| Background and objectivesType 2 diabetes mellitus(T2DM)is an endocrine disease characterized by non-insulindependent hyperglycemia,which poses a significant public health risk worldwide.Among the complications associated with T2DM,poor bone regeneration is of particular concern as it leads to reduced bone mass,abnormal bone microarchitecture,and disrupted bone homeostasis,thereby increasing the risk of fractures.Maresin1(MaR1),a metabolite derived from ω-3 fatty acids,exhibits various physiological roles,including antioxidant activity and promotion of tissue regeneration.However,the specific mechanisms underlying these effects remain unclear.Recent evidence suggests that MaRl regulates the nuclear factor erythroid 2-related factor 2(NRF2),a key antioxidant factor,and inhibits cellular ferroptosis by modulating iron metabolism,glutathione(GSH)levels,lipid peroxidation,and other related processes.In light of these findings,the present study aims to investigate the impact of MaRl on osteogenic function in the T2DM state and further explore the involvement of NRF2 and ferroptosis in the regulation of bone regeneration by MaR1.The findings from this study will contribute to a better understanding of the underlying mechanisms and provide novel therapeutic strategies for promoting bone regeneration in individuals with T2DM.Materials and methods1.The effect of Maresin1 on osteoblast function under high-glucose conditionsMC3T3-E1 osteoblast cell lines were cultured and divided into the following groups:Normal group,T2DM group,T2DM+MaR1(1 nM)group,and T2DM+MaR1(10 nM)group.Cell proliferation was detected by EdU fluorescent staining and the CCK-8 assay.Osteogenic mineralization ability was evaluated through protein blotting(western blot),alkaline phosphatase(ALP)staining,and alizarin red S(ARS)mineralization nodule assay.2.The role of NRF2 in improving osteoblast function by Maresin1 in hyperglycemia stateNRF2 was knocked down by small interfering RNA(siRNA)technology,and the knockdown efficiency was determined using immunofluorescence staining and Western blot analysis.The following groups were established:Normal group,T2DM group,T2DM+MaRl group,T2DM+MaR1+siNC group,and T2DM+MaR1+siNRF2 group,to investigate osteoblast proliferation and osteomineralization ability.3.The impact of NRF2-ferroptosis axis on osteoblast function by Maresin1 in high-glucose conditionsTo investigate the pharmacological mechanism of MaRl and its effect on the NRF2ferroptosis axis,ferroptosis promoters were added to the T2DM+MaR1+Era group.To investigate the pharmacological mechanism of MaRl and its effect on the NRF2-ferroptosis axis,ferroptosis promoters were added to the T2DM+MaR1+Era group.Mitochondrial morphology was observed using transmission electron microscopy.The cellular osteogenic capacity was evaluated comprehensively,as described in Part 1.4.The effect of Maresin1 on calvarial bone regeneration in type 2 diabetic ratsTwenty 4-week-old male Sprague Dawley rats were used and randomly divided into Normal group,T2DM group,T2DM+Vehicle group,and T2DM+MaRl group.The T2DM rat model was constructed using a high-fat and high-sugar diet for one month,combined with streptozocin intraperitoneal injection.In the T2DM+MaRl group,a gelatin sponge infiltrated with MaRl was placed in the bone defect area.The gelatin sponge in the T2DM+Vehicle group was infiltrated with an equal amount of sterile saline as a control.The study evaluated bone regeneration using micro-CT,double fluorescent labeling,and hematoxylin and eosin staining evaluated bone regeneration at 28 days postoperatively.NRF2 and GPX4 expression was detected using immunohistochemical staining.Results1.Maresinl exerted a significant positive effect on the biological function of osteoblasts in a high-glucose environmentThe impaired proliferation and differentiation abilities of osteoblasts in T2DM were effectively restored by MaRl,leading to enhanced cell proliferation and improved mineralization ability.2.NRF2 played a crucial role in mediating the beneficial effects of Maresinl on osteoblasts under high-glucose conditionsIF and Western blot analyses demonstrated that MaRl upregulated the overall expression of NRF2 and promoted its nuclear translocation.Knockdown of NRF2 using siRNA attenuated the osteogenic effects of MaRl,as evidenced by reduced expression of osteogenic differentiation-related proteins and decreased regions of ALP-staining.3.NRF2-ferroptosis axis was a critical pathway through which Maresinl ameliorated the biological function of osteoblasts in a hyperglycemia stateT2DM medium,ferroptosis promoter,and NRF2-siRNA severely disrupted the GSH redox system,resulting in excessive accumulation of iron ions and a significant reduction in GPX4 and SLC7A11 expression.This further exacerbated lipid peroxidation and induced abnormal cell mitochondrial morphology,leading to reduced osteoblast proliferative activity and cellular impaired cellular osteogenic capacity.4.Maresinl demonstrated a significant capability to promote calvarial bone regeneration in type 2 diabetic ratsIn the calvarial defect area of T2DM rats,MaRl treatment led to a remarkable increase in new bone formation,with enhanced calcium deposition observed at the edges of pre-existing and newly formed bone.Moreover,MaRl treatment significantly enhanced the immunoreactivity of NRF2 and GPX4,indicating the activation of antioxidant and osteogenic pathways.Conclusions1.MaRl improves the impaired proliferation and osteogenic differentiation of osteoblasts.2.NRF2 is identified as a potential key regulator for MaR1 in improving the osteogenic process under high-glucose conditions.3.MaR1 mitigates hyperglycemia-induced ferroptosis in osteoblasts by activating NRF2,and its modulation of the NRF2-ferroptosis axis reinstates osteogenic activity.4.In-situ administration of MaR1 ameliorates the dysregulation of the NRF2 antioxidant system and ferroptosis pathway associated with T2DM,promoting new bone remodeling in a T2DM rat calvarial defect model with impaired bone regeneration. |
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