diabetic rats

目的 观察未甲基化寡聚脱氧核苷酸（CpG-ODN）对链脲佐菌素（STZ）诱导糖尿病大鼠Toll样受体9（TLR9）、核转录因子B（NF-κB）及血清辅助性T细胞1型（Th1）、2型（Th2）炎症因子的影响.方法 取5～6周龄雄性SD大鼠40只,随机分为4组：正常对照组（Ⅰ组）、1型糖尿病组（Ⅱ组）、低剂量CpG-ODN（Ⅲ组）和高剂量CpG-ODN 1型糖尿病组（Ⅳ组）.STZ诱导建立1型糖尿病模型,分别给Ⅲ、Ⅳ组皮下注射不同浓度的CpG-ODN,免疫印迹和荧光定量PCR法检测大鼠脾脏和胰腺TLR9和NF-κB的表达;ELISA法测定各组大鼠血清肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）和白细胞介素-6（IL-6）水平.结果 4组大鼠TLR9蛋白在脾脏的表达均明显高于在胰腺中的表达;脾脏组织中,Ⅲ和Ⅳ组TLR9和NF-κB的mRNA和蛋白的表达较Ⅰ组和Ⅱ组明显升高,Ⅳ组TLR9和NF-κB的mRNA和蛋白表达较Ⅲ组升高;胰腺组织中,Ⅲ组和Ⅳ组TLR9和NF-κB（p65）蛋白的表达较Ⅱ组升高,Ⅳ组TLR9和NF-κB（p65）蛋白表达较Ⅲ组升高;Ⅲ组和Ⅳ组血清TNF-α和IFN-γ水平较Ⅰ和Ⅱ组升高,Ⅳ组血清TNF-α和IFN-γ水平高于Ⅲ组.结论 CpG-ODN激活STZ诱导糖尿病大鼠TLR9通路,胰腺和脾脏NF-κB的表达在一定范围内呈剂量依赖性增加.

Objective To observe the mRNA and protein expression of TLR9,NF-κB and serum level of Th1/Th2 cytokines when stimulated with different concentration of CpG-ODN in STZ-induced diabetic rats.Methods 40 SD male rats were randomly divided into four groups：normal control group （Group Ⅰ）,type 1 diabetes control group （Group Ⅱ）,low-dose CpG-ODN treated type 1 diabetes group （Group Ⅲ）,high dose CpG-ODN treated type 1 diabetes group （Group Ⅳ）.60 μg/kg and 120 μg/kg CpG-ODN were injected to rats of Group Ⅲ and Ⅳ when inducing type 1 diabetes model.mRNA and protein levels of TLR9 and NF-κB in spleen and pancreas were detected by fluorescence quantitative PCR and Western blotting.Serum TNF-α,IL-6 and IFN-γ were measured by ELISA.Results TLR9 protein expression levels were much higher in spleen than in the pancreas among these four groups.In spleen,TLR9 and NF-κB mRNA and protein level were significantly higher in Group Ⅲand Ⅳthan those in Group Ⅰ and Ⅱ,and the expression level was higher in Group Ⅳ than in Group Ⅲ.In pancreas,TLR9 and NF-κB protein level were significantly higher in Group Ⅲ and Ⅳ than those in Group Ⅰ and Ⅱ,and the expression level was also higher in Group Ⅳ than Group Ⅲ.Serum TNF-α and IFN-γ level in Group Ⅲ and Ⅳ significantly higher than in Group Ⅰ and Ⅱ,and serum levels of TNF-α and IFN-γ in Group Ⅳ was higher than in Group Ⅲ.Conclusions CpG-ODN activation of TLR9-MyD88 signaling pathways results in doseeffect NF-κB stimulation in STZ-induced diabetic rats.