Objective: To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD). Methods: Wide type (WT) mice and CCM3(+)/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3(+/-) mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ(1-42) in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR. Results: The levels of blood lead WT (216.07±84.16) and CCM3(+/-) (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3(+/-) (11.79±8.20) μg/L, the difference was statistically significant (t=4.18, P=0.006; t=3.79, P=0.016). The levels of brain lead WT (1.78±0.69) and CCM3(+/-) (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3(+/-) (0.97±0.64) μg/L, the difference was statistically significant (t=3.67, P=0.018; t=3.88, P=0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ(1)-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3(+/-) mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3(+/-): 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3(+/-): 0.59±0.01) was decreased with statistical significance (F=199.27, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3(+/-): 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3(+/-):2.12±0.18), the difference was statistically significant (F=288.29, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3(+)/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t=26.90, P<0.001). Conclusion: The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3(+/-) mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.
目的: 观察脑海绵状血管瘤因子3(CCM3)基因敲除小鼠血脑屏障功能改变的影响,初步探讨铅暴露环境下,CCM3基因敲除与阿尔茨海默病(AD)之间的关系。 方法: 以10周龄野生型小鼠与CCM3(+/-)杂合型小鼠为实验对象。采用单纯随机法,将小鼠分别分为野生型对照组、野生型铅暴露组、杂合型对照组、杂合型小鼠铅暴露组,每组8只小鼠。铅暴露组给予质量分数0.05%醋酸铅饮水染毒12周,对照组无限制自由饮用去离子水。石墨炉原子吸收法检测各组小鼠血铅及脑铅水平;取各组小鼠脑组织制作石蜡切片,HE染色观察组织形态,免疫组化法检测脑组织中Aβ(1-42)的表达并标记脑部微血管;提取各组小鼠脑组织蛋白,使用Western blot法检测蛋白Claudin-5和紧密连接蛋白(ZO-1)的相对表达量,并检测脑部磷酸化Tau蛋白(p-Tau)的相对表达量;提取各组小鼠脑组织RNA,使用qRT-PCR法检测小鼠LRP-1基因mRNA的相对表达量。 结果: 铅暴露组野生型、杂合型小鼠血铅[(216.07±84.16)、(189.64±101.86)μg/L]分别高于对照组野生型和杂合型[(19.52±11.46)、(11.79±8.20)μg/L](t值分别为4.18、3.79,P值分别为0.006、0.016)。铅暴露组野生型和杂合型脑铅[(1.78±0.69)、(1.74±0.66)μg/L]分别高于对照组野生型和杂合型[(1.06±0.87)、(0.97±0.64)μg/L](t值分别为3.67、3.88,P值分别为0.018、0.015)。通过HE染色观察,各组小鼠脑部未出现明显病变;免疫组化结果显示各组小鼠脑部无Aβ(1-42)沉积;铅暴露组杂合型小鼠脑部微血管数目减少。野生型、杂合型铅暴露组小鼠脑部Claudin-5(0.96±0.04、0.59±0.01)均低于对照组小鼠(1.30±0.03、1.07±0.08)(F=199.27,P<0.001)。野生型、杂合型铅暴露组小鼠脑部LRP-1基因mRNA相对表达水平(0.32±0.10、0.06±0.01)均低于对照组(1.00±0.06、2.12±0.18)(F=288.29,P<0.001);铅暴露组杂合型小鼠脑部LRP-1基因mRNA相对表达水平低于野生型小鼠[(0.06±0.01)比(0.32±0.10),t=26.90,P<0.001]。 结论: 成年期小鼠在低剂量铅暴露环境下并未出现显著的AD样病变,但导致小鼠脑部血脑屏障的早期损伤和AD样病变的早期改变。其中CCM3(+/-)小鼠对铅暴露的敏感性强于野生型小鼠,提示CCM3基因的缺失可能加速小鼠在铅暴露下发生AD的潜在危险。.
Keywords: Alzheimer's disease; Blood-brain barrier; CCM3 gene; Lead.