Implications of lysyl oxidase-like protein 3 expression in the periodontium of diabetic rats

Abstract Objectives Diabetes has been strongly associated with periodontal diseases. The periodontal ligament (PDL) has an abundant extracellular matrix (ECM). Lysyl oxidases (LOXs) are closely associated with various diseases caused by abnormal ECM functions, however, the role of LOXs in periodontal diseases induced by diabetes remains unclear. Methodology In this study, 8-week-old Zucker diabetic fatty rats were used to establish a type 2 diabetes mellitus (T2DM) model. After 9 and 16 weeks, hematoxylin and eosin (H&E), Masson’s trichrome, and immunohistochemical staining were performed. Results After 9 weeks, loose collagen fibers were found in the interradicular area of the diabetic group, in opposition to the control group. There were no significant differences in LOX expression between the diabetic and control groups (p>0.05). However, after 16 weeks, the diabetic group presented a disordered arrangement of the PDL, showing decreased collagen content and significantly increased lysyl oxidase-like protein 3 (LOXL3) expression when compared with the control group (p<0.05). This suggests that LOXL3 plays a significant role in periodontal histopathological changes in diabetic rats. Conclusion Our study showed elevated LOXL3 expression in the PDL of diabetic rats after 16 weeks, suggesting that LOXL3 may be involved in the occurrence and development of periodontal histopathological changes in diabetic rats. LOXL3 could be further used as an indicator for the early diagnosis of diabetic periodontitis in T2DM patients in clinical settings.


Introduction
Diabetes mellitus (DM) is a global disease that causes severe morbidity and mortality in humans.
A large proportion of DM cases is characterized as type 2 DM (T2DM), which is attributed to unhealthy eating habits, obesity, and physical inactivity. 1 T2DM can lead to complications in various organs, such as the heart, 2 eyes, 3 kidneys, 4 and nervous system, 5 resulting in high financial and health burden on patients. 6 T2DM complications are difficult to diagnose at the early stages of the disease because there is a gradual increase in blood glucose levels without obvious and typical clinical symptoms. However, during this early stage of the disease, there is considerable damage to related tissues, which could be detected by histopathological examination. 7 In clinical settings, T2DM and periodontal diseases have a positive correlation. 8,9 Patients with more severe diabetes tend to have more serious periodontal diseases and vice versa. Therefore, the histopathological examination of periodontal tissue changes is very significant in the early diagnosis of periodontal diseases induced by diabetes.
Lysyl oxidases (LOXs) are a series of enzymes characterized as copper-dependent enzymes, which can stabilize matrix components. LOXs exist in humans and animals in five different forms: lysyl oxidase (LOX) and lysyl oxidase-like [1][2][3][4]. 10 LOXs are potential biomarkers for diseases such as cardiovascular diseases, neurodegeneration, and cancer metastasis. 10 In our previous studies, abnormal LOX expression was also observed in the kidney tissue and bone matrix of Zucker diabetic fatty (ZDF) rats, suggesting that LOXs play a part in diabetic nephropathy and bone fragility. 7,11 Moreover, LOXs play an important role in remodeling the extracellular matrix (ECM) by cross-linking collagen and elastin. 12,13 Vallet and Ricard-Blum 14 (2019)

Group and processing
In this study, to establish a T2DM model, 8-week-old obese male ZDF rats (ZDF-Leprfa/Crl, fa/fa) (diabetic group) and lean male ZDF rats (fa/+) (control group) were purchased from Beijing Vital River Laboratory Implications of lysyl oxidase-like protein 3 expression in the periodontium of diabetic rats J Appl Oral Sci.

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Animal Technology Co., Ltd. (Beijing, China) and fed with a high-fat diet (Purina 5008, Harlan Teklad, Indianapolis, IN, USA). The total number of rats was 60 and rats were randomly divided into two groups, according to the time of sample collection: 9 weeks and 16 weeks (n=10 in each group). All rats were housed at a temperature of 20-25°C and 65-69% humidity under a 12-hour light/dark cycle with free access to food and water. During the whole experiment, the position of the cages was not changed and the physical conditions of rats were observed every other day. Any abnormality found was treated in time.

Tissue preparation
At 9 and 16 weeks after induction of diabetes, rats were euthanized and then maxillary samples were extracted. After removing the soft tissue, the maxillary samples were fixed in 4% paraformaldehyde for 48 hours and decalcified for five weeks.

Histopathological examination
In this study, the periodontal ligament of the first molar (M1) was divided into four parts. The upper part is the apical area, the middle part is the oblique area, and the lower part is the interradicular and alveolar crest areas (Figure 1). After 9 and 16 weeks, H&E and Masson's trichrome staining were performed to evaluate periodontal histopathological changes.
The mean integrated optical density (OD) was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
The mean optical density (OD) was considered the analysis index and the OD of LOX, LOXL1, LOXL2, and LOXL3 is presented as mean±standard deviation. The SPSS software (SPSS Inc., USA) was used to compare data between different groups. The Mann-Whitney U test was used for nonparametric data and U>1.96 was considered statistically significant.

Results
Histopathological changes in the periodontal tissue In this study, histopathological changes were observed by H&E and Masson's trichrome staining.
After 9 weeks, no obvious changes were observed in the control group, however, in the diabetic group, alveolar bone discontinuity and reduction in collagen fiber content (colored in blue) were observed (   In clinical dentistry, it has been observed that, when compared with non-T2DM patients, T2DM patients have a higher risk of gingival inflammation and periodontal disease. 24 Borgnakke, et al. 25 (2013) and Kim, et al. 26 (2014) showed a strong correlation between periodontitis and T2DM. However, the precise mechanism remains unclear.
Periodontitis is a common oral disease associated receding gums, and tooth loss. 29 et al. 30 (2012) and Borgnakke, et al. 25 (2013) showed that T2DM patients have distinct microvascular lesions in gingival vessels and a significantly thickened basement membrane. In this study, after 16 weeks, abnormalities in fibroblasts and collagen fibers in diabetic rats were observed. Moreover, they also presented aggressive alveolar bone destruction, 26 which may be due to the gradual severity of periodontal lesions over time.
It highlights the importance of early diagnosis of periodontal diseases induced by diabetes.
Studies showed a positive correlation between blood glucose levels and the occurrence and progression of periodontitis. 31 Mealey 32 (1996) showed that the prevalence and severity of periodontitis is higher in T2DM patients when compared with T1DM patients. The PDL is susceptible to bacterial infections due to chronic hyperglycemia or poor long-term blood glucose control. [33][34][35] The degree of periodontal tissue damage in T2DM patients is significantly reduced when blood glucose was well controlled. 32 Our previous study showed that the blood glucose of rats in the diabetic group was significantly higher when compared with the

Conflict of interest
The authors declare no potential conflict of interest regarding the authorship and/or publication of this article.

Data availability statements
The datasets generated during this study are available by the corresponding author upon reasonable request.