香芹酚对2型糖尿病小鼠星形胶质细胞损伤的保护作用

Protective effect of carvacrol on astrocytes damage in type 2 diabetic mice

摘要:

目的： 探讨香芹酚对自发性2型糖尿病db/db小鼠星形胶质细胞损伤的保护作用。方法： 选择8周龄雄性db/db小鼠20只，随机分为模型组和香芹酚组，每组10只，随机抽取10只db/m小鼠为对照组。香芹酚组小鼠给予香芹酚（20 mg/kg）灌胃，对照组和模型组分别给予等量生理盐水灌胃，每天灌胃1次，连续给药6周。利用免疫组化法标记胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP），观察不同分组中海马CA1区星形胶质细胞形态的病理改变。免疫组化法检测对照组、模型组和香芹酚组海马CA1区脂质运载蛋白-2（lipocalin 2，LCN2）、水通道蛋白-4（aquaporin 4，AQP4）表达的阳性细胞数量。采用TUNEL法检测海马CA1区LCN2、AQP4与星形胶质细胞凋亡的关系。应用RT-PCR和Western blot检测对照组、模型组和香芹酚组海马CA1区LCN2、AQP4、核转录因子-κB（nuclear factor kappa B，NF-κB）、嗜中性白细胞碱性磷酸酶-3（neutrophilic alkaline phosphatase-3，NALP3）的表达。结果： 模型组空腹血糖（glucose，GLU）、高密度脂蛋白（high density lipoprotein，HDL）、甘油三酯（triglyceride，TG）、总胆固醇（total cholesterol，TC）表达量较对照组明显升高，而香芹酚组较模型组GLU、HDL、TG、TC的表达量明显降低（均P<0.05）。免疫组化显示，模型组GFAP标记的星形胶质细胞数量增加且体积增大，分枝增多、变长；香芹酚组可改善星形胶质细胞的形态，减少星形胶质细胞的数量（均P<0.01）。免疫组化表明，模型组中LCN2、NF-κB、NALP3阳性细胞数量增加，而AQP4阳性细胞数量减少；香芹酚组AQP4阳性细胞数量增加，LCN2、NF-κB、NALP3阳性细胞数量减少（均P<0.01）。TUNEL染色显示，模型组中LCN2与凋亡细胞均增加，AQP4则明显减少；香芹酚组与模型组相比，凋亡细胞、LCN2阳性细胞明显降低，AQP4阳性细胞数量明显增加（均P<0.01）。RT-PCR和Western blot结果显示，与模型组相比，香芹酚组降低海马CA1区中LCN2、NALP3、NF-κB表达，增加AQP4的表达（均P<0.01）。结论： 香芹酚对自发性2型糖尿病小鼠星形胶质细胞损伤具有保护作用，其机制可能与降低LCN2、NF-κB、NALP3和增加AQP4表达有关。

Abstract:

Objective: To investigate the protective effect of carvacrol on astrocytes injury in spontaneous type 2 diabetic db/db mice. Methods: Twenty eight-week-old male db/db mice were randomly divided into model group and carvacrol group, and 10 db/m mice were randomized to the control group. The carvacrol group mice were given 20 mg/kg intragastric administration of carvacrol, and the control group and the model group were respectively given the same amount of intragastric administration of saline, once a day, for 6 weeks. The morphological changes of astrocytes in CA1 area of hippocampus in different groups were observed by labeling glial fibrillary acidic protein (GFAP) through immunohistochemistry. The number of expressed positive cells of lipocalin 2 (LCN2) and aquaporin 4 (AQP4) in CA1 region of control group, model group, and carvarol group were determined by immunohistochemistry. TUNEL was used to detect the relationship among LCN2, AQP4 and astrocyte apoptosis in CA1 region. RT-PCR and Western blot were used to detect the expression of LCN2, AQP4, nuclear factor kappa B (NF-κB) and neutrophilic alkaline phosphatase-3 (NALP3) in CA1 area of hippocampus in control group, model group and carvacrol group. Results: The expression levels of glucose (GLU), high density lipoprotein (HDL), triglyceride (TG) and total cholesterol (TC) in the model group were significantly higher than those in the control group, while the expression levels of GLU, HDL, TG and TC in the carvacrol group were significantly lower than those in the model group (all P<0.05). Immunohistochemistry showed that the number of GFAP labeled astrocytes in the model group increased and the number of branches increased, while the carvacrol group could improve the morphology of astrocytes and reduce the number (all P<0.01). Immunohistochemistry also showed that the number of positive cells of LCN2, NF-κB and NALP3 in the model group increased, while the number of positive cells of AQP4 decreased. However, in the carvacrol group, the number of positive cells of AQP4 increased, and the number of positive cells of LCN2, NF-κB and NALP3 decreased (all P<0.01). TUNEL staining showed that LCN2 and apoptotic cells increased, while AQP4 decreased significantly in the model group, while the number of apoptotic cells and LCN2 positive cells and AQP4 positive cells increased in the carvacrol group compared with the model group (all P<0.01). The results of RT-PCR and Western blot showed that the expression levels of LCN2, NALP3 and NF-κB in hippocampus of carvacrol group were decreased, and the expression level of AQP4 was increased compared with those of model group (all P<0.01). Conclusion: Carvacrol has protective effect on astrocytes injury in spontaneous type 2 diabetic mice, and its mechanism may be related to the decrease of LCN2, NF-κB and NALP3, and the increase of AQP4 expression level.