The Effect of Two Different Doses of Astaxanthin on Alveolar Bone Loss in an Experimental Model of Periodontitis in Diabetic Rats

Abstract

This study evaluated the effects of astaxanthin (ASX) on alveolar bone loss, receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) activity in ligature-induced periodontitis in diabetic rats. Diabetes mellitus (DM) was induced with 50 mg/kg intraperitoneal streptozotocin in 40 male Wistar rats. The Wistar rats were divided into six experimental groups: non-ligated (NL; n = 6); ligature only (L; n = 6); DM only (D; n = 6); DM + ligature (DP; n = 6); DM + ligature + 1 mg/kg/day ASX (ASX 1 group; n = 8); and DM + ligature + astaxanthin 5 mg/kg/day ASX (ASX 5 group; n = 8). Silk ligatures were placed along the gingival margin of the left mandibular first molar tooth. The study duration was 11 days, after which the animals were euthanised. Changes in alveolar bone levels were clinically measured, and RANKL and OPG activities were immunohistochemically examined. Alveolar bone loss was the most significant in the DP group (p < 0.05). Decreased alveolar bone loss was observed in the ASX 5 group (p < 0.05). Although RANKL activity was highest in the DP group, it was observed at lower levels in the groups to which ASX was administered. OPG activity did not differ between groups (p > 0.05). The results of this study suggested that 1 and 5 mg/kg ASX administration reduced RANKL activity and alveolar bone loss in rats with experimentally induced periodontitis.

Keywords

astaxanthin bone loss experimental diabetes experimental periodontitis RANKL

Introduction

Periodontitis is characterized by gingival inflammation, periodontal tissue destruction, alveolar bone loss, and potential tooth loss. Although bacterial plaque is the primary cause of periodontitis, there is speculation that systemic and metabolic factors affect the severity and prognosis, as well as increase the risk of periodontal disease.^1–3 Diabetes mellitus (DM) is a common endocrinopathy diagnosed in humans and domestic animals. Intraoral findings of DM include enlargement of the salivary gland, decreased salivary flow, an increased tendency for infection, delayed and abnormal wound healing, candidiasis, a burning sensation in the mouth, dry mouth, and periodontal disease.⁴ The balance between free radical formation and excretion plays an important role in the pathogenesis of DM and periodontitis,^5–9 as extraradical formation damages the organism. Oxidative stress occurs if there is an increase in free radical formation in the cell, or a decrease in excretion.¹⁰ Oxidative stress occurs when there is an increase in reactive oxygen species (ROS), reactive nitrogen species (RNS), or both.¹¹ Several experimental and clinical trials have shown increased ROS in DM.^12–15

Receptor activator of nuclear factor-κB ligand (RANKL) is a type II homotrimeric transmembrane protein that is expressed in various cells, including osteoblasts, periodontal ligament (PL) cells, lymphocytes, and osteocytes.¹⁶ Its main role in bone is the stimulation of osteoclast differentiation, activity, and inhibition of osteoclast apoptosis.¹⁷ Osteoprotegerin (OPG) is mainly expressed in osteoblasts, and the ratio of RANKL to OPG changes in favor of osteoclastogenesis.¹⁸ Studies have shown that OPG-mediated RANKL inhibition prevents alveolar bone loss in experimental periodontitis.¹⁹

Astaxanthin (ASX) carotenoid is a potential antioxidant known to be a single oxygen destroyer, as well as an effective superoxide anion and hydroxyl radical cleanser.^20,21 Furthermore, it can effectively clean lipid radicals in the cell to protect fatty acids and sensitive membranes, and effectively eliminate peroxide chain reactions.^22,23 One study reported that the antioxidant power of ASX was 800 times stronger than that of coenzyme Q10, and 6000 times stronger than vitamin C.²⁴ ASX's potential as an antioxidant has attracted the attention of many researchers.^25–30

In one study, the authors concluded that dietary intake of ASX at 0.02% improved glycemic control in female db/db mice.³¹ Nuclear erythroid 2-related factor 2 (Nrf2) is an important transcription factor that binds to the antioxidant response element (ARE), as well as regulates the cellular antioxidant and anti-inflammatory defence and mitochondrial protection.³² Other studies suggested that ASX promotes the Nrf2/ARE signaling by upregulating the Connexin 43 which is a transmembrane protein, initially described as a gap junction protein, which participates in cell communication for the prevention of renal fibrosis in diabetic rats³³ and that ASX blocks advanced glycation end product (AGE) formation and prevents inflammation in the diabetic state.³⁴ In our previous study, ASX was found to be effective in reducing the cell apoptosis and osteoclastogenesis, and in increasing bone formation in an experimental periodontitis model.²⁵ Based on all known biological activities of ASX, the aim of this study was to investigate the effect of ASX on alveolar bone loss in diabetic rats via RANKL and OPG immunohistochemistry.

Materials and Methods

Animals

Forty male Wistar rats (350-400 g) were used in the experimental periodontitis design. The rats were acclimatized for 10 days before the experiment and housed in individual cages in a room with 12-hour light/dark cycles. Water and food were available ad libitum. The experimental procedure was approved by the Pamukkale University's Animal Experiments Ethics Committee (document number PAUHADYEK-2017/22).

Induction of Diabetes

Streptozotocin^a (STZ) at 50 mg/kg body weight was dissolved in 4° C pure water and administered intraperitoneally.³⁵ Animals were fed 5% glucose solution for 24 hours to prevent possible hypoglycemia. Blood glucose levels were measured with a glucometer^b after 3 days, and rats with glucose levels greater than 300 mg/dl were divided randomly into six groups.

Induction of Experimental Periodontitis Model

The procedure was carried out under general anesthesia by intraperitoneal administration using 5% ketamine^c (50 mg/kg body weight) and 2% xylazine chloride^d (5 mg/kg body weight). A 4-0 silk suture^e was placed subgingivally around the cervical region of the left mandibular first molar tooth. All ligatures were positioned subgingivally and checked daily by the same operator (ALA.).

Study Groups

Non-ligated (NL; n = 6), control group did not receive any treatment. Ligature only (L; n = 6), experimental periodontitis only was induced in this group. DM only (DM; n = 6). DM + periodontitis (DP; n = 6). DM + ligature + ASX 1 mg/kg (ASX 1; n = 8). DM + ligature + ASX 5 mg/kg (ASX 5; n = 8) The DM, ASX 1, and ASX 5 groups received both the experimental diabetes and experimental periodontitis models. The ASX 1 and ASX 5 groups received 1 or 5 mg/kg ASX dissolved in olive oil, administered by gastric gavage for 11 days. All rats were euthanised on the twelfth day.²⁵

Alveolar Bone Measurements

Following humane euthanasia, samples were collected and preserved in 10% neutral buffered formalin for 24 hours before all soft tissues were removed. The mandibles were stained with 1% aqueous methylene blue to reveal the cemento-enamel junction (CEJ). The images were obtained using a compatible camera^f at 16 × magnification and were measured at six points of the tooth using digital imaging software^g integrated with a stereomicroscope and camera system.

RANKL and OPG Immunohistochemistry

After stereomicroscopic analysis, the formalin-immersed samples underwent a decalcification procedure using 10% EDTA solution. When decalcification was complete, all samples were dehydrated through an ethanol series and embedded in paraffin, then 3 µm serial sections were prepared for RANKL and OPG immunohistochemistry. The sections were incubated with an anti-RANKL antibody^h, and an anti-OPG antibodyⁱ for 32 minutes in a fully automated slide preparation device^j. Slides were rinsed with water and immersed in a series of alcohol baths (80%, 90%, and 100%) prior to incubation at 65⁰ C for 1.5 hours. The slides were clarified with xylene prior to examination with a light microscope. Finally, it was made transparent with xylene, covered, and analyzed using a light microscope. Sections containing alveolar bone and the right mandibular first molar tooth were graded according to staining intensity as follows: - (minus) staining = 0–10% immunopositivity; + (plus) staining = 10%–25%; + + staining = 25%–50%; +++ staining = 50%–70%; and ++++ staining = at or above 75%.³⁵

Statistical Analysis

Data were presented as mean ± SD. Statistical analyses were performed with SPSS® software (vs21.00). Alveolar bone loss, RANKL, and OPG values were analyzed using the Kruskal-Wallis test, and p < 0.05 was considered statistically significant.

Results

Morphometric resultsconfirmed experimental periodontitis was successfully achieved, and silk ligation application around the mandibular first molar teeth caused periodontal destruction with alveolar bone loss. One rat in each of the diabetes groups died, presumably as a consequence of the disease.

Morphometric Analyses

The extent of bone loss was greatest in the DP group at 11 days (p < 0.05). No significant difference was found between DM and NL groups in terms of alveolar bone loss (p > 0.05). Both L and DP groups showed significantly higher bone loss values compared to the ASX 5 group (p < 0.05). Furthermore, the ASX 1 group experienced mild alveolar bone loss compared to that of the L and DP groups, but the value was not statistically significant (p > 0.05; Figure 1). According to these results, both 1 and 5 mg/kg doses of ASX decreased alveolar bone loss, but an AXS dose of 5 mg/kg, alone, achieved statistically significant results (Figure 2).

RANKL, OPG Immunohistochemistry

The NL group showed the lowest RANKL immunoreactivity among the groups (p < 0.05). Both the L and DM groups had similar RANKL activity (p > 0.05). RANKL activity was higher in the DP group compared to all other groups (p < 0.05). Both doses of ASX decreased RANKL levels compared to the DP group; furthermore, the ASX 5 group experienced a significant decrease in RANKL activity compared to the DP group (p < 0.05), but not compared to the L or DM groups (p > 0.05; Figure 3). Immunoreactivity to RANKL was predominantly observed in osteocytes, osteoclasts, and mononuclear inflammatory cells in the alveolar bone and PL (Figure 4).

OPG levels exhibited a similar pattern among the groups, and the lowest values were observed in the DP group. OPG levels of the ASX 5 group were higher than those of the DP group, but the difference was not significant (p > 0.05; Figure 3). OPG demonstrated immunolabelling in mononuclear inflammatory cells, osteocytes, and in osteoblasts belonging to the alveolar bone border and PL (Figure 5).

Discussion

This was the first study to evaluate the effect of two different doses of ASX on alveolar bone destruction in an experimental periodontitis model using diabetic rats. The results revealed that both dosage levels of ASX may be effective in slowing down alveolar bone destruction and periodontal inflammation via the reduction of RANKL activity.

Ligature-induced periodontitis has been practised extensively in previous research.^36,37 Accumulation of bacterial plaque around the first molar due to the silk suture initiates an inflammatory reaction, causing a significant amount of bone loss at days 11 and 30. In this study, an 11-day period was used to observe the early progress of periodontal disease. In the light of the morphometric analysis, all ligatured molars experienced bone loss. However, factors such as trauma to the tooth during ligation and microbial accumulation on the adjacent tooth may accelerate the destruction process. These factors may also affect the inflammatory process.^25,38

Carotenoids produced from phytoplankton, algae, and plants are responsible for many of the different colors seen in nature. ASX, a carotenoid pigment, is found in salmon, trout, and other aquatic organisms.³⁹ Humans can acquire it through their diet by consuming seafood containing ASX, or from dietary supplements.⁴⁰ ASX is mainly used as a pigmentation source in the food industry, but it also has applications in nutraceuticals and pharmaceuticals.⁴¹ Animals are generally incapable of synthesizing ASX, and therefore depend on dietary intake.⁴¹

ASX provides a variety of biological advantages, such as potent antioxidant properties – both in vitro and in vivo – protection against asthma, therapeutic effects in ischemia-reperfusion injury, protection against liver damage, inhibition of the proliferation of lung cancer cells, such as A549, and suppressive effects on neuroinflammation.^42–47

There is only one study evaluating ASX's effect on alveolar bone destruction, and this study presented promising results of ASX in the field of periodontology.²⁵ However, the effect of ASX on osteoclast differentiation and bone loss in experimental periodontitis model in diabetic rats had not been previously studied. Therefore, for this study, we evaluated the amount of alveolar bone loss, as well as RANKL and OPG activity levels, in both diabetic and non-diabetic rats.

Natural carotenoids, one of them being ASX, possess the unique ability to deactivate ROS, and to increase cellular capacity to block oxidative stress. ASX has been shown to reduce lipid peroxidation damage by protecting membrane structures using a membrane model enriched with a polyunsaturated fatty acid.⁴⁸ Antioxidant activity of ASX was 10 times more than that of zeaxanthin, lutein, canthaxanthin, and β-carotene, and 100 times higher than that of α-tocopherol.⁴⁹ Recent studies have shown reduction in DNA damage and increased immune response in people who consume ASX.^50,51

ASX has many applications in veterinary science, such as enhancing the color and quality of meat in animals, reducing complications from oxidative stress, and reducing infection. Some researchers have suggested administering ASX or ASX-rich raw materials as dietary supplements to animals bred for human consumption, such as broiler chickens, pigs, and ducks, because of ASX's effects on the oxidative stability and quality of meat.^29,30 ASX-rich chewable tablets, drops, or sprays are available on the market as supplementary food for cats and dogs. There is evidence that ASX has a significant impact on the reproductive performance, egg production, and egg quality of aquatic animals.^52–54 One study administered 25 mg/kg ASX in addition to a commercial milk-replacer on suckling lambs for 22 days. The results revealed that ASX improved meat and fat color, increased the lipid stability of frozen meat, and also reduced levels of the additive butylated hydroxytoluene (BHT), which, in large amounts, can cause centrilobular necrosis and hemorrhage in the human liver.³⁰ Some aspects of the biokinetic uptake of ASX in dogs and cats are similar to those in humans.⁵⁵ Another study investigated the effect of ASX on immune response and inflammation in beagle dogs by administering 10, 20, or 40 mg ASX for 16 weeks. ASX reduced the C-reactive protein (CRP), which is an acute-phase protein that responds to inflammation in the body, and it increased concentrations of IgG, IgM, and B cell population. Therefore, ASX has shown to improve humoral immune response and inflammation.⁵⁶ ASX supplementation (0.3 mg/kg body weight/day) decreased plasma triglyceride and malondialdehyde (MDA) concentrations, as well as dehydrogenase (LDH) levels in obese and healthy dogs compared to the control group.⁵⁷ In one study, 75 mg/kg ASX was administered to horses for 8 weeks, and the results revealed that muscle damage decreased significantly due to the decrease in creatine kinase (CK) activity compared to the control group.⁵⁸

The preferred dose of ASX in studies generally ranges from 3 to 50 mg/kg.^59–61 No genotoxic or mutagenic effect of ASX given in doses of 40, 200, and 1000 mg/kg/day, in addition to diet, has been reported.⁶² Furthermore, administration of high doses of ASX (100, 200, and 400 mg/kg/day) did not negatively affect foetal development in rabbits.⁶³ The safety of ASX dosage was discussed and reported by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). Up to 100 mg/kg in addition to a normal diet was considered a safe dosage. For female rats, a concentration of 3.4 mg/kg of body weight per day was found to be equal to 2.0 mg ASX per 60-kg person per day.⁶⁴ The recommended dose of ASX for humans is 2–4 mg/day.⁴⁰ In studies performed with ASX, results showed that both dosage levels of ASX (1 and5 mg/kg/day) are within the confidence interval and can be used. In addition, no adverse effects of ASX in diabetic rats were observed during the study.

It has been reported that ASX suppresses the synthesis of inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), prostaglandins, leukotriene and interleukins, nitric oxide, cyclooxygenase-1 and −2 (COX-1 and COX-2), and interleukin-1β (IL-1β) and is used to prevent and treat inflammatory conditions and diseases.^65,66 The anti-inflammatory effects of ASX on chronic inflammatory lesions caused by oral lichen planus were evaluated and the results of this study showed that ASX suppressed inflammation by preserving the biomembrane in the early stages of the lesion. They attributed this to the inflammatory mediators acting on the biomembrane, which suppressed curative doses of ASX. They reported that ASX may be useful in chronic inflammation associated with oral lichen planus.⁴⁴ In our study, both ASX dose levels were found to reduce periodontal inflammation and subsequent alveolar bone destruction. The effects of ASX using an experimental periodontitis model in rats in one study showed that both dose levels of ASX (1mg/kg and 5mg/kg) reduced alveolar bone loss by reducing osteoclastic activity and increasing osteoblastic activity.²⁵ Similar to this study, the researchers concluded that, in the experimental periodontitis model in diabetic rats, ASX application with similar doses reduced alveolar bone destruction. In contrast to the previous study, only 5mg/kg ASX reached statistical significance in diabetes.

The RANKL-OPG system's contribution to bone and mineral metabolism is well researched. Bone resorption is activated by increased levels of RANKL, and OPG inhibits that function.⁶⁷ A recent study featured evidence from clinical studies and animal models. It showed that bone-resorption mediators, such as RANKL-OPG, are an important pathway to the periodontal pathogenesis of diabetes, and this pathway is associated with alveolar bone homeostasis.⁶⁸ A number of studies focusing on factors associated with osteoclastogenesis have reported that high RANKL levels in periodontal tissues are associated with diabetes.^69–71 These studies have suggested that hyperglycemia may modulate the RANKL/OPG ratio in periodontal tissues, which explains, in part, the enhanced alveolar bone destruction that accompanies diabetes.

In this study, the effect of ASX on RANKL and OPG was investigated in diabetic rats due to the negative effects of diabetes on bone metabolism. Another study found that Heamatococcus pluvialis fractions rich in ASX ameliorate bone loss in rats with experimentally induced osteoporosis, likely due to the down-regulation of serum OPG in concurrence with up-regulation of serum RANKL.⁷² Based on the results of our study, we suggest that ASX administration decreases alveolar bone loss by decreasing RANKL activity.

There is still no effective treatment for eliminating diabetes-related complications due to the complex pathogenesis of diabetes. Therefore, employing antioxidants to suppress oxidative stress and inflammation are discussed. ASX has the ability to protect pancreatic β-cells against glucose toxicity.⁴⁰ It has also been shown to play a role in the recovery of lymphocyte dysfunctions associated with diabetic rats.⁷³ ASX has the capacity for a unique molecular structure in the presence of hydroxyl and keto moieties on each ionone ring, which gives it its high antioxidant properties.⁷⁴ ASX has been shown to have protective effects on the diabetic nephropathy model of T2DM rats by inhibiting expression of oxidative stress and inflammatory mediators.³¹ It has been found that ASX increased the level of superoxide dismutase (SOD) and decreased the level of MDA in the hippocampus of T2DM rats. In addition, the levels of pro-inflammatory mediators, such as IL-1β and IL-6, were reduced in ASX treatment.⁷⁵ Another studydemonstrated the protective capacity of ASX (3 mg/kg/day for 8 weeks) against oxidative damage in the ocular tissues of diabetic Wistar rats. According to the results, ASX exhibited a protective effect on diabetic retinopathy through the down-regulation of nuclear transcription factor-kappa B (NF-κB) activity, as well an increase in antioxidant enzymes, and a reduction in downstream inflammatory mediators’ expression.⁷⁶

Diabetes, chronic inflammation, and increased oxidative stress occur in both humans and animals. There are many mechanisms in diabetes that cause alveolar bone loss. Therefore, antioxidant usage in diabetes may provide additional benefits.

Conclusion

Within the limitations of this study, we suggest that administering either 1 or 5 mg/kg, especially 5 mg/kg, of ASX reduces RANKL activity, and results in decreased alveolar bone loss in diabetic rats with experimental periodontitis. To better understand the mechanism of ASX in diabetes, investigation of anti-inflammatory activity and osteoblastic activity with specific markers should be considered for further study.

Footnotes

Acknowledgments

RANKL and OPG immunohistochemistry procedure was performed in a private pathology laboratory.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Pamukkale Üniversitesi, (grant number 2019KKP022).

Materials

a. Zanosar Teva Pharmaceuticals, Irvine, CA

b. Contour plus, Ilfov, Romania

c. Eczacibasi Ilac Sanayi, Istanbul, Turkey

d. Virbaxil®, Virbac do Brasil Ind. Com. Ltda, São Paulo, SP, Brazil

e. Dogsan Ilac Sanayi, Istanbul, Turkey

f. Canon EOS 1000, Tokyo, Japan

g. Zeiss, Stemi 2000, Oberkochen, Germany

h. Novus Biological, NB100-80849

i. Novus Biological, NB100-92279

j. Ventana Benchmark XT, Roche, Germany

ORCID iDs

Aysan Lektemur Alpan

Metin Çalışır

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