基于PI3K/Akt/GPX4铁死亡信号通路探究补肾壮骨汤含药血清外泌体对H2O2处理成骨细胞增殖分化的影响OA北大核心CSTPCD

Objective To investigate the effects of serum exosomes containing the active ingredient from Bushenzhuanggu Decoction on the proliferation and differentiation of hydrogen peroxide(H2O2)-treated osteoblasts and its mechanism.Methods Serum exosomes containing the active ingredient from Bushenzhuanggu Decoction were ex-tracted and identified.MC3T3-E1 osteoblasts were cultured in vitro,and the cells were divided into control group,H2O2 group,serum exosomes containing the active ingredient from Bushenzhuanggu Decoction group(50 and 100 μg/mL),LY294002+serum exosomes containing the active ingredient from Bushenzhuanggu Decoction group(100 μg/mL).Cell counting kit-8(CCK-8)assay was used for cell viability.5-Ethynyl-2′-deoxyuridine(EDU)stai-ning was taken to detect cell proliferation.Phosphorylation levels of phosphoinositide 3-kinase(PI3K)and protein ki-nase B(Akt)and the protein expression of glutathione peroxidase 4(GPX4)were detected by Western blot.Colori-metric assay was taken to detect malondialdehyde(MDA)and glutathione(GSH)levels as well as alkaline phosphatase(ALP)activity.Dihydroethidium(DHE)staining was used to detect reactive oxygen species(ROS)levels.Ferrousion(Fe2+)level was detected by FerroOrange probe.Alizarin red staining was used to observe the mineralization of osteoblast.Osteoclastogenesis inhibitory factor(OCIF),type Ⅰ collagen(COL1)and bone morphogenetic protein-2(BMP2)mRNA expression were detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR).Results Compared to the control group,cell survival rate,proliferation rate,GSH level,ALP activity,mineralized nod-ule formation and OCIF,COL1 and BMP2 mRNA levels and phosphorylated(p)PI3K(p-PI3K),p-Akt,and GPX4 protein expression were significantly reduced(P<0.01),and ROS,Fe2+and MDA levels were significantly higher in the H2O2 group(P<0.01).In comparison with the H2O2 group,cell survival rate,proliferation rate,GSH level,ALP activity,mineralized nodule formation and OCIF,COL1 and BMP2 mRNA levels and p-PI3K,p-Akt and GPX4 protein expressions were significantly elevated(P<0.01)and ROS,Fe2+and MDA levels were significantly reduced(P<0.01)in the serum exosomes containing the active ingredient from Bushenzhuanggu Decoction group.Akt signaling inhibitor LY294002 attenuated the protective effect of serum exosomes containing the active ingredient from Bushenz-huanggu Decoction on H2O2-treated MC3T3-E1 cells.Conclusion Serum exosomes containing the active ingredient from Bushenzhuanggu Decoction attenuate the inhibitory effect of H2O2 on cell proliferation and osteogenic differentiation by activating the PI3K/Akt/GPX4 signaling pathway.