基于二氢卟吩e6的光动力疗法联合替硝唑对牙周炎大鼠协同治疗作用的研究

In vitro and in vivo anti-periodontitis effects of combination treatment of photodynamic and antibiotic therapies

目的探讨基于二氢卟吩e6(chlorin e6,Ce6)的光动力疗法(photodynamic therapy,PDT)联合抗生素替硝唑(tinidazole,TNZ)对牙周炎大鼠的体内外抑制作用。方法采用正畸钢丝结扎法对18只SD大鼠建立牙周炎模型,建模成功后按随机数字表法随机分为以下6组(每组3只):阳性对照组、Ce6组、TNZ组、混合物Ce6/TNZ组、Ce6加光照(Ce6+L)组、Ce6/TNZ加光照(Ce6/TNZ+L)组;采用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)实验考察Ce6(质量浓度为0~20 mg/L)、TNZ(质量浓度为0~16.6 mg/L)及其混合物Ce6/TNZ对成纤维细胞L929的毒性;采用荧光探针法检测Ce6、TNZ和Ce6/TNZ及结合635 nm激光照射(功率0.5 W/cm2,时间5 min)对牙周炎大鼠牙菌斑生物膜中活性氧生成的触发效应,评价Ce6的光动力性能;采用细胞计数试剂盒(cell counting kit-8,CCK-8)实验和活菌/死菌染色法考察各种治疗的体外抗菌性能,并计算Ce6介导的PDT联合TNZ的合用指数(combination index,CI)。采用凋亡试剂盒流式检测各种治疗对鼠巨噬细胞RAW264.7凋亡的诱导效应。各组大鼠局部给药后行激光照射(功率0.5 W/cm2,时间5 min),治疗结束后用显微CT评价PDT联合TNZ对牙周炎大鼠牙槽骨吸收的抑制效应。结果Ce6、TNZ、Ce6/TNZ在预设浓度范围内L929细胞存活率均在90%以上。激光照射后,Ce6与Ce6/TNZ的牙菌斑生物膜中呈现出非常显著的绿色荧光,触发了菌斑内活性氧大量生成;但在不同浓度下,Ce6/TNZ的牙菌斑存活率分别为(85.4±5.5)%、(76.0±8.9)%、(61.7±0.6)%、(56.3±2.6)%、(43.5±0.6)%,均显著低于Ce6和TNZ组(P<0.01),且各药物浓度点对应的CI<1.0,具有显著的协同效应。Ce6+L与Ce6/TNZ+L具有显著强于Ce6与Ce6/TNZ的细胞凋亡诱导活性(P<0.01)。牙周炎大鼠体内,Ce6/TNZ联合激光照射能有效抑制牙槽骨的吸收,牙槽骨体积、骨体积分数分别为(1.49±0.07)mm3和(47.08±0.71)%,颊、腭侧釉质牙骨质界至牙槽嵴顶距离分别减小至(2.13±0.07)和(1.94±0.10)mm。结论基于Ce6的PDT联合抗生素TNZ对牙周炎大鼠有协同治疗作用,能有效杀伤牙菌斑,诱导巨噬细胞凋亡,抑制牙槽骨吸收。

Objective To investigate the effects of combination treatment of photodynamic therapy(PDT)based on photosensitizer chlorin e6(Ce6)and antibiotic agent tinidazole(TNZ)against periodontitis both in vitro and in vivo.Methods The Sprague-Dewley(SD)rat periodontitis model was constructed using the method of orthodontic wire ligation.After successful modeling,SD rats were randomly divided into the following 6 groups(3 rats in each group):positive control(Ctrl+),Ce6,TNZ,a mixture of Ce6 and TNZ(Ce6/TNZ),Ce6 with laser irradiation(Ce6+L),a mixture of Ce6 and TNZ with laser irradiation(Ce6/TNZ+L).Methyl thiazolyl tetrazolium(MTT)assay was used to assess the cytotoxic activities of Ce6(concentration range:0-20 mg/L),TNZ(concentration range:0-16.6 mg/L)and their mixture(Ce6/TNZ)in mouse fibroblast L929 cells.Fluorescence probe method was applied to measure the production of reactive oxygen species in the dental plaque biofilms after various treatments with and without 5-minute laser irradiation at 635 nm at a power density of 0.5 W/cm2(Ce6+L and Ce6/TNZ+L groups),thus to evaluate the PDT performances.Cell counting kit-8(CCK-8)and live/dead staining were used to assess the antibacterial activity in each of the groups and the combination index(CI)of PDT combined with TNZ was calculated subsequently.Flow cytometry was utilized to detect the apoptosis-inducing effects of these treatments in macrophage RAW264.7 cells after processing with the apoptosis detection kit.The inhibitory effects of various treatments on the absorption of alveolar bone of SD rats were further evaluated in the periodontitis rats by using the micro-CT.Results The survival rates of L929 cells in the preset concentration range were all above 90%in Ce6,TNZ and Ce6/TNZ groups.Upon laser irradiation,the plaque biofilms in Ce6 and Ce6/TNZ groups showed significant green fluorescence,indicating that large amounts of reactive oxygen species were triggered and generated significantly in the dental plaque biofilms.However,the survival rates of dental plaque microorganisms in 5 Ce6/TNZ concentrations were(85.4±5.5)%,(76.0±8.9)%,(61.7±0.6)%,(56.3±2.6)%and(43.5±0.6)%respectively,which were significantly lower than that in Ce6 only and TNZ only groups(P<0.01).The CI levle of each drug concentration group was less than 1.0,which showed a significant synergistic antibacterial efficiency.Stronger apoptotic activities were observed in Ce6+L and Ce6/TNZ+L groups compared with those in Ce6 only and Ce6/TNZ only groups(P<0.01).In periodontitis rats,Ce6/TNZ combined laser irradiation could effectively inhibit the absorption of alveolar bone.The alveolar bone volume and the ratio of bone volume and tissue volume were(1.49±0.07)mm3 and(47.08±0.71)%,respectively.The distances between cementoenamel junction to alveolar bone crest on buccal and palatal sites decreased to(2.13±0.07)mm and(1.94±0.10)mm respectively,showing a high inhibition efficiency.Conclusions Ce6-mediated PDT combined with TNZ possessed notable synergistic effects against periodontitis,reflecting in the efficient antibacterial effect,the apoptosis-inducing action on macrophages,and the inhibitory efficacy on the alveolar bone absorption in vivo.