鼻阻塞模型大鼠下颌骨自噬水平的变化

Autophagy level of the mandible in nasal obstruction rats

背景:低氧状态下细胞自噬状态易被激活,而自噬水平对骨代谢的调控作用已经被证明。睡眠呼吸暂停使机体处于间歇低氧状态,而睡眠呼吸暂停诱发的低氧是否影响下颌牙槽骨及牙周膜的自噬水平未见报道。目的:观察机体低氧状态下对大鼠下颌自噬水平的影响。方法:将30只1周龄雄性Wistar大鼠随机分为3组,每组10只,单侧鼻阻塞组、双侧鼻阻塞组分别构建幼年大鼠单侧及双侧鼻阻塞模型,麻醉30 min后用高频电刀电灼阻塞大鼠的左鼻孔,1周后同样方法阻塞双侧鼻阻塞组右侧鼻孔;对照组不处理鼻孔。单侧鼻阻塞建模成功4周后,模型大鼠5周龄时麻醉处死取下颌骨,制备下颌第一磨牙牙周骨组织切片,进行苏木精-伊红染色和LC3免疫组化染色,利用Image-J图像分析系统对染色后的切片做定位定量分析。Western blot法检测下颌牙槽骨缺氧标志蛋白低氧诱导因子1α、自噬相关蛋白p62的蛋白表达水平,以及自噬标志蛋白LC3亚型LC3-Ⅱ/LC3-Ⅰ的比值。结果与结论:(1)苏木精-伊红染色显示,单侧鼻阻塞组、双侧鼻阻塞组模型大鼠下颌牙槽骨骨小梁排列混乱;(2)免疫组化结果显示,单侧鼻阻塞组、双侧鼻阻塞组下颌第一磨牙牙槽骨细胞及牙周膜细胞胞浆LC3表达升高,提示自噬主要发生在牙周膜及牙槽骨区;(3)Western blot结果显示,与对照组相比,单侧鼻阻塞组、双侧鼻阻塞组模型大鼠下颌牙槽骨组织低氧诱导因子1α蛋白的表达水平显著升高,p62蛋白的表达水平显著下降(P<0.05),LC3-Ⅱ/LC3-Ⅰ比值显著升高(P<0.05);而双侧鼻阻塞组LC3-Ⅱ/LC3-Ⅰ比值较单侧鼻阻塞组稍下降(P>0.05);(4)提示大鼠单侧、双侧鼻阻塞导致的低氧使下颌低氧诱导因子1α蛋白的表达水平升高,下颌第一磨牙牙周膜及牙槽骨组织自噬水平升高,但自噬升高的水平并不与鼻阻塞的严重程度呈正相关。

BACKGROUND:Autophagy is easily activated under hypoxia and has been demonstrated to regulate bone metabolism.Sleep apnea keeps the body in a state of intermittent hypoxia,and whether sleep apnea-induced hypoxia affects the autophagy levels of mandibular alveolar bone and periodontal ligament has not been reported.OBJECTIVE:To observe the autophagy level in the rat mandible under hypoxic conditions.METHODS:Thirty 1-wee k-old male Wistar rats we re randomly divided into three groups(n=10 per group).In unilate ral and bilateral nasal obstruction groups,the left nostril was blocked with high-frequency electrocaute ry after 30 minutes of anesthesia,and 1 week late r,the right nostril in the bilateral nasal obstruction group was blocked using the same method.Rats in control group were with no nostril bloc king.Four wee ks after the successful modeling of unilateral nasal obstruction,the model rats at the age of 5 weeks were anesthetized and sacrificed,and the mandibles were removed to prepare periodontal bone tissue sections of the mandibular first molar for hematoxylin-eosin staining and LC3 immunohistochemical staining.Localization of the stained sections was quantified using the image-J image analysis system.Western blot assay was used to detect the expression of hypoxia-inducible factor 1αand autophagyrelated protein p62 in mandibular alveolar bone,as well as the ratio of autophagy marker protein LC3 isoforms LC3-Ⅱ/LC3-Ⅰ.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results revealed disordered arrangement of trabecules of the mandibular alveolar bone in the unilateral and bilateral nasal obstruction groups.Immunohistochemical results indicated an increased expression of LC3 in the alveolar bone cells and periodontal ligament cells of mandibular first molars in the unilate ral and bilateral nasal obstruction groups,suggesting that autophagy mainly occurred in the periodontal ligament and alveolar bone.Western blot results showed that compared with the control group,the expression level of hypoxia-inducible factor1αprotein in the mandibular alveolar bone tissue was significantly increased,the expression level of p62 protein was significantly decreased(P<0.05),and the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly increased in the unilateral and bilateral nasal obstruction groups(P<0.05).Moreover,the ratio of LC3-Ⅱ/LC3-Ⅰin the bilateral nasal obstruction group was slightly lower than that in the unilateral nasal obstruction group(P>0.05).All these findings indicate that hypoxia caused by unilate ral and bilateral nasal obstruction increases the expression level of hypoxia-inducible factor 1αprotein in the mandible and the level of autophagy in the periodontal ligament and alveolar bone of mandibular first molars in rats.However,the increase in autophagy has no positive correlation with the severity of nasal obstruction.