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H_(2)O_(2)处理成骨细胞增殖分化的影响

Effects of serum exosomes containing the active ingredient from Bushenzhuanggu Decoction on the proliferation and differentiation of H_(2)O_(2)-treated osteoblasts based on PI3K/Akt/GPX4 ferroptosis signaling pathway

目的探究补肾壮骨汤含药血清外泌体对过氧化氢(hydrogen peroxide,H_(2)O_(2))处理成骨细胞增殖分化的影响及作用机制。方法提取与鉴定补肾壮骨汤含药血清外泌体。体外培养MC3T3-E1细胞,将细胞分为对照组、H_(2)O_(2)组、补肾壮骨汤含药血清外泌体组(50和100μg/mL)、LY294002+补肾壮骨汤含药血清外泌体(100μg/mL)组。细胞计数试剂盒8(cell counting kit-8,CCK-8)法检测细胞存活率。5-乙炔基-2′-脱氧尿苷(5-Ethynyl-2′-deoxyuridine,EDU)染色检测细胞增殖情况。免疫印迹检测磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)与蛋白激酶B(protein kinase B,Akt)的磷酸化水平和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达。比色法检测丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)水平及碱性磷酸酶(alkaline phosphatase,ALP)活性。二氢乙锭(dihydroethidium,DHE)染色检测活性氧(reactive oxygen species,ROS)水平。FerroOrange探针检测二价铁离子(ferrousion,Fe^(2+))水平。茜素红染色观察成骨细胞矿化。荧光定量逆转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction,qRT-PCR)检测破骨细胞抑制因子(osteoclastogenesis inhibitory factor,OCIF)、Ⅰ型胶原蛋白(collagen typeⅠ,COL1)和骨形态发生蛋白-2(bone morphogenetic protein-2,BMP2)mRNA表达。结果与对照组比较,H_(2)O_(2)组细胞存活率、增殖率、GSH水平、ALP活性、矿化结节形成及OCIF、COL1和BMP2 mRNA水平和磷酸化(phosphorylated,p)PI3K(p-PI3K)、p-Akt、GPX4蛋白表达显著降低(P<0.01),ROS、Fe^(2+)和MDA水平则显著升高(P<0.01)。与H_(2)O_(2)组比较,补肾壮骨汤含药血清外泌体组细胞存活率、增殖率、GSH水平、ALP活性、矿化结节形成及OCIF、COL1和BMP2 mRNA水平和p-PI3K、p-Akt、GPX4蛋白表达显著升高(P<0.01),ROS、Fe^(2+)和MDA水平显著降低(P<0.01)。Akt信号抑制剂LY294002减弱了补肾壮骨汤含药血清外泌体对H_(2)O_(2)处理MC3T3-E1细胞的保护作用。结论补肾壮骨汤含药血清外泌体通过激活PI3K/Akt/GPX4信号通路,减弱H_(2)O_(2)对细胞增殖和成骨分化的抑制作用。

Objective To investigate the effects of serum exosomes containing the active ingredient from Bushenzhuanggu Decoction on the proliferation and differentiation of hydrogen peroxide(H_(2)O_(2))-treated osteoblasts and its mechanism.Methods Serum exosomes containing the active ingredient from Bushenzhuanggu Decoction were extracted and identified.MC3T3-E1 osteoblasts were cultured in vitro,and the cells were divided into control group,H_(2)O_(2) group,serum exosomes containing the active ingredient from Bushenzhuanggu Decoction group(50 and 100μg/mL),LY294002+serum exosomes containing the active ingredient from Bushenzhuanggu Decoction group(100μg/mL).Cell counting kit-8(CCK-8)assay was used for cell viability.5-Ethynyl-2′-deoxyuridine(EDU)staining was taken to detect cell proliferation.Phosphorylation levels of phosphoinositide 3-kinase(PI3K)and protein kinase B(Akt)and the protein expression of glutathione peroxidase 4(GPX4)were detected by Western blot.Colorimetric 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(Fe^(2+))level was detected by FerroOrange probe.Alizarin red staining was used to observe the mineralization of osteoblast.Osteoclastogenesis inhibitory factor(OCIF),typeⅠcollagen(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 nodule 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,Fe^(2+)and MDA levels were significantly higher in the H_(2)O_(2) group(P<0.01).In comparison with the H_(2)O_(2) 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,Fe^(2+)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 Bushenzhuanggu Decoction on H_(2)O_(2)-treated MC3T3-E1 cells.Conclusion Serum exosomes containing the active ingredient from Bushenzhuanggu Decoction attenuate the inhibitory effect of H_(2)O_(2) on cell proliferation and osteogenic differentiation by activating the PI3K/Akt/GPX4 signaling pathway.