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

Background:The prevalence of osteoporosis in China’s elderly population over 60 years of age is about36%,with the proportion of female patients being more than two times higher than that of men.The decrease of estrogen level in the body after menopause is the most important cause of osteoporosis in middle-aged and elderly women.Osteoblast-led bone formation is an important positive factor in bone remodeling,and impaired bone formation accelerates bone loss,which in turn aggravates osteoporosis.Recent studies have revealed that G protein-coupled receptor 81（GPR81）is closely related to bone metabolism and may be involved in the regulation of bone remodeling.Lactate is the only endogenous ligand of GPR81 in vivo,and high-intensity interval training（HIIT）can significantly increase blood lactate concentration in vivo.However,it is not clear whether the lactate produced by HIIT can act on GPR81 receptors on the osteoblast membrane through the circulatory system and participate in regulating the process of bone formation.Based on the above study,it is proposed that lactic acid produced by HIIT can act on GPR81 receptors on osteoblast membranes in bone tissues through the circulatory system and activate intracellular downstream signaling pathways to participate in the regulation of bone formation and improve the bone loss caused by estrogen deficiency.Objective:This study focuses on analyzing the effects of HIIT on bone formation in de-ovalized（Ovariectomy,OVX）mice and further reveals the molecular mechanism of lactate produced by HIIT and its receptor GPR81 in the regulation of bone formation by exercise,which provides potential therapeutic targets for the prevention and treatment of osteoporosis and other related bone metabolic diseases.Methods:（1）Experimental animal grouping:（1）8-week-old wild-type female mice were randomly divided into four groups:sham-operated group（S）,OVX group（O）,OVX+HIIT group（OH）,and OVX+lactic acid injection group（OL）;（2）8-week-old Gpr81 knockout(Gpr81^-/-)female mice were randomly divided into four groups:sham-operated group（K-S）,OVX group（K-O）,OVX+HIIT group（K-OH）,and OVX+lactic acid injection group（K-OL）.OVX+HIIT group（K-OH）,and OVX+lactic acid injection group（K-OL）.（2）In vitro cell differentiation experiments:BMSC induced osteogenic differentiation from 8-week-old wild-type female rats,divided into control group（WT）and lactic acid stimulation group（WT+LA）;BMSC induced osteogenic differentiation from 8-week-old Gpr81^-/-female rats,divided into control group（KO）and lactic acid stimulation group（KO+LA）.（3）Gpr81 knockout mouse model:Gpr81gene was knocked out using CRISPR/Cas9 technology to obtain a Gpr81 whole-body knockout mouse model.（4）OVX surgery:8-week-old female mice underwent bilateral de-ovarianization surgery to construct a mouse model of bone loss.（5）Exercise protocol:HIIT protocol was used,i.e.,mice were trained on a 0-degree inclined treadmill for 1 hour with HIIT 5 times/week for 8 weeks.One HIIT consisted of 10 alternating cycles,in which the high-intensity speed was 80%-90%of the maximum speed for 4 min,and the low-intensity speed was 40%-50%of the maximum speed for 2 min.（6）Lactate injection protocol:1 g/kg of sodium L-lactate（concentration of 0.2 g/ml）was injected intraperitoneally,5 times/week for 8 weeks.（7）Blood lactate concentration assay:Blood was taken from the tail vein of mice and the blood lactate concentration was measured using a Scout 4 lactate meter.（8）Bone histomorphometric index measurements:Micro CT was used to detect cortical bone and cancellous bone morphometric indexes（BMD,BV/TV,Tb.N,Tb.Sp）in mice femurs,and 100frames under the growth plate were taken for analysis.（9）Bone strength measurement:three-point mechanical bending test was used to detect the maximum bearing load on the tibia.（10）Induction of BMSC osteogenic differentiation in vitro:BMSC was extracted from the bone marrow cavity of mouse femur for osteogenic directed differentiation after 8 weeks of intervention.（11）In vitro lactate stimulation:BMSC were extracted from 8-week-old wild-type female mice and Gpr81^-/-female mice for osteogenic directed differentiation,and lactate was added during the culture process to compare the osteoblast differentiation function with/without lactate stimulation.（12）Osteoblast differentiation function assay:Osteoblast differentiation function was analyzed by ALP staining,Von Kossa staining and Alizarin Red staining on days 7,14 and 21 of osteogenic differentiation,respectively.（13）Real-time quantitative PCR:Total RNA of osteoblasts was extracted,and the RNA was reversed to c DNA using My Cycler PCR cycler,amplified using Thermo Fisher Scientific PCR instrument,and the amplification curves were viewed and the correspondingΔCT andΔΔCT values of each gene were calculated.（14）WB experiments:Osteoblasts were lysed using RIPA lysis solution,total proteins were extracted,and electrophoresis was performed to transmembrane and detect the expression of target proteins.Results:（1）Body weight was significantly increased in OVX mice（P<0.01）,and both HIIT exercise intervention and lactate injection were able to significantly increase blood lactate concentration in mice（P<0.01）and inhibit the increase in body weight in OVX mice（P<0.01）.（2）Compared with the S group,cancellous bone BMD,BV/TV and Tb.N were significantly lower（P<0.001）and Tb.Sp was significantly higher（P<0.001）in the O group;after 8 weeks of HIIT training,compared with the O group,cancellous bone BMD and BV/TV were significantly higher（P<0.01）,Tb.N was significantly higher（P<0.05）and Tb.Sp was significantly lower（P<0.05）;after 8 weeks of lactate injection,cancellous bone BMD,BV/TV and Tb.N indexes were significantly higher（P<0.05）in the OL group compared with the O group;compared with the K-S group,cancellous bone BMD,BV/TV and Tb.N were significantly lower（P<0.001）and Tb.Sp was significantly higher（P<0.05）in the K-O group;compared with the K-O group Compared with the K-O group,cancellous bone BMD,BV/TV,and Tb.N were significantly higher（P<0.05）and Tb.Sp was significantly lower（P<0.05）in the K-OH group;compared with the K-O group,cancellous bone BMD,BV/TV,Tb.N,and Tb.Sp were not significantly different in the K-OL group.In terms of cancellous bone BMD and BV/TV,the difference between the OH and O groups was higher than that between the K-OH and K-O groups（P<0.05）.（3）Compared with group S,group O showed lighter staining of ALP staining,Von Kossa staining and Alizarin Red staining of osteoblasts,significantly lower m RNA expression ofβ-catenin,c-myc,Runx2 and ALP（P<0.05）,significantly higher m RNA expression of DKK1（P<0.01）,and significantly higher m RNA expression ofβcatenin,OCN protein expression was significantly decreased（P<0.001）and GSK-3βprotein expression was significantly increased（P<0.05）;compared with the O group,the staining of ALP,Von Kossa staining and Alizarin Red staining of osteoblasts in the OH and OL groups were deepened,and GPR81,β-catenin,c myc,Runx2,ALP m RNA expression were significantly higher（P<0.05）,DKK1m RNA expression were significantly lower（P<0.01）,GPR81,β-catenin,OCN protein expression were significantly higher（P<0.05）,GSK-3βprotein expression were significantly lower（P<0.01）.（4）In vitro lactic acid stimulation experiments showed that compared with the WT group,the ALP staining and Von Kossa staining of BMSC-induced differentiated osteoblasts from the KO group were significantly lighter,and the m RNA expression of Runx2 and OSX were significantly lower（P<0.05）;after lactic acid stimulation,compared with the WT group,the ALP staining and Von Kossa staining of the WT+LA group were deepened,m RNA and protein expression ofβ-catenin were elevated（P<0.05）,m RNA expression of DKK1 was significantly reduced（P<0.05）,m RNA expression of Runx2 and OSX were significantly elevated（P<0.05）,and protein expression of OCN was significantly elevated（P<0.05）;after lactic acid stimulation,compared with the KO group,osteoblasts in the KO+LA group There were no significant changes in ALP staining and Von Kossa staining,and no significant differences in the expression ofβ-catenin,DKK1,Runx2,OSX,and OCN.Conclusions:（1）Ovariectomization can increase the weight of mice,and 8-week HIIT exercise and lactic acid injection can inhibit the weight gain of mice induced by ovariectomization.（2）Ovariectomization can decrease bone density and bone mass in mice;Both 8-week HIIT exercise and lactic acid injection inhibited bone loss due to ovariectomization in mice by promoting bone formation.（3）The molecular mechanism of exercise-regulated bone formation may be that the lactic acid produced by HIIT exercise acts on the GPR81 receptor on the membrane of osteoblasts through the circulatory system,activating the intracellular downstream Wnt/β-catenin pathway,promoting osteoblast differentiation,and ultimately regulating the process of bone formation.