The Effects And Mechanism Of FSTL1 On Periodontitis Associated With Type 2 Diabetes Mellitus

Objective: Previous studies have found that type 2 diabetes can be involved in the regulation of circulating FSTL1 levels and the expression of FSTL1 increased in processes of tissue damage repair.However,the expression of FSTL1 in periodontitis with type 2 diabetes mellitus and its function is still unclear.By detecting the m RNA and protein expression levels of FSTL1 in the periodontal tissues of patients with concomitant type 2 diabetic periodontitis and the expression levels of FSTL1 in the periodontium of db/db periodontitis mice,compared with the control group,we initially explored the changes in the expression of FSTL1 in the periodontal tissues of patients with concomitant type 2 diabetic periodontitis.Methods: Periodontal tissues were collected from normal people,patients with periodontitis,and patients with periodontitis with type 2 diabetes mellitus.The m RNA expression level of FSTL1 in the periodontal tissues of each group were detected by RTPCR.The protein expression level of FSTL1 in the periodontal tissues of each group were detected by Western blot.In 6-week-old male C57BL/6J mice and db/db mice,periodontitis models were constructed by silk ligation between the first and second molars for 14 days.2 weeks later the expression levels of FSTL1 protein in the periodontal tissues of normal C57 mice,C57 periodontitis mice,and db/db periodontitis mice were detected by immunohistochemistry.Results: RT-PCR and Western blot results showed that the m RNA and protein levels of FSTL1 were elevated in periodontal tissues of patients with periodontitis and patients with type 2 diabetic periodontitis compared with those of normal people,and the expression levels were increased in type 2 diabetic periodontitis compared with those in normal periodontitis.Immunohistochemical staining of mice showed that the expression of FSTL1 was elevated in the periodontal tissues of C57 periodontitis mice and db/db periodontitis mice compared with controls.The highest expression of FSTL1 was found in db/db periodontitis mice.Conclusion: The protein and m RNA levels of FSTL1 were elevated in periodontal tissues of periodontitis and periodontitis with type 2 diabetes mellitus,suggesting that it may be involved in the regulation of periodontitis and periodontitis with type 2 diabetes mellitus.Objective: To perform raw confidence analysis of published single-cell sequencing data of periodontal tissues to clarify the differences in the expression patterns of FSTL1 in periodontal tissues of normal controls and periodontitis patients.Methods: The published single-cell transcriptome sequencing data of periodontal tissues from normal people and patients with periodontitis were analyzed by raw confidence analysis,and the sequencing data were analyzed using analytical methods such as downscaling and clustering analyses,and gene differential expression analyses.Results: The expression of FSTL1 was elevated in the cells of periodontitis group compared with control group.Cluster analysis showed that FSTL1 was mainly expressed in fibroblasts and vascular endothelial cells,both of which are mesenchymal stem cell lineages.The expression levels of MSC markers CD73,CD90,CD105,CD146 and Periostin were significantly higher in FSTL1-expressing cells compared to non-FSTL1-expressing cells.Cells expressing and not expressing FSTL1 were divided into two clusters,and the differential genes were enriched.The results of KEGG analysis enriched to the Wnt signaling pathway,TGF-β signaling pathway,and AGE-RAGE signaling pathway.The results of the GO analysis showed that the expression of FSTL1 regulated the Wnt signaling pathway.Conclusion: The proportion and expression level of cells expressing FSTL1 increased in periodontal tissues in periodontitis.FSTL1 was mainly expressed in MSC lines.The expression of FSTL1 was highly co-expressed with stem cell markers.FSTL1-expressing cells were mainly involved in the regulation of the Wnt signaling pathway.Objective: To clarify the role of FSTL1 in periodontitis and periodontitis with type 2 diabetes mellitus,it is necessary to successfully construct a model of periodontitis with type 2 diabetes mellitus in vitro.Type 2 diabetes mellitus(T2DM),a risk factor for periodontitis,is characterized by insulin resistance.Therefore,it is necessary to construct a cellular model with insulin resistance to mimic the type 2 diabetes microenvironment.High glucose(HG),glucosamine and palmitic acid(PA)are commonly used in T2 DM studies to mimic insulin resistance in vitro to construct in vitro models of type 2 diabetes.In this section,human periodontal stem cells(h PDLSCs)were treated with each of the insulin resistance-inducing reagents to detect the level of insulin resistance,the effect on inflammatory response,and to assess their role on osteogenic differentiation.To explore the construction method of periodontitis model with type 2 diabetes mellitus applicable to h PDLSCs,and to lay the foundation for subsequent exploration of the role and mechanism of FSTL1 in periodontitis with type 2 diabetes mellitus.Methods: h PDLSCs were validated by flow cytometry.h PDLSCs were treated with different concentrations of HG(0,15,25,35,45 m M),glucosamine(0,0.8.8,18,28,38 m M)or PA(0,100,200,400,800 μM),and 10 μg/m L LPS for 48 hours,respectively.The insulin signaling pathway and inflammatory factors and inflammatory signaling pathways were detected by WB and RT-PCR.h PDLSCs underwent osteogenic induction for 7-21 days,and the cells were treated with 35 m M HG,18 m M glucosamine,or 200 μM PA combined with 10 μg/m L LPS during the osteogenic induction period.The effects of each reagent on osteogenic differentiation of h PDLSCs were assessed by alkaline phosphatase(ALP)staining,alizarin red staining,RT-PCR and Western blot.Results: High glucose did not mimic insulin resistance.High glucose alone elevated the inflammation but had the effect of mitigating LPS inflammatory responses.High glucose alone did not inhibit osteogenic differentiation.Glucosamine alone induced insulin resistance,which significantly inhibited osteogenic differentiation,but was unable to promote inflammatory response.PA exhibited significant insulin resistance,proinflammatory effects,and enhanced the pro-inflammatory effects of LPS in h PDLSCs.Additionally,PA was able to inhibit osteogenic differentiation.Conclusion: PA mimics the insulin resistance level,elevated inflammatory response and inhibited osteogenic differentiation.It can be used as a reagent to correctly mimic periodontitis with type 2 diabetes in h PDLSCs.Objective: Previous studies have clarified that the expression of FSTL1 is elevated in periodontal tissues in type 2 diabetic periodontitis,but the specific regulatory role of FSTL1 in type 2 diabetic periodontitis remains unclear.Type 2 diabetic periodontitis exhibits an increased inflammatory.To clarify the regulatory role of FSLT1 on type 2 diabetic periodontitis,this vignette explored the regulatory role of FSTL1 on inflammation in type 2 diabetic periodontitis in vitro by overexpressing and knocking down FSTL1 in h PDLSCs.Methods: Primary culture of h PDLSCs and transfection of lentivirus to overexpress FSTL1 or transfection of Si RNA to knock down FSTL1 were combined with LPS and PA treatment of cells to establish in vitro models of inflammation and type 2 diabetes mellitus.The expression level of inflammatory factor of m RNAs was detected by RT-PCR,and activation of inflammation-related pathways was detected by Western blot,and the activation of inflammation-related pathways was detected by enzyme-linked adsorption reaction(ELISA)to detect the level of IL-β in the culture medium.Results: Overexpression of FSTL1 in h PDLSCs promoted the reduction of phosphorylation levels of NFκB,JNK and ERK in inflammatory signaling pathways and inhibited the expression of TNF-α,IL-1β,IL-6,IL-13,and IL-18 m RNAs in basal cultures,in periodontitis mimicking by LPS,and in type 2 diabetic periodontitis model.The results of ELISA showed that overexpression of FSTL1 in h PDLSCs inhibited the expression levels of IL-β in the culture medium and the activation state of the inflammation-related pathways.ELISA results showed a decrease in IL-1β expression level in the culture medium after overexpression of FSTL1.Knockdown of FSTL1 showed the opposite result.Conclusion: FSTL1 has an inhibitory effect on the inflammation of h PDLSCs in basal state,inflammatory state,and type 2 diabetes with inflammatory state.Objective: It was shown that FSTL1 inhibited the inflammatory response of h PDLSCs in basal state,inflammatory state,and type 2 diabetes with inflammatory model states in vitro.In order to further clarify its role in the regulation of inflammation in periodontal tissues,the aim of this section of the study was to verify the role of FSTL1 in regulating the inflammatory response to periodontitis and periodontitis with type 2 diabetes mellitus in vivo by overexpressing and knocking down Fstl1 in periodontal tissues of mice in vivo.Methods: Adeno-associated virus(AAV)localized periodontal injection was used to overexpress Fstl1 in periodontal tissues.6-week-old C57BL/6J male mice were chosen and randomly divided into 6 groups of 6 mice each.4 groups were fed with normal diet for 12 weeks,and the other 2 groups were fed with high-fat diet for 12 weeks.And then injected with STZ intraperitoneally to establish type 2 diabetes mellitus model.The mice were randomly assigned to the normal diet and the type 2 diabetic mice.model mice to control mice and Fstl1 overexpression group.The overexpression groups were injected with overexpression of Fstl1 adeno-associated viral virus(AAV-Fstl1)between the first and second molar teeth,and the control group was injected with control airborne adenoassociated virus(AAV-Gfp).The subgroups were(1)control group(AAV-Gfp CON);(2)periodontitis group(AAV-Gfp PD)(3)periodontitis with type 2 diabetes mellitus group(AAV-Gfp T2DM+PD)(4)overexpressing Fstl1 group(AAV-Fstl1 CON)(5)overexpressing Fstl1 periodontitis group(AAV-Fstl1 PD)(6)overexpressing Fstl1 with type 2 diabetes mellitus periodontitis group(AAV-Fstl1 T2DM+PD).After 1 week,the periodontitis model was constructed using silk ligation between the first and second molars.Two weeks after periodontitis modeling,the expression m RNA of inflammatory factor in periodontal tissues were detected by RT-PCR.The expression levels of IL-1β protein in periodontal tissues were detected by immunofluorescence staining.AAV overexpression of cyclized recombinase(Cre)was used in the periodontal tissues of Fstl1flox/flox mice to inhibit Fstl1 expression in the periodontal tissues.6-week-old Fstl1flox/flox mice were randomly divided into six groups:(1)control group(Fstl1flox/flox CON);(2)periodontitis group(Fstl1flox/ flox PD);(3)periodontitis with type 2 diabetes group(Fstl1flox/flox T2DM+PD);(4)knockout Fstl1 group(Cre;Fstl1flox/flox CON);(5)knockout Fstl1 periodontitis group(Cre;Fstl1flox/flox PD);(6)knockout Fstl1 with type 2 diabetes periodontitis group(Cre;Fstl1flox/flox T2DM+PD).Type 2 diabetes and periodontitis models were constructed as before.After 2 weeks of periodontitis model construction,the m RNA expressions of inflammatory factor in periodontal tissues were detected by RT-PCR.The expression levels of IL-1β protein in periodontal tissues were used to detected by immunofluorescence staining.Results: Overexpression of Fstl1 in periodontal tissues suppressed the m RNA expressions of inflammatory factors including Tnf-α,Il-1β,Il-6,Il-13,and Il-18 in periodontitis and periodontitis with type 2 diabetes mellitus in mice.The immunofluorescence staining results suggested that the expression levels of IL-1β was reduced in periodontal tissue after overexpressing Fstl1.Knockdown of Fstl1 in periodontal tissues promoted the expression of m RNAs of the above inflammatory factors in periodontal tissues of mice in PD and T2DM+PD group.Immunofluorescence staining results suggested that the expression levels IL-1β were elevated in periodontal tissues.Conclusion: FSTL1 inhibited the inflammatory response of periodontal tissues in periodontitis and periodontitis with type 2 diabetes mellitus.Objective: The osteogenic differentiation ability of h PDLSCs is inhibited in periodontitis with type 2 diabetes mellitus.Improving the osteogenic differentiation ability of h PDLSCs in periodontitis with type 2 diabetes could help the reconstruction of periodontal tissues in this disease.Previous studies have clarified the high overlap of FSTL1 with stem cell markers,suggesting that it may be involved in the regulation of stem cell differentiation.In order to clarify its role in the regulation of osteogenic differentiation of stem cells,this vignette explored the regulatory role of FSTL1 on osteogenic differentiation in periodontitis and periodontitis with type 2 diabetes cell models in vitro by overexpressing and knocking down FSTL1 in h PDLSCs.Methods: h PDLSCs were cultured in situ and transfected with lentivirus to overexpress FSTL1,or transfected with Si RNA to knock down FSTL1,and the cells were treated with LPS and PA to establish in vitro models of T2 DM and periodontitis,and the function of osteogenic differentiation was examined by ALP staining and alizarin red staining,the m RNA expressions of osteogenesis-related genes were examined by RT-PCR.The expression level of osteogenesis-related proteins was detected by western blot.Results: After hPDLSCs overexpressed FSTL1,the expressions of osteogenesisrelated proteins including COL-1,OPN,RUNX2,and ALP were elevated.The m RNA expressions of ALP,OCN,OPN,RUNX2,and COL-1 decreased,when compared with the homozygous cell models without overexpression of FSTL1 in basal cultures,the LPSmimicking periodontitis state,and the state of the periodontitis with type 2 diabetes mellitus in vitro model.m RNA levels were reduced.Knockdown of FSTL1 showed the opposite results.Conclusion: FSTL1 has a promoting effect on osteogenic differentiation of h PDLSCs in basal state,inflammatory state and type 2 diabetes and inflammatory state.Objective: It has been demonstrated that FSTL1 promotes osteogenic differentiation of h PDLSCs under basal,inflammatory,and type 2 diabetes mellitus with inflammatory states in vitro study.To further clarify its regulatory role on osteogenesis,we verified the effect of FSTL1 on the bone mass of periodontal tissues in periodontitis and periodontitis with type 2 diabetes mellitus through in vivo studies.Methods: AAV localized periodontal injection was used to overexpress Fstl1 in periodontal tissues.6-week-old C57BL/6J male mice were chosen and randomly divided into 6 groups of 6 mice each.4 groups were fed with normal diet for 12 weeks,and the other 2 groups were fed with high-fat diet for 12 weeks.And then injected with STZ intraperitoneally to establish type 2 diabetes mellitus model.The mice were randomly assigned to the normal diet and the type 2 diabetic mice.model mice to control mice and Fstl1 overexpression group.The overexpression groups were injected with overexpression of Fstl1 adeno-associated viral virus(AAV-Fstl1)between the first and second molar teeth,and the control group was injected with control airborne adenoassociated virus(AAV-Gfp).The subgroups were(1)control group(AAV-Gfp CON);(2)periodontitis group(AAV-Gfp PD)(3)periodontitis with type 2 diabetes mellitus group(AAV-Gfp T2DM+PD)(4)overexpressing Fstl1 group(AAV-Fstl1 CON)(5)overexpressing Fstl1 periodontitis group(AAV-Fstl1 PD)(6)overexpressing Fstl1 with type 2 diabetes mellitus periodontitis group(AAV-Fstl1 T2DM+PD).After 1 week,the periodontitis model was constructed using silk ligation between the first and second molars.Two weeks after periodontitis modeling,the morphology and height of alveolar bone were detected by Micro-CT.The inflammatory cell infiltration,alveolar bone resorption,collagen maturation and new bone neogenesis in periodontal tissues were detected by HE staining and Masson staining.The osteoblastic activities were detected TRAP staining.AAV overexpression of cyclized recombinase(Cre)was used in the periodontal tissues of Fstl1flox/flox mice to inhibit FSTL1 expression in the periodontal tissues.6-week-old Fstl1flox/flox mice were randomly divided into six groups:(1)control group(Fstl1flox/flox CON);(2)periodontitis group(Fstl1flox/ flox PD);(3)periodontitis with type 2 diabetes group(Fstl1flox/flox T2DM+PD);(4)knockdown Fstl1 group(Cre;Fstl1flox/flox CON);(5)knockout Fstl1 periodontitis group(Cre;Fstl1flox/flox PD);(6)knockout Fstl1 with type 2 diabetes periodontitis group(Cre;Fstl1flox/flox T2DM+PD).Type 2 diabetes and periodontitis models were constructed as before.After 2 weeks of periodontitis model construction,the assay was performed in the same way as the in vivo overexpression of Fstl1 assay.Results: After overexpression of Fstl1 in periodontal tissues,alveolar bone height was higher than that in the control group,regardless of the presence or absence of PD and T2DM+PD.HE staining results suggested that periodontal tissues with overexpression of Fstl1 showed less infiltration of inflammatory cell,and the alveolar bone height was higher than that of the control group.the Masson staining results suggested that the overexpression of Fstl1 led to greater collagen maturation and increased the formation of new bone.The TRAP staining results suggested that the overexpression of Fstl1 led to a decrease of TRAP positive cells.Overexpression of Fstl1 resulted in fewer TRAPpositive cells and decreased osteoclastic activity.Knockout of Fstl1 in periodontal tissues showed the opposite result.Conclusion: FSTL1 has a mitigating effect on periodontal bone destruction and promotes its osteogenic differentiation in periodontitis and periodontitis with type 2 diabetes status.Objective: To explore the mechanism of FSTL1 regulation in periodontitis with type 2 diabetes mellitus.Methods: h PDLSCs overexpressing FSTL1 were subjected to RNA-seq sequencing.Differential gene analysis was performed on the sequencing results.KEGG enrichment analysis and GO analysis were performed on the differential genes to explore the major signaling pathways regulated by FSTL1 in h PDLSCs.h PDLSCs were primary cultured and transfected with lentivirus to overexpress FSTL1 or si RNA to knock down FSTL1,and an inflammation model and an in vitro type 2 diabetes model were constructed.The enriched key signaling pathways were verified by Western blot.Overexpress and knock down Fstl1 in periodontal tissues in mice and construct a periodontitis model and an animal model of periodontitis with type 2 diabetes mellitus.The expression of key proteins of the relevant pathways in periodontal tissues were detected by immunofluorescence staining.And the interaction between FSTL1 and key proteins of this signaling pathway was explored by co-immunoprecipitation(Co-IP).Results: Transcriptome sequencing results showed that hPDLSCs overexpressing FSTL1 upregulated a total of 150 genes and downregulated 470 genes.KEGG and GO enrichment analysis of the differential genes,revealed that the differential genes were enriched to the Wnt signaling pathway,a key signaling pathway that regulates stem cell function.h PDLSCs overexpressing FSTL1 in the basal,inflammatory,and type 2 diabetes+inflammatory states promoted p-GSK3β and β-catenin expression.The opposite results were seen after knockdown of FSTL1.Overexpression of Fstl1 In periodontal tissue in mice resulted in elevated β-catenin levels in the basal state,periodontitis model,and periodontitis model with type 2 diabetes state.Knockdown of Fstl1 in periodontal tissues in mice showed the opposite results.Co-IP results demonstrated an interaction between FSTL1 and with β-catenin in the Wnt signaling pathway.Conclusion: FSTL1 can promote Wnt signaling pathway activation to promote the osteogenic differentiation ability of periodontal stem cells by interacting with β-catenin.