Biological Effects Of Overexpression Of CGRP On Rat Bone Mesenchymal Stem Cells And RAW264.7

Background and Purposes:Periodontitis is a chronic infectious disease caused by microorganisms in dental plaque.It results in periodontal tissue damage such as attachment loss and alveolar bone resorption.At present,the treatment of periodontitis is mainly limited to the control of disease instead of periodontal tissues regeneration.The difficulty of periodontal bone regeneration lies in its continuous relatively open inflammatory microenvironment,compared with jaw regeneration and implant bone proliferation which are in a closed environment.Mesenchymal stem cells have been widely used in the study of tissue regeneration,but the function of stem cells in inflammatory microenvironment is affected,and the ability of osteogenic differentiation is reduced.It is difficult to obtain enough bone regeneration.Porphyromonas gingivalis(P.gingivalis)is one of the main pathogenes of periodontitis,lipopolysaccharide(LPS)is a key virulence factor to induce alveolar bone tissue damage and promote the development and progress of periodontitis.LPS can directly inhibit the differentiation of periodontal stem cells and increase the resorption of alveolar bone.In addition,LPS can induce a variety of cells to secrete inflammatory factors such as IL-1β and TNF-α,which could reduce the formation of matrix,induce apoptosis,and lead to the destruction of periodontal tissues.Calcitonin gene-related peptide(CGRP)is a 37-amino-acid neuropeptide secreted by sensory nerve endings.CGRP participates in the formation and repair of bone tissue.Besides,some studies show that CGRP can inhibit inflammation.Our previous study showed that overexpression of CGRP significantly enhanced the osteogenic differentiation of rat bone mesenchymal stem cells(rBMSCs)under normal conditions.The present study was to investigate the effects of CGRP on the immune response and osteogenic differentiation of rBMSCs under inflammatory environment.The effects of rBMSCs with overexpression of CGRP on inflammatory infiltration and bone repair were further studied in LPS treated rat periodontal defects,which may provide a theoretical support for further study of periodontal repair and regeneration under inflammatory microenvironment in clinical.In the meantime,this study also tried to investigate the effects of CGRP on the immune response and osteoclastic differentiation of RAW264.7,which could help us to learn more about the function of CGRP on osteogenisis and osteoclatic differentiation.Materials and methods:1.Isolation,culture and identification of rat bone mesenchymal stem cells.rBMSCs were isolated and cultured in vitro.The characteristics of rBMSCs were identified by detection of surface markers and multiple differentiation potential.2.Construction of rBMSCs and RAW264.7 with overexpressing CGRP gene.1)rBMSCs were infected with lentivirus.The experiment was divided into three groups:c-rBMSCs group(CGRP gene overexpressed rBMSCs),empty vector group(empty vector transfected rBMSCs),rBMSCs group(normal rBMSCs).The cells of three groups were detected the mRNA and protein expressions of CGRP separately.2)RAW264.7 were infected with lentivirus.The experiment was divided into three groups:c-raw group(CGRP gene overexpressed RA W264.7),empty vector group(empty vector transfected RAW264.7),raw group(normal RAW264.7).The cells of three groups were detected the mRNA and protein expressions of CGRP separately.3.Effects of overexpression of CGRP on proliferation,immune response and osteogenesis in LPS-stimulated rat bone mesenchymal stem cells.1)The proliferations of cells were detected by CCK-8.2)The mRNA expressions of IL-1β and TNF-α were detected by Realtime PCR;The secretion levels of of IL-1β and TNF-α were detected by ELISA.3)The mRNA expressions of ALP、BSP and RUNX2 were detected by Realtime PCR;The protein expressions of ALP、BSP and RUNX2 were detected by Western Blot.4)Effects of rBMSCs with overexpression of CGRP on inflammatory infiltration and bone repair in LPS treated periodontal defects were detected by HE staining and Micro-CT.4.Effects of overexpression of CGRP on immune response and osteoclastic differentiation of RAW264.7.1)The mRNA expressions of IL-1β and iNOS were detected by Realtime PCR.pp65/p65 ratio was also detected by Western Blot after LPS-stimulation.2)The protein expressions of CTSK、c-FOS and NFATc-1 were detected by Western Blot.Results:1.rBMSCs were cultured in vitro and identified the cell characteristics.rBMSCs were successfully isolated and cultured in vitro,and they were mainly spindle cells.Flow cytometry analysis showed the surface makers of rBMSCs,and the expressions of CD29(98.9%)、CD44(98.4%)、CD90(98.8%)、CD45(0.20%)、CD11b(0.41%)accord with MSCs.After induction of adipogenic and osteogenic,oil red O and Alizarin Red staining of rBMSCs were positive,which proved the multiple differential potential of rBMSCs.2.Construction of rBMSCs and RAW264.7 with overexpressing CGRP gene.The detection of protein and mRNA expressions of CGRP showed obviously high expression of CGRP in c-rBMSCs and c-raw groups(P<0.05).The protein and mRNA expression of CGRP had no significant difference between another two groups(P>0.05).3.Effect of overexpression of CGRP on proliferation,immune response and osteogenesis of LPS-stimulated rBMSCs.1)Overexpression of CGRP could promote proliferation of LPS-stimulated rBMSCs.2)After 24-hour stimulation of LPS,the mRNA and secretions of IL-1β and TNF-α in the c-rBMSCs group were lower compared with the rBMSCs group(P<0.05).3)Under inflammatory conditions,the mRNA and protein expressions of ALP,BSP and RUNX2 in the c-rBMSCs group at 2w and 3w were all higher compared with the rBMSCs group(P<0.05).4)HE staining of periodontal defects showed that the inflammatory infiltration in c-rBMSCs group was remarkablely less than that in Control group and rBMSCs group.Micro-CT showed that the mineralization density of the new bone in c-rBMSCs group was significantly higher than that in Control group and rBMSCs group(P<0.05),and the new formed bone had filled the majority of the periodontal defects in c-rBMSCs group.4.Effect of overexpression of CGRP on immune response and osteoclatic differentiation of RAW264.7.1)After 24-hour stimulation of 1 μg/ml LPS,the mRNA expressions of IL-1β and iNOS in the c-raw group were lower compared with the raw group(P<0.05);the pp65/p65 ratio was lower in the c-raw group than in the raw group(P<0.05)after LPS-stimulation.2)After 1-week induction of 30ng/ml RANKL,the mRNA and protein expressions of CTSK、c-FOS and NFATc-1 in the c-raw group were all lower than in the raw group(P<0.05)Conclusions:1.Overexpression of CGRP downregulates the expressions of IL-1β and TNF-α in LPS-stimulated rBMSCs and upregulates osteogenic differentiation of LPS-stimulated rBMSCs.rBMSCs with overexpression of CGRP can significantly reduce inflammatory infiltration and promote bone formation in LPS treated rat periodontal defects.2.Overexpression of CGRP downregulates the expressions of IL-1β and iNOS in LPS-stimulated RAW264.7 and downregulates osteoclast differentiation factors of RANKL-induced RAW264.7.