尾静脉注射牙龈卟啉单胞菌对野生大鼠海马神经干细胞和神经元标志分子表达的影响

Effects of Porphyromonas gingivalis injected through tail vein on the expressions of biomarkers in neural stem cells and neurons of wild-type rats hippocampus

目的观察尾静脉注射牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对野生成年大鼠海马神经干细胞和神经元标志分子表达水平的影响,初步探讨Pg入血对海马神经发生的作用。方法建立尾静脉注射Pg大鼠模型:18只雄性Sprague-Dawley(SD)大鼠按随机数字表法随机分为3组(每组6只)。低剂量组、高剂量组大鼠分别经尾静脉注射1.0×10^(3)和1.0×10^(8)菌落形成单位(colony forming unit,CFU)的Pg菌液200μl,假手术组大鼠注射等体积磷酸盐缓冲液(phosphate buffer saline,PBS),3组大鼠均每周注射3次,连续8周。行为学检测:应用莫里斯水迷宫(Morris water maze,MWM)定位航行实验、空间探索实验检测大鼠学习和记忆能力。免疫组织化学法检测各组大鼠海马颗粒下区(subgranular zone,SGZ)神经干细胞标志分子神经上皮干细胞蛋白(nestin)、神经母细胞和未成熟神经元标志分子双皮质素(doublecortin,DCX)、成熟神经元标志分子神经元核抗原(neuronal nuclei,NeuN)的阳性细胞分布。蛋白质印迹法检测各组大鼠海马组织nestin、DCX、NeuN的表达水平。结果学习和记忆能力:MWM定位航行实验结果显示,第5天到达平台时间高剂量组[22.83(16.00,38.34)s]显著长于假手术组[5.59(5.41,6.17)s](t=-11.17,P<0.001),低剂量组[9.85(8.75,21.01)s]与假手术组相比差异无统计学意义(t=-6.83,P=0.080);MWM空间探索实验中60 s内穿越原平台位置次数,高剂量组[1.50(1.00,2.00)次]显著少于假手术组[4.00(2.75,4.00)次](t=9.75,P=0.003),低剂量组[2.50(2.00,3.00)次]与假手术组相比差异无统计学意义(t=4.50,P=0.382)。免疫组织化学法结果显示,对于nestin阳性细胞密度,低剂量组[(35.36±4.32)个/mm^(2)]和高剂量组[(26.51±5.89)个/mm^(2)]均显著低于假手术组[(59.58±14.15)个/mm^(2)](t=24.21,P=0.018;t=33.07,P=0.005);DCX阳性细胞平均吸光度值,低剂量组(0.007±0.002)和高剂量组(0.006±0.002)均显著低于假手术组(0.011±0.001)(t=0.004,P=0.018;t=0.006,P=0.005);NeuN阳性神经元密度,高剂量组[(0.75±0.08)×10^(3)个/mm^(2)]显著低于假手术组[(1.13±0.14)×10^(3)个/mm^(2)](t=0.38,P=0.017),低剂量组[(0.88±0.19)×10^(3)个/mm^(2)]与假手术组相比差异无统计学意义(t=0.25,P=0.075)。蛋白质印迹法结果显示,与假手术组相比,高剂量组海马中nestin、DCX、NeuN表达水平均显著降低(t=0.74,P<0.001;t=0.18,P=0.014;t=0.35,P=0.008),低剂量组上述3个指标与假手术组相比差异均无统计学意义(t=0.18,P=0.108;t=0.08,P=0.172;t=0.19,P=0.077)。结论尾静脉注射Pg后呈剂量依赖性降低野生大鼠学习和记忆能力,并显著降低大鼠海马神经干细胞和神经元标志分子nestin、DCX、NeuN阳性表达细胞数量和表达水平。

Objectives To study the effects of Porphyromonas gingivalis(Pg)injected through tail vein on the molecular expression levels of biomarkers of neural stem cells(NSC)and neurons in the hippocampus of wild-type adult rats,and the effects on hippocampal neurogenesis.Methods Eighteen male Sprague-Dawley(SD)rats were randomly divided into 3 groups based on the table of random numbers(n=6 in each group).In low-intensity group and high-intensity group,rats were injected intravenously through tail vein with 200μl Pg ATCC33277[1.0×10^(3) and 1.0×10^(8) colony forming unit(CFU),respectively]3 times per week for 8 weeks.In the sham group,200μl of phosphate buffer saline(PBS)was given instead.Behavioral tests:the navigation and the exploration tests using Morris water maze(MWM)were applied to evaluate learning and memory ability of rats.Immunohistochemistry was performed to detect cells positively expressing nestin,doublecortin(DCX)and neuronal nuclei(NeuN)in the subgranular zone(SGZ)of rats in each group.Western blotting was used to evaluate the expression levels of nestin,DCX and NeuN in rat hippocampus.Results Learning and memory abilities:on day 5 of navigation test,the lagency time was 22.83(16.00,38.34)s in the high-intensity group,significantly longer than the sham group[5.59(5.41,6.17)s](t=-11.17,P<0.001).There were no significant differences between the low-intensity group[9.85(8.75,21.01)s]and the sham group(t=-6.83,P=0.080).Results in the exploration test showed that,in the high-intensity group,the number of fime crossing over the previous platform area within 60 s was 1.50(1.00,2.00),significantly less than the sham group[4.00(2.75,4.00)](t=9.75,P=0.003);no significant differences between the low-intensity group[2.50(2.00,3.00)]and the sham one(t=4.50,P=0.382).Immunohistochemistry showed that the nestin+cell density in the low-intensity group[(35.36±4.32)cell/mm^(2)]and high-intensity group[(26.51±5.89)cell/mm^(2)]were significantly lower than the sham group[(59.58±14.15)cell/mm^(2)](t=24.21,P=0.018;t=33.07,P=0.005);as for the mean absorbance of DCX+cells,the low-intensity group(0.007±0.002)and the high-intensity group(0.006±0.002)were significantly lower than the sham group(0.011±0.001)(t=0.004,P=0.018;t=0.006,P=0.005);compared with the sham group[(1.13±0.14)×10^(3) cell/mm^(2)],the density of NeuN+neurons in the high-intensity group[(0.75±0.08)×10^(3) cell/mm^(2)]was significantly reduced(t=0.38,P=0.017),and was not significantly changed in the low-intensity group[(0.88±0.19)×10^(3) cell/mm^(2)](t=0.25,P=0.075).Western blotting results showed that,compared with the sham group,the expression levels of nestin,DCX,and NeuN were significantly reduced in the high-intensity group(t=0.74,P<0.001;t=0.18,P=0.014;t=0.35,P=0.008),but were not statistically changed in the low-intensity group(t=0.18,P=0.108;t=0.08,P=0.172;t=0.19,P=0.077).Conclusions Pg injected through tail vein may reduce learning and memory abilities of wild-type rats,and may reduce the number of nestin,DCX,and NeuN-positive cells,and the protein expression levels of the above molecules in the hippocampus.