| Objective: To detect the expression of Fibroblast activation protein α(FAPα)and Integrin α5(ITGA5)in inflammatory tissues by collecting apical granulation tissues from patients with chronic apical periodontitis.The inflammatory microenvironment model of Raw264.7 cells,osteoclasts and RAW264.7-MC3T3-E1co-cultured cells was established to explore the expression of FAPα and ITGA5 under inflammatory conditions in vitro.By confirming the formation of FAPα/ITGA5 protein complex and its mechanism of regulating the destruction of the bone of apical periodontitis,it provides a new idea for clinical treatment of apical periodontitis.Methods:(1)To verify the expression of FAPα and ITGA5 in human apical periodontitisCases clinically diagnosed as chronic apical periodontitis were collected,the apical granulation tissue requiring apical surgery or tooth extraction and the periodontal membrane tissue of healthy permanent teeth requiring tooth extraction for orthodontic treatment were divided into experimental group(5 cases)and control group(5 cases)according to chronic apical periodontitis tissue and healthy periapical tissue,respectively.The expressions of FAPα and ITGA5 in the experimental group and the control group were detected by immunohistochemical staining.(2)To verify the expression of FAPα and ITGA5 in LPS-induced inflammatory microenvironment1)After induction of RAW264.7 cells in vitro,TRAP staining and Real-time qPCR were used to verify the successful induction of osteoclasts.2)RAW264.7 cells and osteoclasts were stimulated by 100 ng/ml LPS for 1 day and 7 days,respectively.The mRNA expression levels of FAPα,ITGA5 and Acp5 genes related to osteoclasts were detected by Real-time qPCR.The protein expression levels of FAPα,ITGA5 and TRAP were detected by Western blot.3)RAW264.7 and MC3T3-E1 cells were directly co-cultured with 100 ng/ml LPS for 24 h,and the mRNA expression levels of FAPα,ITGA5 and bone-related genes Acp5 and ALP were detected by Real-time qPCR.Western blot was used to detect the protein expression levels of FAPα,ITGA5,TRAP and ALP of bone related genes.(3)To verify the formation and mechanism of FAPα/ITGA5 protein complex1)Protein molecular docking: ZDOCK software predicted the possible binding mode of FAPα and ITGA5 protein,and PDBe PISA software analyzed the binding force of their interaction surface.2)Cell assay: The localization of FAPα and ITGA5 in RAW264.7,osteoclasts was observed by immunofluorescence staining.The interaction between FAPα and ITGA5 in RAW264.7 cells and 293-T cells was verified by immunoprecipitation assay.After transfection of FAPα-siRNA and ITGA5-siRNA in RAW264.7 cells,LPS was added for 24 hours to further verify the effect of FAPα/ITGA5 protein complex on osteoclast differentiation in inflammatory microenvironment.The mRNA expression levels of FAPα,ITGA5 and Acp5 genes related to osteoclasts were detected by Real-time qPCR,and the protein expression levels of FAPα,ITGA5 and TRAP genes related to osteoclasts were detected by Western blot.Results:(1)The expression of FAPα and ITGA5 in human apical periodontitisThe results of immunohistochemical staining showed that the expressions of FAPαand ITGA5 in the chronic apical periodontitis tissues were higher than those in the control tissues(p < 0.0001).(2)The expression of FAPα and ITGA5 in LPS-mediated inflammatory microenvironment1)After 7 days of RANKL treatment,RAW264.7 cells showed multinucleation and morphological changes,with Pseudopod Protrusion.TRAP staining results showed that the cytoplasm contained wine red acid phosphatase particles,and Real-time qPCR results showed increased Acp5 gene expression(p < 0.01).All the above results indicated that osteoclast induction was successful.2)After 1 day and 7 days of LPS treatment,Real-time qPCR results showed that the growth rate of FAPα and ITGA5 mRNA in RAW264.7 cells decreased with time,which was higher than that in negative control group,while the growth rate of Acp5 mRNA increased with time.They were higher than those in negative control group(p <0.05).Western blot results showed that the protein expressions of FAPα,ITGA5 and TRAP increased gradually with the extension of time after 1 day and 7 days of LPS treatment,and were higher than those of negative control group(p < 0.05).3)After 1 and 7 days of LPS treatment,Real-time qPCR results showed that the mRNA growth rates of FAPα,ITGA5 and Acp5 in osteoclasts increased gradually with the passage of time,and were all higher than those in negative control group(p < 0.05).Western blot results showed that,after 1day and 7 days of LPS treatment,the growth rate of the protein expression of FAPα and ITGA5 decreased gradually as time went on,and the growth rate of the protein expression of TRAP increased gradually as time went on,and the growth rate was higher than that of the negative control group(p < 0.05).4)After LPS was applied to RAW264.7-MC3T3-E1 co-cultured cells for 24 hours,the Real-time qPCR results showed that the mRNA expressions of FAPα,ITGA5 and Acp5 were higher than those in the control group,while the mRNA expression of ALP was lower than that in the control group(p < 0.05).Western blot results showed that the protein expressions of FAPα,ITGA5 and TRAP were higher than those of control group,but the protein expressions of ALP were lower than those of control group(p < 0.05).(3)Formation and mechanism of FAPα/ITGA5 protein complex1)Protein molecular docking results showed that the free energy of FAPα/ITGA5 protein complex interaction surface was-14.5 kcal/mol,indicating that it had good binding activity.2)Immunofluorescence staining showed that FAPα and ITGA5 co-located in RAW264.7 cells and osteoclasts.3)The results of co-immunoprecipitation showed that FAPα could bind to ITGA5 to form the FAPα/ITGA5 protein complex.4)After transfection with FAPα-siRNA,LPS was added to RAW264.7 cells for 24 hours.Real-time qPCR results showed that the expression levels of FAPα,ITGA5 and Acp5 mRNA were significantly decreased(p < 0.05).Western blot results showed that the protein expressions of FAPα,ITGA5 and TRAP were significantly decreased(p <0.05).5)After transfection with ITGA5-siRNA,LPS was added to RAW264.7 cells for24 hours.Real-time qPCR results showed that the expression levels of ITGA5,FAPαand Acp5 mRNA were significantly decreased(p < 0.05).Western blot results showed that the protein expressions of ITGA5,FAPα and TRAP were significantly decreased(p< 0.05).Conclusions: In inflammatory microenvironment,FAPα and ITGA5 participate in the regulation of osteoclast differentiation through the formation of protein complex,and then participate in the development of chronic apical periodontitis. |
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