| Objective:This study was conducted to investigate the molecular mechanism of serum exosomes regulating osteoclast bone resorption function and related factor changes through the lectin-like oxidized low-density lipoprotein receptor-1（LOX-1）/tumor necrosis factor receptor-associated factor-6（TRAF6）/nuclear factor-κB（NF-κB）signaling pathway under high glucose,aiming to elucidate the pathological mechanism of diabetic osteoporosis and provide new ideas for the search of targeted drugs for the treatment of diabetic osteoporosis.Methods:Bone marrow macrophages were isolated from C57BL/6 mice in vitro,induced to differentiate into osteoclasts,identified by anti-tartrate acid phosphatase staining,and cultured in DMEM medium;Exosomes were extracted from healthy human serum using Exo Quick TMexosome extraction kit,and observed morphologically by transmission electron microscopy（TEM）and nanosight（NTA）for particle concentration distribution analysis and osteoblast treated with freshly extracted serum exosomes.Lentiviral LOX-1 RNAi silencing vector（LV-LOX-1）and lentiviral control vector（LV-CON）were constructed for transfection of osteoblasts.The experimental groups were as follows:normal control group（NC group）,high glucose group（HG group）,lentiviral LOX-1 RNAi silencing group（LV-LOX-1 group）,and lentiviral control vector group（LV-CON group）.Osteoclasts were treated with TRAF-STOP 6877002（TRAF6 inhibitor）and BAY11-7082（NF-κB inhibitor）,respectively,and cell viability was detected by CCK-8 method.The expression levels of mRNA and protein of LOX-1、TRAF6、NF-κB、matrix metallase-9（MMP-9）and anti-tartrate acid phosphatase（TRAP）were detected by qRT-PCR and Western blotting techniques.Results:1.TRAP staining results showed that osteoclasts were observed to be large and morphologically diverse under an inverted phase contrast microscope,with folded edges and a large number of pseudopods visible around them,and most osteoclasts were stained with strong blue spots and visible≥3nuclei.2.TEM results showed that most of the exosomes were in the shape of round vesicles.3.NTA results showed that most of the serum exoosmes were between 30 and 150 nm in diameter,with the largest distribution of 74.75 nm and a concentration of 3.34E+9particles/ml.4.The expression levels of mRNA and protein of LOX-1,MMP-9 and TRAP were increased in the cells of HG group.Compared with the NC group,the mRNA expression levels in LOX-1,MMP-9 and TRAP in the cells of HG group increased by 134.46%,100.60%and 136.36%,respectively,and the protein expression levels increased by 49.47%,59.35%and 44.18%,with statistically significant differences（P<0.01）.5.The expression levels of mRNA and protein of LOX-1,MMP-9 and TRAP were decreased in the cells of HG+LV-LOX-1 group.Compared with the HG+LV-CON group,the mRNA expression levels of LOX-1,MMP-9 and TRAP in the cells of HG+LV-LOX-1 group were decreased by 27.47%,25.62%and 29.40%,respectively,and the protein expression levels were decreased by 19.10%,23.78%and 23.56%,respectively,with statistically significant differences（P<0.01）.6.The expression levels of mRNA and protein of LOX-1,MMP-9 and TRAP were decreased in cells of EXOs group.Compared with NC group,the mRNA expression levels of LOX-1,MMP-9 and TRAP in cells of NC+EXOs group were decreased by 45.14%,47.18%and 46.11%,respectively,and the protein expression levels were decreased by 40.98%,49.25%,with statistically significant differences（P<0.01）.Compared with the HG group,the mRNA expression levels of LOX-1,MMP-9 and TRAP in the HG+EXOs group decreased by53.61%,54.63%and 60.37%,respectively,and the protein expression levels decreased by49.19%,50.37%and 72.52%,respectively,with statistically significant differences（P<0.01）.7.The expression levels of mRNA and protein of TRAF6,NF-κB,MMP-9 and TRAP were decreased in cells of HG+LV-LOX-1+TRAF-STOP6877002 group and HG+LV-LOX-1+BAY11-7082 group.Compared with HG+LV-LOX-1 group,the mRNA expression levels of TRAF6,NF-κB,MMP-9 and TRAP were decreased by 55.79%、38.46%、37.27%,40.69%,respectively,and protein expression levels were decreased by 36.89%、19.92%、38.74%、32.38%,respectively,in HG+LV-LOX-1+TRAF-STOP6877002 group cells,with statistically significant differences（P<0.01）.Compared with the HG+LV-LOX-1 group,the mRNA expression levels of NF-κB,MMP-9 and TRA P in the HG+LV-LOX-1+BAY11-7082 group decreased by 53.17%、54.75%、52.16%,respectively,and the protein expression levels decreased by34.52%、48.91%、46.62%,respectively,with statistically significant differences（P<0.01）.8.The expression levels of mRNA and protein of MMP-9 and TRAP were decreas ed in cells of HG+LV-LOX-1+TRAF-STOP 6877002+EXOs group and HG+LV-LOX-1+BAY11-7082+EXOs group.Compared with the HG+LV-LOX-1+TRAF-STOP 6877002group,the mRNA levels of MMP-9 and TRAP were decreased by 27.56%and 18.16%,respectively,and the protein expression levels were decreased by 11.76%and 16.42%,respectively,in the cells of HG+LV-LOX-1+TRAF-STOP 6877002+EXOs group,with statistically significant differences（P<0.01）;Compared with the HG+LV-LOX-1+BAY11-7082 group,the mRNA expression levels of MMP-9 and TRAP in cells of HG+LV-LOX-1+BAY11-7082+EXOs group decreased by 30.42%and 22.28%,respectively,and the protein expression levels decreased by 17.25%and 14.13%,respectively,with statistically significant differences（P<0.01）.Conclusions:The expression levels of LOX-1,MMP-9 and TRAP was upregulated in osteoclasts treated by high glucose,while lentiviral LOX-1 RNAi silencing reversed these phenomena and inhibited osteoclast bone resorption function;Serum exosomes inhibited the expression levels of MMP-9 and TRAP in osteoclasts treated by high glucose via the LOX-1/TRAF6/NF-κB pathway,improving osteoclast bone resorption dysfunction and delaying the progression of DOP. |
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