The Mechanism And Function Of LRRc17 In Adolescent Idiopathic Scoliosis Osteopenia

BackgroundAdolescent idiopathic scoliosis(AIS)is the most common type of scoliosis,and its etiology remains unclear.Previous studies have reported that decreased bone mass is closely related to the occurrence and development of AIS.Bone marrow mesenchymal stem cells(BMSCs),as stem cells with multiple differentiation potentials,play an important role in maintaining bone mass.The study found that BMSCs in patients with AIS osteopenia have abnormal differentiation,suggesting that they may be involved in the occurrence of bone mass reduction in AIS.Cell senescence is a cellular state after cells are subjected to various stress factors.For BMSCs,cell senescence will significantly affect their differentiation and proliferation.The increased aging of BMSCs and its abnormal differentiation were observed in elderly patients with osteoporosis and animal models,suggesting that there may be increased aging of BMSCs in patients with reduced bone mass of AIS.However,there is no relevant research report at present.Therefore,the purpose of this study is to clarify whether there is an increase in aging in BMSCs of patients with AIS osteopenia,and to explore the factors and possible mechanisms leading to the increase in aging,in order to provide a new explanation for the abnormal differentiation of BMSCs in patients with AIS osteopeniaMethods:AIS patients were divided into 3 groups according to dual energy Xray(DXA)results,combined with anthropometric data,Cobb angle,apical vertebral deviation and upper endplate inclination,and the apical vertebral HU value was measured by IP ACS software;Compare the differences of the above data between different groups,and explore the correlation between BMD and Cobb angle and between BMD and HU value.The osteogenic and adipogenic differentiation of BMSCs in AIS osteopenia group and control group were observed by alizarin red staining,oil red O staining,Western blot and qPCR.Through SA-β-Gal staining and Western blot were used to detect the aging of BMSCs in AIS osteopenia group and control group.The differentially expressed genes and related pathways of BMSCs in AIS bone mass reduction group and control group were compared by RNA-seq and biological analysis,and the verification was expanded.By lentivirus transfection,LRRc17 was overexpressed in BMSCs of healthy control group,and by SA-β-Gal staining and Western blot were used to detect the senescence of LRRc17 overexpression group and vheicle group.The osteogenic and adipogenic differentiation of BMSCs in LRRc17 overexpression group and vheicle group were observed by alizarin red staining,oil red O staining and qPCR.The expression of autophagy related proteins in LRRc17 overexpression group,LRRc17 overexpression+autophagy enhancer(RAPA)group,Vehicle group and vehicle+RAPA group were detected by Western blot,and the level of intracellular oxidative stress was detected by DHE staining.Western blot was used to detect the autophagy related proteins of BMSCs with AIS osteopnia and healthy control,and AIS group,healthy control group,healthy control+autophagy inhibitor(3MA)group and AIS+RAPA group were set up.Western blot was used to detect the expression of autophagy related genes in each group;The levels of oxidative stress in AIS group,healthy control group,healthy control+3MA group and AIS+RAPA group were detected by reactive oxygen species(ROS)and DHE.Mitochondrial superoxide,mitochondrial membrane potential and ATP content in LRRc17 overexpression group,LRRc17 overexpression+RAPA group,Vehicle group and Vehicle+RAPA group were detected by mitosox,tmre and ATP fluorescence detection.Mitochondrial superoxide,mitochondrial membrane potential and ATP content in AIS group,control group,control+3MA group and AIS+RAPA group were detected by mitosox,JC-1 and ATP fluorescence detection.Western blot was used to detect the protein expression of key genes of PI3K/mTOR pathway in LRRc17 overexpression group,LRRc17 overexpression+RAPA group,Vehicle group and Vehicle+RAPA group.Results:The proportion of patients with osteopenia was 76.71%in our center.Patients with osteopenia had lower BMI and weight,greater Cobb angle of main curve and apical vertebral deviation;The CT HU value of the parietal vertebrae was positively correlated with the minimum Z score,and the HU value of the convex side of the parietal vertebrae was negatively correlated with the Cobb angle.Compared with healthy controls,alizarin red and oil red O staining showed that patients with AIS osteopnia had decreased calcium nodules and increased lipid droplets after induction for 3 weeks;Western blot and qPCR showed that the expression of osteogenic related transcription factors decreased and adipogenic related transcription factors increased,indicating that the differentiation of BMSCs in patients with AIS bone mass decreased(osteogenic differentiation decreased and adipogenic differentiation increased).SA-β-Galactosidase staining showed the increase of galactoglyceride positive cells in BMSCs of patients with AIS osteopenia and Western blot showed the increase of aging related protein expression,indicating the increase of aging in BMSCs of patients with AIS osteopenia.RNA-seq showed that LRRc17,CPT1B and PCDH7 genes were differentially expressed in BMSCs with AIS osteopenia and healthy controls.KEGG enrichment analysis of DEG(differentially expressed genes)showed abnormalities in autophagy and mitochondria autophagy;Extended validation found that LRRc17 gene was significantly increased in AIS osteopenia BMSCs.Compared with vehicle group,SA-βGalactosidase staining showed that the galactosidase positive cells of BMSCs in LRRc17 overexpression group increased significantly,and Western blot showed that the expression of aging related proteins increased,indicating that the aging of BMSCs in LRRc17 overexpression group increased.Alizarin red and oil red O staining showed that calcium nodules decreased and lipid droplets increased in LRRc17 overexpression group after 3 weeks of induction;qPCR showed that the expression of osteogenic related transcription factors decreased and adipogenic related transcription factors increased,indicating that LRRc17 overexpression caused BMSCs differentiation disorder(osteogenic differentiation decreased and adipogenic differentiation increased).Western blot showed that autophagy in LRRc17 overexpression group was significantly inhibited than that in vehicle group,and autophagy in LRRc17 overexpression+RAPA group was higher than that in LRRc17 overexpression group;DHE staining showed that the level of intracellular oxidative stress in LRRc17 overexpression group was higher than that in non transfection group and LRRc17 overexpression+RAPA,indicating that LRRc17 overexpression inhibited autophagy and increased the level of oxidative stress in BMSCs.Western blots showed that the autophagy of BMSCs in patients with AIS osteopenia compared with healthy controls;The autophagy of healthy control+3MA group was weaker than that of healthy control group,and that of AIS group was weaker than that of AIS+RAPA group;It indicates that the autophagy of BMSCs is weakened in patients with AIS osteopenia.ROS and DHE staining showed that the level of oxidative stress in AIS osteopnia group increased compared with healthy control group.Similarly,the level of oxidative stress in AIS+3MA group increased compared with healthy control group and AIS+RAPA group;It indicates that the weakening of autophagy in AIS leads to the increase of oxidative stress.The results of Mitosox,Tmre and ATP showed that compared with the empty transfection group,the mitochondrial oxidative stress,mitochondrial membrane potential and ATP decreased in the LRRc17 overexpression group.Compared with LRRc17 overexpression+RAPA group,LRRc17 overexpression group increased mitochondrial oxidative stress,decreased mitochondrial membrane potential and decreased ATP.This suggests that LRRc17 overexpression leads to mitochondrial dysfunction by affecting autophagy.The results of Mitosox,JC1 and ATP showed that compared with healthy controls,the mitochondrial oxidative stress,mitochondrial membrane potential and ATP in BMSCs with reduced AIS osteopenia.Compared with healthy control group and AIS group and AIS+RAPA group,healthy control+3MA group showed increased mitochondrial oxidative stress,decreased mitochondrial membrane potential and decreased ATP;It indicates that the weakening of autophagy in AIS leads to mitochondrial dysfunction.Western blots showed that the expression of p-PI3K/PI3K and p-mTOR/mTOR increased in/RRc17overexpression group compared with vehicle group.After RAPA inhibited mTOR,the expression of PI3K/PI3K and p-mTOR/mTOR decreased in LRRc17 overexpression+RAPA group compared with LRRc17 overexpression group,indicating that LRRc17 regulates autophagy through PI3K/mTOR pathway.Conclusion:Decreased bone mass is a risk factor for the progression of AIS.There is an increase in aging in BMSCs in AIS osteopenia,which may be a new explanation for the abnormal differentiation of BMSCs in AIS.The specific reason may be related to the increase of oxidative stress in BMSCs caused by autophagy inhibition and mitochondrial dysfunction.LRRc17 may participate in this process through PI3K/mTOR pathway.