转录因子核因子E2相关因子2对小鼠睾丸支持细胞炎性损伤的保护作用及其机制

Protective mechanism of transcription factor nuclear factor e2-related factor 2 on inflammatory damage of mouse testicular sertoli cells

目的探讨转录因子核因子E2相关因子2(Nrf2)对小鼠睾丸支持细胞炎性损伤的保护机制。方法小鼠睾丸支持细胞TM4培养至对数生长期,分为对照组、脂多糖(LPS)组和Nrf2组,Nrf2组细胞采用过表达Nrf2的慢病毒感染TM4细胞过表达Nrf2,对照组和LPS组采用空载慢病毒感染。LPS组和Nrf2组细胞采用1μg/ml LPS处理12 h,细胞计数试剂盒(CCK-8)分析3组细胞活力,采用酶联免疫吸附实验和荧光定量聚合酶链反应(PCR)检测炎性因子[白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)]mRNA和蛋白质表达水平;蛋白质免疫印迹分析Nrf2和炎症相关蛋白p65磷酸化水平。30只小鼠分析对照组,模型组和Nrf2组,Nrf2组小鼠尾静脉富马酸二甲酯50 mg/kg,对照组和模型组小鼠注射等体积生理盐水。预处理12 h后,模型组和Nrf2组小鼠经腹腔注射30 mg/kg LPS诱导损伤12 h。对照组小鼠注射等体积生理盐水。解剖小鼠,取睾丸组织,采用苏木精-伊红(HE)染色分析炎症变化,提取总蛋白,分析炎性因子(IL-6和TNF-α)mRNA和蛋白质表达水平。蛋白质免疫印迹分析转录因子核因子-κB(NF-κB)磷酸化水平。组间计量数据比较采用单因素方差分析。结果Nrf2组细胞吸光度(A)值(1.70±0.10)明显高于LPS组(1.26±0.12),差异有统计学意义(t=6.203,P<0.05)。Nrf2组细胞炎性因子IL-6和TNF-αmRNA水平(2.27±0.12、1.97±0.19)明显低于LPS组(3.06±0.36、2.76±0.13),差异有统计学意义(t=5.127、8.210,P<0.05)。Nrf2组细胞炎性因子IL-6和TNF-α水平[(45.60±5.82)、(48.07±9.06)pg/ml]明显低于LPS组[(87.59±6.16)、(105.12±12.20)pg/ml],差异有统计学意义(t=12.130、9.197,P<0.05)。Nrf2组细胞Nrf2蛋白表达水平(2.12±0.23)明显高于对照组(0.97±0.05),差异有统计学意义(t=14.250,P<0.05)。Nrf2组细胞磷酸化p65蛋白表达水平(1.36±0.11)明显低于LPS组(1.84±0.11),差异有统计学意义(t=7.498,P<0.05)。Nrf2组小鼠睾丸组织炎症水平显著改善。Nrf2组小鼠睾丸组织IL-6和TNF-αmRNA水平(1.78±0.07、1.69±0.07)明显低于LPS组(2.42±0.26、2.31±0.17),差异有统计学意义(t=5.902、8.241,P<0.05)。Nrf2组小鼠睾丸组织磷酸化p65蛋白表达水平(2.00±0.11)明显低于LPS组(2.90±0.14),差异有统计学意义(t=12.180,P<0.05)。结论转录因子Nrf2在睾丸支持细胞炎症过程中发挥重要作用,激活Nrf2课显著抑制炎症发生,保护睾丸支持细胞。

Objective To explore the protective mechanism of transcription factor nuclear factor E2-related factor 2(Nrf2)on inflammatory damage of mouse testicular sertoli cells.Methods Mouse testicular sertoli cells(TM4 cell line)were divided into control group,lipopolysaccharide(LPS)group,and Nrf2 group.The TM4 cells in the Nrf2 group were infected with Nrf2 overexpressing lentivirus,and those in the control group and LPS group were infected with empty lentivirus.The cells in the LPS group and Nrf2 group were treated with LPS(1μg/ml)after 12 h.The cell viability of cells in three groups was analyzed by cell counting kit-8(CCK-8)assay.The mRNA and protein expression levels of inflammatory factors[interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)]were analyzed by enzyme-linked immunosorbent assay and fluorescence quantitative polymerase chain reaction(PCR).Nrf2 and inflammation related protein p65 phosphorylation levels were analyzed by protein immunoblotting analysis.Totally,30 mice were analyzed in the control group,model group,and Nrf2 group,with Nrf2 group mice receiving 50 mg/kg of dimethyl fumarate via the tail vein.The mice in the control group and model group were injected with an equal volume of physiological saline.After 12 h of pretreatment,the mice in the model group and Nrf2 group were intraperitoneally injected with 30 mg/kg LPS to induce injury for 12 h.The mice in the control group were injected with an equal volume of physiological saline.The mice were dissected,testicular tissue was taken,inflammation changes were analyzed using hematoxylin and eosin(HE)staining,total proteins were extracted,and the mRNA and protein expression levels of inflammatory cytokines(IL-6 and TNF-α)were detected.The transcription factor nuclear factor-κB(NF-κB)phosphorylation levels were analyzed by protein immunoblotting.The comparison of inter group econometric data was conducted using one-way analysis of variance.Results The absorbance(A)value of Nrf2 group cells(1.70±0.10)was significantly higher than that of LPS group cells(1.26±0.12,t=6.203,P<0.05).The levels of inflammatory cytokines IL-6 and TNF-αmRNA in the Nrf2 group(2.27±0.12,1.97±0.19)were significantly lower than those in the LPS group(3.06±0.36,2.76±0.13,t=5.127,8.210,P<0.05).The levels of inflammatory cytokines IL-6 and TNF-αin the Nrf2 group[(45.60±5.82),(48.07±9.06)pg/ml]were significantly lower than those in the LPS group[(87.59±6.16),(105.12±12.20)pg/ml,t=12.130,9.197,P<0.05].The expression level of Nrf2 protein in the Nrf2 group cells(2.12±0.23)was significantly higher than that in the control group cells(0.97±0.05,t=14.250,P<0.05).The expression level of phosphorylated p65 protein in Nrf2 group cells(1.36±0.11)was significantly lower than that in LPS group cells(1.84±0.11,t=7.498,P<0.05).The inflammation level of testicular tissue in Nrf2 group mice was significantly improved.The levels of IL-6 and TNF-αmRNA in the testicular tissue of Nrf2 group mice(1.78±0.07,1.69±0.07)were significantly lower than those of LPS group mice(2.42±0.26,2.31±0.17,t=5.902,8.241,P<0.05).The expression level of phosphorylated p65 protein in testicular tissue of Nrf2 group mice(2.00±0.11)was significantly lower than that of LPS group mice(2.90±0.14,t=12.180,P<0.05).Conclusion The transcription factor Nrf2 plays an important role in the inflammatory process of testicular sertoli cells by activating Nrf2,significantly inhibits inflammation and protecting testicular sertoli cells.