Protective Effect of Fufang Yatongding on Periodontitis Model Rats

Xianjin ZENG, Zhijun ZHOU, Jing HU, Shihao LUO

Affliated Hospital of Jinggangshan University, Ji’an 343000, China

Abstract [Objectives] The paper was to investigate the protective effect of Fufang Yatongding on experimental periodontitis in rats. [Methods] Experimental periodontitis rats were randomly divided into blank group (5 rats), model group, control group and experimental group, with 8 rats in each group. The rats in the blank group were fed with normal diet, and those in the model group, control group and experimental group were administered intragastrically with normal saline, minocycline hydrochloride solution and Fufang Yatongding solution, respectively. After 4 weeks, alveolar bone resorption was measured. Serum matrix metalloproteinases (MMPs) and inflammatory factors were detected by ELISA, and the changes in gingival tissue were observed by HE staining. [Results] Compared with the control group, the distance from enamel cementum to alveolar crest in the experimental group was decreased (P<0.05). Compared with the control group, the levels of serum MMPs and inflammatory factors in the experimental group were decreased (P<0.05). The results of HE staining showed that the cells in the gingival tissue of rats in the blank group were normal in morphology and intact in structure, and the cells in the gingival tissue of rats in the model group were damaged and out of order, while the cells in the control group were slightly intact and arranged orderly, and the pathological damage of rats in the experimental group was less than that in the control group. [Conclusions] Fufang Yatongding has protective effect on experimental periodontitis in rats by inhibiting the release of MMPs and inflammatory factors.

Key words Periodontitis, Fufang Yatongding, Matrix metalloproteinase (MMPs), Inflammatory factors, Rats, Sprague-Dawley

1 Introduction

Periodontitis is a chronic infection of periodontal tissue caused by a variety of factors, mainly manifested by gingival inflammation and alveolar bone destruction. In severe cases, it may lead to tooth mobility or even odontoptosis. As the main cause of adult tooth loss, a survey report pointed out that periodontitis is prevalent worldwide, involving all ages, and the patient population accounts for up to 10% of the global population[1]. At present, periodontitis is mainly treated with periodental non-surgical treatment, and cooperated with local or systemic drug therapy. In recent years, it has been found that functional polymorphism of matrix metalloproteinases (MMPs) gene is closely related to an increased risk of periodontitis and plays an important role in bone destruction and remodeling[2]. The level of inflammatory factors is proportional to the severity of periodontitis, and can be used to predict the prognosis of chronic periodontitis[3]. Studies have shown that some Chinese medicines and their purified active components can play a role in treating periodontitis by inhibiting MMPs activity and alleviating inflammatory reaction[3-4]. In this study, the effects of Fufang Yatongding on experimental periodontitis rats were explored with MMPs related proteins and inflammatory factors as the starting point, in order to provide the experimental basis for clinical application.

2 Materials and methods

2.1 Experimental reagents and instrumentsPorphyromonasgingivalis(Beijing BeNa Culture Collection Research Institute); Lipopolysaccharide solution (Wuhan Boster Biological Technology Co., Ltd., 2021A125); Minocycline hydrochloride solution (Shanghai Meiyou Pharmaceutical Factory, H10950348); Fufang Yatongding (Guizhou Tongjitang Pharmaceutical Co., Ltd., Z20025807); Chloral hydrate (China National Pharmaceutical Group Cooperation, 20201206); ELISA kit (Beijing Yita Biotechnology Co., Ltd.); RIPA lysate (Wuhan Boster Biological Technology Co., Ltd.); Microplate reader (TC1000-S, Thermo Fisher, USA); Stereoscopic microscope (Suzhou Nanguang Optical Instrument Co., Ltd.); Pipette (Eppendorf, Germany); SD rats (Hunan SJA Laboratory Animal Co., Ltd.).

2.2 Animal modeling and groupingA total of 35 healthy adult SPF SD rats, 10-12 weeks old, with the body weight of (250±50) g and the male/female ratio of 18∶17 were selected. The use license number of the experimental unit was SYXK (Jiangxi) 2012-0001, and the production license number was SCXK (Hunan) 2016-0002. After 1 week of adaptive feeding in the animal room, the experimental rates were transferred to experimental modeling. The experiment passed the approval of ethical committee. The rats were divided into model group (n=30) and blank group (n=5). The rats in the blank group were fed normally without any treatment. In the model group, periodontitis was induced by molars ligation andP.gingivalisinfection, and the rats were modeled using the operation methods recorded in literature[5-6]. After anesthesia by intraperitoneal injection of 10% chloral hydrate, 4-0 silk thread was taken to ligate the neck area of bilateral maxillary first molars. Then normal saline containing 2 μL ofP.gingivalisand 15 μL of lipopolysaccharide solution was injected evenly into the silk thread of gingival sulcus of rats once every 2 d with pipette. The modeling was conducted for consecutive 4 weeks, and the gingival condition was observed every day. The successful standard for rat modeling referred to the method in the reference[7]. Inflammatory cell infiltration was observed in the gingival tissue of rats; the collagen fibers in connective tissue were destroyed and the junctional epithelium was proliferated towards roots, with deepened periodontal pocket; the alveolar bone structure was loose, the bone resorption lacunae formed, and the number of osteoclasts increased significantly.

A total of 24 rats meeting the requirements of the model were screened out in this experiment. The rats after modeling were stained and labeled, and then divided into 3 groups by random number table method: model group, control group and experimental group, with 8 rats in each group. The rats in the model group were administered intragastrically with equal amount of normal saline; the rats in the control group were administered intragastrically with equal amount of minocycline hydrochloride solution at the dose of 45 mg/kg; and the rats in the experimental group were administered intragastrically with Fufang Yatongding (Guizhou Tongjitang Pharmaceutical Co., Ltd., Z20025807, 50 mL/bottle) at the dose of 500 mg/kg; the administration dosage referred to equivalent dose coefficient conversion method of human and animal body surface area[8]. The rats were administered intragastrically twice a day for consecutive 4 weeks till the sampling nodes.

2.3 Detection method(i) Detection of serum MMPs and inflammatory factors by ELISA. Blood samples were collected from caudal vein of rats in each group, and total protein was extracted with protein lysate. The samples were placed into the wells of ELISA plate, dropped with biotin-labeled antibody, added with ABC working solution, and developed color with TMB. TheODvalue of each well was measured by ELISA plate at the wavelength of 450 nm, and the standard curve was drawn. (ii) Determination of alveolar bone resorption in rats. The rats were executed after anesthesia, and the maxillary bone tissues were separated gradually. Then the maxillary bone tissues were stained in methylene blue solution for 2 min, and the morphological changes of maxilla were observed by stereoscopic microscope. The distance between enamel cementum and alveolar crest from the first to the third molars was measured, and the average value was used as the alveolar bone resorption value of the tooth. (iii) Fluorescent staining. First, picrosirius red dyeing liquor for collagen fiber dyeing was prepared, followed by slice dewaxing, ethanol gradient dehydration, rinsing, dyeing by picrosirius red dyeing liquor, rinsing, dehydrating, transparent and sealing, and finally the slice was observed under a fluorescence polarization microscope. (iv) HE staining. The slices were prepared and stained. The gingival tissues of the maxillary first molars of rats were collected and fixed in 4% paraformaldehyde BS, followed by routine decalcification, dehydration and transparent, paraffin imbedding, and slice preparation. After routine HE staining, the pathological changes of cell morphology were observed under an electron microscope at 10×20 field of vision.

3 Results and analysis

3.1 Comparison of body weight of rats after modelingBefore modeling, there was no significant difference in body weight of rats among all groups (P>0.05). After 4 weeks of modeling, the body weight of rats in the blank group was (274.1±24.1) g and that in the model group was (269.9±23.7) g, and there was no significant difference between the two groups (P>0.05).

3.2 Comparison of gingiva in rats before and after modeling

Fig.1 shows the comparison of experimental periodontitis rats before and after modeling. There was no obvious swelling of gingiva in rats before modeling (Fig.1A). After successful modeling, the gingiva of rats was obviously red and swollen, with deep periodontal pockets and accumulation of food residues (Fig.1B). Before modeling, collagen fibers in periodontal tissues of rats were evenly distributed, dense, with consistent direction and complete boundary (Fig.1C). After successful modeling, collagen fibers in rats were distributed unevenly, loose and disordered, and black absorption holes were observed (Fig.1D). Before modeling, the gingival epithelium of rats was intact, and the junctional epithelium and intrasulcus epithelial structure were normal (Fig.1E). After modeling, there was obvious inflammation and destruction in periodontal tissue, and the junctional epithelium was proliferated towards roots (Fig.1F).

Note: A, B. Visibility graph of gingiva by naked eye; C, D. Periodontal tissue (immunofluorescence staining ×200); E, F. Periodontal tissue (HE staining ×200).

3.3 Determination of alveolar bone resorption of rats in each groupThere was no abnormality in distance between enamel cementum and alveolar crest in the blank group (Table 1). Compared with the blank group, the distance between enamel cementum and alveolar crest was significantly increased in the model group, control group and experimental group (P<0.05). Compared with the model group, the distance between enamel cementum and alveolar crest was decreased in the control group and experimental group (P<0.05). Compared with the control group, the distance between enamel cementum and alveolar crest was decreased in the experimental group (P<0.05).

3.4 MMPs and inflammatory factors in serum of rats in each groupCompared with the blank group, MMPs and inflammatory factors (TNF-α, IL-6, IL-8) in serum of rats in the model group were significantly increased (P<0.05). Compared with the model group, MMPs and inflammatory factors in serum of rats in the control group and experimental group were decreased (P<0.05). Compared with the control group, MMPs and inflammatory factors in serum of rats in the experimental group were decreased (P<0.05, Table 2).

Table 1 The distance from enamel cementum to alveolar crest in rats of each group (mm,

Table 2 MMPs and inflammatory factors in serum of rats in each group (ng/mL,

3.5 HE stainingIn the blank group, the gingival tissue cells were normal in morphology and intact in structure, the startified squamous epithelium was visible, and the collagen fibers in the periodontal membrane were orderly arranged in morphology and structure, as shown in Fig.2A. In the model group, the cellular structure in gingival tissue was damaged and disordered, and the collagen fibers in the periodontal membrane were degenerated and destroyed, accompanied by infiltrating distribution of a large number of inflammatory cells and the formation of microscopic abscess; there were a large number of osteoclasts in the bone and deep periodontal pocket was formed (Fig.2B). In both the control group (Fig.2C) and the experimental group (Fig.2D), the cell structure was slightly intact and orderly arranged, and the destruction of collagen fiber structure was alleviated, but still accompanied by inflammatory cell infiltration, and the number of osteoclasts was reduced. Compared with the control group, the overall pathological damage of rats in the experimental group was reduced.

Note: A. Blank group; B. Model group; C. Control group; D. Experimental group.

4 Discussion

Currently, the etiology and pathogenesis of periodontal disease have not been fully elucidated. Studies have shown that MMPs and immune inflammatory response are closely related to the pathogenesis of periodontal disease. MMPs is considered to be an important regulatory factor in the process of periodontitis, mediating the inflammatory response process[9]. MMPs, with a total of 28 members, is a secretase in zinc and calcium-dependent extracellular proteolytic enzymes, capable of degrading most of the extracellular matrix[7]. MMPs are generally divided into 5 distinct types of enzymes (gelatinase, collagenase, membranous MMPs, matrix proteases, and other proteases). Studies have proved that there are gelatinases (MMP-2 and MMP-9) in dentin, which are produced by odontoblasts and secreted into extracellular matrix[10]. They not

only are involved in the regulation of organic matter formation and mineralization of dentin during the tooth germ stage, but also participate in physiological processes such as tooth age-related changes and dentin repair, as well as the development of some pathological processes, such as caries and periodontal disease[11]. MMPs not only have specific expression in healthy dentin, but also show different expression states and even higher activity in caries-affected dentin[12]. The results of this experiment showed that the expression of MMPs in the model group was significantly increased, which confirmed the above conclusion. MMPs is closely related to periodontal disease injury. First, bacterial acid production can lower the pH of oral environment, and then activate MMP-2 and MMP-9. When the pH is 4.5, its activity can be increased by 4 times, and its collagen decomposition ability is proportional to pH in a certain range. When caries-affected teeth are exposed in the mouth, the buffer effect of saliva makes MMPs in a nearly neutral environment so that MMPs can promote the development of caries to a greater extent[13]. Second,Streptococcusmutanscontaining collagen-binding adhesins in oral cavity can directly activate MMP-9, leading to periodontal injury. At the same time, local periodontal inflammation will destroy the integrity of the epithelium and cause inflammatory transmitters such as interleukin and other cytokines in the periodontal pocket to enter the blood circulation system and peripheral immune organs from the periodontal pocket, causing systemic inflammatory reactions[14]. TNF is an important inflammatory cytokine in periodontal tissue destruction, involving in the migration of inflammatory cells from the periodontitis microenvironment to tissue destruction. It enhances immune response by stimulating the migration of chemokine cells to infection and inflammation sites and promotes the expression of MMPs by up-regulating the expression of IL-1β and IL-6, and further increases osteoclast activity and decreases osteoblast activity, leading to periodontitis tissue destruction[15-16]. IL-6 is a magnifying factor that stimulates inflammatory responses to induce pro-inflammatory cytokines, which can promote lymph B cell differentiation and inflammation, playing a pro-inflammatory role in immune responses[17]. IL-8 releases a series of active products through the activation of neutrophils, ultimately leading to local inflammatory reactions in the body[18]. In this experiment, the damage of rats was most obvious in the model group. The experimental group and the control group could inhibit the gingival injury caused by the molding agent, and the experimental group was superior to the control group in alleviating the gingival symptoms of rats, with lower levels of MMPs and inflammatory factors, suggesting that Fufang Yatongding plays a role by inhibiting the release of MMPs and inflammatory factors.

Minocycline hydrochloride has high antibacterial effect and easy penetration, which is often used clinically for the treatment of gingivitis. The drug belongs to tetracycline derivatives antibiotics, with potent antimicrobial effect and long-lasting duration, which has good inhibitory effect against a variety of pathogens such as gram-negative bacteria and actinomyces, and also interferes with collagenase activity and reduces gingival tissue damage[19]. Periodontal disease belongs to the category of "gingival atrophy, desiccated teeth" in traditional Chinese medicine. It was first recorded in Huangdi Neijing: "exhaustion of qi of foot yin deficiency brings about atrophy of bones…the muscles contract and the teeth look long and dull", which describes the manifestations of periodontitis, such as gingival atrophy, root exposure and tartar attachment. Diet preference or compulsive drinking and smoking will impair the spleen and stomach, resulting in wet accumulation, hindrance of functional activities of qi and gradual congestion, while damp-heat accumulation in orifices will cause periodontitis, and the treatment should be dominated by dispelling wind and eliminating dampness, relieving heat and detoxification, dispersing stasis and relieving pain[4]. In Fufang Yatongding,Valerianaoffeinalisplays the role of dispelling wind and eliminating dampness, promoting qi, invigorating blood and relieving pain;Impatiensbalsaminaplays the role of dispelling wind and removing obstruction in the meridians, clearing away heat and toxic materials, invigorating blood circulation and alleviating pain;Carthamustinctoriouscan promote blood circulation, dissipate blood stasis and relieve pain;Cinnamomumcamphoraplays the role of clearing heat and killing pesticides, clearing qi, dredging collaterals and relieving pain; the combined use of all drugs cooperatively play the role of invigorating the circulation of scattered stasis, relieving swelling and pain. Yang Jialongetal.[20]showed that Fufang Yatongding has bactericidal effect on standard strains of common oral pathogens, and has the strongest inhibitory effect onP.gingivalis. Clinical studies have also confirmed that Fufang Yatongding has good anti-inflammatory and analgesic effects. Our research results showed that Fufang Yatongding has better comprehensive curative effect in the treatment of periodontitis than minocycline hydrochloride.

In summary, Fufang Yatongding inhibits the release of MMPs and inflammatory factors, which may be a mechanism of Fufang Yatongding.