| Background and objectivePeriodontitis is a chronic inflammatory disease that occurs in periodontal tissues such as gingiva,periodontal ligament,cementum,and alveolar bone.It is the sixth most common disease in the world,and is closely related to cardiovascular,diabetes and other systemic diseases.In inflammatory environment,the imbalance of periodontal bone metabolism,active osteoclast differentiation and bone absorption lead to continuous destruction of alveolar bone,and insufficient bone repair leads to gradual loosening or even tooth loss.The existing treatment measures include periodontal basic treatment,surgical treatment,as well as periodontal tissue engineering therapy based on material research and development,stem cell technology and gene technology.However,many of the treatments are incremental remedial treatments for the missing alveolar bone,and the prognosis is still uncertain.It is better to prevent a disease from occurring than to treat it.Therefore,it is of positive basic research value and clinical significance to study the regulatory mechanism of periodontal bone metabolism imbalance under inflammatory environment and to find new targets for bone metabolism regulation that can reduce inflammatory bone destruction and promote bone repair.Regulation of bone metabolism is a relatively complex physiological activity in human body,among which RANKL/RANK osteoclast activation signaling pathway is a relatively classical pathway in bone metabolism.According to this classic osteoclast activation signaling pathway,drugs for inhibiting bone resorption therapy have serious adverse reactions such as jawbone necrosis,hypocalcemia,and mineral metabolism imbalance.There may be unclear signaling pathways outside of RANKL/RANK at work,and a non-RANKL osteoclast mechanism exists in the process of alveolar bone resorption,which deserves further in-depth research.In recent years,non-RANKL dependent pathways for bone metabolism diseases have become a research hotspot in the field of osteoclasis.Transforming growth factor-β1(TGF-β1)is one of the non-RANKL pathway osteoclast differentiation factors,and plays an important role in bone metabolism.In the process of bone resorption,the release of bone-derived TGFβ1 into the microenvironment can act on osteoblasts through the paracrine pathway,but also affect osteoclasts themselves through autocrine,so that the signaling coupling between osteogenesis and osteoclast can be carried out.On the one hand,TGF-β1 activates the differentiation of osteoblasts through Smad3 pathway and promotes osteogenesis.On the other hand,osteoclasts are inhibited by inhibiting Smad1 pathway.Periodontal ligament-associated protein-1(PLAP-1)(also known as Asporin)is a highly expressed extracellular matrix protein in the periodontal membrane and plays an important regulatory role in oral,cartilage,and tumor tissues.The specific mechanism of action of PLAP1 in articular cartilage is to inhibit the phosphorylation of Smad3 through TGF-β1/Smad pathway,thus inhibiting cartilage formation.It promotes the proliferation and migration of tumors by regulating TGF-β1/Smad3 signaling pathway.At present,there are few studies on PLAP-1 in the field of stomatology,but literature reports show that PLAP-1 can negatively regulate the osteogenic differentiation of periodontal membrane cells.In addition,PLAP-1 can be detected in osteoclasts of alveolar bone of experimental periodontitis,and the protein level of PLAP-1 is higher than that of normal periodontal tissue.This suggests that PLAP-1 may play an important role in alveolar bone resorption.However,no studies have systematically elucidated the effect of PLAP-1 on osteoclast differentiation and its relationship with TGFβ1/Smad signaling pathway.Based on the mechanism of TGF-β1 in bone regulation and the possible role of PLAP-1 in alveolar bone metabolism,it is speculated whether PLAP-1 regulates downstream Smad1 signal transduction through TGF-β1 signaling pathway in alveolar bone metabolism,thereby affecting osteoclast differentiation,and regulates Smad3 signal transduction to affect osteoblast differentiation.In conclusion,the purpose of this study was to investigate:1.The effects of PLAP-1 on the differentiation and function of osteoclasts and osteoblasts in normal,inflammatory and recovery environments were studied in vitro,and the possibility of TGF-β1/Smad pathway participating in its effects was verified;2.The effects of Plap-1 gene knockout mice on alveolar bone imbalance(bone resorption and formation)in experimental periodontitis models,and to reveal the association of PLAP-1 with TGF-β1/Smad pathway.Methods1.The effects and mechanism of PLAP-1 on osteoclast and osteoblast differentiation and its association with TGF-β1/Smad pathway(1)The effects of PLAP-1 on osteoclast differentiation and functionPlap-1 knockout mice(C57BL/6N-Plap-1-/-)were constructed and genotypes were identified by mRNA and protein levels.The primary bone marrow-derived macrophages(BMMs)of C57BL/6N-Plap-1-/-mouse and C57BL/6N-Plap-1-/-mouse were cultured and induced.The experiment was divided into PLAP-1-/-and PLAP-1+/+ groups.The inflammatory environment was constructed with 1 μg/ml Porphyromonas gingivalis lipopolysaccharide(P.gingivalis LPS).The experiment was divided into LPS+PLAP-1-/-and LPS+PLAP-1+/+groups.PLAP-1 gene knockout BMMs were transfected with PLAP-1 overexpression lentiviral vector to detect the rescue effect of PLAP-1.The experiment was divided into LV-PLAP-1 and LV-CMV groups.The expression levels of osteoclast related markers were detected by qRTPCR and Western blot.Tartrate-resistant acid phosphatase staining(TRAP)and cytoskeleton staining were used to analyze the changes in osteoclast differentiation ability,and to clarify the role of PLAP-1 in BMMs differentiation into osteoclasts in normal and inflammatory environments.The BMMs were inoculated on the surface of fresh bovine cortical bone respectively,and osteoclast differentiation was induced under normal and inflammatory conditions.After 14 days of incubation,the bone resorption area of each group was analyzed by scanning electron microscopy(SEM).(2)The roles of PLAP-1 in osteoblast differentiation and functionBone marrow mesenchymal stem cells(BMSCs)of C57BL/6N-Plap-1-/-and C57BL/6NPlap-1+/+ mice were isolated and cultured in normal(PLAP-1-/-and PLAP-1+/+ groups)and inflammatory(LPS+PLAP-1-/-and LPS+PLAP-1+/+groups)environments.Osteoblast related markers were detected by ALP staining,alizarin red staining,qRT-PCR and Western blot after mineralization induction at 1 w and 4 w under normal culture and inflammatory conditions.respectively.After 4 weeks of mineralization induction in BMSCs,the calcified nodules were detected by alizarin red staining.(3)TGF-β1/Smad may be involved in the effect of PLAP-1 on osteoclast and osteoblast differentiationThe colocalization of PLAP-1 and TGF-β1 in osteoclasts and osteoblasts was observed by confocal laser scanning microscope(CLSM)and co-immunoprecipitation(CoIP)technique.TGF-β1 signaling pathway was inhibited by LY 364947.Phosphorylation levels of Smad1 and Smad3 after PLAP-1 gene knockout were detected.After adding TGF-β1 inhibitor LY 364947.TGF-β1/Smadl inhibitor SB 501542 and TGF-β1/Smad3 inhibitor SIS3 to inhibit the signal pathway,the expression changes of osteoclastic and osteogenic differentiation indicators were detected by qRT-PCR and Western blot.2.The effect of PLAP-1 on the coupling of osteoclasts and osteoblasts via TGF-β1/Smad pathway in co-culture system(1)PLAP-1 regulated the coupling of osteoclasts and osteoblastsBMSCs of C57BL/6N-Plap-1-/-and C57BL/6N-Plap-1+/+mice were inoculated and osteogenic induced for 7 days in lower chamber of indirect co-culture system(2 × 106/cm2).BMMs were inoculated on fresh beef bone slices.After osteoclast induced for 7 days,bone slices were placed in the upper chamber of indirect co-culture system(1.5×105/cm2).BMMs and BMSCs from the same genetic mice were inoculated in the same culture system:BMSCs were divided into C-PLAP-1+/+group(from C57BL/6N-Plap-1+/+mice)and C-PLAP-1-/-group(from C57BL/6N-Plap-1-/-mice).BMMs were divided into B+C-PLAP-1+/+and B+C-PLAP1-/-groups.The osteoclasts and osteoblasts were obtained after 7 days of co-culture under inflammatory condition,and the mRNA and protein levels of osteoclast and osteogenic markers were detected by qRT-PCR and Western blot.The bone resorption area of beef bone slices in each group was analyzed by SEM after 14 days of co-culture.After 4 w of co-culture,alizarin red staining was used to evaluate the mineralization nodules formation ability of the BMSCs,and it was clear that PLAP-1 was involved in the mutual regulation of osteoclasts and osteoblasts.(2)Relationship between TGF-β1/Smad pathway and PLAP-1 coordination the coupling of osteoclasts and osteoblastsWestern blot was used to detect the phosphorylation levels of Smad1 in BMMs and Smad3 in BMSCs in the co-culture system.LY 364947 and SB 431542(TGF-β1/Smad blocker)were added to detect the expression levels of osteoclast and osteogenic markers,and to clarify the possible role of PLAP-1 in regulating osteoclast and osteoblast differentiation through TGFβ1/Smad pathway.3.The role and mechanism of PLAP-1 in affecting alveolar bone imbalance in experimental periodontitis by regulating TGF-β1/Smad pathway(1)The effect of PLAP-1 on alveolar bone imbalance in experimental periodontitisBefore establishing the experimental periodontitis model,the differences between C57BL/6N-Plap-1-/-mice and C57BL/6N-Plap-1+/+ mice were analyzed in terms of alveolar bone level,femur volume and mouse body weight.Experimental periodontitis models were established in 8-week-old C57BL/6N-Plap-1-/-and C57BL/6N-Plap-1+/+ mice.The experiment was divided into C57 PLAP-1+/+ and C57 PLAP-1-/-groups.Maxillary tissue was collected from the two groups at 7 and 14 days after operation,respectively.The level of alveolar bone resorption was observed by micro-CT.Hematoxylin-eosin staining(H&E),TRAP,immunohistochemical staining,qRT-PCR and Western blot were performed to study the expressions of osteogenic and osteoclast related markers in the two groups.The experimental model of subcutaneous osteogenesis in nude mice was established to verify the effect of PLAP1 on bone formation,and the experiment was divided into Nu PLAP-1+/+ and Nu PLAP-1-/groups.BMSCs(1 × 107 cells)in LPS+PLAP-1+/+ and LPS+PLAP-1-/-groups were implanted subcutaneously with hydroxyapatite/tricalcium phosphate(HA/TCP)compound after 1 week of mineralization induction.Bone tissue complexes were taken at 6 w after operation,and the differences in bone formation were detected by H&E and Masson staining.(2)TGF-β1/Smad may be the key pathway for PLAP-1 to affect alveolar bone imbalanceThe expression and localization of PLAP-1/TGF-β1 in periodontal tissues were studied by immunofluorescence staining.The expression levels of phosphorylated Smad1 and Smad3 downstream of TGF-β1 were detected by protein levels,confirming that PLAP-1 affects bone imbalance in experimental periodontitis by regulating TGF-β1/Smad pathway.Results1.PLAP-1/TGF-β1/Smad1 signaling pathway promoted the osteoclast differentiation and functionThe results of agarose gel electrophoresis,qRT-PCR and Western blot showed that C57BL/6N-Plap-1-/-mice were successfully constructed.In normal,inflammatory and recovery environments,the osteoclast differentiation ability of BMMs and bone resorption area decreased after Plap-1 gene knockout.During osteoclast differentiation,a large area of coexpression of PLAP-1 and TGF-β1 was observed by immunofluorescence.The results of coimmunoprecipitation showed the interaction between the two proteins.The phosphorylation level of Smad1 decreased in the osteoclasts of Plap-1 knockout mice,and the addition of LY 364947 and SB 501542 prevented PLAP-1 from affecting the differentiation and function of osteoclasts.These results suggest that TGF-β1/Smad1 signaling pathway is involved in the effect of PLAP-1 on osteoclast differentiation.2.PLAP-1/TGF-β1/Smad3 signaling pathway inhibited the osteoblast differentiation and functionCompared with normal BMSCs,the results of qRT-PCR and Western blot showed that the indicators related to osteogenic differentiation of Plap-1 gene knockout BMSCs were significantly increased,ALP staining and ALP activity detection technology also showed the same trend.Alizarin red staining results showed that the number of mineralized nodules was significantly increased.Co-immunoprecipitation confirmed that there was interaction between PLAP-1 and TGF-β1 during osteogenic differentiation of BMSCs in LPS+PLAP-1+/+group.Compared with LPS+PLAP-1+/+ group,Smad3 phosphorylation level was increased in LPS+PLAP-1-/-group,and TGF-β1 and TGF-β1/Smad3 inhibitors blocked the effect of PLAP1 on osteoblast differentiation.The expression of Smad protein complex and the addition of inhibitors suggest that PLAP-1 may regulate osteoblast differentiation and function through TGF-β1/Smad3 signaling pathway.3.PLAP-1 regulated osteoclast/osteoblast differentiation by TGF-β1/Smad pathway in coculture systemThe results of mRNA and protein levels showed that in the inflammatory indirect coculture system,the osteoclast-related differentiation indexes of Plap-1 knockdown BMMs were significantly decreased,and the osteogenic differentiation related indexes of BMSCs were increased.Scanning electron microscopy results showed that the area of bone resorption was reduced in B+C-PLAP-1-/-group.The phosphorylation level of Smad1 in B+C-PLAP-1-/-group decreased,and the phosphorylation level of Smad3 in C-PLAP-1-/-group increased.After blocking TGF-β1 and TGF-β1/Smad signaling pathways,the effects of PLAP-1 on osteoclastic differentiation of BMMs and osteogenic differentiation of BMSCs were reduced.The difference of Smad protein complex and the addition of pathway inhibitors suggest that TGF-β1/Smad pathway may be involved in PLAP-1 regulation of osteoclast/osteoblast differentiation.4.TGF-β1/Smad signaling pathway was a key mechanism by which PLAP-1 affects bone imbalance in experimental periodontitisThere was no significant difference between C57BL/6N-Plap-1+/+ mice and C57BL/6NPlap-1+/+ mice by analyzing alveolar bone level,femur volume and body weight of mice.In the mouse experimental periodontitis model,firstly,micro-CT scanning results showed that alveolar bone resorption level in the C57 PLAP-1-/-group was significantly decreased compared with that in the C57 PLAP-1+/+group.Secondly,mRNA and protein levels and histochemical staining results showed that the expression of osteoclastic differentiation related markers in C57 PLAP-1-/-group was significantly decreased,while the expression of osteogenic differentiation related markers was significantly increased.At the same time,the results of H&E and Masson staining showed that the Nu PLAP-1-/-group had enhanced bone formation ability(consistent with the results of experimental periodontitis model).A large area of co-expression of PLAP-1 and TGF-β1 around alveolar bone was observed by immunofluorescence staining.The expression of Smad protein complex:compared with C57 PLAP-1+/+group,the phosphorylation level of Smad1 in C57 PLAP-1-/-group decreased,and the phosphorylation level of Smad3 increased.These results suggest that TGF-β1/Smad may be one of the key pathways of PLAP-1 affecting alveolar bone imbalance during periodontitis.Conclusion1.Through the culture of primary bone marrow-derived mononuclear macrophages and bone marrow mesenchymal stem cells,it is demonstrated that PLAP-1 promotes osteoclast differentiation and inhibits osteoclast differentiation after osteoclast induction and osteogenic induction,respectively.It reveals that TGF-β1/Smad1 signaling pathway is involved in the process of PLAP-1 affecting osteoclast differentiation.At the same time,TGF-β1/Smad3 signaling pathway is involved in the process of PLAP-1 affecting osteoblast differentiation.2.Through indirect co-culture of osteoblasts and osteoclasts in an inflammatory environment,the role of PLAP-1 in promoting osteoclast differentiation and inhibiting osteoblast differentiation is confirmed,and the important correlation between TGF-β1/Smad signaling pathway and PLAP-1 in coordinating osteoclast and osteoblast differentiation is verified.3.Experimental periodontitis models and subcutaneous osteogenesis models in nude mice show that PLAP-1 promotes alveolar bone resorption while inhibiting alveolar bone formation,and TGF-β1/Smad pathway may be a key pathway in the effect of PLAP-1 on alveolar bone imbalance. |
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