Effects Of LncRNA MEG3/P62/Runx2 Pathway In Promoting Bone Formation In T2DM Mice During Exercise

Objective:(1)The effect of inhibiting the LncRNA MEG3/p62/Runx2 pathway in T2DM mouse bone on osteoblast differentiation and bone formation and metabolism.(2)To explore the effects of different exercise modes on bone formation and metabolism in T2DM mice,and how the LncRNA MEG3/p62/Runx2 pathway changes during this process.Methods:Fifty-five 4-week-old C57BL/6 male mice(initial weight 16.2±0.8g)were fed one week later and randomly divided into a normal control group(NC,n=12)and 43 T2DM models.After 8 weeks of high-fat diet feeding,24 hours on an empty stomach,STZ(110mg/kg)was injected into the T2DM model,and citric acid/sodium citrate solution was injected in the NC group(refer to the same concentration of STZ at 110mg/kg).After 3 days of fasting for 12 hours,the blood glucose concentration in the tail vein was measured.T2DM mice with blood glucose concentration≥ 16.7mmol/L were successfully modeled.They were randomly divided into T2DM control group(TC,n=13),T2DM+downhill running exercise group(TP,n=14),T2DM+high intensity intermittent exercise group(TG,n=13).T2DM mice continued to have a high-fat diet.Mice in the NC group were fed with common diets,and raised and trained according to the protocol.After 8 weeks,the weight of each group of mice and wet bone weight were measured;calipers were used to measure the tibia bone morphological indicators of each group of mice;Micro-CT scan of the femurs of each group of mice was used to detect the bone morphometric indicators;Morphological structure of rat bone;AKP staining to observe the activity of OB in each group of mice;serum from each group of mice was taken to detect AKP activity,calcium and phosphorus concentrations in serum;RT-PCR was used to detect LncRNA MEG3,miR-16 in mouse bone,PI3K,Akt,mTOR,p62,NF-κB,and Runx2 mRNA expressions;Western Blot was used to detect Akt,mTOR,p62,and Runx2 protein expressions in bones of mice in each group.Results:(1)Compared with the NC group,the mice in the TC group had significantly increased body weight(P<0.01);the tibial wet weight,distal coronary axis width,medial sagittal axis width,and proximal coronary axis width all significantly decreased(P<0.01 or P<0.05);BMD and Tb.Th of the cortical bone decreased significantly(P<0.05),and BMD,BV/TV,Tb.Th,Tb.Sp,and Tb.N of the cancellous bone decreased significantly(P<0.05 or P<0.01);HE staining results and AKP staining results decreased significantly.(2)Compared with the NC group,the AKP activity in the serum of the TC group mice decreased significantly,and the calcium ion concentration and phosphorus ion concentration increased significantly(P<0.01 or P<0.05);LncRNA MEG3,PI3K,Akt,mTOR and NF-κB mRNA expression increased significantly,miR-16 and Runx2 mRNA expression decreased significantly(P<0.01 or P<0.05);Akt,mTOR,and p62 protein expressions were significantly up-regulated;Runx2 protein expression was significantly reduced(P<0.05 or P<0.01).(3)Compared with the TC group,the body weight of the mice in the TG group decreased significantly;the tibial wet weight increased significantly(P<0.05),the cortical bone BMD increased significantly(P<0.05),and cancellous bone BMD,BV/TV and Both Tb.N increased significantly and Tb.Sp decreased significantly(P<0.05);both HE staining results and AKP staining results increased significantly.The body weight of mice in the TP group decreased significantly;the tibial wet weight,tibial length,and median sagittal axis width increased significantly(P<0.05);cortical bone BMD,BV/TV,and Tb.Th significantly increased(P<0.05),Cancellous bone BMD,BV/TV,Tb.Th,and Tb.N all increased significantly,and Tb.Sp decreased significantly(P<0.05);HE staining results and AKP staining results increased significantly.Compared with the TG group,the width of the sagittal axis of the tibia in the TP group increased significantly(P<0.05);BMD,BV/TV,and Tb.N of cancellous bone increased significantly(P<0.05);HE staining results and AKP staining The results have risen significantly.(4)Compared with the TC group,the AKP activity in the serum of mice in the TG group increased significantly,the concentration of phosphorus ion and calcium ion decreased significantly(P<0.05);the expression of miR-16 and Runx2 mRNA in bone increased significantly,and mTOR mRNA The expression decreased significantly(P<0.05);the expression of Runx2 protein increased significantly,and the expression of p62 protein decreased significantly(P<0.05).The AKP activity in the serum of the TP group mice significantly increased,and the phosphorus ion concentration decreased significantly(P<0.05);the expression of LncRNA MEG3,PI3K,mTOR,and NF-κB mRNA in bone decreased significantly,and the expression of miR-16 and Runx2 mRNA increased significantly(P<0.05);Akt,mTOR and p62 protein expressions were significantly down-regulated,and Runx2 protein expression was significantly increased(P<0.05).Compared with the TG group,the AKP activity in the serum of the TP group mice increased significantly,and the calcium ion concentration decreased significantly(P<0.05);the expression of LncRNA MEG3,PI3K,and mTOR mRNA in bone decreased significantly,and the expression of Runx2 mRNA increased significantly(P<0.05);The expression of p62 protein in the bone of mice in the TP group decreased significantly,while the expression of Runx2 protein increased significantly(P<0.05).Conclusion:(1)When LncRNA MEG3/p62/Runx2 is activated in the bones of T2DM mice,the expression of LncRNA MEG3 is up-regulated,activating p62,thereby inhibiting bone autophagy,osteogenic differentiation and bone formation metabolism.(2)Both downhill running and high-intensity intermittent exercise can promote bone formation in T2DM mice,and the former is better than the latter.The mechanism may be that exercise may down-regulate the expression of LncRNA MEG3 and negatively regulate its downstream miR-16,and further inhibit the PI3K/Akt/mTOR pathway,enhance bone autophagy through mTORC1,down-regulate p62 and NF-κB expression,and ultimately increase Runx2 expression to promote bone formation.