The Role And Mechanism Of GPR81 In Exercise-mediated Bone Formation

Research Background:In recent years,the association of G protein-coupled receptors（GPCRs）with bone formation has attracted much attention.Recent studies have found that G protein-coupled receptor 81（GPR81/ HCAR1）is a kind of hydroxyl carboxylic acid receptor widely expressed in tissues and cells and has a variety of physiological functions.Lactic acid is the only endogenous ligand of GPR81/ HCAR1 under known physiological conditions.Studies have shown that high-intensity exercise can stimulate the production of lactic acid in skeletal muscle cells and increase the concentration of lactic acid in the body’s microenvironment（bone marrow cavity）.At present,although studies have shown that exercise can participate in the regulation of bone formation,the specific molecular mechanism remains unclear.In addition,it has not been confirmed whether lactic acid,an exercise metabolite,participates in the process of exercise regulating bone formation.Based on this,we by the high lactic acid threshold（above）and low(below the lactate threshold intensity of exercise intervention,explore GPR81 in mediating role in the process of bone formation and the possible mechanism,so as to treat bone metabolism related disease targets to offer reference to the research and development,at the same time to prevent chronic metabolic bone diseases such as osteoporosis to provide scientific basis for the establishment of exercise prescription.Research Objectives:To explore the new physiological functions of GPR81 through exercise intervention of high（above lactate threshold）and low（below lactate threshold）intensity,and to further reveal the possible molecular mechanism of GPR81 in the process of exercise regulation of bone formation,providing reference for the development of new targets of bone metabolity-related diseases.Research methods:（1）Exercise intervention method: 3 groups of mice,2 groups of exercise intervention group and 1 group of quiet control group.Exercise intervention group,using High（High,above lactate threshold,18m/min）and Low（Low,below lactate threshold,12m/min）intensity treadmill exercise,8 weeks of age mice for 12 weeks（6times/week;60 min/time）exercise intervention;Quiet control group（Ctrl）mice normal diet,no exercise intervention;（2）Detection of femur morphometric indicators:Micro CT instrument（Company: Brock）was used to measure and analyze the morphometric indicators of mouse femur(BV/TV,BMD,Tb.N,Tb.（3）Cell culture experiment: BMSCs from both femur and tibia of mice were taken for primary cell culture and induced to differentiate into osteoblasts to detect the expression of Marker genes and proteins during differentiation;（4）Osteoblast differentiation function experiment: BMSCs were induced to differentiate into osteoblasts by 24-well plates,and the differentiation levels of osteoblasts were analyzed by ALP staining,Von Kossa staining and Alizarin Red S staining on the 7th,14 th and 21 st day of differentiation,respectively.（5）Double standard calcein test: the mice were injected twice with calcein reagent at an interval of 14 days before death.Then the vertebrae were taken and the Osteomeaure instrument was used to analyze the osteogenesis function and mineralysis level.（6）Serum indicators: a portable blood lactate tester was used to detect the serum lactate concentration of mice immediately after exercise;（7）Real-time PCR: Total RNA of osteoblasts was extracted by Trizol extraction method and reversely transcribed into c DNA,which was tested and analyzed by Thermo Fisher Scientific.（8）Wes TM automatic protein expression analysis:Osteoblast cells were lysed with Ripa and total proteins were collected to detect the expression levels of related proteins in osteoblast differentiation and signaling pathways.Research results:（1）The average body weight of mice in the three groups increased slowly.Compared with the Ctrl group,the body weight of mice in the exercise intervention group increased at a slower rate,and the average body weight of mice in the High group was the lowest.In addition,compared with Ctrl group（3.56±0.57mmol/L）,the blood lactic acid concentration of mice in High group（7.41±0.47 mmol/L）was significantly increased（P<0.01）,while that in Low group（4.21±0.70mmol/L）was not significantly different.Compared with Low group,blood lactic acid concentration in High group was significantly increased（P<0.05）.（2）Compared with Ctrl group,the indexes of cancellous bone BMD,BV/TV,Tb.N,Tb.Th of mice in High group were significantly increased（P<0.01）,while the indexes of cancellous bone BMD,BV/TV,Tb.Compared with Low group,the indexes of cancellous bone BMD,BV/TV,Tb.N,Tb.Th in High group were significantly increased（P<0.01）;Compared with Ctrl group,cortical bone BMD,BV/TV,Tb.N,Tb.Th of mice in High and Low groups had no significant difference.Compared with Ctrl group,BFR of mice in High group was significantly increased（P<0.01）,while there was no significant difference in BFR of mice in Low group.Compared with Low group,BFR of mice in High group was significantly increased（P<0.05）;Compared with Ctrl group,MAR of mice in High group was significantly increased（P<0.01）,while MAR of mice in Low group had no significant difference.Compared with Low group,MAR of mice in High group was significantly increased（P<0.01）.（3）Compared with Ctrl group and Low group,ALP staining,Vonkossa staining and Alizarin Red S staining of mice in High group were significantly deepened.In addition,compared with Ctrl group,the expression levels of ALP mRNA and OCN protein in High group were significantly increased（P<0.01）.There was no significant difference in ALP mRNA and OCN protein expression in Low group.Compared with Low group,the expression levels of ALP mRNA and OCN protein in High group were significantly increased（P<0.05）.（4）Compared with Ctrl group,the mRNA expression levels of GPR81,PKC,Akt,Osterix and Runx2 in High group were significantly increased（P<0.05）.There were no significant differences in GPR81 mRNA,PKC mRNA,Akt mRNA,Osterix mRNA and Runx2 mRNA expression levels in Low group.Compared with Low group,the expression levels of GPR81 mRNA,PKC mRNA,Akt mRNA and Osterix mRNA in High group were significantly increased（P<0.05）.In addition,compared with Ctrl group,the expression levels of GPR81 protein and P-Akt/ Akt and P-CREb/CREB protein in High group were significantly increased（P<0.05）,while the expression levels of GPR81 protein and P-Akt/ Akt and P-CREb /CREB protein in Low group were not significantly different.Compared with Low group,the protein expression of GPR81 in High group was significantly increased（P<0.05）.Research Conclusions:（1）Exercise higher than lactate threshold intensity can significantly increase the blood lactate concentration of mice,exercise can inhibit the weight gain of mice,high intensity exercise is significantly higher than low intensity exercise.（2）Exercise above lactate threshold increased cancellous bone mass and bone mineral density and promoted bone formation in mice,while exercise below lactate threshold had no significant effect on bone mass,bone mineral density and bone formation in mice.（3）Exercise higher than lactate threshold intensity can promote the expression of ALP,OCN and other specific markers of osteogenic differentiation and promote the differentiation of BMSCs into osteoblasts,while exercise lower than lactate threshold intensity has no significant effect on the differentiation process of osteoblasts.（4）Exercise above lactate threshold intensity may activate lactate receptor GPR81 on osteoblastic membrane by increasing lactic acid concentration in the body,and promote osteoblastic differentiation and bone formation through intracellular downstream PKC-Akt-CREB signaling pathway,thus improving bone mass and increasing bone density.