Protective mechanism of transcription factor nuclear factor e2-related factor 2 on inflammatory damage of mouse testicular sertoli cells
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 tes-ticular 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(1 μg/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[in-terleukin-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 pro-tein 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 anal-ysis 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 cyto-kines IL-6 and TNF-α mRNA 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 inflam-matory cytokines IL-6 and TNF-α in the Nrf2 group[(45.60±5.82),(48.07±9.06)pg/ml]were sig-nificantly 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 signifi-cantly 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-α mRNA 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.
Nuclear factor E2 related factor 2Testicular supporting cellsInflammatory responseAgonists
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