Effects of vitamin C local application on ligature-induced periodontitis in diabetic rats

Abstract Objective: This study evaluated the effects of local vitamin C treatment on tissue advanced glycation end products (AGE), interleukin (IL)-6, 8-hydroxy-2-deoxyguanosine (8-OHdG), and matrix metalloproteinases (MMP)-8 in tissues; serum C-terminal telopeptide fragments (CTX); and alveolar bone loss (ABL) in rats. Methodology: 35 male Sprague Dawley rats were divided equally into five groups: 1) control (C), 2) experimental periodontitis (P), 3) experimental diabetes (D), 4) experimental diabetes and experimental periodontitis (D + P), and 5) experimental diabetes–experimental periodontitis–locally applied vitamin C (D + P + LvitC). Diabetes was induced in rats with alloxan monohydrate, after which periodontitis was induced by ligature placement in the right mandibular first molar teeth for 11 days. In the treatment group, vitamin C was administered locally three times with two-days interval after ligature removal. The animals were sacrificed, and the samples were analyzed histometrically and immunohistochemically. Results: CTX, 8-OHdG, and AGE values significantly decreased in the treatment group compared to the D + P group. IL-6 and MMP-8 values decreased in the treatment group compared to the D + P group, but this is not significant. ABL was significantly reduced by the local delivery of vitamin C. Conclusion: This study reveals that vitamin C treatment may be beneficial to reduce serum CTX and gingival MMP-8 levels, oxidative stress, inflammation, and AGE accumulation in periodontal tissue. Vitamin C may be an immunomodulator and antioxidant locally applied in the treatment of periodontitis to reduce the adverse effects of diabetes in periodontal tissues.


Introduction
A large body of studies regarding the potential effect of diabetes mellitus (DM) on periodontal disease are available in the literature. However, the question of which biologic mechanism triggers the destruction of periodontal tissue in DM has not been exactly answered. 1 One of the potential mechanism is that advanced glycation end products (AGEs) lead to hyperinflammatory response, oxidative stress, and deterioration of the relationship between bone destruction and repair. [2][3][4] AGEs are formed by the irreversible nonenzymatic reaction between amine residues and reducing sugars in nucleic acids, lipids, or proteins as a result of long-term hyperglycemia. 5 AGEs may implement their biological effects on tissues with their cross-link formation or receptor antigen recognition. The most extensively studied receptor for AGE recognition is the multi-ligand receptor for AGE (RAGE). The engagement of RAGE by AGEs in cells stimulates an inflammatory response by upregulating the expression of molecules, such as matrix metalloproteinases (MMPs) and osteolytic activators, potentially damaging the periodontal ligament and alveolar bone, resulting in periodontal disease. 6,7 Inflammation is involved in the pathogenic mechanism of both DM and periodontitis. The inflammatory process is upregulated in the periodontal tissues of patients with DM. AGE-RAGE interaction in monocytes increases the generation of adhesion molecules and cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. 8 IL-6 is a significant proinflammatory cytokine in DM and periodontitis pathogenesis, bone resorption, and osteoclast development. 9 The increase in these proinflammatory cytokines induces the production of MMPs. 10 MMP-8 (neutrophil collagenase or collagenase-2) is a significant enzyme that plays a role in the destruction and remodeling of periodontal connective tissue during pathological events. MMP-8 has been noted as a key factor in the degradation of type 1 collagen, which is the most common type of collagen in supportive tissue in chronic periodontal diseases and DM. 11 C-terminal telopeptide fragments (CTX) are formed as degradation products during bone resorption, and they are released into the serum. Serum CTX is considered an extremely specific and sensitive marker of bone destruction. 12 In addition to insulin resistance, endothelial dysfunction, and hyperinflammatory responses, it has been suggested that hyperglycemia-induced oxidative stress plays a role in periodontal tissue destruction. 3,4 Activation of RAGE increases reactive oxygen species (ROS) by activating signal pathways, including nuclear factor kappa B (NF-κB), mitogenactivated protein (MAP) kinase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. 8 High concentrations of ROS can lead to direct tissue damage, including excessive DNA damage in periodontal tissue cells, periodontitis, and diabetes. 13 8-hydroxy-2-deoxyguanosine (8-OHdG)-excreted in the bodily fluids during DNA repair and is an oxidized nucleoside-is the most determinant biomarker exhibiting DNA damage. 14 The use of several antioxidants has been suggested to prevent adverse effects of oxidative damage in periodontal tissues, 4,15 .
Vitamin C is a powerful reducing agent and antioxidant. It is known to affect periodontal health, with an inverse relationship between periodontitis and serum/plasma vitamin C concentrations occurring. 13 In recent years, an increasing number of studies have reported the significance of vitamin C as an antioxidant against periodontal diseases. 16,17 This study aimed to evaluate the effects of local application of vitamin C in tissue levels of AGE, in  standard rat chow and water throughout the study.

Experimental design
The rats were randomly divided into five equal groups as following: 1) Group C (healthy control group), 2) Group P (periodontitis was induced and saline solution was administered), 3) Group D (diabetes was induced and saline solution was administered), 4) Group D+P (diabetes and periodontitis were induced and saline solution was administered), 5) Group D+P+LvitC (diabetes and periodontitis were induced and vitamin C was locally administered. with fasting blood glucose level higher than 250 mg/dl were considered as diabetic and therefore they were included in the study. 19

Induction of experimental periodontitis
The animals in the experimental periodontitis groups were anesthetized with xylazine hydrochloride (0.1 ml/kg i.p. Rompun, Bayer, Istanbul, Turkey) and ketamine hydrochloride (1 ml/kg i.p. Ketalar, Pfizer, Istanbul, Turkey). Then, 3-0 aseptic silk ligature was placed in a subgingival position around the right mandibular first molars in order to retain oral bacterial and to induct experimental periodontitis. The ligatures were removed on day 11. 20 Placebo and vitamin C administration After diabetes and periodontitis were induced, vitamin C (50 μL, Redoxon amp 500 mg/5 mL; Bayer Chemical Industry, Istanbul, Turkey) was locally administered into the subperiosteum at the right buccal gingiva of the mandibular first molar teeth three times, on two-days intervals with insulin needle (0.5 ml, 30 gauge; Becton Dickinson, Franklin Lakes, NJ).
Physiological saline (50 μL) was locally administered in the P, D, and P+D groups. [21][22][23] Blood sampling and measurements of serum CTX After 27 seven days of experimental period, 10 cc blood was collected from the rats by conducting a cardiac puncture. Animals were sacrificed under anesthesia. Blood samples were centrifuged at 1500g for 10 min within 1 h after collection. Until analysis day, the serum samples were stored in a -80 °C freezer.
Serum CTX concentrations were determined using a rat-specific CTX enzyme-linked immunosorbent assay

Biochemical results
Serum CTX levels were significantly higher in the P group compared to C and D groups (p<0.05). Serum CTX values were significantly higher in D+P group compared to D group (P<0.05) and significantly lower after vitamin C administration in the D+P+LvitC group (p<0.05) (Figure 3).

IL-6 values
The

8-OHdG values
The number of 8-OHdG positive cells in P group was significantly higher than the number in C and Clinical attachment loss  Figure 4).

Periodontal bone loss
Distal periodontal bone support (DPBS) and mesial periodontal bone support (MPBS) were inferior in P group than in D and C groups (p<0.05). The values decrease in D+P group were higher than in the D and P groups (p<0.05). The mean values of MPBS and DPBS were statistically lower in the D+P+LvitC group than in the D+P group (p<0.05) (Table 1, Fig. 4).  Inflammation has been associated with pathogenic mechanisms of both DM and chronic periodontitis. An increase in serum IL-6 levels has also been reported for DM and periodontitis, which are both chronic diseases. 2 In an experimental rat study, Elburki, et al. 32 (2017) found that IL-6 levels were higher in gingival tissue and serum samples of diabetic and periodontitis groups than in those of a control group. Ross, et al. 33 (2010) reported that IL-6 values were higher in immunohistochemical staining of gingival tissue samples of subjects with diabetes and periodontitis than in those of controls and subjects with only periodontitis. We found significantly higher numbers of IL-6-positive cells in the gingival tissue of the D and P groups than in the control group. We also found a statistically significant difference in the increase of IL-6 values in the D+P group compared to both D and P groups. Vitamin C has been reported to present anti-inflammatory properties capable of modulate DNA-binding activity of NF-κB. 34 Ellulu, et al. 35 (2015) reported that serum IL-6 levels significantly decreased in DM patients treated with 500 mg of vitamin C twice a day for eight weeks compared to control. Jang, et al. 36 (2014) suggested that vitamin C decreased the plasma levels of TNF-α and IL-6 by downregulating hepatic mRNA expression. In our study, we observed a decrease in the density of IL-6-positive staining in the treatment group compared to the D+P group, although the difference was insignificant (p>0.05). Based on these results, it can be concluded that vitamin C exerts anti-inflammatory activity by decreasing the levels of inflammatory cytokines.
In this study, we compared the changes in the destruction of extracellular matrix and bone resorption between the groups by measuring CTX in serum and MMP-8 in gingival samples. CTX is a marker of bone resorption among biochemical markers of bone turnover and is a marker of collagen that can reflect early changes in bone turnover. 37 It has been reported that there is no significant difference between CTX levels of diabetic patients and healthy subjects and the resorption phase remains unchanged during diabetic bone turnover. 38 Arabacı,et al. 39 (2015)

Conclusions
This study demonstrates that local vitamin C application positively affects inflammatory cytokine, oxidative stress, and AGE levels. Our results also suggest that the anti-glycolytic, anti-inflammatory, and antioxidant activity of vitamin C may reduce bone and periodontal tissue degradation. Despite the limitations of this study, it suggests that vitamin C may be used as a locally administered host modulatory agent.