Mechanism Of GLP-1RA Liraglutide Regulating Bone Resorption In Diabetic Osteoporosis Through GLP-1R

With the change of lifestyle,diabetes mellitus(DM)has gradually become the most common endocrine and metabolic disorder.According to the report of the International Diabetes Federation(IDF),the total number of patients with diabetes in the world will reach 463 million in 2019,and this number will reach 720 million by 2045.Diabetes has become the seventh most fatal disease in the world.Many complications of diabetes seriously affect the health and quality of life of patients.In recent years,the research on diabetic cardiovascular disease,diabetic retinopathy and diabetic nephropathy is gradually deepening,while the research on diabetic osteoporosis,a kind of "hidden" complications,is still relatively weak.Osteoporosis(OP)is a systemic metabolic bone disease characterized by destruction of bone microstructure,damage of bone trabeculae,decrease of bone mineral density,increase of bone fragility and easy fracture.Osteoporosis and diabetes are often considered as two separate diseases,but in fact they are closely related.As early as 1927,Morrison et al.Found that with the extension of diabetes,children's bone growth will be more and more slow,and even cause bone atrophy.In 1948,the concept of "diabetic osteoporosis"(DOP)was first mentioned.Diabetes mellitus is an independent risk factor of osteoporosis.It can directly or indirectly affect bone remodeling and bone strength through the changes of hormone levels such as hyperglycemia,oxidative stress,insulin level and parathyroid hormone(PTH)balance.In addition,diabetic patients with diabetic retinopathy and other factors lead to increased risk of fracture.Once the fracture,the quality of life of patients will be further reduced,which will bring serious burden to patients,families and society.Although diabetic complications of diabetic osteoporosis are not widely known,the incidence rate is very impressive.It has been reported that the incidence rate of osteoporosis complications in DM patients is 40% to60%,and this data increases with the duration of diabetes.Therefore,it is necessary to further explore the pathogenesis and possible treatment strategies of diabetic osteoporosis,so as to provide the basis for the clinical management of diabetic osteoporosis.Osteoclast bone resorption and osteoblast bone formation are the key processes of bone reconstruction,which run through the whole life process.In short,this process of bone reconstruction can be divided into three basic links.First,osteoclast differentiation begins the process of bone resorption.Next,osteoclast apoptosis is accompanied by osteoblast aggregation and differentiation in the bone lacuna.Finally,osteoid formation and mineralization completely repair bone resorption.In healthy state,bone resorption and bone formation maintain dynamic balance and maintain bone integrity.Once the balance between them is broken,a variety of bone metabolic diseases such as osteoporosis will occur.In diabetic patients,bone resorption was enhanced,bone formation was decreased,and bone remodeling was unbalanced.Diabetic hyperglycemia inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and induces the apoptosis of osteoblasts through a variety of mechanisms;advanced glycation end products(AGES)formed by hyperglycemia promote the synthesis and secretion of bone resorption factors TNF-? and IL-6,It can increase the expression of RANKL,TNF-?,VEGF-A and M-CSF,enhance the activity of osteoclasts and promote bone resorption.In addition,metabolic abnormalities in diabetic patients can lead to increased parathyroid hormone(PTH)levels and insufficient insulin secretion,further promoting bone resorption and bone loss.With the development of gut bone brain axis research,the effect of incretin on extrapancreatic bone tissue has attracted much attention.Liraglutide,a new hypoglycemic drug,plays a multi-target role by binding to GLP-1R,which is widely expressed in many tissues and organs.GLP-1RA plays an important role in maintaining bone health.Animal studies show that GLP-1RA liraglutide and exendin-4 can reduce the serum levels of tartrate resistant acid phosphatase(TRACP)and type I collagen(CTX-1)in ovariectomized rats,reduce the number of osteoclasts,increase bone mineral density,and have bone protective effect.At the cellular level,GLP-1RA can promote the osteogenic differentiation of BMSCs.In addition,GLP-1RA up regulates the expression of GLP-1R in MC3T3-E1 cells,activates PI3K-Akt,Camp-PKA,MEK-ERK and Hedgehog/Gli1 signaling pathways to promote the differentiation of MC3T3-E1 cells into osteoblasts.However,there are few studies on the molecular level of the effect of GLP-1RA on osteoclasts.Some studies have shown that GLP-1 can inhibit osteoclast differentiation,but the mechanism may be through binding with GLP-1 receptor on thyroid C cells,promoting the secretion of calcitonin by thyroid C cells,indirectly inhibiting bone resorption.GLP-1RA works by binding to GLP-1R on cells.However,it is still controversial whether GLP-1R is expressed on osteoclasts,and the mechanism of GLP-1RA liraglutide on bone resorption of osteoclasts is less studied.NFATc1(nuclear factor of activated T cells type C1,NFATc1)is a key factor for osteoclast gene transcription.It regulates the expression of osteoclast specific genes c-fos,cathepsin K,TRACP under the action of NF-? B,Ca2 +,MAPK and other signaling pathways.Studies have shown that GLP-1RA can inhibit bone resorption by inhibiting LPS induced TNF-?expression,and TNF-? can activate NF-?B signaling pathway to promote osteoclast differentiation and regulate bone remodeling.TNF-? can regulate the polarization of monocyte macrophages and affect bone resorption.Whether GLP-1RA liraglutide can directly affect NF-?B and MAPK signaling pathways to affect osteoclast differentiation remains to be further explored.Compared with other types of osteoporosis,the pathogenesis of diabetic osteoporosis is more complex.Oxidative stress mediated by reactive oxygen species(ROS)plays an important role in this process.Under oxidative stress,the increase of ROS destroys the balance between oxidative and antioxidant systems,thus inhibiting bone formation and inducing osteoblast apoptosis,resulting in imbalance of bone remodeling.However,the effect of high glucose on the function of osteoclasts is still controversial.Some studies have found that high glucose promotes the differentiation of osteoclasts,but some scholars believe that the differentiation of osteoclasts is inhibited and the bone resorption function of osteoclasts is damaged under high glucose environment.It has been reported that GLP-1RAcan improve the oxidative stress state of cardiovascular system under high glucose.Can liraglutide change the differentiation and apoptosis of osteoclasts under high glucose by improving the ROS content in osteoclasts?In conclusion,this study aims to elucidate the potential molecular mechanism of glp-1ra liraglutide in regulating osteoclast differentiation at both cellular and animal levels,which is of great significance to provide a new basis for clinical management of diabetic osteoporosis.Part One GLP-1RA liraglutide regulates osteoclast differentiation through GLP-1R-NF-?B/ MAPK signaling pathwayObjectives:To verify the expression of GLP-1R in primary BMMs cells and RAW264.7 cells,and to observe the effect of different concentrations of liraglutide on osteoclastic differentiation of mouse osteoclasts as well as the mechanism.Methods:1.The m RNA and protein levels of GLP-1R in primary BMMs and RAW264.7 cells were detected by RT-PCR and Western blot analysis,and the expression of GLP-1R in primary BMMs and RAW264.7 cells was qualitatively analyzed by immunofluorescence.2.Primary BMMs and RAW264.7 cells were induced to osteoclastic differentiation by 30 ng / L M-CSF combined with 50 ng / L RANKL and 50ng/L RANKL,respectively.The cells were treated with different concentrations of liraglutide(0,10,100,1000 n M)respectively.The cells were collected on the fourth day after the intervention,and the specific differentiation markers of osteoclasts were detected by fluorescent quantitative PCR The m RNA levels of TRACP,NFATc1 and cathepsin K were detected;the osteoclasts in each group were stained and counted by trap staining method;the activity of trap in each group was detected.3.Western blot analysis was used to determine the phosphorylation levels of MAPKs and NF-? B signaling pathway at 20 min to determine the inhibition of liraglutide on MAPKs and NF-? B signaling pathway.4.Small interfering RNA(si RNA)was used to silence GLP-1R.The effects of GLP-1R silencing on the phosphorylation of MAPKs and NF-? B signaling pathway induced by liraglutide were detected.Results:1.GLP-1R exists in primary BMMs cells and RAW264.7 cells.RT-PCR results showed that GLP-1R specific bands were found in mouse primary bmms and RAW264.7 cells at 111 BP.Western blot analysis and immunofluorescence detection further confirmed that mouse primary BMMs cells and RAW264.7 monocyte macrophages expressed GLP-1R protein.2.The changes of osteoclast differentiation marker genes TRACP,NFATc1 and cathepsin K m RNA indicated that mouse primary BMMs cells and monocyte macrophage RAW264.7 experienced a specific osteoclast differentiation and maturation process under the action of osteoclast inducer.Liraglutide significantly inhibited the m RNA expression of TRACP,NFATc1 and cathepsin K.Trap staining showed that liraglutide reduced the number of mature osteoclasts and the activity of TRAP.3.Liraglutide down regulates the phosphorylation of NF-?B as well as p38 and JNK in MAPKs signaling pathways,indicating that NF-?B and MAPKs signaling pathways are involved in the process of liraglutide on osteoclastic differentiation.4.After silencing GLP-1R by si RNA,liraglutide significantly decreased the inhibition of NF-?B and MAPKs signaling pathways,indicating that liraglutide regulates downstream signaling pathways through GLP-1R.Part Two GLP-1RA liraglutide affects osteoclast differentiation and apoptosis by regulating ROS in high glucose environmentObjectives:To investigate the effect of liraglutide on the differentiation and apoptosis of osteoclasts in high glucose environment and its mechanism..Methods:1.RAW264.7 cells were cultured in 5.5 m M,15 m M,25 m M and 35 m M high glucose environment respectively.The cell activity was detected by CCK8 method at 1,2,3 and 4 days during culture.RAW264.7 cells were induced to differentiate into osteoclasts under 5.5 m M,15 m M,25 m M and35 m M high glucose medium.The m RNA levels of TRACP,NFATc1 and cathepsin K were analyzed by real-time RT-PCR;2.RAW264.7 cells were cultured in 5.5 mm,15 mm,25 mm and 35 mm high glucose environment respectively,and the content of ROS in each group was detected by ROS kit.The apoptosis of RAW264.7 cells was detected by Hoechst 33258 staining and flow cytometry.3.Different concentrations of liraglutide(0,100,1000 n M)were used to interfere with RAW264.7 cells cultured in high glucose.The content of ROS in each group was detected by ROS kit,and the apoptosis of each group was analyzed by flow cytometry.4.Liraglutide was used to intervene the osteoclastic differentiation of RAW264.7 cells under the condition of high glucose.To determine the inhibition of liraglutide on osteoclastic differentiation and MAPKs and NF-?B signaling pathway under high glucose,the m RNA levels of NFATc1 and cathepsin K were detected by RT-PCR,and the phosphorylation levels of MAPKs and NF-?B in 20 min were determined by Western blot analysis.5.Small interfering RNA(si RNA)was used to silence GLP-1R in high glucose environment.The effect of silencing GLP-1R on the content of reactive oxygen species(ROS)in liraglutide treated cells was detected..Results:1.15 m M glucose increased the activity of RAW264.7 cells,35 m M glucose inhibited the activity of RAW264.7 cells.15 m M glucose increased the m RNA expression of osteoclast differentiation marker genes TRACP,NFATc1 and cathepsin K,while 25 m M and 35 m M glucose inhibited the m RNA expression of osteoclast differentiation marker genes TRACP,NFATc1 and cathepsin K.2.Glucose increased ROS content and promoted apoptosis of RAW264.7cells in a dose-dependent manner.3.Liraglutide can inhibit the increase of ROS and apoptosis induced by high glucose,and the effect of high dose liraglutide is better.4.Liraglutide decreased the m RNA expression of TRACP,NFATc1 and cathepsin K,and decreased the phosphorylation of MAPKs and NF-?B signaling pathway.5.After silencing GLP-1R by si RNA,liraglutide significantly decreased the inhibition of NF-?B and MAPKs signaling pathways,indicating that liraglutide regulates downstream signaling pathways through GLP-1R.Part Three GLP-1RA liraglutide regulates osteoclastic differentiation in diabetic osteoporosis miceObjectives:To investigate the effect of glp-1ra liraglutide on bone resorption in diabetic osteoporosis mice.Methods:1.Diabetic osteoporosis model was established in mice,and liraglutide was used for intervention to verify the changes of blood glucose,insulin,blood calcium,blood phosphorus and femur weight among the groups.2.The levels of TRACP-5b and CTX-1 in diabetic osteoporosis mice and liraglutide treated mice were detected by ELISA.3.HE staining and trap staining were used to detect the changes of bone microstructure and the number of osteoclasts in diabetic osteoporosis mice and liraglutide treated mice.4.The contents of GLP-1R and ROS in bone tissue of diabetic osteoporosis mice and liraglutide treated mice were detected by immunohistochemistry and fluorescence.Results:1.The diabetic osteoporosis mouse model was successfully established.Liraglutide intervention can reduce blood glucose,increase insulin,and increase femoral mass in diabetic osteoporosis mice,but has no significant effect on blood calcium and phosphorus.2.The levels of TRACP-5b and CTX-1 in blood of diabetic osteoporosis mice increased,and liraglutide intervention could significantly reduce the levels of TRACP-5b and CTX-1.3.Compared with the normal control group,the bone structure of diabetic osteoporosis mice was disordered,trabecular bone decreased,the content of bone parenchyma decreased,and the number of osteoclasts were increased.Liraglutide intervention can reduce the number of osteoclasts in diabetic state and improve the bone microstructure.4.Liraglutide can increase the expression of GLP-1R and inhibit the content of ROS in bone tissue.Conclusions:1.Liraglutide inhibits osteoclast differentiation by regulating NF-?B and MAPKs signaling pathways through GLP-1R.2.Liraglutide can inhibit apoptosis and osteoclastic differentiation by inhibiting ROS in RAW264.7 cells under high glucose condition.3.Liraglutide can increase the level of GLP-1R in bone tissue to inhibit the content of ROS in bone tissue,inhibit the number of osteoclasts and improve bone microstructure.