The Role Of Nrf2 In Chronic Apical Periodontitis

Objectives: The expression of Nrf2 in chronic periapical periodontitis was detected by collecting periapical tissues of patients with chronic periapical periodontitis.To study the expression of Nrf2 in osteoclasts and osteoblasts and its effect on proliferation and differentiation of these two kinds of cells,and to explore the mechanism of Nrf2 in bone destruction of chronic periapical periodontitis.Methods:1.The experimental samples were collected from clinical patients admitted to the Stomatological Hospital of Dalian Medical University from January 2019 to December2020.The experimental group was clinically diagnosed with chronic periapical periodontitis and needed apical surgical treatment or tooth extraction surgery.In the control group,healthy premolars were removed for orthodontic needs or the periodontal tissue of the apical root of the third molar was removed because of the impacted third molar.All samples were collected with the approval of the Ethics Committee of Dalian Medical University and the informed consent of the patients.The collected tissues were divided into experimental group(n=32)and control group(n=22)according to the granulation tissues of chronic periapical periodontitis and healthy periapical periodontitis,respectively.The expression of Nrf2 in 6 samples of the experimental group and 6samples of the control group was detected by immunohistochemical assay.The expressions of Nrf2 and TRAP in the experimental group(n=20)and the control group(n=10)were detected by real-time quantitative PCR.The expression of Nrf2 and TRAP in6 samples of experimental group and control group were detected by Western blot assay.2.The effect of Nrf2 on the proliferation and differentiation of osteoclasts and osteoblasts in LPS-mediated inflammatory microenvironment.(1)Osteoclasts and osteoblasts cell induction and identification1)Culture RAW264.7 cells in vitro,induce them with 0.1 μg/ml RANKL induction solution,detect TRAP gene by light microscope,q PCR and TRAP staining to identify whether the osteoclasts are successfully induced.2)MC3T3-E1 cells were induced to identify the induced OB,q PCR was used to detect the expression of ALP gene,ALP staining and Alizarin red staining.(2)The expression of Nrf2 in osteoclasts and osteoblasts under LPS-mediated inflammation.1)100 ng/ml LPS acts on osteoclasts,q PCR detects the gene expression of Nrf2,TRAP and CTSK,Wb detects Nrf2,TRAP and CTSK protein expression;2)1000 ng/ml LPS acts on osteoblasts,q PCR detects the gene expression of Nrf2,ALP and Runx2,Wb detects the protein expression of Nrf2,ALP and Runx2.(3)Under LPS-mediated inflammation,down-regulating the effect of Nrf2 on the proliferation and differentiation of osteoclasts and osteoblasts.1)After transiently transfecting osteoclasts and osteoblasts with Nrf2-si RNA,the transfection efficiency was verified.2)CCK-8 experimental detection of down-regulation of Nrf2 on the proliferation of osteoclasts and osteoblasts.3)q PCR detection of down-regulated Nrf2 osteoclast TRAP and CTSK gene expression,osteoblast ALP and Runx2 gene expression.4)Wb detects the protein expression of TRAP and CTSK in osteoclasts after downregulation of Nrf2,and the protein expression of ALP and Runx2 in osteoblasts.Results:1.Immunohistochemical staining showed that the expression of Nrf2 in 6 cases of chronic periapical periodontitis was higher than that in 6 cases of healthy periodontium(P<0.0001).Real-time PCR results showed that the gene expression levels of Nrf2 and TRAP in 20 cases of chronic periapical periodontitis were higher than that in 10 cases of healthy periodontal membrane(P<0.05).Western blot also showed that the protein expressions of Nrf2 and TRAP in 6 cases of chronic periapical periodontitis were higher than those in 6 healthy controls(P<0.05).2.The effect of Nrf2 on the proliferation and differentiation of osteoclasts and osteoblasts in the LPS-mediated inflammatory microenvironment.(1)Induction and identification of osteoclasts and osteoblasts1)Culture RAW264.7 cells in vitro and add RANKL induction solution five days after the induction,the RAW264.7 cells were found to be round,irregular,with three or more nuclei.q PCR test results showed that the level of TRAP m RNA increased(p<0.05).TRAP staining showed dark brown acid phosphatase particles in the cytoplasm.The results showed that RAW264.7 was successfully induced into osteoclasts.2)After 5 days of induction of MC3T3-E1 cells,q PCR assay showed that the expression of ALP m RNA was increased(p<0.05).The results of ALP staining showed that the cytoplasm was red.Alizarin red staining showed the formation of flaky dark red calcified nodules.The results showed that MC3T3-E1 was successfully induced into OB.(2)The expression of Nrf2 in osteoclasts and osteoblasts under LPS-mediated inflammation.1)q PCR results show that after 100 ng/ml LPS acts on OC,the expression of Nrf2,TRAP and CTSK are increased(p<0.05),Wb results showed that the expression of Nrf2,TRAP and CTSK increased(p<0.05).2)q PCR results showed that after 1000 ng/ml LPS acted on OB,the m RNA level of Nrf2 increased,and ALP and Runx2 decreased(p<0.05).Wb results showed that the expression of Nrf2 increased,ALP and Runx2 protein expression decreased(p<0.05).(3)Under LPS-mediated inflammation,down-regulating the effect of Nrf2 on the proliferation and differentiation of osteoclasts and osteoblasts.1)Verification of transfection efficiency: After transiently transfecting osteoclasts and osteoblasts with Nrf2-si RNA,fluorescence microscopy.The observation results showed that the si RNA fragments were successfully transferred into the cells,and q PCR results showed that the expression of Nrf2 in OC and OB decreased(p<0.05).2)CCK-8 results showed that Nrf2-si RNA transfection promoted the proliferation of osteoclasts and osteoblasts.3)q PCR results showed that TRAP and CTSK expression increased in OC with Nrf2-si RNA(p<0.05),and ALP and Runx2 expression in OB increased(p<0.05).4)Wb results showed that TRAP and CTSK expression in OC increased with Nrf2-si RNA(p<0.05),and ALP and Runx2 expression in OB increased(p<0.05).Conclusions:Nrf2 is involved in the disease process of chronic periapical periodontitis.In the inflammatory environment mediated by LPS,Nrf2 may participate in the occurrence and development of bone destruction in periapical periodontitis by regulating the proliferation and differentiation of osteoclasts and osteoblasts.