| Objective: In recent years,the incidence of Diabetes Mellitus(DM)in China has been increasing with the development of testing methods,the dietary habits of the population and China’s gradual entering of an aging society.At present,China has the largest number of diabetes patients in the world.Diabetes mellitus is characterized by high glucose toxicity,which can lead to systemic vascular,microvascular,nerve tissue,bone tissue and other systemic complications.Diabetic Osteoporosis(DOP)is a systemic metabolic bone disease characterized by decreased bone mass,microstructural changes of bone tissue,and decreased bone strength.This series of changes enhances bone fragility and predisposes to fracture.DOP is a complication of chronic diabetes mellitus,but due to its hidden occurrence,there was a lack of adequate understanding of DOP in the past.In recent years,the incidence of T2 DM in China has been increasing year by year,so the incidence of DOP is also increasing.Osteoporotic fracture will first lead to large area of bone defect due to its bone microstructure changes,which leads to the difficulty of surgical fixation.The incidence of surgical complications such as internal fixation failure,bone malunion,and internal fixation fracture is high.Such as bedsore infection,hypostatic pneumonia,urinary system infection,lower extremity deep vein thrombosis,etc.,high disability and mortality bring great difficulties and challenges to clinical treatment.Therefore,it is of positive significance to clarify the regulatory mechanism of the upstream of the disease and explore ways to improve bone conditions for the treatment of DOP.A number of studies have shown that high glucose seriously hinders the migration,proliferation and mineralization of osteoblasts.The main effect of type 2 diabetes on bone metabolism is that the function of osteoblasts(OB)is reduced,and bone formation is reduced and slow.Osteoblasts are mainly differentiated from Mesenchymal Stem cells(MSC)in the inner and outer periosteum and bone marrow matrix,which are the main cells for bone growth and development.Their synthesis and secretion are regulated by multiple neural and humoral regulations,and they can specifically secrete a variety of bioactive substances related to bone function.It is involved in regulating and affecting bone formation and reconstruction.Therefore,we hypothesized that clarifying the endocrine regulation mechanism of osteoblasts in the pathological environment of type 2 diabetes is the key to improve the osteogenic ability of osteoblasts.Orexin,also known as hypocretin,is A hypothalamic neuropeptide that signals through G protein-coupled receptors,including orexin A and orexin B isoforms.Orexin has a variety of biological functions and participates in the process of glucose and lipid metabolism and energy homeostasis.Its receptors not only exist in the central nervous system,but also widely act on peripheral tissues including adrenal gland,gastrointestinal tract,pancreas,bone tissue and so on.A number of studies have confirmed that orexin can inhibit the secretion of inflammatory products and participate in anti-oxidation through NF-κB related pathways.However,whether orexin plays an important role in type 2 diabetes combined with osteoporosis as an endocrine regulatory mechanism,and the specific regulatory effect and mechanism of orexin on OB osteogenic function are still unknown.Based on the above background,this study aims to investigate the effects of orexin on mitochondrial function and osteogenesis of OB in the diabetic microenvironment,and to further explore the effects and mechanisms of orexin on inflammatory response and oxidative stress in osteoblasts under high glucose environment,so as to provide new targets and therapeutic methods for the prevention and treatment of DOP.Methods: 1.Study on the relationship between Orexin and type 2 diabetic osteoporosis:(1)We detected Orexin A and Orexin B in the peripheral blood of 100 male patients with type 2 diabetes mellitus with osteoporosis and 100 male patients with osteoporosis by ELISA,and observed the changes of orexin in DOP;(2)SD rats were fed with high-fat and high-sugar diet and injected with low-dose streptozotocin(STZ)intraperitoneally to establish type 2 diabetic animal model.The biomechanical properties of tibial tissue of rats were detected by maximum stress test and elastic modulus test.Bone Micro-CT and HE staining were used to determine the success of the DOP model.ELISA was used to detect Orexin A and Orexin B in the peripheral blood of 15 SD rats with type 2 diabetes and osteoporosis and 15 SD rats in the control group,and the changes of orexin in the DOP model were observed.Laser confocal microscopy was used to observe the co-localization of OX1 R,OX2R and OSX,and to determine the expression of orexin receptor on the surface of osteoblasts.According to the results,Orexin A was selected for subsequent research.The mechanism of Orexin A on type 2 diabetic osteoporosis:(1)To determine the regulatory effect of Orexin A on the inflammatory pathway SIRT1/NLRP3 under high glucose environment,the human osteoblast cell line h FOB1.19 was used as the in vitro study object.In order to select a suitable high glucose environment,we set the control group(glucose concentration 17.5m M)as 1 times the glucose concentration,and the glucose concentration gradient was set at 1,1.5,2,2.5 and 3 times.The osmotic pressure was adjusted by sucrose to exclude the effect of osmotic pressure on cell viability.Thus,the treatment conditions simulating the high glucose environment were determined.(2)h FOB1.19 cells were treated with different concentrations of Orexin A(0,2.5,5μM)in high glucose environment for 72 hours,and the protein expressions of NLRP3 and SIRT1 were detected by Western-blot.(3)The control group,Orexin A group,Orexin A group,Orexina + SIRT1 group,Western-blot method was used to detect the expression of NLRP3,and ELISA method was used to detect the expression of inflammatory factors IL6 and IL8.To determine the upstream and downstream relationship between orexin A and SIRT1 and NLRP3 in osteoblasts under high glucose environment.(4)In order to determine the regulatory effect of Orexin A on ROS level in osteoblasts under high glucose environment,we set up control group,Orexin A group,Orexin A+ activated NLRP3 group,and h FOB1.19 cells treated with 5μM Orexin A under high glucose environment.The intracellular ROS level was detected by ROS fluorescent probe.3.Effect of Orexin A on type 2 diabetic osteoporosis:(1)In order to test the regulatory effect of Orexin A on mitochondrial function in osteoblasts under high glucose environment,the control group,Orexin A group and Orexin A+ SIRT1 group were set up.Mitochondrial fluorescent probe staining,mitochondrial DNA detection,ATP production and oxygen consumption were detected.The mitochondrial morphology was observed by transmission electron microscope.(2)To test the effect of orexin A on the osteogenic potential of h FOB1.19 cells,HFOB1.19 cells were treated with 5μM Orexin A for 72 hours in high glucose environment,and a control group and a SIRT1 silencing group were set up.The m RNA levels of OPG,OCN,ALP,Runx2,and Collagen I were detected by PCR,and mineralized nodules were observed by alizarin red staining.(3)We used the method of local injection of Orexin A and SIRT1 silencing lentivirus into the proximal tibia of type 2diabetic rats with osteoporosis.Micro CT,HE staining and biomechanical test were performed on the control group,orexin-A treatment group and orexin-A + SIRT1 silencing group after one month of treatment.The therapeutic effect of Orexin A on type 2 diabetes mellitus with osteoporosis in high glucose environment.Results:1.Elisa results showed that the levels of Orexin A and Orexin B in the peripheral blood of type 1 and type 2 diabetic patients with osteoporosis were lower than those in the peripheral blood of simple osteoporosis patients,suggesting that orexin A and orexin B are decreased in T2 DM.2.The DOP model was successfully established.The levels of Orexin A and Orexin B in the peripheral blood of type 2 diabetic rats with osteoporosis were decreased by Elisa,suggesting that orexin A and orexin B were decreased in the DOP model.3.Immunofluorescence co-localization results showed that OX1 R was mainly expressed in osteoblasts in tibial tissue sections of SD rats,and orexin receptor 1 was the specific receptor of orexin A,suggesting that orexin A could act on osteoblasts.4.According to the results of CCK8,the appropriate high glucose condition in the appropriate cell experiment was selected.In the high glucose environment,with the increase of Orexin A concentration,the expression of SIRT1 protein increased,while the expression of NLRP3 protein decreased,indicating that orexina A can promote the expression of SIRT1 and inhibit the expression of NLRP3.5.Western blot results showed that the inhibitory effect of Orexin A on NLRP3 was abolished after lentivirus silencing SIRT1,indicating that Orexina can inhibit NLRP3-mediated inflammatory response through SIRT1 in high glucose environment.Elisa results showed that the content of inflammatory products IL-6 and IL-8 decreased after NLRP3 inhibition,indicating that the inflammatory response was alleviated.6.ROS fluorescent probe results showed that Orexin A could inhibit ROS production in osteoblasts under high glucose environment through SIRT1/NLRP3 signaling pathway,indicating that Orexin A could inhibit inflammatory response through SIRT1,thereby inhibiting NLRP3-mediated oxidative stress.7.Under high glucose environment,the number of mitochondria,the number of mitochondrial DNA detected,ATP production and oxygen consumption of cells in the Orexin A group increased,and the promoting effect was abolished after SIRT1 silencing,suggesting that Orexin A can enhance mitochondrial function of osteoblasts through SIRT1 under high glucose environment.8.In the Orexin A group,the m RNA levels of OPG,OCN,ALP,Runx2,Collagen I and other osteogenic related indicators were increased under high glucose environment,and alizarin red staining showed that the number of mineralized nodules increased,and the promoting effect was relieved after SIRT1 silencing.These results indicated that Orexin A could enhance the osteogenic function of OB through SIRT1.9.After one month of Orexin A treatment,Micro CT and HE staining showed that the density and number of trabecular bone in the Orexin A treatment group increased,and the maximum stress under biomechanical test increased,indicating that Orexin A could have therapeutic effect on type 2 diabetic rats with osteoporosis through SIRT1.Conclusions:1.Orexin is decreased in peripheral blood of type 2 diabetic patients with hyperostosis.Orexin A can act on osteoblasts.2.Orexin A can regulate the inflammasome NLRP3 through SIRT1 in the microenvironment of type 2 diabetes,thereby inhibiting the production of ROS in osteoblasts,thereby improving the mitochondrial function and osteogenic ability of osteoblasts.3.Orexin A may be an important regulator of inflammatory response and oxidative stress in the microenvironment of type 2 diabetes,and it is a key source innovative treatment for type 2 diabetes with osteoporosis. |
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