柚皮素缓解高糖对雪旺细胞损伤的作用机制分析

Analysis of the mechanism of naringenin in relieving the injury of Schwann cells caused by high glucose

目的分析柚皮素缓解高糖对雪旺细胞损伤的作用机制。方法使用Bockes改良法从新生3 d的Wistar乳鼠的坐骨神经组织内分离纯化雪旺细胞,将其随机分为5组,A组细胞使用DMEM培养基培养72 h,B组细胞使用含5.6 mmol/L葡萄糖的DMEM培养基培养72 h,C组细胞使用含5.6 mmol/L葡萄糖和20μmol/L柚皮素的DMEM培养基培养72 h,D组细胞使用含5.6 mmol/L葡萄糖和40μmol/L柚皮素的DMEM培养基培养72 h,E组细胞使用含5.6 mmol/L葡萄糖和80μmol/L柚皮素的DMEM培养基培养72 h。对各组细胞培养不同时间的细胞活力和细胞凋亡率、培养72 h后培养液内炎症因子和氧化应激指标水平、培养72 h后NF-κB相关蛋白表达及其mRNA水平进行比较。结果与A组相比,B组、C组、D组、E组细胞培养24、48、72 h的吸光度(A)值均降低;与B组相比,C组、D组、E组细胞24、48、72 h的A值均升高;且随着柚皮素剂量的升高细胞24、48、72 h的A值升高,差异均有统计学意义(P<0.05)。与A组相比,B组、C组、D组、E组细胞培养24、48、72 h的凋亡率均升高;与B组相比,C组、D组、E组细胞24、48、72 h的A值均降低;且随着柚皮素剂量的升高细胞24、48、72 h的凋亡率降低,差异均有统计学意义(P<0.05)。与A组相比,B组、C组、D组、E组细胞培养72 h后培养液内白细胞介素(IL)-4、IL-5、IL-13、丙二醛水平均升高,超氧化物歧化酶(SOD)、基质金属蛋白酶(MMP)水平均降低;与B组相比,C组、D组、E组细胞培养72 h后培养液内IL-4、IL-5、IL-13、丙二醛水平均降低,SOD、MMP水平均升高;且随着柚皮素剂量的升高细胞培养72 h后培养液内IL-4、IL-5、IL-13、丙二醛水平均降低,SOD、MMP水平均升高,差异均有统计学意义(P<0.05)。与A组相比,B组、C组、D组、E组细胞培养72 h后NF-κB p65、NF-κB p50相对表达及其mRNA水平均升高,IκBα相对表达及其mRNA水平均降低;与B组相比,C组、D组、E组细胞培养72 h后NF-κB p65、NF-κB p50相对表达及其mRNA水平均降低,IκBα相对表达及其mRNA水平均升高;且随着柚皮素剂量的升高细胞培养72h后NF-κB p65、NF-κB p50相对表达及其mRNA水平均降低,IκBα相对表达及其mRNA水平均升高,差异均有统计学意义(P<0.05)。结论柚皮素可能通过抑制NF-κB信号通路缓解高糖损伤雪旺细胞过程中的炎症反应和氧化应激反应,进而提高细胞活力,抑制细胞凋亡,且作用效果随剂量的升高而提升。

Objective To analyze the mechanism of naringenin in relieving the injury of Schwann cells caused by high glucose.Methods The modified Bocks method was used to separate and purify Schwann cells from the sciatic nerve tissue of newborn 3 d Wistar suckling mice,and randomly divide them into 5 groups.Cells in group A were cultured in DMEM medium for 72 h,and cells in group B were cultured with 5.6 mmol/L.Glucose-containing DMEM medium was cultured for 72 h.Group C cells were cultured for 72 h with DMEM medium containing 5.6mmol/L glucose and 20μmol/L naringenin.Group D cells were cultured with 5.6mmol/L glucose and 40μmol/L naringenin.The cells in group E were cultured in DMEM medium for 72 h,and cells in group E were cultured in DMEM medium containing 5.6 mmol/L glucose and 80μmol/L naringenin for 72 h.The cell viability and apoptosis rate of each group at different times,the levels of inflammatory factors and oxidative stress indexes in culture medium after 72 h,and the expression and mRNA levels of NF-κB related proteins in each group after 72 h were compared.Results Compared with group A,the absorbance(A)values of cells in groups B,C,D and E all decreased after 24,48 and 72 h;compared with group B,cells in group C,D,and E were 24,48 h the A values of cells at 72 h and 72 h all increased;and with the increase of the dose of naringenin,the A values of cells at 24,48 and 72 h increased,and the differences were statistically significant(P<0.05).Compared with group A,the apoptotic rate of cells in groups B,C,D and E was increased after 24,48 and 72 h;compared with group B,the A values of cells in group C,D and E at 24,48 and 72 h were all decreased;and with the increase of the dose of naringenin,the apoptotic rate of cells at 24,48 and 72 h decreased,and the differences were statistically significant(P<0.05).Compared with group A,cells of group B,C,D and E contained interleukin(IL)-4,IL-5 and IL-13,malondialdehyde(MDA)levels increased,superoxide dismutase(SOD),matrix metallo proteinase(MMP)levels decreased;compared with group B,group C,group D,group E after the cells were cultured for 72 h,the levels of IL-4,IL-5,IL-13,and MDA in the culture medium decreased,and the levels of SOD and MMP increased;and with the increase of the dose of naringenin,the cells were cultured for 72 h in the culture medium.The levels of IL-4,IL-5,IL-13,and MDA decreased,while the levels of SOD and MMP increased,and the differences were statistically significant(P<0.05).Compared with group A,the relative expression of NF-κB p65,NF-κB p50 and its mRNA level increased after cells in group B,C,D,and E were cultured for 72 h,while the relative expression of IκBαand its mRNA level decreased;compared with group B,the relative expression of NF-κB p65,NF-κB p50 and its mRNA level decreased after cells in group C,D,and E were cultured for 72 h,and the relative expression of IκBαand its mRNA level increased;and the increase of the dose of naringenin the relative expression of NF-κB p65,NF-κB p50 and its mRNA level decreased after 72 h of cell culture,and the relative expression of IκBαand its mRNA level increased,and the differences were statistically significant(P<0.05).Conclusion Naringin may alleviate the inflammatory response and oxidative stress response of schwann cells in the process of high glucose injury by inhibiting the NF-κB signaling pathway,thus improving cell viability and inhibiting apoptosis,and the effect of naringin increased with the increase of dose.