枸杞多糖对人子宫内膜上皮细胞的氧化损伤及激活Nrf2/HO-1通路的影响

Effects of lycium barbarum polysaccharides on oxidative damage and activation of Nrf2/HO-1 pathway in human endometrial epithelial cells

目的分析枸杞多糖对过氧化氢刺激引起的人子宫内膜上皮细胞(endometrial epithelial cells,EECs)氧化损伤的影响及对核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)信号通路蛋白的激活情况。方法体外培养人的原代EECs,分为正常组、枸杞多糖组、过氧化氢组及干预组4组。正常组:孵育液;枸杞多糖组:孵育液+100 mg/L的枸杞多糖;过氧化氢组:孵育液+100μmol/L的过氧化氢;干预组:100 mg/L的枸杞多糖提前干预24 h,再加入100μmol/L的过氧化氢。4组均作用24 h。比较各组细胞的形态变化,噻唑蓝实验用于细胞数量分析,流式细胞仪分析细胞的凋亡率,比色法测量细胞内容物中丙二醛(malondialdehyde,MDA)的含量和超氧化物歧化酶(superoxide dismutase,SOD)的活性,蛋白免疫印迹分析细胞中B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X,Bax)、Nrf2和HO-1的含量。结果枸杞多糖组和正常组比较,细胞的存活率、凋亡率、细胞内容物中MDA含量和SOD活性、细胞中Bcl-2、Bax、Nrf2和HO-1含量的差异均无统计学意义(均P>0.05);过氧化氢组和正常组比较,细胞缩小变圆,漂浮细胞较多,存活率[(50.85±3.57)%]减少而凋亡率[(52.47±4.93)%]升高,MDA含量[(5.86±0.70)mmol/L]增加而SOD活性[(10.18±0.97)U/L]下降,胞质中Bcl-2(0.153±0.027)、Nrf2(0.582±0.058)和HO-1(0.814±0.072)含量降低,Bax含量(1.056±0.054)升高(均P<0.05);干预组和过氧化氢组比较,细胞呈伸展状,漂浮细胞较少,存活率[(85.53±3.69)%]增加而凋亡率[(19.33±2.56)%]降低,MDA含量[(2.69±0.40)mmol/L]减少而SOD活性[(17.49±2.24)U/L]上升,胞质中Bcl-2(0.863±0.075)、Nrf2(0.819±0.069)和HO-1(0.814±0.072)含量上升,Bax含量(0.200±0.020)下降(均P<0.05)。结论枸杞多糖能抑制过氧化氢导致的EECs氧化损伤,降低氧化应激产物,与激活Nrf2/HO-1信号通路密切相关。

Objective To explore the effect of lycium barbarum polysaccharides(LBP)oxidative damage of human endometrial epithelial cells(EECs)stimulated by hydrogen peroxide and the activation of nuclear factor erythroid-2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)signal pathway protein.Methods The primarily incubated EECs were arranged into four groups:normal group,LBP group,hydrogen peroxide group and intervention group.Different solutions were added to the four groups.Normal group:incubation solution,LBP group:incubation solution+100 mg/L LBP,hydrogen peroxide group:incubation solution+100 mg/L LBP,intervention group:100 mg/L LBP was added in advance for 24 hours,and then 100μmol/L hydrogen peroxide was added.All groups were treated for 24 hours.The morphology of cells in each group was compared.Thiazole blue test was used to analyze the cell proliferation,the flow cytometry was used to analyze the apoptosis percentage,the colorimetry was used to measure the content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD),and the Western blot was used to measure the contents of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(Bax),Nrf2 and HO-1.Results There were no statistically significant differences in cell survival rate,apoptosis rate,MDA content and SOD activity,the content of Bcl-2,Bax,Nrf2 and HO-1 in cells between normal group and LBP group(all P>0.05).Compared with the normal group,the cells in hydrogen peroxide group shrank and became round,with more floating cells and lower survival rate[(50.85±3.57)%],the apoptosis rate increased[(52.47±4.93)%],the content of MDA[(5.86±0.70)mmol/L]increased and the activity of SOD[(10.18±0.97)U/L]decreased,the contents of Bcl-2(0.153±0.027),Nrf2(0.582±0.058)and HO-1(0.814±0.072)decreased,and the content of Bax(1.056±0.054)increased(all P<0.05).Compared with the hydrogen peroxide group,the cells in the intervention group were stretched with less floating,the survival rate[85.53±3.69)%]increased and the apoptosis rate[(19.33±2.56)%]decreased,the content of MDA[(2.69±0.40)mmol/L]decreased and the activity of SOD[(17.49±2.24)U/L]increased,the contents of Bcl-2(0.863±0.075),Nrf2(0.819±0.069)and HO-1(0.814±0.072)increased,and the content of Bax(0.200±0.020)decreased(all P<0.05).Conclusion Lycium barbarum polysaccharides can inhibit the oxidative damage of EECs induced by hydrogen peroxide and reduce the oxidative stress products,which is closely related to the activation of Nrf2/HO-1 signaling pathway.