Targeting LRRc17 Prevents Senescence Of BMSCs Via Mitophagy And Enhances The Therapeutic Effects In Osteoporosis

Background and ObjectiveMesenchymal stem cells（MSCs）are typical adult stem cells.For a long time,MSCs have been paid attention to for their pluripotency and potential clinical therapeutic value.MSCs exist in connective tissues and come from a wide range of sources,including umbilical cord and blood,amniotic membrane and amniotic fluid,placenta,bone marrow.MSCs can differentiate into varieties of somatic cell lines,and migrate and homing to injury and inflammatory tissues or organs.In addition,based on its regenerative ability and immunomodulatory properties,MSCs have been widely used to treat various degenerative and inflammatory diseases.More and more evidences have shown the clinical therapeutic value of bone marrow mesenchymal stem cells（BMSCs）.Systemic MSCs transplantation has been successfully implied to treat a variety of diseases,including myocardial infarction and systemic lupus erythematosus（SLE）.However,the therapeutic effect of allogeneic BMSCs is greatly weakened due to immune rejection after transplantation.At present,based on the development of expansion and cultivation of BMSCs,large amounts of studies associated with autologous BMSCs transplantation have been investigated.Aging is an inevitable and gradual process of functional degradation,leading to tissue and organ dysfunction.This process is driven by strict and complex interactions between genes and internal and external factors.Decreased differentiation and replication capacity of stem cells are generally considered to be hallmarks of aging.Recent studies have demonstrated that along with the development of aging,the regenerative ability and immunomodulatory properties of BMSCs were significantly decreased,which greatly hinders the clinical application of BMSCs.At the same time,the mechanism of stem cell senescence is still unclear.Therefore,elucidating the potential molecular mechanisms regulating the biological functions of senescent BMSCs is very important for delaying the senescence of BMSCs and improving clinical treatment effects.More and more evidences show that BMSCs aging is an inherent characteristic of body aging and is closely related to the process of chronic aging diseases including OP.Age-related OP is not only caused by the increase in bone resorption activity,but also related to the dysfunction of BMSCs,which related to the alterations in adipogenic differentiation capacity and decreased cell proliferation capacity.Recent studies have shown that after intramedullary infusion,BMSCs can reduce bone loss through systemic immune regulation or local anabolic effects of cell homing,but the underlying mechanism is not fully understood.It is suggested that targeted inhibition of BMSCs senescence may be of great significance in delaying or treating OP.LRRc17 is an important regulator of osteoclast differentiation.As a negative regulator of osteoclasts,it participates in the interaction between osteoclasts and osteoblasts,and inhibits RANKL-induced osteoclasts by blocking PLC signaling.Autophagy is essential for cell metabolism,function and homeostasis.This process is achieved through the lysosomal degradation pathway.The defect of autophagy is related to the impaired osteogenic differentiation ability of senescent BMSCs in osteoporosis.Therefore,both autophagy and LRRc17 are essential for bone homeostasis.However,whether there is a correlation between them,and the specific molecular mechanism has not been reported yet.Based on the above background,this article studies the differential expression of LRRc17 in the aging process of BMSCs from the transcription level.In vitro,establishes H₂O₂-induced BMSCs senescence models and natural senescence models of BMSCs to further explore whether LRRc17 affects differentiation potential and mitophagy.In addition,we further explored whether LRRc17 regulates bone microenvironment homeostasis by improving the senescence of BMSCs to alleviate OP.Our research will lay a foundation for understanding the internal mechanism of BMSCs senescence and the treatment of OP.Materials and Methods1.In vitro1)Transcriptome sequencing of BMSCs in young,middle-aged,and old C57/BL6mice and age-related changes in LRRc17The bone marrow cavity mononuclear cells of young,middle-aged and old C57/BL6 mice were isolated by bone marrow cavity flushing.The primary BMSCs were cultured by adherent screening method and cultured to passage3 respectively,and the immunophenotype was identified.Then the cytoplasmic histone-related DNA fragments of dead cells were detected by enzyme immunoassay,the abundance of telomerase Telo TAGGG fragments was detected by PCR/ELISA,the senescence-relatedβ-galactosidase（SA-β-Gal）staining was used to detect cell senescence,and the total cellular proteins were extracted to detect senescence-related proteins p16,p21and p53.Transcriptome sequencing of Y-BMCSs and O-BMSCs at P3 were carried out respectively.After quality control,gene quantitative analysis and analysis based on gene expression level were performed,including KEGG pathway significant enrichment analysis,GO functional significant enrichment analysis,weighted gene co-expression network analysis（WGCNA）assessment of targeting proteins,time series analysis of the expression level of LRRc17 in different ages.2)Targeting the senescence and differentiation potential of LRRc17 on BMSCs.Two cell senescence models were used to explore the effects of LRRc17 on BMSCs senescence:BMSCs were induced to produce stress senescence models by H₂O₂ treatment for 72 hours,and the natural senescence BMSCs model used P3generation BMSCs isolated from old C57/BL6（24 months）.Then the cell proliferation was detected.After further silencing and over-expressing LRRc17 on O-BMSCs and Y-BMSCs,respectively,we explored whether LRRc17 affects its differentiation potential through osteogenic and adipogenic induction.Alizarin red staining was conducted 14 days after induction of osteogenesis,and oil red O staining was conducted 21 days after adipogenesis induction,followed by microscopic examination.3)Silencing LRRc17 activates mitochondrial autophagy through the PI3K/m TOR pathway to improve mitochondrial damage induced by senescenceThe mitochondria of Y-BMSCs and O-BMSCs were specifically labeled with MitoTrackerGreen,and the morphology of mitochondria was observed.At the same time,the expressions of OPA1 and Drp1 were detected by WB.Then,the mitochondrial abundance and structural continuity were evaluated by transfecting LRRc17 and adding RAPA for 72 hours with the H₂O₂ induced BMSCs senescence model.The mitochondrial biogenesis was detected to explore whether LRRc17 can postpone cell senescence by restoring BMSCs mitochondrial fusion/fission and energy synthesis.For H₂O₂-induced BMSCs aging model and natural aging BMSCs model,after silencing LRRc17,WB was used to detected autophagy-related proteins,to verify whether LRRc17 inhibited aging by autophagy capacity elevation in BMSCs.Furthermore,lysosomal inhibitor HCQ was added to detect the expression levels of LC3Ⅱand P62,and the alterations of autophagy flux were determined by mitochondrial and LC3Ⅱco-localization.Then mitochondrial biogenesis was detected;SA-β-Gal staining was used to detect cell senescence,and evaluate the mechanism of the effect of LRRc17 on autophagy capacity of BMSCs.4)LRRc17 expressed by BMSCs regulates bone homeostasis by inhibiting OCs differentiation.The femur of 8-week C57/BL6 was flushed,and the monocytes were obtained by washing the bone marrow cavity.After adhering to the wall overnight,the suspension cells were cultured with 25 ng/ml M-CSF for 3 days to induce the BMMs.After the identification of BMMs by F4/80,the Actin ring was identified by phalloidin staining.OCs and BMSCs were co-cultured in Transwell chamber at the ratio of 1:3.OCs were seeded in the lower layer and BMSC in the upper layer.Five days later,OCs was stained with TRAP,SEM was used to observe the bone resorption lacunae,and the total RNA of OCs was extracted to detect the osteoclast related genes.Combined with transcriptome and proteome analysis,to determine whether the LRRc17 expressed by BMSCs regulates the osteoclast process by inhibiting the differentiation of OCs.2.In vivo1)Establishment of mouse OP model induced by OVX6-month-old female C57BL/6 mice were ovariectomized to establish the model of osteoporosis.3 month later,the bone mineral density（BMD）was measured by Micro-CT,and the osteoclast factor CTX,TRAP,ICTP,were detected by blood biochemistry,and osteogenic factor ALP was detected at the same time.2)Bone marrow cavity transplantation of BMSCs improves OVX-induced bone loss in OP miceThe experiment was divided into 5 groups:Sham,OVX,Y-BMSCs+OVX,O-BMSCs^LV-GFP+OVX,O-BMSCs^sh-LRRc17+OVX,8 mice in each group.The mice were sacrificed 1 month after transplantation,and Calcein was injected intraperitoneally with 20 mg/kg 2 days and 16 days before sacrifice,respectively.The BMD,Tb.Th,Tb.N,Bv/Tv,Tb.Sp and Ct.Th were measured by Micro-CT;the ratio of new bone formation was observed under fluorescence microscope;the pathological structure of metaphysis of femur was observed by HE and Masson staining.The number of osteoclasts and osteoblasts was detected by TRAP staining and toluidine blue staining,and the distribution and expression of osteoclast factor NFATC1 and osteogenic factor RUNX2 in femur were detected by immunohistochemical staining.Results1.In vitro1)Transcriptome sequencing of BMSCs and age-related changes of LRRc17 in young,middle-aged and old C57/BL6The BMSCs isolated and cultured in young,middle-aged and old C57/BL6 mice were expressed with high ratios of positive markers and low ratios of negative markers,and there was no significant difference between Y-BMSCs and O-BMSCs.Cell death analysis showed that the number of dead cell fragments in BMSCs and culture supernatant increased with age,while telomerase activity assay also showed that BMSCs telomerase activity decreased with age.SA-β-Gal staining showed that the positive rate of SA-β-Gal increased with age.At the same time,compared with Y-BMSCs,the morphology of O-BMCS was flatter,the volume of cells was larger,the shape of irregular cells was more obvious,and the number of vacuolar granules in the cytoplasm elevated.Additionally,the expression level of age-related proteins p16,p21and p53 significantly increased with age.A total of 2,711 differentially expressed genes were obtained by transcriptional sequencing,of which 1,467 genes were up-regulated and 1,244 gens were obviously down-regulated.GO function enrichment and KEGG Pathway were enriched in pathways correlated with cell proliferation and cell senescence,in which cell growth and death pathway,cell senescence was enriched,and the cell senescence signal pathway was ranked firstly in the total enrichment pathway.Gene co-expression network analysis showed that LRRc17 interacted with multiple aging proteins,and time series analysis showed that LRRc17 was positively correlated with age.Therefore,we speculated that the elevated expression of LRRc17 may be correlated with the senescence of BMSCs.2)Targeting LRRc17 alleviates senescence of BMSCs and affects the potential of cell differentiationY-BMSCs overexpression of LRRc17 accelerated cell senescence,characterized as the increased protein expression levels of p16,p21 and p53.In natural senescence model,the expression levels of p16,p21 and p53 in LRRc17 silenced O-BMSCs were significantly lower than those in GFP transfection group.Moreover,comparing with the H₂O₂ induced-senescent Y-BMSCs,the protein expression of p16,p21 and p53were decreased when the LRRc17 was silenced.These results suggest that LRRc17knockdown can effectively alleviate BMSCs senescence.Furthermore,LRRc17silencing also decreases the accumulation of ROS,and the proportion of apoptotic cells,while improved the cell proliferation capacity.These results showed that LRRc17silencing significantly improved the proliferation ability of impaired BMSCs.The osteogenic ability of BMSCs decreased gradually from youth,middle age to old age,while the adipogenic ability increased.The osteogenic ability of Y-BMSCs decreased and the adipogenic ability increased after LRRc17 overexpression.In addition,after LRRc17 knockdown,the osteogenesis ability of O-BMSCs was improved and the adipogenesis ability decreased.In addition,the expression levels of key proteins of BMSCs osteogenesis and adipogenic transcription factor were consistent with the above staining results.3)LRRc17 silencing activates mitochondrial autophagy through inhibition of PI3K/m TOR pathway to improve mitochondrial dysfunction in senescent BMSCsCompared with Y-BMSCs,the mitochondrial morphology of O-BMSCs was obviously fragmentation,and the incompleteness of mitochondrial results was also observed in H₂O₂-induced-senescent BMSCs.While the morphology of senescent BMSCs was significantly improved after LV^sh-LRRc17transfection.In addition,the expression of OPA1 was increased,while the expression of Drp1 was decreased.At the same time,after silencing LRRc17,the accumulation of mt ROS was decreased,while the membrane potential,ATP content and OCR were elevated in senescent BMSCs,indicating that LRRc17 silencing can not only improve mitochondrial fusion/fission,but also effectively increase mitochondrial bioenergetics.To further explore whether LRRc17 affects aging by regulating BMSCs autophagy,we knockdown LRRc17 in BMSCs.Here,we found that the expression of LC3Ⅱand Beclin1 protein were increased,while the expression of P62 protein was decreased in BMSCs transfected with LV^sh-LRRc17.Meanwhile the ratio of p-PI3K/PI3K and p-m TOR/m TOR were also significantly decreased.Based on the above results,we speculated that the inhibition of BMSCs senescence by LRRc17silencing may depend on the activation of autophagy.To determine the role of autophagy in LRRc17-mediated BMSCs senescence,lysosomal inhibitor HCQ was used to block the degradation of autophagosomes.After HCQ treatment,LC3Ⅱaccumulated significantly and P62 increased significantly.At the same time,the co-localization of LC3Ⅱand mitochondria were increased significantly.Consequently,the mitochondrial bioenergetics were decreased.Additionally,the expression of p16,p21 and p53 were significantly increased.The above results confirmed that cell senescence led to mitochondrial dysfunction,and the ameliorative role of LRRc17 silencing in BMSCs senescence was contributed to the activation of autophagy.4)LRRc17 expressed by BMSCs regulates bone homeostasis by inhibiting OCs differentiation.After co-culturing,compared with OC group,the size of TRAP positive cells in OCs+BMSCs group was significantly decreased.And after LRRc17 upregulation,the blocking effect of BMSCs on OCs was further elevated.Moreover,the area and number of TRAP positive OCs were decreased,the area of bone resorption pits were decreased,and the expression of osteoclast differentiation transcription factors also were significantly decreased.Combined transcriptome and proteome analysis showed that LRRC17 silencing affects autophagy related PI3K-AKT pathway and osteoclastic differentiation regulatory pathway.It is suggested that LRRc17 inhibited the differentiation of OCs through increased osteogenesis to participates in the regulation of bone microenvironment homeostasis in vitro.2.In vivo1)The establishment of mouse OP model induced by OVX.At 3 m after ovariectomy,the biochemical indexes of caudal vein blood showed that the expression level of osteoclast factor increased significantly,while the expression level of osteogenic factor ALP decreased significantly.Micro-CT analysis revealed that the BMD of OVX group significantly elevated compared with sham group.Collectively,these data demonstrated that the mouse osteoporosis model induced by OVX was established successfully.2)Bone marrow cavity transplantation of BMSCs ameliorates OVX-induced bone loss in OP mice.Micro-CT analysis showed that compared with OVX group,O-BMSCs could effectively improve bone loss in osteoporotic mice when LRRc17 gene was knockdown,and the parameter related to new bone growth rate increased significantly.The results of OBs and OCs pathological staining of femur showed that the number of osteoblasts labeled with toluidine blue increased.On the contrary,the number of OCs specifically labeled by TRAP decreased significantly,indicating that the down-regulation of LRRc17 gene was beneficial to BMSCs to improve bone loss in osteoporotic mice.HE and Masson staining showed that bone loss was improved by BMSCs transplantation,but the therapeutic effect of O-BMSC was significantly weaker than that of Y-BMSC+OVX group.After silencing LRRc17 in BMSCs,the number of bone trabeculae increased,the expression of collagen in new bone increased,while the expression of NFATc1 decreased and the expression of RUNX2 increased.Based on the above results,it is suggested that LRRc17 silencing regulated osteogenesis and osteoclast in ovariectomized mice by improving O-BMSCs senescence.ConclusionsIn this study,we first found that LRRc17 was a key aging regulator,and its overexpression was positively correlated with the aging process of BMSCs.The accumulation of LRRc17 led to the transition of Y-BMSCs from osteogenic differentiation to adipogenic differentiation and promoted the ell senescence.At the same time,LRRc17 knockdown reversed BMSCs senescence,mitochondrial dysfunction and activated autophagy activity via inhibiting PI3K/m TOR pathway.In addition,after silencing LRRc17,O-BMSCs improved OP-induced bone loss by activating autophagy and inhibiting osteoclast formation in vivo.Collectively,this study revealed the significant role of LRRc17 in the BMSCs senescence,and targeting inhibition of LRRc17 may lay a foundation for alleviating the senescence of BMSCs and the clinical treatment of OP.