Effect And Mechanism Of GPR81 On Bone Resorption In OVX Mice Induced By Exercise

Background: Osteoporosis is a systemic bone metabolic disease characterized by a decrease in bone mass and microstructural deterioration of bone tissue.In the population of patients with osteoporosis,females account for up to 70%.The main factor causing osteoporosis in women is the decrease in estrogen levels caused by menopause.It is generally believed that regular exercise can improve bone remodeling processes and increase bone density and mass in the body.Osteoclast-mediated bone resorption is an important link in the process of bone remodeling and plays a key role in regulating bone mass.However,the specific mechanism by which exercise regulates osteoclast-mediated bone resorption is currently unclear.Studies have found that G protein-coupled receptor 81(GPR81)plays an important role in regulating osteoclast differentiation and function,and lactate is the only endogenous ligand of GPR81.Therefore,our research hypothesis proposes that lactate produced by exercise acts on the GPR81 receptor on the membrane of osteoclasts through the circulatory system,thereby regulating the function and bone resorption process of osteoclasts,improving bone remodeling,and preventing and treating osteoporosis induced by decreased estrogen levels.Research purposes: This study established a Gpr81 gene knockout mouse model and an ovariectomy(OVX)-induced bone loss mouse model.High-intensity interval training(HIIT)was used as the exercise intervention program to produce lactate.Through in vivo tissue and in vitro cell experiments,the role of exercise in regulating bone resorption in bone loss mice was analyzed.Furthermore,potential molecular mechanisms were revealed to provide potential therapeutic targets for the prevention and treatment of bone metabolism-related diseases.Research methods:(1)Experimental groups: The experimental mice were divided into 8 groups:WT+Sham(wild type + sham surgery),WT+OVX(wild type + ovariectomy),WT+OVX+HIIT(wild type + ovariectomy + high-intensity interval training),WT+OVX+Lac(wild type + ovariectomy + lactate injection),Gpr81-KO+Sham(Gpr81 knockout + sham surgery),Gpr81-KO+OVX(Gpr81 knockout + ovariectomy),Gpr81-KO+OVX+HIIT(Gpr81 knockout + ovariectomy + high-intensity interval training),Gpr81-KO+OVX+Lac(Gpr81 knockout + ovariectomy + lactate injection);(2)Gpr81 knockout mouse model: The CRISPR/Cas9 technology was used to knock out the Gpr81 gene,and the Gpr81-KO mouse model was obtained;(3)Construction of bone loss mouse model: Bilateral ovariectomy was performed on 8-week-old female mice,and at the 10 th week after ovariectomy,the bone density of the mice was reduced by about 50%,reaching the modeling standard of a 20% reduction;(4)Exercise intervention method: HIIT intervention was performed 5 times a week,2 hours each time.Each exercise intervention consisted of 10 cycles of high-intensity exercise at 80%~90% of maximum speed,lasting 4 minutes each time,with an interval exercise of 2 minutes at 40%~50% of maximum speed;(5)Exogenous lactate injection: Sodium L-lactate was injected into the peritoneal cavity at a dose of 1 g/kg according to the time and frequency of exercise intervention,and the blood lactate concentration was ≥5 mmol/L;(6)Bone tissue morphometric index: bone mass,bone density,and bone trabeculae were measured by taking fresh femurs from mice and using Micro-CT for detection,and 100 frames under the growth plate were analyzed using CTAnalysis software;(7)Osteoclast differentiation function experiment: Bone marrow-derived macrophages(BMMs)were inoculated in a 96-well plate at a density of 1 × 10^4 cells per well and induced to differentiate into osteoclasts by adding M-CSF and RANKL at final concentrations of 10 ng/ml and 50 ng/ml,respectively,in α-MEM medium containing 10% FBS.After changing the medium the next day,the osteoclast differentiation function was analyzed by TRAP staining after 7 days of directional differentiation;(8)Protein extraction and WB: After treatment under different conditions,the osteoclasts were washed with PBS and added to RIPA lysis buffer.The cell lysate was collected and the protein was extracted using a kit.After determining the protein concentration using the BCA method,SDS-PAGE protein electrophoresis was performed and transferred to a PVDF membrane.After blocking,the antibodies were incubated,and then incubated with a blocking solution containing HRP-labeled Ig G.ECL ultra-sensitive luminescence was used for imaging,and X-ray film was exposed.Results: 1)Compared with the WT+Sham group,the body weight of mice in the WT+OVX group significantly increased(P<0.01);compared with WT+OVX mice,the body weight of mice in both the WT+OVX+Lac and WT+OVX+HIIT groups significantly decreased(P<0.01).In addition,the blood lactate levels of mice in the WT+OVX+HIIT and WT+OVX+Lac groups were significantly higher than those in the WT+OVX group(P<0.01).2)In wild-type mice,compared to the WT+Sham group,the trabecular bone morphometric parameters of the WT+OVX group were significantly reduced,including BMD(P<0.01),BV/TV(P<0.01),TB.N(P<0.01),and Tb.Th(P<0.05).Compared to the WT+OVX group,the trabecular bone morphometric parameters of the WT+OVX+HIIT group were significantly increased,including BMD(P<0.01),BV/TV(P<0.01),and TB.N(P<0.05).In addition,there was no significant change in cortical bone morphometric parameters among the WT+Sham,WT+OVX,WT+OVX+Lac,and WT+OVX+HIIT groups of mice.3)Compared with Gpr81-KO+Sham group,the trabecular bone morphometric parameters of Gpr81-KO+OVX group mice were significantly decreased,including BMD(P<0.01),BV/TV(P<0.01),and TB.N(P<0.01).Compared with Gpr81-KO+OVX group,the BMD and BV/TV levels of trabecular bone in Gpr81-KO+OVX+HIIT group mice were significantly increased(P<0.05).In addition,there were no significant changes in cortical bone morphometric parameters among the four groups including Gpr81-KO+Sham,Gpr81-KO+OVX,Gpr81-KO+OVX+HIIT,and Gpr81-KO+OVX+Lac.4)In in vitro experiments,it was found that compared to the WT+Sham group,osteoclast activity was enhanced in the WT+OVX group,while the osteoclast activity in the WT+OVX+HIIT group was significantly reduced compared to the WT+OVX group.In TRAP staining experiments on bone tissue slices of gene knockout mice,it was found that HIIT and lactate injection could significantly inhibit the enhanced osteoclast activity caused by OVX surgery.5)In vitro cell experiments showed that compared to WT+Sham,the number and area of BMMs from WT+OVX group that were directionally induced to differentiate into osteoclasts significantly increased(P<0.01).Compared to WT+OVX group,the number of osteoclasts from WT+OVX+HIIT group decreased significantly(P<0.01).In addition,the number of BMMs from WT mice that were directionally induced to differentiate into osteoclasts significantly decreased after lactate stimulation(P<0.01),while the number of osteoclasts differentiated from BMMs from KO mice did not show a significant change.6)Molecular mechanism: Compared to the WT+Sham group,there was no significant difference in the expression of GPR81 in osteoclasts from WT+OVX mice.Compared to the WT+OVX group,the expression of GPR81 was significantly increased(P<0.01)in osteoclasts differentiated from WT+OVX+HIIT and WT+OVX+Lac mice.Compared to the WT+Sham group,the expression of NF-k B(pp65)was significantly increased(P<0.01)in osteoclasts differentiated from WT+OVX mice.Compared to the WT+OVX group,the expression of NF-k B(p-p65)was significantly decreased(P<0.01)in osteoclasts differentiated from WT+OVX+HIIT and WT+OVX+Lac mice.There was no significant difference in the expression of NFk B(p-p65)in osteoclasts differentiated from Gpr81-KO+OVX+Lac mice compared to Gpr81-KO+OVX+HIIT mice.Compared to the WT+Sham group,the expression level of Tak1 was significantly decreased(P<0.05)in osteoclasts differentiated from WT+OVX mice.Compared to the WT+OVX group,the expression of Tak1 protein was significantly increased(P<0.01)in osteoclasts differentiated from WT+OVX+HIIT and WT+OVX+Lac mice.There was no significant difference in Tak1 expression in osteoclasts differentiated from Gpr81-KO+OVX+Lac mice compared to Gpr81-KO+OVX+HIIT mice.Conclusions:(1)OVX can increase the weight of mice and cause bone loss,while HIIT exercise can increase the level of lactate in mice and inhibit weight gain and bone loss caused by OVX.(2)8 weeks of HIIT exercise intervention and in vitro lactate injection can both inhibit osteoclast differentiation,suppress bone resorption,and increase bone density and mass.(3)HIIT exercise can increase the circulating level of lactate,activate the lactate receptor Gpr81 on the membrane of osteoclasts,inhibit the downstream NF-k B signaling pathway in the cells,increase the expression of Tak1 protein,and suppress osteoclast differentiation.