| BackgroundPeriodontitis is a common oral disease and the primary cause of tooth loss.It has been well established that periodontal disease is a bacterial inflammation induced by the Lipopolysaccharide(LPS)released from microorganism,which furthermore arouse immune response and result in alveolar bone lose and inflammatory gingival destruction.It has been proven that LPS could mediate the deacetylation of high mobility group box-1(HMGB1).After deacetylation,HMGB1 would be transferred into cytoplasm and finally be released into excellular.Above-mentioned process suggests that HMGB1 may be the downstream cytokine of LPS and affect the disease secondary to LPS.However,the role of HMGB1 in the pathological mechanism of periodontitis remains largely unknow.At the beginning of inflammation,macrophages are recruited to the the periodontal tissue to participate in the immunal response and give rise to the gingivitis.Macrophages can mediate the phagocytosis in innate immunity and release inflammatory cytokines.They exert different function according to their different states of activation or polarization.In the later stages of inflammation,macrophages could differentiate into pre-osteoclasts under certain stimuli to mediate bone resorption.Both macrophages and pre-osteoclasts are derived from hematopoietic monocyte and they have homology to some extent.Pre-osteoclasts,are considered as another stage of macrophages,have the function of both osteoclasts and macrophages.Hence,macrophages and pre-osteoclasts are central players in the pathogenesis of periodontitis.Recent studies proved that macrophages are functionally heterogeneous cell populations,which could polarize into different phenotypic under different micro environmental stimulation.According to the environment that the macrophages are exposed to,the cytokines they release,and cellular functions they exhibit,macrophages can be classified as two distinct phenotypic.Classically activated/inflammatory macrophages(M1)are associated with increased microbicidal activity and antigen-presenting function which contributes to the inflammatory response.Alternatively activated/regenerative macrophages(M2)are associated with anti-inflammatory and homeostatic functions linked to wound healing,fibrosis and tissue repair.The ratio of M1/M2 type macrophages affect the progress of the disease.Our research aims to investigate how LPS and its downstream cytokine HMGB1 affect the progression of periodontal inflammatory diseases by mediating the polarization of macrophages and the expression of polarization-related genes in pre-osteoclasts,and explore the possible signaling pathways and underlying mechanisms invoved.The object of this project is to provide new ideas for the clinical treatment of periodontitis.Chapter 1 Detection of HMGB1,IL-6 and TNF-a Expressions in GingivalCrevicular Fluid ObjectiveIt’s well known that LPS is related to periodontitis,and HMGB1 released by macrophages is the downstream cytoine of LPS.Does HMGB1 play a role in periodontitis?IL-6 and TNF-a are the markers of M1 polarization.In view of the clinical significance of gingival crevicular fluid,the expression of HMGB1 in gingival crevicular fluid was detected to investigate the relationship between HMGB1 and periodontiris.The expression of IL-6 and TNF-a in gingival crevicular fluid were detected to explore the relationship between periodontitis and cell polarization.Method1.Experimental subject were chosen from the Department of Periodontitis,Stomatological Hospital,Southern Medical University.10 patients with chronic periodontitis were selected as the experimental group,with 5 patients were diagnosed as moderate degree and another 5 patients were diagnosed as severe degree.At the same time,10 persons without periodontal disease were selected as the control group.2.General conditional and systematic periodontal examinations were conducted.3.Filter paper were lightly inserted into the buccal gingival crevice for 30s to collect the gingival crevicular fluid.4.Weight gingival crevicular fluid,and converted it into volume according to the weight-volume ratio of gingival crevicular fluid 1 mg/μl.5.Per microliter of sample was dissolved and diluted into 50 μ1 of PBS solution.6.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of HMGB1,TNF-a and IL-6 protein in normal control group,moderate periodontitis group and severe periodontitis group.7.Statistical analysis was conducted.Results1.The HMGB1 protein expression level was 6.76 ng/ml in the control group,compared to 15.71 ng/ml in the experimental group.The HMGB1 protein expression level was 12.97 ng/ml in the moderate periodontitis group,compared to 18.44 ng/ml in the severe periodontitis group.2.The expression level of IL-6 was 0.11 ng/ml.in the control group,compared to 1.40 ng/ml in the experimental group.The expression level of IL-6 was 0.78 ng/ml in the moderate periodontitis group,compared to 2.02 ng/ml in the severe periodontitis group.3.The expression level of TNF-a was 0.28 ng/ml.in the control group,compared to 1.86ng/ml in the experimental group.The expression level of TNF-a was 1.45ng/ml in the moderate periodontitis group,compared to 2.27 ng/ml in the severe periodontitis group.Conclusion:The expression level of HMGB1,IL-6,TNF-a in gingival crevicular fluid increased progressively higher from control group,moderate periodontitis group to severe periodontitis group.The periodontitis promotes the expression and release of HMGB1,IL-6,TNF-a.Chapter 2 Effect of Lipopolysaccharide on the M1/M2 Types Related GeneExpression of Murine Macrophages RAW264.7 and RAW264.7 Pre-osteoclastsObjectiveLPS is the main component that induces periodontal inflammation.As major effector cells,macrophages and pre-osteoclasts participate in the pathological process of periodontal disease.There have been many studies on the relationship between LPS and periodontitis,but there are few studies on the role of macrophages,especially on pre-osteoclasts.The purpose of this experiment was to investigate the role of LPS in macrophages polarization and polarization related gene expression in pre-osteoclasts to explore its mechanism in periodontal inflammatory diseases,which helps to know how LPS cause periodontal bone destruction..Method1.The mouse macrophages cell line RAW264.7 was cultured in DMEM high glucose medium containing 10%fetal bovine serum,100 U/ml penicillin,and 100μg/ml streptomycin in 37 ℃,5%carbon dioxide incubator.And medium was changed every 2～3 d.2.With the stimulation of 100 ng/ml RANKL and 1%MCSF for 3 d,the macrophages were differentiated into RAW264.7 pre-osteoclasts,their morphology was observed under a microscope.3.RAW264.7 macrophages and pre-osteoclasts are devived into two groups.The experimental group was stimulated with LPS(Pg)1μg/ml in DMEM medium for 18 h,while the control group was cultured in DMEM medium for the same time.4.The cellular RNA was extracted and collected.The sample RNA concentration was measured and reverse transcribed into cDNA.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of M1 type macrophages polarization related genes,including:Inducible Nitric Oxide Synthases 2(NOS2),Tumor Necrosis Factor-a(TNF-a),Interleukin-6(IL-6)and CXC Motif Chemokine 11(CXCL-11);M2 type macrophages polarization related gene:Arginase 1(ARG-1),Resistin-like Molecule Alpha(RELM-a/FIZZ 1).GAPDH was used as the reference gene,and the final Cq value was calculated.And statistical analysis were performed.Results1.Marcrophages:The expression of M1 related mRNA were significantly upregulated after the stimulation of LPS(Pg).The expression of NOS2 was upregulated by the fold change of 19.34(P<0.01).The expression of IL-6 was upregulated by the fold change of 61.78(P<0.01).The expression of TNF-a was upregulated by the fold change of 2.12(P<0.01).The M2 related mRNA were downregulated after the stimulation of LPS(Pg).The expression of ARG-1 was downregulated by the fold change of 0.67(P>0.05).The expression of RELM-a was downregulated by the fold change of 0.42(P>0.05).2.Pre-osteoclasts:The expression of M1 related mRNA were significantly upregulated after the stimulation of LPS(Pg).The expression of NOS2 was upregulated by the fold change of 3.45(P<0.01).The expression of IL-6 was upregulated by the fold change of 112.27(P<0.01).The expression of TNF-a was upregulated by the fold change of 2.42(P<0.01).The expression of IL-1 was upregulated by the fold change of 1.30(P<0.05).The expression of CXCL-11 was upregulated by the fold change of 1.01(P>0.01).While the expression of M2 related mRNA,ARG-1 was downregulated by the fold change of 0.90(P>0.05),RELM-a was upregulated by the fold change of 1.20(P>0.05)after the stimulation of LPS(Pg).ConclusionThe stimulation of LPS(Pg)increased the expression of M1 related genes in both RAW264.7 macrophages cell line.and RAW264.7 pre-osteoclasts,suggesting promoted inflammatory destruction by LPS(Pg)stimulation.Chapter 3 Effect of High Mobility Group Box-1 on the M1/M2 Types RelatedGene Expression of Murine Macrophages RAW264.7 and RAW264.7Pre-osteoclastsObjectiveIt’s suggested that HMGB1 is involved in the progression of periodontitis in chapter 1.It’s found that LPS promotes the polarization of M1 macrophages and the expression of M1 related polarized genes in the pre-osteoclasts in chapter 2.It has been shown that LPS could induce the synthesis,translocation,and release of HMGB1 in macrophages,which suggested that HMGB1 might be one of the downstream cytokine of LPS and could affect the disease secondary.The purpose of this experiment was to further explore how HMGB1 affects the progression of periodontal disease by affecting the polarization of macrophages and the related polarization gene expression levels of pre-osteoclasts in this signaling pathway.Method1.The mouse macrophages cell line RAW264.7 was cultured in DMEM high glucose medium containing 10%fetal bovine serum,100 U/ml penicillin,and 100μg/ml streptomycin in 37 ℃,5%carbon dioxide incubator.And medium was changed every 2-3d.2.With the stimulation of 100 ng/ml RANKL and 1%MCSF for 3 d,the macrophages were differentiated into RAW264.7 pre-osteoclasts and their morphology was observed under a microscope.3.RAW264.7 macrophages and pre-osteoclasts were devived into two groups.The experimental group was stimulated with HMGB1 1μg/ml in DMEM medium for 18 h,while the control group is cultured in DMEM medium for the same time.4.The cellular RNA was extracted and collected.The sample RNA concentration was measured and reverse transcribed into cDNA.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of M1 type macrophages polarization related genes,including:Inducible Nitric Oxide Synthases 2(nOS2),Tumor Necrosis Factor-a(TNF-a),Interleukin-6(IL-6)and CXC Motif Chemokine 11(CXCL-11);M2 type macrophages polarization related gene:Arginase 1(ARG-1),Resistin-like Molecule Alpha(RELM-a/FIZZ 1).GAPDH was used as the reference gene,and the final Cq value was calculated.And statistical analysis were performed.Results1.Marcrophages:The expression of M1 related mRNA were significantly upregulated after the stimulation of HMGB1.The expression of NOS2 was upregulated by the fold change of 4.02(P<0.01).The expression of IL-6 was upregulated by the fold change of 2.50(P>0.05).The expression of TNF-a was upregulated by the fold change of 1.29(P<0.01).The M2 related mRNA were upregulated after the stimulation of HMGB1.The expression of ARG-1 was upregulated by the fold change of 1.25(P>0.05).The expression of RELM-a was upregulated by the fold change of 13.61(P<0.01).2.Pre-osteoclasts:The expression of M1 related mRNA had no significantly change after the stimulation of HMGB1.The expression of NOS2 was downregulated by the fold change of 0.80(P<0.05).The expression of IL-6 was upregulated by the fold change of 11.22(P>0.05).The expression of TNF-a was upregulated by the fold change of 1.04(P>0.05).The expression of IL-1 was upregulated by the fold change of 0.81(P>0.05).The expression of CXCL-11 was upregulated by the fold change of 0.94(P>0.05).While the expression of M2 related mRNA,ARG-1 was upregulated by the fold change of 2.42(P>0.05),RELM-a was upregulated by the fold change of 3.52(P<0.01),MRC-1 was upregulated by the fold change of 7.28(P<0.01)after the stimulation of HMGB1.ConclusionThe M1/M2 polarization of RAW264.7 macrophages were both promoted by the stimulation of HMGB 1 signi,ficantly.As for the pre-osteoclasts,HMGB1 stimulation had no significant effect on the expression level of M1 polarization-related genes,but it had a significant promotion effect on the expression of M2 polarization-related genes,which could work as the negative feedback regulation,and play a role on promoting tissue repair. |
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