Roles And Mechanisms Of Calcitonin Gene-Related Peptide Mediating Pg-LPS-induced Apoptosis In Osteoblasts

Bone loss is caused by the imbalance of bone remodeling.Pathological changes in bone tissue not only depend on the number of osteoclasts and osteoblasts but also are closely associated with the apoptosis-mediated lifecycle of these cells.Extensive studies in recent years have shown that apoptosis participates in the development and progression of periodontitis.Periodontal pathogens mainly include Gram-negative anaerobic bacteria.LPS is a major component of the cell wall of Gram-negative bacteria.LPS is considered to be a pivotal factor for inducing the destruction of alveolar bone tissue and for the development and progression of periodontitis.It can directly inhibit the differentiation of periodontal target cells and increase the absorption of periodontal tissues.Therefore,LPS is of growing concern for its role in alveolar bone resorption and periodontitis incidence.Research has verified that LPS and the metabolites of periodontal pathogens can affect matrix formation and cellular apoptosis.Regarding this imbalanced process,the biological behavior of osteoblasts is particularly important.LPS can cause inflammatory responses in osteoblasts and induce abnormal osteoblast apoptosis,which can lead to a disorder in the number of cells coupled between bone resorption and formation,thereby causing bone loss.Currently there are few effective treatments for bone destruction caused by bacteria.A major objective of bone loss prevention is to investigate the protection of osteoblast function and activity in the imbalanced state and search for potential drug targets.Calcitonin gene-related peptide(CGRP)was first extracted from medullary thyroid carcinoma tissue in 1971.CGRP comprises 37 amino acids and includes two isomers,a-cgrp and b-cgrp.Originally,CGRP was found to be often stored as secretory granules in sensory nerve endings.Recently,CGRP has been shown to be widely distributed in the nervous,cardiovascular,respiratory,and digestive systems.CGRP has diverse physiological effects in various tissues and controls the immune response.Exogenous CGRP can increase the number and size of osteoblast colonies.In bone tissues,CGRP is not only produced in sensory nerve fibers and endings but also generated by osteoblasts,and it functions in both autocrine and paracrine signaling.It has been recently reported that CGRP,as the intersection of the nervous and immune systems,has a potential role in regulating inflammation.CGRP can directly affect CD4+ T helper cells and can influence the function of antigen-presenting cells in order to regulate adaptive immune responses.Additionally,CGRP can suppress the release of inflammatory cytokines,such as interleukin(IL)-1β,tumor necrosis factor(TNF)-α,and carbon tetrachloride,by monocyte-macrophage cells and dendritic cells upon stimulation by inactivated bacteria or Toll-like receptor agonists.Extensive studies allowed us to ascertain that CGRP is a potential regulator of inflammation that can inhibit the production of pro-inflammatory cytokines;CGRP plays a major regulatory role in the inflammatory process.However,the effect of CGRP on Pg-LPS-induced osteoblast apoptosis is unclear.Here,we observed that CGRP blocks Pg-LPS-induced cytostatic activity and apoptosis,while TNF-α plays an important reverse role in this process.The aim of the present study was to assess the in vitro effect of CGRP on Pg(Porphyromonas gingivalis)LPS-induced osteoblast apoptosis.Materials and Methods1:Cell culture and reagentsThe primary osteoblasts were obtained from the calvaria of BALB/C mice and cultured in Dulbecco’s modified Eagle medium(DMEM;Gibco,11965)containing 10% fetal bovine serum(FBS;Gibco,10100),1.5 g/L sodium bicarbonate(Gibco,25080),0.11 g/L sodium pyruvate(Gibco,11360)and 100 μg/ml penicillin/streptomycin(Gibco,15140).Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.Calcitonin gene related peptide(CGRP,C0167),and tumor necrosis factor-α(TNF-a,T6674)were purchased from Sigma–Aldrich.Pg-LPS(Invivogen,San Diego,CA)was reconstituted in distilled and deionized water according to the manufacturer’s recommendation.2:Cell viability(CCK8)assayCell viability was evaluated using a Cell counting kit-8(CCK8;Beyotime Biotechnology,C0038)according to the manufacturer’s instructions.Briefly,Osteoblasts(1.0 × 104 per well)were seeded in 96-well plates and were cultured overnight.After treatment as indicated for 48 h dependent on experimental conditions.10 μL of CCK8 was added to each well and incubated for 2 h,the absorbance of each well was measured by microplate reader(Bio Rad,iMark 680)at 450 nm.All experiments were performed independently and repeated for three times.The cell viabilities were normalized to the control group.3:Enzyme-linked immunosorbent assay(ELISA)The ELISA kit for human TNF-a(MTA00B),IL-1β(MLB00C),IL-6(M6000B),MCP-1(MJE00)and MCP-2(DY790)were all purchased from R&D Systems.The concentrations of TNF-a,IL-1β,IL-6,MCP-1 and MCP-2 in cell culture supernatants were determined using the properly ELISA kit according to the manufacturer’s instructions.The optical density of each well was recorded by using microplate reader at 450 nm and normalized to the control group.4:Apoptosis assayApoptosis was assessed by flow cytometry using an Annexin V-FITC/PI staining kit(BD Biosciences,556547)according to the manufacturer’s instructions.Briefly,cells were collected and washed twice with cold phosphate-buffered saline(PBS),and then resuspended in staining buffer containing Annexin V-FITC(0.025 μg/ml)and PI(1 μg/ml)and incubated for 15 min in dark.After washing twice by PBS,apoptotic cells were analyzed by flow cytometry(FACScan,Becton Dickinson).5:Western blot analysisCells were treated and collected by centrifugation at 2,000 rpm at 4°C,after washing by PBS,cells were lysed in RIPA buffer(50 m M Tris(p H 7.4),150 mM NaCl,1% sodium deoxycholate,1 mM sodium orthovanadate,1% Triton X-100,0.1% SDS,10 μg/ml aprotinin,10 μg/ml leupeptin,1 m M PMSF)on ice for 30 min.The supernatants were collected by centrifugation at 13,000 rpm for 5 min at 4?C.The protein concentrations were measured using an Enhanced BCA Protein Assay Reagent(Beyotime Biotechnology,P0010).Equal amounts of cellular protein were boiled for 10 min and separated on 12% SDS-PAGE gel,and then transferred to polyvinylidene difluoride(PVDF)membrane(Bio-Rad,162-0177)at 50 V for 3 h at 4?C.After blocking with 5% nonfat dried milk for 2 h at room temperature,membranes were incubated with appropriate primary antibodies as follows: CRGP(Cell Signaling Technology,14959;1:1000),Cleaved-Caspase-8(Cell Signaling Technology,9505;1:500),Cleaved-Caspase-3(Cell Signaling Technology,9664;1:500),TNF-a(Abcam,ab6671;1:1000)and Actin(Santa Cruz Biotechnology,sc-8432;1:1000)overnight at 4°C,and then washed three times in 1×TBST buffer(Tris-buffered saline containing 0.1% Tween-20),membranes were followed by incubation with Horseradish peroxidase-conjugated goat anti-rabbit(Kirkegaard and Perry Laboratories,474-1506)or Horseradish peroxidase-conjugated goat anti-mouse(Kirkegaard and Perry Laboratories,474-1806)secondary antibodies for 2 h at room temperature.After washing by 1×TBST buffer,the bands were visualized using the enhanced chemiluminescence kit(Bio-Rad,170-5061)according to the manufacturer’s instructions.Densitometric analysis of the blots were performed using Quantity One software(Bio-Rad,Germany)and normalized to the the actin level.Results1.Pg-LPS induces the cell vability inhibition and apoptosis.To assess the effect of Pg-LPS on the activity of osteoblasts,we treated osteoblasts with different concentrations of Pg-LPS and then analyzed the cellular activity after 48 h.that osteoblast activity was significantly reduced by Pg-LPS at concentrations of 50,100,500 and 1000 ng/mL(P < 0.05).activity of the Osteoblasts after being stimulated with 500 ng/mL Pg-LPS for various time periods.As the stimulation time increased,the activity of the Osteoblasts significantly decreased(P <0.01).The apoptosis analysis revealed that the rate of OSTEOBLAST cell apoptosis increased with increasing concentrations of Pg-LPS.At the same concentration of Pg-LPS,the rate of osteoblasts apoptosis was positively correlated with treatment time.2.Calcitonin gene-related peptide(CGRP)attenuate the cell viability inhibition and apoptosis induced by Pg-LPS.Western blot results showed that a transient increase occurred in CGRP protein expression at 6 h;thereafter,the protein levels gradually decreased over time.The results of a CCK8 assay showed that pre-treatment of Pg-LPS-stimulated osteoblasts with 100 nM CGRP significantly reduced the cytostatic activity of Pg-LPS on osteoblasts.Next,we determined the apoptosis rate using flow cytometry.Consistent with the above results,100 n M CGRP markedly suppressed the Pg-LPS-induced apoptosis of osteoblasts.Additionally,CGRP suppressed the Pg-LPS-induced upregulation of cleaved caspase(c-caspase)3 and c-caspase 8 protein levels.3.CGRP blocks Pg-LPS-induced TNF-a expression in Osteoblasts.LPS is a classic endotoxin and has long been considered to be a trigger of periodontal diseases.LPS can induce cells to release large amounts of inflammatory cytokines,such as TNF-α,IL-1β and IL-6 and cause a series of inflammatory reactions.ELISA data show that compared with the control,Pg-LPS promoted TNF-α,IL-1β,IL-6,monocyte chemotactic protein(MCP)-1 and MCP-2 production in a time-dependent manner.Pre-treatment with CGRP did not effectively reduce IL-1β,IL-6,MCP-1 and MCP-2 production,while only TNF-α production was significantly inhibited.Western blot analysis also showed that the Pg-LPS-induced increase in the protein level of TNF-α was suppressed by CGRP.4.TNF-a is a key molecule in osteoblasts viability inhibition and apoptosis induced by Pg-LPS and reversed by CGRP.CCK8 assay and flow cytometry results revealed that CGRP mitigated Pg-LPS-induced cytostatic activity and apoptosis in the cells;however,this effect was not observed in the presence of TNF-α.Furthermore,the analysis results for c-caspase 8 and c-caspase 3 confirmed that CGRP inhibited the Pg-LPS-induced upregulation of c-caspase 8 and c-caspase 3 levels;however,CGRP could not inhibit the upregulation of c-caspase 8 and c-caspase 3 expression in the presence of both Pg-LPS and TNF-α.TNF-α is an important pro-inflammatory cytokine and a major bone resorption factor.TNF-α mainly acts on osteoclasts and osteoblasts,and it can cause osteoblast apoptosis.The current results demonstrate that CGRP inhibited Pg-LPS-induced apoptosis;however,this phenomenon was reversed by TNF-α.We speculate that TNF-α is likely the key factor playing an opposing role in the CGRP inhibition of Pg-LPS-induced osteoblast apoptosis.Conclusion:1: Pg-LPS induces the cell vability inhibition and apoptosis.2:Calcitonin gene-related peptide(CGRP)attenuate the cell viability inhibition and apoptosis induced by Pg-LPS.3.CGRP blocks Pg-LPS-induced TNF-a expression in Osteoblasts.4.TNF-a is a key molecule in osteoblasts viability inhibition and apoptosis induced by Pg-LPS and reversed by CGRP.