The Study On The Relevance Of Endocannabinoid Receptor 2 To Bone Abnormalities In Eldly Population And Its Effect And Mechanism To Osteoclast Formation

Objective:In this study,we analyzed the relationship between cannabinoids receptor 2of the endocannabinoids system and bone abnormalities in the elderly population,and explored the mechanism of cannabinoids receptor 2 to the process of osteoclast formation,in order to provide new targets for the further treatment of osteoporosis in the elderly population.Methods:1.Clinical data collection:All 255 subjects of the study were randomly selected from the Geriatrics department of The First Affiliated Hospital of China Medical University.Registration requirements for the subjects were as follows:≥60years and no history of disease as follows:acute cardiovascular diseases,mental and psychological diseases,rheumatic immune system diseases,blood diseases,and malignant tumors which can cause secondary osteoporosis.Bone mineral density（BMD）was measured by dual-energy X-ray absorptiometry（DXA）,and the subjects were divided into normal BMD group（T≥-1.0）and abnormal BMD group（T<-1.0）according to T score of the Lumbar spine BMD.Blood samples were collected after all subjects had fasted for 6-8h,and then sent to Inspection Center of The First Affiliated Hospital of China Medical University for analysis included the following:albumin,prealbumin,alkaline phosphatase,serum calcium,serum phosphorus,low density lipoprotein cholesterol,total cholesterol,total triglyceride,serum uric acid and markers of bone metabolism.Meanwhile,23 subjects and 9 subjects were selected according to the above criteria.Bone mineral density（BMD）was measured by dual-energy X-ray absorptiometry（DXA）,and the subjects were divided into normal BMD group（T≥-1.0）,osteopenia group（-2.5<T<-1.0）and osteoporosis group（T≤2.5）according to T score of the Lumbar spine BMD.Blood samples were collected after all subjects had fasted for 6-8h,and then peripheral blood mononuclear cell（PBMC）extraction was performed.Reverse transcription-PCR（RT-PCR）and WB were used to observe the expression of cannabinoid receptor 2 of PBMC,and its relationship with LBMD was analyzed.2.Experimental cells and process:（1）Mouse monocyte RAW264.7 was used in this research,which was induced by receptor activator of the Nfk B ligand（RANKL）to establish osteoclast differentiation model.CB₂ receptor agonist AM1241and CB₂ receptor antagonist AM630 were used in the differentiation process,and tartrate-resistant acid phosphatase（TRAP）staining and TRAP activity assay were performed.Western blotting was performed in order to detect the protein expressions of nuclear factor E2-related factor 2（Nrf₂）and heme oxygenase-1（HO-1）.（2）RAW264.7 cells were transfected with si-Nrf₂ lentiviral vector,and then induced into osteoclast.AM1241 and AM630 were used to intervene the differentiation process.TRAP staining and TRAP activity assay were performed,and western blotting was performed in order to detect the protein expressions of Nrf₂ and HO-1.3.Statistical analysis:All statistical analyses of the clinical data were made by SPSS version 22.0.According to different types of data distribution,T-test,Kruskal Wallis rank test,Pearson’s X² test,Fisher’s exact test or one-way analysis of variance（ANOVA）were used as appropriate.Continuous variables were characterized as mean±standard deviation（（?）±S）or median（inter-quartile range）,while categorical or ranked data were presented as count and proportion.The correlation between LBMD and other variables was analyzed by Pearson correlation analysis.Multiple regression analysis was adopted using the LBMD as the dependent variable,and bone metabolism markers were used as the independent variable.Meanwhile,the subjects were divided into quartiles（1-4）ofβ-Crosslaps,Osteo C and T-P1NP,with quartile 1 presented the lowest levels ofβ-Crosslaps,Osteo C and T-PINP.Binary logistic regression was performed using LBMD as the dependent variable,and quartiles ofβ-Crosslaps,Osteo C and T-P1NP as the independent variable to analyze associations between them.Two models for linear trend were further used.Model 1 was not adjusted for any variable,and Model 2 was adjusted for age,sex,BMI,ALB,PAB,TC,LDL-C and SUA.Multiple regression analysis was adopted using the LBMD as the dependent variable,and CB₂R-m RNA content of PBMC was used as the independent variable.P value less than 0.05 was regarded to be statistically significant.Results of the cells were denoted by mean±SEM from 3 separate determinations.We analyzed data by Graph Pad Prism-5 package.One-way ANOVA along with Dunnett’s Multiple comparison Test were employed to test significances.P value less than 0.05 was regarded to be statistically significant.Results:（1）In the 255 subjects,the proportion of female increased,while BMI、PAB、SUA、TG decreased with the decline of LBMD,and the comparison between groups was statistically significant（P<0.05）.PAB（R=0.14,P<0.05）and SUA（R=0.29,P<0.01）were positive correlation with LBMD.Serum calcium（R=-0.15,P<0.05）and TG（R=-0.16,P<0.05）were negative correlation with LBMD.β-Crosslaps,Osteo C and T-PINP were lower in normal LBMD groups,and the comparison between groups was statistically significant（P<0.05）.β-Crosslaps（R=-0.22,P<0.01）,Osteo C（R=-0.22,P<0.01）and T-P1NP（R=-0.13,P<0.05）were negative correlation with LBMD.When multiple linear regression analysis using LBMD decline as the dependent variable and bone metabolic marker as the independent variable,β-Crosslaps、Osteo C and T-P1NP had significantly correlation with LBMD.After adjustment for confounding variables including age,sex,BMI,ALB,PAB,TC,LDL-C and SUA,β-Crosslaps was still significantly correlated LBMD（P<0.05）,and the correlation of Osteo C and T-P1NP with LBMD disappeared.When binary logistic regression analysis using LBMD decline as the dependent variable and quartiles ofβ-Crosslaps as the independent variable,it was found that the odds ratios values of quartile 2 group、3 group and 4 group were 0.64（95%CI 0.30-1.36,P=0.254）、1.10（95%CI 0.53-2.26,P=0.801）and 2.78（95%CI 1.37-5.76,P=0.005）comparing with the quartile 1 group,with P=0.002 for the trend.After adjustment for confounding variables including age,sex,BMI,ALB,PAB,TC,LDL-C and SUA,the odds ratios values of quartile 4 group was 2.35（95%CI 1.02-5.52,P=0.047）.When binary logistic regression analysis using LBMD decline as the dependent variable and quartiles of Osteo C or T-P1NP as the independent variables respectively,it was found that the odds ratios values of Osteo C quartile 2 group、3 group and 4 group were 1.75（95%CI 0.85-3.64,P=0.132）、1.16（95%CI 0.54-2.48,P=0.709）and 3.05（95%CI 1.48-6.41,P=0.003）comparing with the Osteo C quartile 1 group,with P=0.011 for the trend.After adjustment for confounding variables including age,sex,BMI,ALB,PAB,TC,LDL-C and SUA,the correlation between Osteo C and LBMD disappeared.Whether the variable was adjusted or not,no correlation between T-P1NP and LBMD was found.（2）In the 23 subjects,CB₂R-m RNA level of PBMC extracted from peripheral blood was down-regulated in both osteopenia group and osteoporosis group compared with the normal group,and the comparison among three groups was statistically significant（P<0.05）.CB₂R-m RNA level（R=0.675,P<0.05）was significantly positive correlation with LBMD.Multiple linear regression analysis showed that CB₂R-m RNA level was significantly correlated with LBMD（P<0.05）.The correlation between CB₂R-m RNA level and LBMD was not affected after adjustment for confounding variables including age and gender.In the 9 subjects,CB₂R protein level of PBMC extracted from peripheral blood was also down-regulated in both osteopenia group and osteoporosis group compared with the normal group.（3）RAW264.7 cells were induced by RANKL for 5 days.In RANKL group,the number of osteoclasts and TRAP activity significantly increased comparing with the control group（P<0.05）.In AM1241+RANKL group,the number of osteoclasts and TRAP activity increased comparing with RANKL group and the normal group（P<0.05）.In AM630+RANKL group,the number of osteoclasts and TRAP activity increased comparing with the normal group（P<0.05）,while the number of osteoclasts and TRAP activity decreased comparing with RANKL group（P<0.05）.Western blotting showed that the expression of Nrf₂ and HO-1 were significantly increased in AM1241+RANKL group and AM630+RANKL group（P<0.05）.si Nrf₂-RNA lentiviral plasmid was constructed to intervene the above process.Western blotting confirmed that the expression levels of Nrf₂ and HO-1 in si Nrf₂ groups were lower than those in si RNA groups,and the expression levels of Nrf₂ and HO-1 were significantly lower after AM1241 and AM630 intervention（P<0.05）.In si Nrf₂ groups,the number of osteoclasts and TRAP activity increased than that in si RNA groups.In si Nrf₂+RANKL+AM630 group,the number of osteoclasts and TRAP activity increased comparing with si RNA+RANKL+AM630 group（P<0.05）.The inhibiting effect of AM630 on RANKL-induced osteoclast differentiation of RAW264.7 cells disappeared.Conclusion:In the elderly population,osteoporosis is associated with female,BMI,PAB,SUA andβ-Crosslaps of bone metabolism markers,as well as altered CB₂R expression.Intervention of CB₂ receptor could affect the RANKL-induced osteoclast differentiation of RAW264.7 cells.AM630 could inhibit the effect of RANKL-induced osteoclast differentiation of RAW264.7 cells,and its mechanism may be related to the redox reaction mediated by Nrf₂.CB₂ receptor related bone mechanism may become a new target for the treatment of osteoporosis in the elderly population.