The Mechanism Of Diabetic Osteoporosis Mediated By The Activation Of Inflammatory Pathway And Inhibition Of Efferocytosis In Osteoclast

With the improvement of living standards,Diabetes mellitus(DM)has become the most common endocrine and metabolic disorder disease that is threatening the health of both humans and animals.The clinical symptoms of pet diabetes were similar with humans.And there were often accompanied by severe complications.Among them,diabetic osteoporosis(DOP)is widely considered as a hot discussion problem.which is a kind of systemic metabolic bone disease.It is a bone disease characterized by decreased bone mass and degradation of bone microstructure.It has been found that DOP was related to hyperglycemia,disorder of calcium and phosphorus metabolism and insulin deficiency.However,its pathogenesis was unclear,which seriously affects its effective prevention and treatment.There is a certain relationship between osteoporosis and inflammation.Reactive oxygen species(ROS)are produced as a natural byproduct of the normal metabolism of oxygen.When their concentrations are beyond a certain range,they could disturb the oxidant–antioxidant balance.This disturbance could lead to many inflammatory diseases.The NLRP3 inflammasome is a protein complex,which is implicated not only in inflammatory disorders but also in numerous metabolic diseases.And a recent study reported that the NLRP3 inflammasome played an important role in bone resorption.Nuclear factor kappa B(NF-κB)is a transcription factor that regulates the expression of multiple genes and governs various cellular functions,including inflammatory signaling.In addition.Mitogen-activated protein kinases(MAPKs)can control various cellular activities,including gene expression and mitosis.In particular,MAPKs(ERK,p38 and JNK)have important roles in regulating inflammatory and immune responses.Macrophages are key immune cells,when the organization was damaged,neutrophils accumulate in the tissue and rapidly undergo apoptosis.The process of clearing apoptotic cells by macrophages is called efferocytosis.The failure to clear apoptotic cells exacerbates inflammation in vivo,therefore,efferocytosis plays a crucial role in modulating the inflammatory response in macrophages to promote inflammation resolution.Osteoclasts are a giant multinucleated cell that originates from monocyte macrophages/monocytes,and it considered one of the target cells for treating osteoporosis and it play an important role in bone resorption.The model of spontaneous diabetes in companion animals is more similar to that in rodents and human clinical diseases,and dogs and humans have more similar chromosomes and genetic structures,which is more suitable as a medical model for human disease translation.Therefore,in this study,osteoclasts were used as target cells,artificially induced diabetic osteoporotic rats and spontaneous diabetic osteoporotic dogs were used as disease models together with human clinical samples to investigate whether high glucose could enhance osteoclast bone resorption through the production of osteoclast ROS and NLRP3-mediated inflammatory activation and the inhibition of efferocytosis,then further explored the pathogenesis of DOP in humans and animals in the molecular and pathological levels.First,we first discovered that osteoclast NLRP3 inflammasome-mediated inflammatory activation is a causative factor for diabetic osteoporosis at home and abroad.We used an inverted microscope to detect the induction of isolated osteoclasts.And used a fluorescence microplate reader to detect the ROS production.Western blot was used to analysis the inflammation-related pathway proteins MAPKs(p-ERK,p-p38,and p-JNK),NF-κB(nuclear NF-κB,p-IκB,and IKK)and NLRP3inflammasome(ASC,Caspase-1,IL-18,IL-1β,and NLRP3)expression levels.In addition,ELISA was used to detect the secretion of IL-18 and IL-1β.And the pathway inhibitors were used to observe the relationship between ROS,MAPKs pathway,NF-κB pathway and NLRP3 inflammasome.The results showed that all of the isolation and induced cells were mature osteoclasts detected by TRAP staining.HG could induce ROS production in osteoclasts and the optimal induction time was 36 h,the optimal induction concentration was 35 mM and the production were NOX2-dependent.HG also could induce the expression of MAPKs-related pathway proteins,NF-κB-related pathway proteins and NLRP3 inflammasome proteins and the secretion of IL-18 and IL-1β,and the optimal induction time was 36 hours,the optimal induction concentration was 35 mM.And then DPI(ROS inhibitor)could significantly inhibited the expression of MAPKs-related pathway proteins,NF-κB-related pathway proteins and NLRP3 inflammasome proteins and the secretion of IL-18 and IL-1β.And PD98059(ERK inhibitor),SP600125(JNK inhibitor)and SB203580(P38 inhibitor)can significantly inhibited the expression of NF-κB-related pathway proteins and NLRP3 inflammasome proteins and the secretion of IL-18,IL-1β,but it could not inhibit ROS production.BAY11-7082(NF-κB inhibitor)could significantly inhibit the expression of NLRP3 inflammasome proteins and the secretion of IL-18 and IL-1β,but could not inhibit the production of ROS and the expression of MAPKs-related pathway proteins.Ac-YVAD-cmk(Caspase-1 inhibitor)was no inhibitory effect on other pathways.In summary,HG induces osteoclast inflammation by inducing osteoclast ROS production and activating the MAPKs/NF-κB/NLRP3 inflammation pathway.And ROS production occurs upstream of MAPKs,NF-κB and NLRP3;MAPKs are upstream of NF-κB and NLRP3;and NF-κB is upstream of NLRP3 inflammasome.Second,internationally,we are the first to clarify that the activation of osteoclast inflammation and the inhibition of efferocytosis could enhance the bone resorption of osteoclasts,which are the main pathogenic factors of diabetic osteoporosis.We used flow cytometry to detect the occurrence of osteoclast efferocytosis.ELISA was used to detect the expression of LXA 4.Western blot technology was used to detect the expression level of efferocytosis-related protein MertK.And the relationship between the HG-induced osteoclast inflammation and efferocytosis was observed by pretreated with the Ac-YVAD-cmk.Scanning electron microscopy was used to observe the bone resorption capacity of osteoclasts,and the relationship between the efferocytosis,inflammation and bone resorption was observed through the Ac-YVAD-cmk,insulin and LXA 4,silencing the NLRP3 gene.The results showed that HG could significantly inhibited the occurrence of osteoclast efferocytosis.And the HG significantly inhibited the secretion of the LXA4 and expression of osteoclast MertK protein,and Ac-YVAD-cmk could significantly reversed the inhibited the effect of HG-induced.And HG could induce the enhancement of osteoclast bone resorption capacity,but Ac-YVAD-cmk,insulin and LXA 4,silencing the NLRP3 gene could significantly inhibit HG-induced osteoclast bone resorption capacity.To sum up,HG could enhance osteoclast bone resorption capacity by activating NLRP3 inflammasome mediated inflammation and inhibiting osteoclast efferocytosis.Third,we established a diabetic osteoporosis rat model by streptozotocin(STZ)induction,and verified that the osteoclast inflammatory response and inhibition of efferocytosis are the mechanisms of diabetic osteoporosis in vivo.And we are the first time to find that LXA 4,an efferocytosis enhancer,could be used as a potential drug in the treatment of diabetic osteoporosis at home and abroad.ELISA was used to detect the osteoclasts and bone tissue markers in the model rats.Detection of bone density and bone minerals with dual-energy X-ray absorptiometry.HE staining was performed to detect the bone trabeculae and other integrity of bone microstructures in the model rats.TRAP staining was performed to detection of osteoclast content in bone tissue of model rats.And co-localization of cathepsin K and p-ERK,NF-κB,NLRP3 and MertK was detected in bone tissue using laser confocal microscopy.And then western blot and ELISA were used to detect the bone tissue inflammation-related pathway proteins p-ERK,p-p38,p-JNK,nuclear NF-κB,p-IκB,IKK,ASC,Caspase-1,IL-18,IL-1β,NLRP3 and MertK protein and inflammatory cytokines IL-18,IL-1β and TNF-α.The results showed that the contents of TRACP-5b,cathepsin K,and deoxypyridoline in urine were significantly increased in the STZ-induced group,while the levels of insulin and osteocalcin were significantly reduced.And the bone mineral density and bone mineral salt content were significantly reduced.And the pathological tissue section showed that the bone trabeculae in the bone tissue were more discrete and the number reduced and irregularly arranged,and the osteoclast content was significantly increased in bone tissue of rats in the STZ-induced group.Osteoclasts in the STZ-induced group can co-localize with inflammatory pathway-related proteins and MertK proteins.The expression of inflammatory pathway-related protein and the secretion of inflammatory cytokines was significantly increase in the tissue sections,but the MertK was decrease in STZ-induced group.While the LXA 4 and insulin treatment group reversed the above phenomenon.In summary,the activation of the NLRP3 inflammation pathway and the inhibition of efferocytosis in osteoclasts to mediate the development of diabetic osteoporosis in rats.Finally,we aimed at clinical samples of diabetic osteoporosis in humans and dogs,and further verified that the osteoclast inflammation and the inhibition of efferocytosis in vivo are the main mechanisms of diabetic osteoporosis.We use the same experimental method as the above STZ-induced diabetic osteoporosis rat model to detect and analyze relevant indicators on clinical samples of diabetic osteoporosis in humans and dogs,respectively.The results showed that the number of osteoclasts in the bone tissue of patients and dogs with diabetic osteoporosis increased significantly,and both the activation of inflammation and the inhibition of efferocytosis could be detected.This result was consistent with the results of the previous experiments in vitro,and consistent with diabetic osteoporosis rat model.In summary,our results indicate that HG activation of ROS/MAPKs/NF-κB/NLRP3 inflammatory pathways and inhibition of efferocytosis to mediate the enhanced of osteoclast bone resorption capacity are the main causes of diabetic osteoporosis.And we found that LXA 4,an efferocytosis,could be used as a potential drug in the treatment of diabetic osteoporosis.This study will lay a foundation for the further study of the pathogenesis of diabetic osteoporosis and the targeted treatment of diabetic osteoporosis in the future.