archives of oral biology 59 (2014) 1301–1306
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Insulin modulates cytokines expression in human periodontal ligament cells Yingying Wu a,1, Fuwei Liu b,1, Xiang Zhang b,1, Lei Shu c,* a
Department of Dentistry, Chinese PLA General Hospital, Beijing 100853, China Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China c Department of Dentistry, 101 Hospital of Chinese People’s Liberation Army, Wuxi 214044, China b
article info
abstract
Article history:
Objective: Periodontal disease (PD) has recently been recognized as the ‘sixth’ major com-
Accepted 13 July 2014
plication of diabetes but the mechanisms involved in diabetic PD remain unclear. This study was to elucidate the potential bone-sparing effect of insulin in diabetic PD conditions.
Keywords:
Design: The human periodontal ligament (hPDL) cells were obtained from the healthy hPDL
Glucose
tissue and were treated with high concentrations (25 or 45 mM) of glucose with or without
Insulin
different concentrations (10 6, 10
Cytokine
Results: High concentrations of glucose increased the production of pro-inflammatory
7
Periodontal ligament cells
cytokines interleukin-1 beta (IL-1b), tumour necrosis factor alpha (TNF-a), Interleukin-6
or 10
8
mM) of insulin.
(IL-6) at both mRNA and protein levels, and receptor activator of NF-kB ligand (RANKL) at mRNA levels in hPDL cells. Insulin treatment alleviated the stimulatory effects of high glucose on pro-inflammatory cytokines and RANKL expression by hPDL cells. Moreover, insulin up-regulated OPG expression and therefore attenuated the reduction of OPG vs. RANKL ratio. Conclusion: Insulin plays significant roles in modulating the periodontal tissue responses to hyperglycemia, and thus exerts its bone-sparing effects on periodontal tissues via altering the inflammatory cytokines expression in hPDL cells. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Both diabetes mellitus and periodontal disease (PD) are chronic illnesses. There have been increased interests in a possible link between diabetes and PD.1 PD has recently been recognized as the ‘sixth’ major complication of diabetes. Epidemiologic studies have confirmed a high prevalence of PD with diabetes. Clinical evidence has also shown a positive correlation between diabetes and PD. The majority of clinical and epidemiological evidence demonstrated that individuals
with diabetes (type I and type II) tend to have a higher prevalence of PD as well as more severe and more rapidly progressing forms of PD than nondiabetics.2 Although diabetes is a putative risk factor for PD, its possible effects on periodontal tissue at the cellular and molecular levels are not known. PD is a chronic infectious inflammatory disease characterized by the destruction of the tooth-supporting structures, with the presence of periodontopathogens required for disease development.3 It is believed that much of the periodontal tissue destruction is host-mediated through
* Corresponding author. Tel.: +86 13665101460. E-mail addresses: [email protected], [email protected] (L. Shu). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.archoralbio.2014.07.002 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
1302
archives of oral biology 59 (2014) 1301–1306
pro-inflammatory cytokines by local tissues and immune cells in response to the bacterial flora and its products/metabolites, especially lipopolysaccharide (LPS).4–6 Recently, some of the molecular mechanisms underlying periodontal tissue destruction were elucidated, and several inflammatory mediators were considered to play important roles in determining periodontal disease progression.7,8 The most convincing cytokines that have been strongly implicated in periodontal tissue destruction involve interleukin-1 beta (IL-1b), tumour necrosis factor alpha (TNF-a), Interleukin-6 (IL-6), osteoprotegrin (OPG) and receptor activator of NF-kB ligand (RANKL). These inflammatory cytokines all contribute to the progression of periodontal disease, either attenuate or promote tissue destruction.9–11 Diabetes is a group of metabolic disorders characterized by abnormal fuel metabolism, which results most notably in hyperglycemia and dyslipidemia, due to defects in insulin secretion, insulin action, or both.12 Recently, several studies suggested that even mild hyperglycemia is detrimental and it aggravates endotoxemic shock and hypermetabolism.13 Hyperglycemia may further contribute to morbidity and mortality after burns or surgery.8 Furthermore, some study demonstrated that insulin had anti-inflammatory effects by decreasing pro-inflammatory signal transcription factors and pro-inflammatory cytokines, while increasing anti-inflammatory cytokines.1 The periodontal ligament (PDL) is a noncalcified connective tissue consisting mainly of PDL cells and collagen. The PDL cells play a vital role in maintaining the homeostasis of periodontal tissues via affecting coordinated balance of boneforming and bone-resorbing activities.1,14 However, whether diabetes-induced hyperglycemia has any effects on the expression of inflammatory cytokines in PDL cells remains unraveled. Moreover, it is not known whether insulin exerts its effects directly through modulating inflammatory mediators’ expression in PDL cells. Therefore, the present study was designed to investigate whether high glucose conditions affects the inflammatory cytokines expression in PDL cells, and if so, to explore the potential modulatory effects of insulin in PDL cells.
detached with 0.25% trypsin and 0.2% EDTA and subcultured in 1:3 ratios. Cell cultures between the fifth and the eighth passages were used in this study.
2.2.
Treatment of hPDL cells with glucose and insulin
The hPDL cells were seeded in six-well plates at a density of 1 106 cells per well and were grown in DMEM containing 5% FBS until confluence. The cells were then silenced with serumfree DMEM overnight. After silencing, they were stimulated by 5.5 mM (normal culture condition), 25 mM and 45 mM glucose with or without insulin (10 6, 10 7 or 10 8 mM, Sigma) for 6, 12 and 24 h.16–18 The hPDL cells of all treatment groups were lysed in 0.2% Triton X-100 and the cell lysates were collected and centrifugated. The cell-free supernatants were stored at 70 8C until being assayed.
2.3.
Inflammatory cytokines measurement by ELISA
Secreted TNF-a, IL-6 and IL-1b were measured by commercially available ELISA kits (R&D Systems Co., Minneapolis, MN, USA) according to the manufacturer’s directions. Experiments were always performed in triplicate.
2.4. PCR
mRNA expression detection by quantitative real-time
Total RNA from hPDL cells was extracted using the Total Extraction KitI (Omega BioTek, Norcross, GA) according to the manufacture’s protocols and was quantified spectrophotometrically. cDNA was synthesized with 2 mg RNA in a 20 ml reaction mixture containing 2.5 mM random hexamers and Superscript II reverse transcriptase (GIBCO BRL). To quantify the mRNA expression of TNF-a, IL-6, IL-1b, OPG and RANKL, real-time PCR was performed on an ABI PRISM 7500 real-time PCR System (Applied Biosystems, Foster City, CA) with SYBR1 Premix Ex TaqTM II (TaKaRa Biotechnology) using the previously reported primer sequences.19,20 Reaction products were quantified (Roche Quantification Software, Roche Diagnostics) with GAPDH as the reference gene.
2.
Material and methods
2.5.
2.1.
Cell culture
Data were presented as mean SEM. All data were subjected to analysis of variance (ANOVA) followed by the Scheffe’s correction for post hoc t test comparisons. P-values
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Insulin modulates cytokines expression in human periodontal ligament cells Yingying Wu a,1, Fuwei Liu b,1, Xiang Zhang b,1, Lei Shu c,* a
Department of Dentistry, Chinese PLA General Hospital, Beijing 100853, China Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China c Department of Dentistry, 101 Hospital of Chinese People’s Liberation Army, Wuxi 214044, China b
article info
abstract
Article history:
Objective: Periodontal disease (PD) has recently been recognized as the ‘sixth’ major com-
Accepted 13 July 2014
plication of diabetes but the mechanisms involved in diabetic PD remain unclear. This study was to elucidate the potential bone-sparing effect of insulin in diabetic PD conditions.
Keywords:
Design: The human periodontal ligament (hPDL) cells were obtained from the healthy hPDL
Glucose
tissue and were treated with high concentrations (25 or 45 mM) of glucose with or without
Insulin
different concentrations (10 6, 10
Cytokine
Results: High concentrations of glucose increased the production of pro-inflammatory
7
Periodontal ligament cells
cytokines interleukin-1 beta (IL-1b), tumour necrosis factor alpha (TNF-a), Interleukin-6
or 10
8
mM) of insulin.
(IL-6) at both mRNA and protein levels, and receptor activator of NF-kB ligand (RANKL) at mRNA levels in hPDL cells. Insulin treatment alleviated the stimulatory effects of high glucose on pro-inflammatory cytokines and RANKL expression by hPDL cells. Moreover, insulin up-regulated OPG expression and therefore attenuated the reduction of OPG vs. RANKL ratio. Conclusion: Insulin plays significant roles in modulating the periodontal tissue responses to hyperglycemia, and thus exerts its bone-sparing effects on periodontal tissues via altering the inflammatory cytokines expression in hPDL cells. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Both diabetes mellitus and periodontal disease (PD) are chronic illnesses. There have been increased interests in a possible link between diabetes and PD.1 PD has recently been recognized as the ‘sixth’ major complication of diabetes. Epidemiologic studies have confirmed a high prevalence of PD with diabetes. Clinical evidence has also shown a positive correlation between diabetes and PD. The majority of clinical and epidemiological evidence demonstrated that individuals
with diabetes (type I and type II) tend to have a higher prevalence of PD as well as more severe and more rapidly progressing forms of PD than nondiabetics.2 Although diabetes is a putative risk factor for PD, its possible effects on periodontal tissue at the cellular and molecular levels are not known. PD is a chronic infectious inflammatory disease characterized by the destruction of the tooth-supporting structures, with the presence of periodontopathogens required for disease development.3 It is believed that much of the periodontal tissue destruction is host-mediated through
* Corresponding author. Tel.: +86 13665101460. E-mail addresses: [email protected], [email protected] (L. Shu). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.archoralbio.2014.07.002 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
1302
archives of oral biology 59 (2014) 1301–1306
pro-inflammatory cytokines by local tissues and immune cells in response to the bacterial flora and its products/metabolites, especially lipopolysaccharide (LPS).4–6 Recently, some of the molecular mechanisms underlying periodontal tissue destruction were elucidated, and several inflammatory mediators were considered to play important roles in determining periodontal disease progression.7,8 The most convincing cytokines that have been strongly implicated in periodontal tissue destruction involve interleukin-1 beta (IL-1b), tumour necrosis factor alpha (TNF-a), Interleukin-6 (IL-6), osteoprotegrin (OPG) and receptor activator of NF-kB ligand (RANKL). These inflammatory cytokines all contribute to the progression of periodontal disease, either attenuate or promote tissue destruction.9–11 Diabetes is a group of metabolic disorders characterized by abnormal fuel metabolism, which results most notably in hyperglycemia and dyslipidemia, due to defects in insulin secretion, insulin action, or both.12 Recently, several studies suggested that even mild hyperglycemia is detrimental and it aggravates endotoxemic shock and hypermetabolism.13 Hyperglycemia may further contribute to morbidity and mortality after burns or surgery.8 Furthermore, some study demonstrated that insulin had anti-inflammatory effects by decreasing pro-inflammatory signal transcription factors and pro-inflammatory cytokines, while increasing anti-inflammatory cytokines.1 The periodontal ligament (PDL) is a noncalcified connective tissue consisting mainly of PDL cells and collagen. The PDL cells play a vital role in maintaining the homeostasis of periodontal tissues via affecting coordinated balance of boneforming and bone-resorbing activities.1,14 However, whether diabetes-induced hyperglycemia has any effects on the expression of inflammatory cytokines in PDL cells remains unraveled. Moreover, it is not known whether insulin exerts its effects directly through modulating inflammatory mediators’ expression in PDL cells. Therefore, the present study was designed to investigate whether high glucose conditions affects the inflammatory cytokines expression in PDL cells, and if so, to explore the potential modulatory effects of insulin in PDL cells.
detached with 0.25% trypsin and 0.2% EDTA and subcultured in 1:3 ratios. Cell cultures between the fifth and the eighth passages were used in this study.
2.2.
Treatment of hPDL cells with glucose and insulin
The hPDL cells were seeded in six-well plates at a density of 1 106 cells per well and were grown in DMEM containing 5% FBS until confluence. The cells were then silenced with serumfree DMEM overnight. After silencing, they were stimulated by 5.5 mM (normal culture condition), 25 mM and 45 mM glucose with or without insulin (10 6, 10 7 or 10 8 mM, Sigma) for 6, 12 and 24 h.16–18 The hPDL cells of all treatment groups were lysed in 0.2% Triton X-100 and the cell lysates were collected and centrifugated. The cell-free supernatants were stored at 70 8C until being assayed.
2.3.
Inflammatory cytokines measurement by ELISA
Secreted TNF-a, IL-6 and IL-1b were measured by commercially available ELISA kits (R&D Systems Co., Minneapolis, MN, USA) according to the manufacturer’s directions. Experiments were always performed in triplicate.
2.4. PCR
mRNA expression detection by quantitative real-time
Total RNA from hPDL cells was extracted using the Total Extraction KitI (Omega BioTek, Norcross, GA) according to the manufacture’s protocols and was quantified spectrophotometrically. cDNA was synthesized with 2 mg RNA in a 20 ml reaction mixture containing 2.5 mM random hexamers and Superscript II reverse transcriptase (GIBCO BRL). To quantify the mRNA expression of TNF-a, IL-6, IL-1b, OPG and RANKL, real-time PCR was performed on an ABI PRISM 7500 real-time PCR System (Applied Biosystems, Foster City, CA) with SYBR1 Premix Ex TaqTM II (TaKaRa Biotechnology) using the previously reported primer sequences.19,20 Reaction products were quantified (Roche Quantification Software, Roche Diagnostics) with GAPDH as the reference gene.
2.
Material and methods
2.5.
2.1.
Cell culture
Data were presented as mean SEM. All data were subjected to analysis of variance (ANOVA) followed by the Scheffe’s correction for post hoc t test comparisons. P-values
