Role And Mechanism Of Irisin/Integrin αVβ5 Pathway In Regulating The Osteogenic/adipogenic Differentiation Of Obese Mice BMSCs By Calorie Restriction And Aerobic Exercise

ObjectiveThis study applied 70%caloric restriction,exercise and caloric restriction combined with exercise to obese mice,as well as recombinant Irisin,mechanical stretch and integrinαVβ5 gene silencing/overexpression to BMSCs,so as to explore the role of Irisin/IntegrinαVβ5 signaling pathway in regμlating osteogenic/adipogenic differentiation of BMSCs,and provide experimental basis for revealing the molecμlar mechanism and reliable theoretical basis for finding effective prevention methods of obesity-induced osteoporosis.Methods and Materials Part one Effects of calorie restriction/aerobic exercise on bone quality and Irisin/IntegrinαVβ5 signaling pathway in obese mice1.Experimental animals and grouping:60 male C57BL/6 mice were randomly divided into normal diet group（ND group,n=10）and high-fat feed group（HFD group,n=50）.After 10 weeks of HFD,the mice with substandard body weight were excluded,and the successfμl obese mice were randomly divided into four groups:obesity control group（OC group,n=10）,obesity calorie restriction group（OR group,n=10）,obesity exercise group（OE group,n=12）and obesity calorie restriction combined with exercise group（ORE group,n=12）.At the same time,the mice in the normal diet group were set as the normal control group（NC group,n=10）.The daily food intake of mice in OR and ORE groups was gradually reduced from the 11th week.After the 13th week,the daily food intake of mice in OR and ORE groups was 70%of the diet of OC group until the end of the intervention at the 18th week.The treadmill exercise load of mice in OE and ORE groups was increased by increasing the exercise speed and time from the 11th week.After the 12th week,the exercise load was fixed at 20m/min,60min/day and the slope was 0 degrees until the end of the intervention at the 18th week.2.Test method:BV,TV,BV/TV and TB were obtained by micro-CT scanning of mouse femur Tb.N、Tb.Th、Tb.Sp and SMI.3D cancellous bone structure reconstruction was performed;The maximum load,failure load,bending strength and elastic modμlus of femur were measured by three-point bending test;ELISA kit was used to determine the concentrations of Irisin,OCN,BALP and tracp-5b in mouse serum;The morphology of mouse femoral bone marrow adipocytes was observed by H&E staining,and the number and volume of bone marrow adipocytes were counted;The levels of FNDC5/Irisin and IntegrinαVβ5,TGFβ1,p38MAPK,Runx2,PRDM16,CIDEαand UCP1 factors in mouse tibia were detected by PCR and Western blot.Part two Role and mechanism of Irisin and mechanical stretch in regμlating osteogenic/adipogenic differentiation of BMSCs1.IntegrinαVβ5 expression of BMSCs:C57BL/6 mouse bone marrow mesenchymal stem cells（BMSCs）were cμltured in complete medium,osteogenic medium and adipogenic medium respectively for 7 days,and then the presence of IntegrinαVβ5expression in BMSCs was detected by Western blot and PCR2.Irisin dose screening:different concentrations of Irisin（0,50,100 and 500 ng/ml）were used to intervene BMSCs cells.After 48h,cell proliferation was detected by CCK-8 experiment.The osteogenesis factor（TGFβ1/p38MAPK/Runx2）and brown fat factor（PRDM16/Cideα/UCP1）was detected by PCR to screen the best dose of Irisin,which was used to intervene BMSCs.3.Screening of mechanical stretch strength:0%,2%,4%and 8%mechanical stretch groups were set up,and FX-5000 tension system was used to perform mechanical stretch once a day on BMSCs.After the experiment,cell proliferation was detected by CCK-8 test kit,and the above osteogenesis/brown fat factors mRNA were detected by PCR to screen the best intensity of mechanical stretch,which was used to intervene BMSCs.4.Irisin and mechanical stretch intervene BMSCs differentiation:control,Irisin,stretch and Irisin+stretch groups were set up.After adding Irisin containing osteogenic/adipogenic differentiation medium,stretch was carried out once every two days.After the experiment,the osteogenic differentiation group was stained with ALP and alizarin red,and the mRNA levels of the above osteogenic factors were detected by PCR.The adipogenic differentiation group was stained with oil red O,and the mRNA levels of brown fat and white fat factors were detected by PCR.5.BMSCs Integrinβ5 gene overexpression/silencing,simμltaneous Irisin and mechanical stretch intervention:pEx-BLANK,pEx-Integrinβ5,pEx-Integrinβ5+Irisin,pEx-Integrinβ5+Stretch,pEx-Integrinβ5+Irisin+stretch,scr-siRNA,si-Integrinβ5,si-Integrinβ5+Irisin,si-Integrinβ5+stretch and si-Integrinβ5+Irisin+stretch groups were set up.After the intervention,the expressions of osteogenic/brown fat factors were detected by PCR and Western blot.Resμlts:Part one Effects of calorie restriction/aerobic exercise on bone quality and Irisin/IntegrinαVβ5 signaling pathway in obese mice1.Obesity group:（1）Compared with NC group,BV/TV,and Tb.N and Conn.Dn were decreased significantly（P<0.05）,Tb.Sp was increased significantly in OC group（P<0.05）.3D reconstruction images showed that the bone microstructure of OC group mice was seriously damaged.（2）Compared with NC group,the maximum load,failure load and bending strength of OC group were significantly lower（P<0.05）.（3）Compared with NC group,the concentrations of serum Irisin,OCN and BALP in OC groups were decreased significantly（P<0.05）,while the concentration of serum TRACP-5b was increased significantly（P<0.05）.（4）Compared with NC group,the number and volume of bone marrow adipocytes in OC group increased significantly（P<0.05）.（5）Compared with NC group,FNDC5 mRNA and Irisin protein in bone tissue of OC group were decreased significantly（P<0.05）,and osteogenesis factors（TGFβ1/p38MAPK/Runx2）and brown fat factor（PRDM16/Cideα/UCP1）also were decreased significantly（P<0.05）,but mRNA and protein levels of integrinαV and integrinβ5were no significant changed（P>0.05）.2.Calorie restriction/aerobic exercise groups:（1）Compared with OC group,BV/TV,Tb.N and Conn.Dn were increased significantly in OR group（P<0.05）;BV,BV/TV,Tb.N and Conn.Dn in OE and ORE groups were increased significantly（P<0.05）;and in OR group Tb.Sp was decreased significantly;and Tb.Sp and SMI in OE and ORE groups were decreased significantly（P<0.05）.Compared with OR group,BV/TV and Tb.N and Conn.Dn were increased more significantly in ORE group（P<0.05）.3D reconstruction images showed that the destruction of bone microstructure in OR,OE and ORE groups was significantly alleviated.（2）Compared with OC group,the maximum load and flexural strength in OR group were increased significantly（P<0.05）,while the maximum load,failure load,flexural strength and elastic modμlus in OE and ORE groups were increased significantly（P<0.05）.（3）Compared with OC group,the concentrations of Irisin and OCN in serum of OR,OE and ORE groups were significantly increased（P<0.05）,the level of BALP in serum of ORE group was also significantly increased（P<0.05）,and the concentration of TRACP-5b in serum of OE and ORE groups was significantly decreased（P<0.05）.Compared with ORE and OE groups,the concentrations of serum Irisin,BALP and OCN in ORE group were significantly higher（P<0.05）.The level of serum TRACP-5b in ORE group was significantly lower than that in OR group（P<0.05）.（4）Compared with OC group,AV and AN parameters of bone marrow adipocytes in OR,OE and ORE groups were significantly lower（P<0.05）.Compared with OR and OE groups,the parameters of AV and AN of bone marrow adipocytes in ORE group were significantly lower（P<0.05）.（5）Compared with OC group,FNDC5 mRNA and Irisin protein in bone tissue of OR,OE and ORE groups were significantly increased（P<0.05）,and the levels of osteogenic/brown fat factors were also significantly increased.Compared with OR and OE groups（P<0.05）,the levels of FNDC5/Irisin and these osteogenic/brown fat factors in ORE group were higher（P<0.05）.But the levels of levels of integrinαV and integrinβ5 were no significant changed（P>0.05）.Part two Role and mechanism of Irisin and mechanical stretch in regμlating osteogenic/adipogenic differentiation of BMSCs1.There is IntegrinαVβ5 in BMSCs cells.The best dose of Irisin is 100 ng/ml,and the best strength of tensile stress is 4%mechanical stretch.2.Under osteogenic differentiation conditions:（1）Compared with control group,ALP enzyme activity,number of calcium nodμles and OD₅₇₀value in Irisin,stretch and Irisin+stretch groups were significantly higher（P<0.05）.Compared with Irisin and stretch groups,the ALP enzyme activity,the number of calcium nodμles and OD₅₇₀value in Irisin+stretch group were higher（P<0.05）.（2）Compared with control group,FNDC5,TGFβ1,p38MAPK and Runx2 mRNA levels were increased significantly in Irisin,stretch and Irisin+stretch groups（P<0.05）.There was no significant change in IntegrinαV and Integrinβ5 mRNA in each intervention group（P>0.05）.Compared with Irisin and stretch groups,TGFβ1,p38MAPK and Runx2 mRNA levels were higher in Irisin+stretch group（P<0.05）.3.Under the condition of adipogenic differentiation:（1）Compared with control group,lipid droplet formation and OD₅₂₀value in Irisin,stretch and Irisin+stretch groups were significantly reduced（P<0.05）.Compared with Irisin and stretch groups,the lipid droplet formation was least（P<0.05）and the OD₅₂₀value was lowest in Irisin+stretch group（P<0.05）.（2）Compared with control group,FNDC5,PRDM16,cideαand UCP1 mRNA levels of Irisin,stretch and Irisin+stretch groups were increased significantly,and Asc-1,Hoxc8 and Hoxc9 mRNA levels were decreased significantly（P<0.05）.But integrinαV and integrinβ5 mRNA were no significant changed in each intervention group（P>0.05）.Compared with Irisin and stretch groups,the mRNA levels of brown fat factor in Irisin+stretch group were significantly higher and that of white fat factor were significantly lower（P<0.05）.4.BMSCs overexpressing Integrinβ5 gene,with Irisin and mechanical stretch intervention at the same time.The resμlts showed that,compared with the p EX-blank group,the levels of IntegrinαV,Integrinβ5,TGFβ1,p38MAPK,Runx2,PRDM16,CIDEαand UCP1 were increased significantly in p EX-Integrinβ5 group（P<0.05）.Compared with p EX-Integrinβ5 group,the levels of TGFβ1,p38MAPK,Runx2,PRDM16,CIDEαand UCP1 were significantly higher in p EX-Integrinβ5+Irisin,p EX-Integrinβ5+stretch,p EX-Integrinβ5+Irisin+stretch group（P<0.05）.FNDC5mRNA and Irisin protein levels were also significantly higher in p EX-Integrinβ5+stretch and p EX-Integrinβ5+Irisin+stretch groups（P<0.05）.There was no significant difference in mRNA and protein levels of IntegrinαVβ5 in each group（P>0.05）.Compared with p EX-Integrinβ5+Irisin and pEx-Integrinβ5+stretch,levels of these osteogenic/brown fat factor were higher in pEx-Integrinβ5+Irisin+stretch group（P<0.05）.5.Silence Integrinβ5 gene,with Irisin and mechanical stretch intervention at the same time.The resμlts showed that,compared with scr-siRNA group,the levels of IntegrinαV,Integrinβ5,TGFβ1,p38MAPK,Runx2,PRDM16,CIDEαand UCP1were decreased significantly in si-Integrinβ5 group（P<0.05）.Compared with si-Integrinβ5 group,the expressions of IntegrinαV,Integrinβ5,TGFβ1,p38MAPK,Runx2,PRDM16,CIDEαand UCP1 were not significant different than in si-Integrinβ5+Irisin,si-Integrinβ5+stretch and si-Integrinβ5+Irisin+stretch（P>0.05）.However,the levels of FNDC5 mRNA and Irisin protein in si-Integrinβ5+stretch and si-Integrinβ5+Irisin+stretch groups were significantly increased（P<0.05）.Conclusion:1.Obesity caused the damage of Irisin/IntegrinαVβ5 signaling pathway,the imbalance of osteogenic/adipogenic differentiation of BMSCs,the destruction of bone microstructure and the decline of mechanical properties,which may be one of the reasons for obesity-induced osteoporosis.2.Caloric restriction,aerobic exercise and combined intervention may alleviate the imbalance of osteogenic/lipid differentiation of BMSCs,improve the abnormal increase of bone marrow fat and the destruction of bone microstructure,improve bone formation and bone strength,and reverse obesity-induced osteoporosis through improving the signal damage of Irisin/IntegrinαVβ5 signaling pathway in bone.The effect of combined intervention is better than that of single intervention.3.Mechanical stretch up-regμlated the expression of osteogenic factors（TGFβ1/p38MAPK/Runx2）and brown fat factors（PRDM16/Cideα/UCP1）,regulate the differentiation of BMSCs through Irisin/IntegrinαVβ5 signaling pathway.