Pg-LPS对束缚应激大鼠腹腔巨噬细胞凋亡的影响

目的：检测牙龈卟啉菌内毒素（Pg-LPS）对束缚应激大鼠腹腔巨噬细胞凋亡情况的影响,初步探讨应激影响牙周疾病的机制。方法：采用束缚应激方式,将大鼠随机分为正常对照组、应激组,于应激结束时处死动物,常规收集腹腔液。巨噬细胞于贴壁纯化后分别用RPMI-1640培养液,RPMI-1640培养液＋0．01μg／ml Pg-LPS,RPMI-1640培养液＋1μg／ml Pg-LPS继续培养。于24h后收集细胞爬片,采用荧光染色法、原位末端标记法（TUNEL）观察巨噬细胞凋亡情况。结果：1μg／ml Pg-LPS刺激后,正常对照组及应激组巨噬细胞的凋亡率明显增加,应激组巨噬细胞凋亡率显著高于正常对照组。结论：一定浓度的即LPS刺激后,大鼠腹腔巨噬细胞发生凋亡;束缚应激能加重这种异常。     

内毒素诱导单核—巨噬细胞产生细胞因子的作用机理

内毒素刺激巨噬细胞产生，ＴＮＦ－α，ＩＬ－１，ＩＬ－６，ＩＬ－８，补体Ｃ３前列腺素（ＰＧ）等生物活性物质，在机体内发挥多种生物学，免疫学效应，细胞膜蛋白家族成人员之一的ＣＤ１４在内毒素诱导单核－巨噬分泌细胞因子的过程中起着重要作用，ＣＤ１４与ＬＰＳ－ＬＰＳ结合蛋白复合体（ＬＰＳ－ＬＢＰ）的结合发挥受体功能，抗ＣＤ１４抗体能够阻断ＬＰＳ－ＬＢＰ与单核细胞膜表面的结合，从而抑制细胞因子的产生。     

牙龈卟啉单胞菌脂多糖(Pg-LPS)对破骨细胞EphA2基因表达的调控

目的:研究小鼠单核巨噬细胞系RAW264.7在破骨分化过程中,Pg-LPS对破骨细胞EphA2表达的影响。方法:用终浓度10 mg/L的Pg-LPS 刺激RAW264.7 细胞后,分别在1、3、5 d,应用RT-PCR检测破骨细胞中EphA2基因和破骨细胞相关基因(破骨细胞内基质金属蛋白酶( MMP9)、ACP5、c-fos、组织蛋白酶K(CtsK)、NFATc1)的表达,并且通过酒石酸抗酸性磷酸酶(TRAP)染色观察实验组和对照组破骨细胞的分化成熟情况。结果:RT-PCR检测10 mg/L的Pg-LPS在第3天和5天,实验组比对照组EphA2基因表达分别增高2.4倍和1.2倍,两组之间存在显著差异(P<0.01);同时也能够促进破骨相关基因c-fos、NFATc1、CtsK、ACP5、MMP9的表达,实验组与对照组相比差异有显著性;TRAP染色结果显示:实验组比对照组的TRAP阳性多核细胞数目明显增多。结论:10 mg/L的Pg-LPS对小鼠单核巨噬细胞系RAW264.7,在破骨分化的中期和晚期均能够促进EphA2基因的表达,但是在破骨分化早期对EphA2基因的表达无明显作用。     

IL-10对Pg-LPS刺激下兔枯否氏细胞凋亡的影响

目的:探讨IL-10对Pg-LPS刺激下兔枯否氏细胞凋亡的影响。方法:新西兰兔3只,收集枯否氏细胞(Kupffer cell,KC),分为空白对照组:RPMI1640+KC,Pg-LPS组:1mg/LPg-LPS+KC,IL-10+Pg-LPS组:0.1mg/L IL-10+1mg/L Pg-LPS+KC,刺激24h后,荧光定量PCR法检测凋亡相关基因Caspase-3、Fas、p53的表达量。结果:1mg/L Pg-LPS可促进兔KC的凋亡,Caspase-3、Fas、p53表达量增多(P<0.05);IL-10干预处理可减少兔KC的凋亡,抑制Caspase-3、Fas的表达(P<0.05),但对p53的表达无明显作用。结论:Pg-LPS可通过外源性/内源性凋亡途径导致兔KC凋亡,而IL-10可能主要通过抑制外源性死亡受体途径从而达到抑制KC凋亡的作用。     

牙龈蛋白酶对M1型巨噬细胞表达抑菌细胞因子的调控作用

目的:探讨牙龈蛋白酶对M1型巨噬细胞表达抑菌细胞因子的调控作用。方法:采用sheets改良法从Pg ATCC 33277培养物上清中制备牙龈蛋白酶,应用质谱法鉴定牙龈蛋白酶,底物发色法测定酶活性,鲎试剂检测牙龈蛋白酶中LPS残留量。体外细胞模型实验评价牙龈蛋白酶对大肠杆菌脂多糖(Ec-LPS)诱导的M1型巨噬细胞表达抑菌细胞因子的影响。实验分为3组:阴性对照组、Ec-LPS组和Ec-LPS+牙龈蛋白酶组,采用qRT-PCR和ELISA法检测M1型巨噬细胞表达的抑菌细胞因子白细胞介素12(IL-12)、一氧化氮合酶(iNOS)和白细胞介素10(IL-10)在基因和蛋白水平的表达情况。结果:制备的牙龈蛋白酶为RgpA,活性20 U/L,LPS残留量<0.01EU/mL。qRT-PCR和ELISA结果显示,与阴性对照组相比,Ec-LPS组IL-12和iNOS基因和蛋白的表达水平明显升高,IL-10的表达降低,即成功诱导M1型巨噬细胞,组间有显著差异(P<0.01);Ec-LPS+牙龈蛋白酶组IL-12、iNOS和IL-10基因和蛋白的表达较Ec-LPS组显著降低,但高于阴性对照组,组间有显著差异(P<0.01)。结论:牙龈蛋白酶具有抑制M1型巨噬细胞表达抑菌细胞因子IL-12、iNOS的作用。     

情绪应激对大鼠咀嚼肌疼痛敏感度的影响

目的建立交流箱大鼠心理应激模型,研究情绪应激对大鼠咀嚼肌机械疼痛阈值的影响。方法50只SD大鼠,随机分为5组：空白对照组、药物对照组、盐水对照组、足部电击组和情绪应激组,实验时后4组大鼠均同处在交流箱内,同条件饲养。参照Ren方法,根据von Frey纤维粗细和动物痛觉反应次数,确定咬肌和颞肌疼痛分值。结果1）应激后大鼠咬肌和颞肌疼痛阈值出现变化,在7 d的时候出现最高峰,12~14 d的时候达到稳定状态,但与空白对照组相比疼痛阈值还是降低了。2）应激后药物对照组疼痛阈值变化趋势同情绪应激组,但与情绪应激组相比疼痛阈值要高。结论情绪应激可以导致咬肌和颞肌痛觉敏感,抗抑郁药物可以降低应激导致的疼痛敏感度。     

情绪应激对大鼠咬肌超微结构的影响

目的：应用电镜观察情绪应激对大鼠咬肌超微结构的影响。方法：48只Wistar大鼠，随机分为3组：情绪应激组Ⅰ（3周应激组），情绪应激组Ⅱ（5周应激组）和空白对照组。前两组大鼠同处交流箱内，3组大鼠同条件饲养，分别于3、5周处死大鼠，电镜观察咬肌超微结构变化。结果：3周情绪应激组大鼠咬肌线粒体出现水肿，基质密度降低，线粒体嵴减少，肌纤维微血管内出现充血性改变；5周情绪应激组大鼠咬肌线粒体则出现严重空泡性变，肌纤维微血管出现更为严重的充血性改变。结论：情绪应激可导致咬肌纤维问微血管充血性改变和线粒体损伤，并且这种变化随时间的延长而加重。这种影响可能是咀嚼肌紊乱（masticatorymusclesdysfunction。MMD）的原因之一。     

情绪应激对大鼠咬肌能量代谢的影响

目的：研究情绪应激对大鼠咬肌能量代谢的影响。方法：48只Wistar大鼠,随机分为情绪应激组和空白对照组各24只,应激组大鼠依应激时间分为1周组（Ⅰ组）、3周组（Ⅱ组）和5周组（Ⅲ组）：另外24只大鼠仅接受足部电击,在实验中作为应激源,不进入实验处理。应激组大鼠和接受足部电击大鼠同处交流箱内,所有大鼠同条件饲养,分别于1周、3周、5周处死应激组和对照组大鼠,检测咬肌Na^＋-K^＋ATP酶活性,Ca^2＋-ATP酶活性,乳酸脱氢酶（LDH）活性以及肌肉乳酸（LD）含量的变化。结果：应激源刺激发生后,随着应激时间的延长,咬肌Na^＋-K^＋ATP酶活性与Ca^2＋-ATP酶活性呈现逐渐下降趋势,同时,大鼠咬肌组织中LD含量逐渐增高,LDH活性呈逐渐升高的趋势。结论：情绪应激可以引起大鼠咬肌能量代谢发生变化,这种变化可能是引起咬肌超微结构发生改变、导致咀嚼肌紊乱疾病的原因之一。     

生命早期负性应激对大鼠实验性牙周炎的影响

目的:探究生命早期负性应激对大鼠成年后实验性牙周炎进展的影响。方法:在3周龄雄性SD大鼠建立慢性温和不可预知应激(unpredictable chronic mild stress, UCMS)模型,以正常饲养大鼠为对照。应激5周后对所有大鼠右上第二磨牙进行实验性牙周炎处理。通过行为学实验评估大鼠情绪状态;ELISA检测血清皮质酮、IL-1β和TNF-α水平;HE染色观察破骨细胞数目,测量牙槽骨丧失量。结果:UCMS大鼠出现糖水偏好下降(P<0.001)、强迫游泳实验中不动时间增加(P<0.01)等抑郁样行为;血清皮质酮及炎性细胞因子水平升高(P<0.05)。HE染色可见UCMS大鼠牙周炎侧牙槽骨吸收量最大,其次为对照组牙周炎侧,再次为对照组正常侧(P<0.001);破骨细胞数目变化趋势与之类似(P<0.001)。血清皮质酮含量与牙周破坏程度呈正相关关系(P<0.05)。结论:生命早期负性应激可能通过HPA轴增强体液免疫,加剧大鼠实验性牙周炎病变程度。     

牙龈卟啉单胞菌脂多糖对高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响

目的 比较牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis on lipopolysaccharide,Pg-LPS)对健康和高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响.方法 将健康新西兰兔12只随机分为2组,分别给予普通饲养喂养(健康兔)和高脂饲料喂养,6周后建立高脂血症模型(高脂兔).采用腹腔灌洗法分别获得健康和高脂兔巨噬细胞,将健康和高脂兔巨噬细胞均随机分为对照组、Pg-LPS组(1μg/mL)及阳性对照组大肠杆菌-脂多糖(Escherichia,E.coli-LPS)组(1μg/mL),24 h后采用实时PCR法分别检测各组巨噬细胞中C-反应蛋白(C-reaction protein,CRP)、白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)、白介素8(interleukin-8,IL-8)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)mRNA的表达水平.结果 高脂兔对照组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α水平高于健康兔对照组巨噬细胞;相较于对照组,高脂兔和健康兔Pg-LPS组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α表达均升高,且高脂兔Pg-LPS组巨噬细胞以上因子的表达水平高于健康兔Pg-LPS巨噬细胞(P<0.05).结论 Pg-LPS可增强高脂血症兔巨噬细胞炎症相关因子mRNA的表达.     

Cytokine secretion of periodontal ligament fibroblasts derived from human deciduous teeth: effect of mechanical stress on the secretion of transforming growth factor-beta 1 and macrophage colony stimulating factor

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF-Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation, 3-cycles/min for 24 h on d 7 in culture using a Flexercell strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF-beta 1) and macrophage colony stimulating factor (M-CSF) contents in the conditioned medium. The TGF-beta 1 level in the conditioned medium of HPLF was significantly higher than that of HPLF-Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF-Y and HGF. M-CSF secretion was inhibited by the stretching on all of HPLF, HPLF-Y and HGF. 1 alpha, 25 dihydroxy vitamin D3 (D3) stimulated M-CSF secretion into the culture medium of both HPLF and HPLF-Y, but the stretching inhibited M-CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.     

Effect of mineral trioxide aggregate on cytokine production by peritoneal macrophages

AIM: To test the effect of two commercial brands of grey mineral trioxide aggregate (ProRoot and MTA-Angelus) on cytokine production by M1 and M2 inflammatory macrophages. METHODOLOGY: M1 (from C57BL/6 mice) and M2 peritoneal inflammatory macrophages (from C57BL/6 IL12p40-/- mice) were obtained and cultured in vitro in the presence of MTA. The cellular viability and the production of tumour necrosis factor-alpha, interleukin (IL)-12 and IL-10 in response to stimulation with interferon-gamma and Fusobacterium nucleatum or Peptostreptococcus anaerobius were evaluated. Data were analysed by Mann-Whitney, Kruskal-Wallis and anova tests. RESULTS: The cements did not interfere with cellular viability or with cytokine production by either type of macrophage. However, M2 macrophages produced higher levels of IL-10 when stimulated with F. nucleatum than M1 macrophages (P < 0.05). CONCLUSIONS: The brands of MTA evaluated did not interfere in the cytokine response by M1 or M2 macrophages to the two bacteria tested. However, a difference in cytokine production between the two types of macrophages was found.     

Effects of exercise training on gingival oxidative stress in obese rats

Objective

The purpose of the present study was to investigate the effects of exercise training on serum reactive oxygen species (ROS) level and gingival oxidative stress in obese rats fed a high-fat diet.

Design

Rats were divided into three groups (n = 14/group): one control group (fed a regular diet) and two experimental groups (fed a high-fat diet with and without exercise training [treadmill: 5 days/week]). The rats were sacrificed at 4 or 8 weeks. The level of serum reactive oxidative metabolites (ROM) was measured as an indicator of circulating ROS. The level of 8-hydroxydeoxyguanosine (8-OHdG) and reduced-form glutathione (GSH)/oxidised-form glutathione (GSSG) ratio were determined to evaluate gingival oxidative stress.

Results

The obese rats fed a high-fat diet without exercise training showed higher serum ROM levels [Carratelli Units (CARR U)] (mean ± SD; 413 ± 64) than the control (333 ± 12) at 4 weeks (p = 0.023). Such a condition resulted in higher 8-OHdG levels (ng/mg mtDNA) (0.97 ± 0.18) (p < 0.05) and a lower GSH/GSSG ratio (17.0 ± 3.1) (p < 0.05) in gingival tissues, compared to the control (0.55 ± 0.13 for 8-OHdG and 23.6 ± 5.8 for GSH/GSSG ratio) at 8 weeks. In addition, the obese rats fed a high-fat diet with exercise training showed lower serum ROM (623 ± 103) (p < 0.001) and gingival 8-OHdG levels (0.69 ± 0.17) (p = 0.012) than those without exercise training (1105 ± 95 for ROM and 0.55 ± 0.13 for 8-OHdG) at 8 weeks.

Conclusions

Obesity prevention by exercise training may effectively suppress gingival oxidative stress by decreasing serum ROS in rats.     

Effect of compressive force on the expression of inflammatory cytokines and their receptors in osteoblastic Saos-2 cells

OBJECTIVE: In orthodontic tooth movement, some cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal processes of bone remodelling, resulting in physiological bone resorption. We examined the effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the expression of inflammatory cytokines that promote osteoclast formation, as well as on their receptors, in osteoblastic Saos-2 cells. DESIGN: The cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum with or without continuous compressive force (0.5-3.0 g/cm(2)) and/or IL-1ra for up to 24h. The gene expression levels of the cytokines and their receptors were estimated by determining mRNA levels using real-time PCR; the protein levels were determined using ELISA or immunohistochemical staining. RESULTS: The expression of IL-1beta, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and tumor necrosis factor-alpha (TNFalpha) increased depending on the strength and duration of the compressive force, whereas the expression of IL-8, IL-11 receptor and TNFalpha receptor did not change with the application of compressive force. The expression of cytokines and their receptors produced by 3.0 g/cm(2) of compressive force decreased with the simultaneous addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFalpha. CONCLUSION: These results indicate that mechanical stress induces the production of inflammatory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by the autocrine action of IL-1beta, which is increased in amount by mechanical stress.     

Effects of cytokines on the production of nitric oxide in a chondrogenic cell line established from human osteogenic sarcoma

OBJECTIVES: The purpose of this study was to examine the effects of various cytokines and/or lipopolysaccharide (LPS) on nitric oxide (NO) production from USAC, a newly established clonal cell line derived from human osteogenic sarcoma that expressed chondrocytic phenotypes. MATERIALS AND METHODS: No production was measured by Griess method. Inducible nitric oxide synthase (iNOS) mRNA was detected by PCR analysis. Western blotting analysis and immunocytochemistry was used to detect iNOS protein. RESULTS: Although USAC cells treated without any stimulants produced only small amounts of NO, exposure to cytokines and/or LPS induced iNOS in USAC cells and produced high levels of NO. The stimulatory effects of cytokines and/or LPS on NO production required TNF-alpha. TNF-alpha alone neither induced iNOS in USAC cells nor caused production of NO, but addition of TNF-alpha to USAC cells pretreated with LPS and IFN-gamma enhanced the expression of iNOS mRNA, induced iNOS protein and produced NO. Dexamethasone inhibited the stimulatory effect of TNF-alpha. CONCLUSIONS: The responsiveness of USAC cells to cytokines and/or LPS and steroid hormone on NO production was quite different from that reported for rabbit and human articular cartilaginous cells. The differences in responsiveness between articular cartilaginous chondrocytes and USAC cells might have been because USAC cells were established from a malignant tumor.