盐酸小檗碱对实验性牙周炎牙周组织及相关细胞因子的影响_英文_赵玮

中国组织工程研究与临床康复 第14卷 第2期 2010–01–08出版Journal of Clinical Rehabilitative Tissue Engineering Research January 8, 2010 Vol.14, No.2P .O. Box 1200, Shenyang 110004 370 1Department of Stomatology,People’s Hospital of Peking University, Beijing 100044, China; 2College of Stomatology,Lanzhou University, Lanzhou 730000, Gansu Province, ChinaZhao Wei ☆, Doctor, Attending physician, Department of Stomatology,People’s Hospital of Peking University, Beijing 100044, Chinarmzhaowei@ Correspondence to: Gao Cheng-zhi, Doctor, Chiefphysician, Master’s supervisor, Department of Stomatology,People’s Hospital of Peking University, Beijing 100044, Chinagaochengzhi@ Received: 2009-07-19 Accepted: 2009-09-20 (20090619009/W)Zhao W, Yu ZH, Gao CZ.Effects of berberinehydrochloride on periodontal tissues and cytokineexpression in rats with experimental periodontitis. Zhongguo ZuzhiGongcheng Yanjiu yu Linchuang Kangfu. 2010;14(2): 370-374.[ ]Effects of berberine hydrochloride on periodontal tissues and cytokine expression in rats with experimental periodontitis ☆Zhao Wei 1, Yu Zhan-hai 2, Gao Cheng-zhi 1AbstractBACKGROUND: The broad-spectrum antibacterial action of berberine hydrochloride mainly contributes to recurrent aphtha,periapical periodontitis, radioactive mucositis and pericoronitis, however a little evidence support the action mechanism underlying periodontitis treatment.OBJECTIVE: To determine the effects of berberine hydrochloride on the expressions of related cytokines in periodontal tissues of experimental periodontitis rats, to reveal and understand the action pathway of berberine hydrochloride on oral tissue repair.METHODS: Sixty Wistar rats weighing 160-200 g, aged 3 months Models, were involved in this study. Models of experimental periodontitis were established in rats through a use of local steel-wire ligation and systemic injection of prednisone acetate. Forty successfully established models were randomized into periodontitis model group (n =8) and periodontitis treatment group (n =32), at the same time, 10 normal rats served as control group. The treatment group of animals were fed with 0.06 g/kg berberine hydrochloride daily and medicated to death over the 1, 2, 3, 4 weekends (8 rats each). The model group was fed with isodose normal saline. The model group and normal control group were killed at the fourth weekend. Main observations: ①Oral gross observation and X-ray film examination; ②Pathological assay of periodontal tissues; ③Immunohistochemical SABC method was conducted to determine the expressions of tumor necrosis factor-α (TNF-α), bone gla protein (BGP), interleukin-1β (IL-1β), interleukin-6 (IL-6) in periodontal tissues in rats.RESULTS AND CONCLUSION: ①Following hormone injection, gum tissue exhibited erosion and pyorrhea in model group of rats; the above-mentioned symptoms were relieved in rats of treatment group; there was no abnormality in periodontal tissues of normal rats. X-ray examination revealed alveolar crest resorption and obvious interradicular shadow in the model group. ②Rats of model group showed obvious pathologic changes in periodontal tissues, the levels of TNF-α, IL-1β, IL-6 weresignificantly higher and the level of BGP was dramatically lower than those in normal group (P < 0.05); Treatment with berberine hydrochloride decreased the levels of TNF-α, IL-1β, IL-6 in periodontal tissues and increased the level of BGP compared with model group (P < 0.05). The periodontal tissues in groups treated with berberine hydrochloride exhibited pathological changes at inflammatory repair stage. Results showed that berberine hydrochloride inhibits the expressions of TNF-α, IL-1β, IL-6 in periodontal tissues in experiment rat models of periodontitis, and promotes the expression of BGP and repair of periodontal tissue.INTRODUCTIONPeriodontitis is a kind of non-specific inflammatory disease in teeth supporting tissues, caused bysuch microorganisms as Gram negative anaerobe, monotrichous, helicoids and helicoids [1]. Dental plaque is the predominant etiological factor of Periodontitis, host immune system can act a defensive role and release varying inflammation mediators, such as tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), which may result in secondary injury of periodontal tissues [2].Therefore, facilitating local plaque formation and reducing defensive capacity of host may be involved into the considerations of establishing experimental periodontitis models [3]. This study aimed to observe the pathological changes of periodontal tissues following berberinehydrochloride perfusion into the experimental periodontitis rat models. Immunohistochemical SABC method was conducted to determine the expressions of TNF-α, bone gla protein (BGP), IL-1β, IL-6 in periodontal tissues in rats [4], thusproviding experimental evidence for berberine hydrochloride treatment of periodontitis.MATERIALS AND METHODSDesignA randomized controlled animal experiment.Time and settingThe experiment was performed at Department of Stomatology in the People’s Hospital of PekingUniversity and at the Medical Experimental Center of Lanzhou University (BSL-2 grade) between May 2007 and May 2008.MaterialsSixty Wistar rats weighing 160-200 g, aged 3 months, of either gender, was offered by Medical Experimental Center of Lanzhou University, China (License No. Medicine14-006). All theexperimental protocols were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals , formulated by the Ministry of Science and Technology of the People’s Republic of China [5].Zhao W, et al. Effects of berberine hydrochloride on periodontal tissues and cytokine expression in…ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 371www.CRTER.orgMain reagents are as follows ：MethodsExperimental periodontitis models established in Wistar rats Sixty rats were involved in this study and 15 of them served as normal control group. The remaining 45 rats were used for model establishment. Briefly rats were successfullyanesthetized using intraperitoneal injection of 10 mL/kg, with limbs and head fixed, left upper mandible second molar was ligated with oral orthodontic steel ligature (0.2 mm diameter), taking the ligature inserting into free gingival but no damage to junctional epithelium as suitable. Intramuscular injection of 5 mg/kg (1 mL/kg) prednisolone acetate was performed twice daily, for 7 times, then once every three days, for twice. Totally 9 times of rechecks were done regularly. Five rat models and five normal rats were killed through cervical dislocation method, then left upper mandible alveolar bone, teeth and periodontal tissue were cutting off within 5 minutes, followed by steel ligature removal, X-ray film, saline washing, fixation,decalcification. Left upper mandible second molar was prepared into buccal palate paraffin sections at a 5-µm thickness for hematoxylin-eosin stain, to observe frontal resorption and periodontal pocket formation.Berberine hydrochloride for treatment of periodontitis in rats Forty successfully established models were randomized into periodontitis model group (n =8) and periodontitis treatment group (n =32), at the same time ten normal rats served as control group. The treatment group of animals were fed with 0.06 g/kg berberine hydrochloride daily by drench andmedicated to death over the 1, 2, 3, 4 weekends (8 rats each). The model group was fed with isodose normal saline. The model group and normal control group were killed over the 4 weekend. All groups were prepared into periodontal tissue section for hematoxylin-eosin stain to observe the pathological changes. Immunohistochemical SABC method was conducted to determine the expressions of TNF-α, BGP , IL-1β, IL-6 in periodontal tissues in rats.SABC method procedureGlass slices were soaked in polylysine 5 minutes and baked 30-60 minutes at 58 ℃-60 ℃, then warm water bath at 40 ℃and dried in 37 ℃ incubator. Xylene dewaxing, water washing, soaked 10 minutes in 3% H 2O 2, water washing, adding 0.01 mol/L citrate buffered solution (pH 6.0), cooking 3 minutes in microwave oven (pyretic stroke), cooled to room temperature. PBS rinsing 5 minutes, adding 5% BSA blocking solution at 20 . Adding TNF ℃-α, BGP , IL-1β, IL-6 monoclonal antibodies, while PBS into normal control group, placing overnight 4 . ℃PBS washing three times, 5 minutes once, adding biotinylated goat anti-mouse, in 20 ℃-37 incubator ℃for 0.5 hour SABC 20 ℃-37 incubator ℃for 0.5 hour. DAB coloration 10-20 minutes, hematoxylin counterstained, alcohol dehydration, xylene douse, neutral gum mounting, stained particles observed undermicroscopy. Image analysis adopted average absorbance value.Main outcome measures①Oral gross observation. ②Macroscopic observation and X-ray film examination; ③Pathological assay of periodontal tissues; Expressions of TNF ④-α, BGP , IL-1β, IL-6 in periodontal tissues.Statistical analysisstatistical analysis was performed using SPSS 10.0 software. Immunohistochemical staining strength was analyzed using Chi-square test. A level of P < 0.05 was considered statistically significant.RESULTSGross observation in model group and normal control groupAt 4 days after hormone injection, rat models exhibited extrados curling, rarefraction and dim hairs, lassitude, decreased appetite, dry stool and so on. These symptoms became severe along with injection times; in the treatment group, these symptoms were improved with the increased medication duration; normal controlled rats exhibit normal manifestations.Macroscopic observation and X-ray examination (Figure 1).a: Normal group Figure 1 X-ray photograph of periodontal tissues Reagent Source Berberine hydrochlorideDimethyl sulfoxide Ketamine Hydrochloride Injection 0.01 mol/L phosphate buffered solution, 0.01 mol/L citrate buffered solution, DAB coloration kitInstant SABC immunohisto-chemical stain kit, rabbitanti-TNF-α, rabbit anti-BGP , rabbit anti-IL-1β, rabbit anti-IL-6 mono-clonal antibody AX80 automatic systemic micros-copy National Institute for the Control of Pharmaceutical and Biological Prod-ucts, China Asia-Pacific Fine Chemical IndustryCo.,Ltd Fujian Gutian Pharmaceutical Co.,Ltd, China Beijing Zhong Shan -Golden Bridge Biological Technology CO.,LTD, ChinaWuhan Boster, ChinaOlympus, Japan b: Model groupZhao W, et al. Effects of berberine hydrochloride on periodontal tissues and cytokine expression in…P .O. Box 1200, Shenyang 110004 372www.CRTER.orgAt 4 days after hormone injection, rat models exhibitededema of left upper mandible second molar, swelling dark red at 7 days, then periodontal pocket formed and deepened, gum tissues presented with erosion and pyorrhea; These symptoms were relieved in the treatment group; periodontal tissues remained unchanged in normal controlled rats. X-ray examination revealed alveolar crest resorption and obvious shadow between roots in the left upper mandible second molar, sever absorption reached half of the root.Pathological observation of periodontal tissuesIn normal control group, there were no pathological symptoms in gingival epithelium and subcutaneous connective tissue, periodontal membrane fiber arranged in order manner,alveolar bone was smooth, no osteoclasts or bone absorption lacuna occurred. In model group, periodontal membrane fiber arranged in disorder manner, alveolar crest absorbed dramatically and were nibble shaped, alveolar bone was osteoporosis-like, with obvious osteoclasts and boneabsorption lacuna. In the treatment group, inflammatory cells reduced and fibroblasts gradually increased, periodontal spaces recovered to normal levels, periodontal membrane and alveolar bone structure were close to normal levels, periodontal principal fibers arranged in order manner, few vasodilatation and lymphocyte exudation in lamina propria, indicating periodontal tissues at inflammation repair stage (Figure 2).Expressions of TNF-α, BGP , IL-1β, IL-6 in periodontal tissues (Table 1)The positive stain sites of TNF-α, BGP , IL-1β, IL-6 were mainly located in cytoplasm, yellow-brown color (Figures 3-5).a: Normal control groupFigure 2 Hematoxylin-eosin staining of periodontal tissues (×20)c: Treatment group at the end of 4 wk b: Model group a:TNF-α expression in modelgroup Figure 3 Expression of tumor necrosis factor-α (TNF-α) and bone gla protein (BGP ) in periodontal tissues of rats (×40) c: BGP expression in model group b: TNF-α expression in treatmentgroup at the end of 4 wk d: BGP expression in treatment group at the end of 4 wk a: IL-1β expression in m odel group b: IL-1β expression in treatmentgroup at the end of 4 wk Figure 4 Expression of interleukin-1β (IL-1β) in periodontal tis-sues of rats (×40)Zhao W, et al. Effects of berberine hydrochloride on periodontal tissues and cytokine expression in…ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 373www.CRTER.orgIn the model group, the BGP expression in periodontal tissues was decreased, while the levels of TNF-α, IL-1β, IL-6 were significantly higher than those in normal group (P < 0.05); from the second week, following treatment with berberine hydrochloride decreased the levels of TNF-α, IL-1β, IL-6 in periodontal tissues and increased the level of BGP compared with model group (P < 0.05).DISCUSSIONAccording to reference [6], local ligation method, that is ligating left upper mandible second molar with oral orthodontic steel ligature and inserting into free gingiva, taking no damage to junctional epithelium as a principal. Accordingly dental plaque can be greatly accumulated and intramuscular injection of prednisolone acetate can reduced the defense function of immune system in experimental animals, resulting in endocrine disturbance and osteoporosis, thus facilitating periodontal disease occurrence. Rats were involved asexperimental animal due to their periodontal tissue structure, histopathology, dental plaque formation and development, multiply and proliferation are close to human being.Pathological changes of periodontal tissues in periodontitis animals, such as bone resorption and destruction, are all similar with periodontitis lesion in human [7]. In this study, intramuscular injection of hormone was given immediately following ligation, animal death rate was shown to be high, which is possible due to great periodontal injury occurred upon ligation and immediately medication of hormone could reduce resistance and result in infective death [8]. Therefore, intramuscular injection of hormone conduced at 2 days after ligation, totally 9 times, then models were confirmed a success through X-ray film and pathological section.IL-1β and TNF-α, as the cytokines determined in this study, aim to induce inflammation, directly or indirectly mediate bone tissue absorption, their varying biological effects correlate with periodontal tissue destruction, thus considered as an important factor to mediate periodontitis [9-10]. Studies reported that, IL-1 and TNF-α exhibited a high concentration in gingival crevicular fluid of periodontitis sites, and then reducedfollowing treatment [11]; the increased inflammation degree of periodontitis relates with the increased IL-1 concentration [12]; in case of primitive model of chronic periodontitis, IL-1 and TNF-α antagons can reduce the inflammation cells in alveolar bone by 80% and bone absorption by 60%[13]. IL-1 and TNF-αin gingival crevicular fluid can be used as an effective and non-invasive method for periodontitis process determination. Periodontal treatment could reduce the levels of IL-1 and TNF-α[14]. IL-6 influences periodontitis through the following aspects [15]: ①induces vascular endothelial growth factor expression in periodontal tissues, promotes vascularization, aggravates energy expenditure and inflammation; ②inhibits periodontal membrane cells growth, influences tissue metabolism and repair function; ③induces osteoblast to generate osteoclast differentiation factor and matrix metalloproteinase, which can promote bone matrix degradation; ④inhibit osteoblast alkaline phosphatase activity and osteogenesis action. Many scholars report that IL-6 expression in periodontal tissues has a positivecorrelation with periodontitis degree, IL-6 expression can serve as an indicator of periodontal tissue destruction, effective controlling IL-6 plays an important significance on periodontitis treatment [16].BGP widely exists in vertebrate bone tissues and is a marker of mature osteoblasts [17-18]. Current evidence indicate that BGP indicative role remains controversial although it is a specific and sensitive biochemical indicator for bonemetabolism. McCracken et al [19] have reported serum BGP concentration increases, then alveolar bone exhibited obvious destruction. King et al [20] have studied the bone transition in the process of orthodontic movement through a combined method of histomorphology and serum BGPdetermination, results showed that serum BGP concentration increased at the peak of bone formation, which wasconsistent with the present study outcomes. Therefore the increased BGP level is not enough to express bone formation or bone resorption, it is suggested to combine with other determination indices to conduct comprehensive analysis, therefore the levels of TNF-α, BGP , IL-1β, IL-6 weredetermined simultaneously in this study to obtain a objective outcome.Berberine hydrochloride, as an active component of traditional Chinese medicine Golden Thread, acts abroad-spectrum antibacterial activity and is usually applied in clinical treatment of intestinal bacteria infection [21-22]. Recent studies have proved anti-arrhythmia, hypoglycemic effect, anti-inflammation and immunoloregulation [23-25]. It has achieved a use in Department of Stomatology to treatrecurrent aphtha, periapical periodontitis, radioactive dental membranitis and pericoronitis [26]. In this study, gross observation and histomorphology of the experimentalperiodontitis rats were shown to improve following berberine hydrochloride treatment, in comparison with model group; the levels of TNF-α, IL-1β, IL-6 in periodontal tissues were significantly decreased, while BGP levels was significantly increased compared with model group (P < 0.05). Indicating the fact that the levels of TNF-α, BGP , IL-1β, IL-6 inperiodontal tissues correlates with the periodontitis degree and bone destruction, also berberine hydrochloride canreduce the levels of TNF-α, IL-1β, IL-6 in periodontal tissues and increase BGP level upon inflammation reaction, avoid inflammatory reaction, directly or indirectly inhibit bone resorption, inhibit inflammatory cells aggregation intoperiodontal tissues, inhibit osteoclast formation, and relieve osteoclast function [27]. It is indicated that berberineFigure 5 Expression of interleukin-6 (IL-6) in periodontal tissuesof rats (×40) a: IL-6 expression in model groupb: IL-6 expression in treatmentgroup at the end of 4 wkZhao W, et al. Effects of berberine hydrochloride on periodontal tissues and cytokine expression in…P .O. Box 1200, Shenyang 110004 374www.CRTER .orghydrochloride can relieve inflammation reaction, inhibitosteoclast formation and promote functional rehabilitation of periodontitis patients in clinical context.REFERENCES[1] Shu R. Research advances in etiology and treatment of periodontal disease. Shanghai Jiaotong Daxue Xuebao: Yixueban. 2007;27(6):625-627.[2] Cochran DL. Inflammation and bone loss in periodontal disease. J Periodontol. 2008;79(8 Suppl):1569-1576.[3] Che YH, Li GY , Zhao LJ. Evaluation of effect of animal models of parodontitis using synthetic methods. Jilin Daxue Xuebao: Yixueban. 2006;32(6):1119-1121.[4] Zhang XH, Zhang GY , Yu ZH. 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Effects of berberinehydrochloride on human periodontal ligament cells cultured in vitro. Shiyong Kouqiang Yixue Zazhi. 2007;23(5):637-640.盐酸小檗碱对实验性牙周炎牙周组织及相关细胞因子的影响☆赵 玮1，余占海2，高承志1 (1北京大学人民医院口腔科，北京市 100044；2兰州大学口腔医学院，甘肃省兰州市 730000)赵 玮☆，女，1975年生，陕西省榆林市人，汉族，2003年四川大学华西口腔医学院毕业，博士，主治医师，主要从事口腔临床研究。