归芪定年方通过调节JAK2/STAT通路活性延缓小鼠肾系膜细胞衰老

Guiqi Dingnian Prescription Delays Senescence of Mouse Mesangial Cells by Regulating JAK2/STAT Pathway Activity

目的:探讨归芪定年方(GDP)对D-半乳糖(D-gal)诱导致衰型小鼠肾系膜细胞中Janus酪氨酸蛋白激酶2(JAK2)/信号转导与转录激活因子(STAT)信号通路相关分子表达水平的影响。方法:以D-gal 10 g·L^(-1)诱导复制小鼠肾系膜细胞衰老模型,经GDP 40 mg·L-1水煎剂处理3 d后,衰老相关β-半乳糖苷酶(SA-β-gal)染色测定细胞衰老状态,流式细胞术检测细胞周期,细胞增殖与活性检测(CCK-8)法检测细胞存活率,实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),核转录因子-κB(NF-κB)和白细胞介素-1α(IL-1α)m RNA表达水平,蛋白免疫印迹法(Western blot)测定细胞中JAK2/STAT信号通路相关分子STAT1,磷酸化STAT1(p-STAT1),STAT3,p-STAT3蛋白水平。结果:CCK-8结果显示GDP最佳药物质量浓度为40 mg·L^(-1)。与空白组比较,模型组SA-β-gal细胞阳性率显著升高(P<0.01),G0/G1期细胞百分数明显升高(P<0.05),G2/M期和S期细胞百分数显著降低(P<0.01),TNF-α,IL-6,NF-κB和IL-1αm RNA表达水平显著上调(P<0.01),STAT1,p-STAT1,STAT3和p-STAT3蛋白水平显著升高(P<0.01);与模型组比较,模型+GDP组SA-β-gal细胞阳性率明显降低(P<0.05),G0/G1期百分数明显降低(P<0.05),G2/M期和S期细胞百分数显著增加(P<0.01),TNF-α,IL-6,NF-κB和IL-1αm RNA表达水平明显下调(P<0.05),STAT1,p-STAT1,STAT3和p-STAT3蛋白水平明显降低(P<0.05)。结论:GDP可延缓小鼠肾系膜细胞衰老的进程,其作用机制可能与下调细胞JAK2/STAT通路相关因子的表达水平相关。

Objective:To investigate the effects of Guiqi Dingnian prescription (GDP) on the expression of related molecules in Janus tyrosine kinase 2(JAK2)/signal transducer and activator of transcription(JAK2/STAT)signaling pathway of D-galactose(D-gal)-induced senescent mesangial cells.Method:The senescent mouse mesangial cells induced by 10 g·L^(-1) D-gal were continuously treated with 40 mg·L^(-1) GDP for three days.The senescence of the treated cells was determined by senescence-associated(SA)-β-gal staining.The cell cycle was detected by flow cytometry.The cell viability was analyzed using the cell counting kit-8(CCK-8).The m RNA expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),nuclear transcription factor-κB(NF-κB),and IL-1αwere detected by real-time polymerase chain reaction(Real-time PCR).The protein expression levels of STAT1,phosphorylated STAT1(p-STAT1),STAT3,and p-STAT3 in the JAK2/STAT signaling pathway were determined by Western blot.Result:CCK-8 results showed that the optimal concentration of GDP was 40 mg·L^(-1).Compared with the blank group,the positive rate of SA-β-gal in the model group was significantly higher(P<0.01),the percentage of cells in G0/G1 phase was significantly increased(P<0.05),the percentage of cells in G2/M and S phase was significantly decreased(P<0.01).The m RNA expression levels of TNF-α,IL-6,NF-κB and IL-1αwere significantly increased(P<0.01).Compared with the model group,the model+GDP group exhibited significantly decreased SA-β-gal-positive cells(P<0.05),reduced cells in the G0/G1 phase(P<0.05),increased cells in the G2/M and S phases(P<0.01),and down-regulated TNF-α,IL-6,NF-κB,and IL-1αm RNA expression(P<0.05)and STAT1,p-STAT1,STAT3,and p-STAT3 protein expression(P<0.05).Conclusion:GDP delays the senescence of mouse mesangial cells possibly by down-regulating the expression of related molecules in the JAK2/STAT pathway.