Objective: Brain and muscle ARNT-like protein 1 (BMAL1) is a core component of hepatocyte molecular clock and plays an important role in the regulation of other related rhythmic genes in the body through a transcriptional-translational feedback loop in molecular circadian oscillations. Therefore, the aim of this study was to investigate the role of BMAL1 in the rat periodontitis-induced liver injury. Methods: Twelve male Wistar rats were divided into the control group and the periodontitis group according to the random number table method. The rats in the control group were untreated. The periodontitis models were established by ligating the necks of the bilateral maxillary first molars in the periodontitis group rats. After 8 weeks, periodontal clinical indexes of rats in both groups were examined and executed. Micro-CT scans of the maxilla were performed and levels of the alveolar bone resorption were analyzed. Pathological changes in periodontal and liver tissue of rats in two groups were detected by HE and oil red O staining. Biochemical kits were used to detect glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total cholesterol (TC) and triglycerides (TG) in serum. The gene and protein expression levels of BMAL1, nuclear factor kappa-B (NF-κB) and tumor necrosis factor-α (TNF-α) in liver tissue were measured by real time fluorescent quantitative-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) assays. Apoptosis was detected in liver tissues by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit staining. Results: The results of HE staining of maxillary first molars and micro-CT results of maxillary bones showed that alveolar bone resorption was significant in the periodontitis group of rats. The liver histopathology results showed infiltrated inflammatory cells in the liver tissue, disorganized liver cords and a large number of lipid droplets formed in the hepatocytes of the periodontitis group compared with the control group. The results of serum biochemical assay showed that the levels of GOT [(62.77±2.59) U/L], GPT [(47.54±1.04) U/L], TC [(3.19±0.23) mmol/L] and TG [(1.11±0.09) mmol/L] in the serum of rats with periodontitis were significantly higher than that in the control group respectively [GOT: (38.66±2.47) U/L, GPT: (31.48±1.57) U/L, TC: (1.60±0.05) mmol/L and TG: (0.61±0.09) mmol/L](P=0.003, P=0.001, P=0.002, P=0.038). qRT-PCR results showed that the mRNA expression level of BMAL1 was significantly decreased in liver tissue of the periodontitis group [(0.60±0.04)%] compared to the control group [(1.01±0.07)%] (t=4.80, P=0.009), while the mRNA expression levels of NF-κB and TNF-α [(1.62±0.12)%, (2.69±0.16)%] were significantly increased compared to the control group [(1.00±0.03)%, (1.03±0.16)%] (P=0.008, P=0.002); IHC results showed that the protein expression level of BMAL1 in liver tissue of the periodontitis group (averaged optical density, AOD) (11.58±2.15) was down-regulated compared to the control group (AOD) (22.66±1.67) (P=0.015), while NF-κB and TNF-α (AOD) (31.77±2.69, 24.31±2.32) were up-regulated compared to the control group (AOD) (19.40±1.82, 11.92±0.94) (P=0.019, P=0.008). WB results showed that the protein expression level of BMAL1 in liver tissue was down-regulated in the periodontitis group [(0.63±0.10)%] compared to the control group [(1.00±0.06)%] (t=3.19, P=0.033), while NF-κB and TNF-α [(1.61±0.12)%, (2.82±0.23)%] were up-regulated compared to the control group [(1.00±0.12)%, (1.00±0.11)%] (P=0.022, P=0.002). TUNEL staining showed increased apoptotic cells in the liver tissue of the periodontitis group of rats compared to the control group. Conclusions: Periodontitis may induce liver injury by down-regulating the BMAL1 expression levels in liver tissue, which in turn activates NF-κB signaling molecules, leading to the elevated levels of inflammation and apoptosis in rat liver.
目的： 探讨脑和肌肉芳香烃受体核转运样蛋白1（brain and muscle ARNT-like protein 1，BMAL1）在大鼠牙周炎诱导肝损伤模型中的作用。 方法： 根据随机数字表法将12只Wistar雄性大鼠随机分为对照组和牙周炎组，每组6只。对照组大鼠不做处理。牙周炎组大鼠通过结扎双侧上颌第一磨牙颈部建立牙周炎模型。建模8周后检测两组大鼠牙周临床指标并处死。显微CT（micro-CT）扫描大鼠上颌骨并分析牙槽骨吸收情况。HE及油红O染色分析两组大鼠牙周组织和肝组织的病理变化。生化试剂盒检测血清中谷草转氨酶（glutamic-oxaloacetic transaminase，GOT）、谷丙转氨酶（glutamic-pyruvic transaminase，GPT）、总胆固醇（total cholesterol，TC）和甘油三酯（triglyceride，TG）等肝功能相关指标。实时荧光定量PCR（real time fluorescent quantitative-PCR，qRT-PCR）、免疫组化和蛋白质印迹法检测肝组织中BMAL1、核因子-κB（nuclear factor kappa-B，NF-κB）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）基因及蛋白的表达水平。原位末端转移酶标记（terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling，TUNEL）试剂盒染色法检测肝组织中的细胞凋亡。 结果： 上颌第一磨牙HE染色和micro-CT结果显示，牙周炎组大鼠牙槽骨吸收明显。肝组织病理学结果显示，与对照组相比，牙周炎组肝组织内炎症细胞浸润、肝索结构紊乱且肝细胞中可见大量脂滴形成。大鼠血清生化检测结果显示，牙周炎组大鼠血清中GOT［（62.77±2.59）U/L］、GPT［（47.54±1.04）U/L］、TC［（3.19±0.23）mmol/L］和TG［（1.11±0.09）mmol/L］均较对照组GOT［（38.66±2.47）U/L］、GPT［（31.48±1.57）U/L］、TC［（1.60±0.05）mmol/L］和TG［（0.61±0.09）mmol/L］含量显著升高（P=0.003，P=0.001，P=0.002，P=0.038）。qRT-PCR结果显示，牙周炎组大鼠肝组织中BMAL1 mRNA的表达［（0.60±0.04）%］较对照组［（1.01±0.07）%］显著下调（t=4.80，P=0.009），NF-κB、TNF-α mRNA的表达［（1.62±0.12）%、（2.69±0.16）%］均较对照组［（1.00±0.03）%、（1.03±0.16）%］显著上调（P=0.008，P=0.002）。免疫组化结果显示：牙周炎组大鼠肝组织中BMAL1蛋白表达（平均光密度值）（11.58±2.15）较对照组（22.66±1.67）显著下调（P=0.015），而NF-κB、TNF-α表达（31.77±2.69、24.31±2.32）均较对照组（19.40±1.82、11.92±0.94）显著上调（P=0.019，P=0.008）。蛋白质印迹法结果显示：牙周炎组大鼠肝组织中BMAL1蛋白表达［（0.63±0.10）%］较对照组［（1.00±0.06）%］显著下调（t=3.19，P=0.033），NF-κB、TNF-α表达［（1.61±0.12）%、（2.82±0.23）%］均较对照组［（1.00±0.12）%、（1.00±0.11）%］显著上调（P=0.022，P=0.002）。TUNEL染色结果显示牙周炎组大鼠肝组织中凋亡细胞较对照组增多。 结论： 牙周炎可能通过下调大鼠肝组织中BMAL1的表达激活NF-κB信号分子，引起大鼠肝脏组织中炎症水平和凋亡水平升高，最终诱导肝损伤。.