观察ERK/mTOR通路在六味地黄丸调控自噬骨细胞应激反应的影响

Observation of the Effect of ERK/mTOR Pathway on the Regulation of Autophagy Osteocyte Stress Response by Liuwei Dihuang Pills

目的:观察以ERK/mTOR通路的角度分析六味地黄丸含药血清的调控自噬,对改善骨细胞应激反应的影响。方法:将60只健康SPF级SD的大鼠作为研究对象,研究人员于模型组大鼠体外培养MC3T3-E1细胞,并进行24h的0.04~0.79mmol/LH 2O细胞干预随机分为3组:六味地黄丸组(给予六味地黄丸治疗)、紫檀芪组(给予紫檀芪治疗)及对照组(无任何治疗方案),各20只,治疗14周。给药结束后检测氧化应激指标、各组自噬相关LC3B mRNA含量、ERK1/2及p-ERK1/2蛋白表达水平。结果:研究结果显示,伴随时间延长,与对照组相比,六味地黄丸组及紫檀芪组的SOD水平数值均得到显著提升,MDA水平数值明显下降(P<0.05)。且六味地黄丸组SOD水平数值高于紫檀芪组,MDA水平数值低于紫檀芪组(P<0.05);与对照组和紫檀芪组相比,六味地黄丸组的自噬相关LC3B mRNA含量明显提升,且紫檀芪组高于对照组,差异存在统计学意义(P<0.05)。说明六味地黄丸可改善应激状态下的成骨细胞自噬水平的提升;与对照组相比,六味地黄丸组和紫檀芪组的ERK1/2及p-ERK1/2蛋白表达水平下降,差异存在统计学意义(P<0.05)。但六味地黄丸组和紫檀芪组的ERK1/2及p-ERK1/2蛋白表达水平对比无统计学意义(P>0.05)。说明六味地黄丸可利用压制ERK/mTOR通路以诱导自噬细胞降低成骨细胞的损伤。结论:六味地黄丸可改善机体应激指标,提升MC3T3-E1细胞的细胞活性,提升LC3B mRNA含量,降低的ERK1/2及p-ERK1/2蛋白表达水平。

Objective:To observe the effect of Liuwei Dihuang Pill containing serum on regulating autophagy and improving bone cell stress response from the perspective of ERK/mTOR pathway.Method:Sixty healthy SPF grade SD rats were selected as the research subjects.MC3T3-E1 cells were cultured in vitro in the model group rats and subjected to 24-hour 0.04~0.79mmol/LH 2O cell intervention.They were randomly divided into three groups:the Liuwei Dihuang Pill group(treated with Liuwei Dihuang Pill),the Zitanqi group(treated with Zitanqi),and the control group(without any treatment regimen),with 20 rats in each group,for 14 weeks of treatment.After administration,oxidative stress indicators,autophagy related LC3B mRNA content in each group,and expression levels of ERK1/2 and p-ERK1/2 proteins were detected.Result:The research results showed that with the prolongation of time,compared with the control group,the SOD levels in the Liuwei Dihuang Pill group and the Zitanqi group were significantly increased,while the MDA levels were significantly decreased(P<0.05).And the SOD level in the Liuwei Dihuang Pill group was higher than that in the Zitanqi group,while the MDA level was lower than that in the Zitanqi group(P<0.05);Compared with the control group and the Zitanqi group,the content of autophagy related LC3B mRNA in the Liuwei Dihuang Wan group was significantly increased,and the Zitanqi group was higher than the control group,with a statistically significant difference(P<0.05).It indicates that Liuwei Dihuang Wan can improve the increase of autophagy level of osteoblasts under stress;Compared with the control group,the expression levels of ERK1/2 and p-ERK1/2 proteins in the Liuwei Dihuang Wan group and the Zitanqi group decreased significantly(P<0.05).However,there was no statistically significant difference in the expression levels of ERK1/2 and p-ERK1/2 proteins between the Liuwei Dihuang Wan group and the Zitanqi group(P>0.05).This indicates that Liuwei Dihuang Wan can induce autophagy cells to reduce osteoblast damage by suppressing the ERK/mTOR pathway.Conclusion:Liuwei Dihuang Wan can improve stress indicators in the body,enhance the cell activity of MC3T3-E1 cells,increase LC3B mRNA content,and reduce the expression levels of ERK1/2 and p-ERK1/2 proteins.