Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model

Abstract Objective The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. Material and Methods Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. Results Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1β, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. Conclusions This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.


Introduction
Periodontitis is a chronic oral infectious disease that results from a dysregulated host immune response towards microorganisms in the dental biofilm. 1,2 The relationship between periodontitis and other pathological conditions could be established by the immunogenic potential of host and/or bacterial products that reach the bloodstream and target distant organs and systems. Periodontal disease (PD) 3 is a known risk factor for diabetes mellitus (DM), and there is evidence that treatment of PD reduces the incidence of inflammation and DM complications. 4 In turn, DM is a risk factor for periodontitis. A bidirectional relationship exists between glycemic control and PD severity. 5 The most common types of diabetes observed in primary care practice are type 1 and type 2. Type 1 diabetes is an autoimmune disease characterized by total destruction of pancreatic beta cells, whereas insulin resistance, impaired insulin secretion, and inappropriate hepatic glucose secretion characterize type 2 diabetes. 6 The use of anti-diabetic drugs to control type 2 DM has secondary pharmacological benefits with regard to inflammatory processes. 7,8 Gliclazide is an anti-diabetic medication, a secondgeneration sulfonylurea. Sulfonylureas release insulin from pancreatic cells and act on insulin-sensitive tissues to enhance glucose uptake. 9,10 While these agents directly stimulate insulin secretion by the β-cell, Sulfonylureas have also been shown to have antiinflammatory effects. 11 Insulin stimulation results in the activation of distinct pathways involved in metabolic regulation, including the phosphatidylinositol-3-kinase (PI3K) cascade. 10 Gliclazide has a direct effect on PI3K insulin-resistant skeletal muscle to enhance insulin signalling. 10 The PI3K signaling pathway affects the inflammatory process, contributing to increased neutrophil survival, 12 and the osteoclast differentiation pattern. 13 PI3K expression is higher in tissue from patients with periodontitis than in healthy gingival tissue. 14 Periodontal bone resorption is induced by osteoclasts. Receptor activator of nuclear factor-κB ligand (RANKL), its receptor RANK, and a decoy receptor osteoprotegerin (OPG) are key molecules that regulate osteoclast differentiation, recruitment, and function. 15 Cathepsin-K is a key protease in the degradation process of bone matrix molecules. There is a positive correlation between cathepsin-K and RANKL levels, suggesting that excess production of RANKL resulted in the formation of active osteoclasts and led to cathepsin-K production in osteoclasts in the periodontium of patients with periodontitis, thus contributing to osteoclastic bone resorption. 16 Other important proteases involved in destructive periodontal diseases are Matrix metalloproteinases (MMPs). MMPs were traditionally thought to degrade extracellular matrix components and grouped according to their substrate specificity in collagenases, gelatinases, stromelysins, matrilysins, and membrane type. Because type I collagen represents the bulk component of periodontal extracellular matrix, special attention has been paid to MMP-2 gelatinases in periodontitis. 17 Gliclazide also decreased the expression of inflammatory markers and endothelial dysfunction in patients with type 2 diabetes. 18 The inhibitory effect of gliclazide on AGE-induced monocyte adhesion involves a reduction in EC adhesion molecule expression and inhibition of nuclear factor kappaB (NF-kappaB) activation. 19 The aim of this study was to investigate the roles of gliclazide in inflammation, bone loss, and PI3K/AKT pathway activation in an experimental PD rat model.  utilized for the assay. 26 Gingival samples (five/group) were used to determine levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using an enzymelinked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's guidelines, as previously described. 27 All samples were within the wavelength range of ultraviolet-visible spectrophotometry (absorbance measured at 490 nm).

RNA extraction and qRT-PCR
Total RNA from gingival tissues was extracted using the Trizol ® reagent (Life Technologies, Los Angeles, USA) and chloroform (VETEC, São Paulo, SP, Brazil), as previously described. 28     1 mg/kg GLI group relative to the L group (p<0.001; Figure 6D). Densitometry analysis confirmed significantly increased NFKB p50 (cytoplasm) immunoreactivity relative NFKB p50 (Nuclear) in the L group (p<0.05; Figure 7). PI3K immunoreactivity was indicated by strong labeling in the periodontal area of samples from the L group (Figure 7). Discrete PI3K labeling was observed in dentin, in samples from the NL group (Figure 7), and weak, diffuse labeling was observed in samples from the 1 mg/kg GLI group (p<0.05; Figure 7).

Oxidative stress, inflammation, and gene expression analysis
Analysis of gingival tissue samples showed increased MDA levels in the L group compared to the NL group (p<0.001). All GLI-treated groups showed reduced MDA levels compared with the L group (1 and 5 mg/kg GLI, p<0.001; 10 mg/kg GLI, p≤0.05; Figure   8A). GSH levels did not differ significantly from those in the L group ( Figure 8C). GLI at a dose of 1 mg/kg prevented the increase of MPO activity (p≤0.05, Figure   8B) and IL-1β and TNF-α content (both p<0.05; Figure   9A      Our data showed that the best results related to bone loss were found when gliclazide administration at the lower doses of 1 mg/kg translates the drug dosage from animal to human dose, 20 as the therapeutic dose of gliclazide in humans occurs in a range of 30 mg-120 mg/day. We find that the dose of 10 mg/  This reduced pathway activation resulted in a lower neutrophil survivability, as confirmed by the reduced MPO activity. Gliclazide seems to dysregulate the PI3K/ AKT pathway, thereby reducing neutrophil survivability.
Early activation of the PI3K/AKT pathway is a major step in the inhibition of apoptosis in Anaplasma phagocytophilum-infected neutrophils, 12 and the role of the NFKB and PI3K pathways in neutrophil apoptosis inhibition in periodontal inflammation has been demonstrated. 14 Treatment with 1 mg/kg GLI resulted in a more dramatic reduction in bone loss compared with the other gliclazide treatments.
The F4/80 glycoprotein is a member of the EGFtransmembrane 7 family and has been established as a specific cell-surface marker for murine macrophages. 39 We observed reduced F4/80 gene expression in the group treated with 1 mg/kg GLI in this study, which was corroborated by the MIF immunofluorescence assay. In the context of PD, MIF has a role in controlling bacterial growth, but it more significantly contributes to the progression of bone loss by directly affecting osteoclast differentiation and activity. 13 High doses of gliclazide (e.g., 10 mg/kg) activate the PI3K/AKT pathway and increase periodontal bone loss. Some cytokines activate the PI3K/AKT pathway, leading to osteoclastogenesis. 40 An increase in the inflammatory cytokine IL-1β was observed in the 10 mg/kg GLI group. IL-1 has been reported to stimulate osteoclastogenesis through two parallel events: direct in gingival tissue from rats without periodontal disease (no ligature) and those with periodontal disease (ligature and 1 mg/kg GLI groups). The expression of NF-κB p50, PI3K, AKT, and F4/80 mRNA was decreased in the 1 mg/kg GLI (p<0.05 and p<0.001) and no ligature (p<0.001) groups. Analysis of variance followed by Bonferroni's test (n=5 animals/group) enhancement of RANKL expression and suppression of OPG expression. 40

Conclusion
We conclude that the use of GLI at a 1-mg/kg dose in rats reduces the formation of lipid peroxidation products, prevents PI3k signaling, and decreases IL-1β and TNF-α levels, which in turn reduce MMP-2, cathepsin K, and RANKL levels. Reduced gene expression of PI3K/AKT pathway genes was observed together with lower neutrophils infiltration, accompanied by lower neutrophil survivability and reduced migration of macrophages (MIF), which may have contributed to the reduction in linear bone loss.