Exploring The Molecular Mechanism Of Oxidative Stress Damage Reduction In Osteoblasts By Liuwei Dihuang Pills Based On ERK/mTOR Pathway Regulation Of Autophagy

Objective:1.To investigate the effects of Liuwei Dihuang Pills-containing serum on the proliferative activity,reactive oxygen species（ROS）and autophagy of MC3T3-E1 osteoblasts under oxidative stress induced by hydrogen peroxide（H₂O₂）.2.To investigate the effects of Liuwei Dihuang Pills-containing serum-regulated autophagy on H₂O₂-induced oxidative damage in MC3T3-E1 osteoblasts from the perspective of ERK/mTOR signaling pathway.Methods:1.MC3T3-E1 osteoblasts were cultured in vitro,and the cells were intervened with0.05-0.80 mmol/L H₂O₂for 24 h and with 5%-30%volumetric concentration of Liuwei Dihuang Pills-containing serum for 24 h.Cell proliferation was detected by cck-8,and the optimal intervention concentrations of H₂O₂and Liuwei Dihuang Pills-containing serum were screened;MC3T3-E1 osteoblasts were MC3T3-E1 osteoblasts were divided into normal group（Blank）,model group（Model）,classical induction group（Control）,blank serum group（H+Blank）and Liu Wei Di Huang Wan group（H+LWDH）,and were cultured under different conditions for induction of differentiation intervention according to the grouping,and ALP staining（calcium-cobalt method）was performed after a total of 14 d of intervention.2.MC3T3-E1 osteoblasts were divided into normal control group（Control）,model group（Model）,blank control group（H+Blank）and Liuwei Dihuang Pills group（H+LWDH）,and the content of ROS in each group was detected by fluorescent probe DCFH-DA after the intervention according to the grouping.3.MC3T3-E1 osteoblasts were divided into normal control group（Control）,model group（Model）,blank control group（H+Blank）,Liuwei Dihuang Pills group（H+LWDH）and autophagy inhibitor group（LWDH+3-MA）.qRT-PCR was used to detect Beclin 1,LC3B mRNA in each group of cells according to the grouping intervention expression.4.MC3T3-E1 osteoblasts were divided into normal control group（Control）,model group（Model）,Liuwei Dihuang Pills group（H+LWDH）,blank control group（H+Blank）,rapamycin group（H+Rap）,U0126 group（H+U0126）,rapamycin+Liuwei Dihuang Pills group（H+Rap+LWDH）,and U0126+Liuwei Dihuang Pills group（H+U0126+LWDH）,and after intervention according to the grouping,the content of ROS in each group of cells was detected by fluorescent probe DCFH-DA,ERK1/2 and mTOR mRNA expression in each group of cells by qRT-PCR,and LC3,ERK1/2,mTOR,LC3,ERK1/2 and mTOR in each group of cells by Western blot.p-ERK1/2,p-mTOR protein expression by Western blot.Results:1.The effect of Liuwei Dihuang Pills-containing serum on the proliferative activity of MC3T3-E1 cells under oxidative stress in the presence of H₂O₂.When the concentration of H₂O₂was 0.10 mmol/L,the average proliferation rate of osteoblasts was 70.65%compared with the control group,and its inhibitory effect on osteoblasts was more significant（P<0.05）;when the concentration of H₂O₂was further increased,cell proliferation was further inhibited or even died,so the H₂O₂concentration of0.10 mmol/L was selected for the subsequent experimental study.The proliferation rate of osteoblasts was positively correlated with the concentration of serum containing Liuwei Dihuang Pills,and the highest proliferation rate of osteoblasts was observed when the concentration of serum containing Liuwei Dihuang Pills was 20%,and the difference was most significant when compared with the blank group（P<0.05）,and when the concentration was greater than 20%,the proliferation of osteoblasts was inhibited,indicating that the serum containing Liuwei Dihuang Pills could promote the proliferation activity of osteoblasts under oxidative stress.The results of ALP staining showed that compared with the Control group,the black-brown granules in the cell membrane and cytoplasm of the Model group were significantly reduced,indicating that H₂O₂inhibited the ALP activity of osteoblasts;compared with the Model group,the black-brown granules in the H+LWDH group were more obvious under the naked eye and inverted microscope,suggesting that Liuwei Dihuang Pillsl can promote the proliferation of osteoblasts.This indicates that the drug-containing serum of Liuwei Dihuang Pills can promote the ALP activity of osteoblasts under oxidative stress.2.The effect of Liuwei Dihuang Pills-containing serum on the intracellular ROS of MC3T3-E1 cells under oxidative stress.Compared with the Control group,the fluorescence intensity of ROS in the Model group was significantly enhanced（P<0.05）,indicating that the osteoblasts were under oxidative stress under the intervention of H₂O₂;the fluorescence intensity in the H+LWDH group was significantly weakened（P<0.05）compared with the Model group,indicating that the drug-containing serum of Liuwei Dihuang Pills could reduce the level of ROS in the cells and mitigate the oxidative damage caused by H₂O₂on The fluorescence intensity was significantly weaker（P<0.05）compared with that of the Model group,indicating that Liuwei Dihuang Pills containing serum could reduce the level of ROS in cells and mitigate the oxidative damage caused by H₂O₂on osteoblasts.3.The effect of Liuwei Dihuang Pills-containing serum on the expression of autophagy-related genes Beclin 1 and LC3B mRNA in MC3T3-E1 cells under oxidative stress.Compared with the Control group,autophagy-related Beclin 1 and LC3B mRNA expression was significantly lower in the Model group（P<0.05）;compared with the Control group,Model group and H+Blank group,autophagy-related Beclin 1 and LC3B mRNA expression was significantly higher in the H+LWDH group（P<0.05）;LWDH+3-MA group showed significantly lower expression of autophagy-related genes Beclin 1 and LC3B mRNA compared with the H+LWDH group（P<0.05）.It can be seen that the expression of cellular Beclin 1 and LC3B mRNA increased under the protective intervention of Liuwei Dihuang Pills containing serum,indicating that Liuwei Dihuang Pills containing serum can promote the autophagy level of osteoblasts under oxidative stress.4.Effects of Liuwei Dihuang Pills-containing serum on ERK/mTOR pathway in MC3T3-E1 cells under oxidative stress.The intracellular ROS levels were reduced in the Rap,U0126,LWDH+Rap and LWDH+U0126 groups compared with the Model group（P<0.05）,indicating that inhibition of the ERK/mTOR pathway could reduce the ROS levels in osteoblasts under oxidative stress and mitigate the oxidative damage of H₂O₂on osteoblasts.Compared with the Model group,protein LC3II/Ⅰincreased in the H+LWDH group,H+Rap group and H+LWDH+Rap group（P<0.05）;compared with the Model group,protein LC3II/Ⅰincreased in the H+U0126 group and H+LWDH+U0126 group,but there was no statistical difference,indicating that both LWDH-containing serum and inhibition of ERK/mTOR pathway can both promote the autophagy level of osteoblasts under oxidative stress,while the LWDH-containing serum promoted the autophagy of osteoblasts under oxidative stress associated with the inhibition of ERK/mTOR pathway.Compared with the Model group,p-mTOR protein expression was down-regulated in the H+LWDH and Rap groups（P<0.05）;compared with the Model group,there was a trend of down-regulation of p-mTOR protein in the H+Rap+LWDH and H+U0126+LWDH groups,but there was no statistical difference;compared with the Model group,the H+LWDH and H+U0126 groups p-ERK1/2 protein expression was down-regulated in the H+Rap group and H+U0126+LWDH group compared with the Model group（P<0.05）;p-ERK1/2 protein expression was down-regulated in the H+Rap group and H+U0126+LWDH group compared with the Model group,but there was no statistical difference.It indicates that Liuwei Dihuang Pills-containing serum can promote autophagy to reduce oxidative damage in osteoblasts by inhibiting ERK/mTOR pathway.Conclusion:H₂O₂can inhibit the proliferative activity of MC3T3-E1 osteoblasts,put the cells in an oxidative stress state,and reduce the expression of autophagy-related Beclin 1 and LC3B mRNA and increase the expression of protein p-ERK1/2 and p-mTOR;while Liuwei Dihuang Pills-containing serum can promote the proliferative activity of osteoblasts under oxidative stress caused by H₂O₂,reduce the intracellular ROS The drug-containing serum promoted the proliferative activity of osteoblasts under H₂O₂-induced oxidative stress,decreased the intracellular ROS level,increased the expression of Beclin 1 and LC3B mRNA,and decreased the expression of p-ERK1/2 and p-mTOR.This indicates that the drug-containing serum of Liuwei Dihuang Pills can reduce the damage of MC3T3-E1 osteoblasts under oxidative stress by inhibiting the ERK/mTOR pathway to induce cell autophagy,and finally achieve the purpose of promoting the proliferative activity of osteoblasts.