6-OHDA诱导的帕金森病小鼠表现出以p16Ink4a上调和星形胶质细胞衰老为特征的衰老表型

doi:10.3969/j.issn.1674-8115.2021.07.005

摘要/Abstract

摘要：

目的·探索6-羟基多巴胺（6-hydroxydopamine，6-OHDA）诱导的帕金森病小鼠模型是否具有衰老表现及胶质细胞的衰老变化。方法·将30只雄性9~10月龄C57BL/6J小鼠随机分为假手术组（Sham组）和6-OHDA组，每组15只。通过6-OHDA纹状体立体定位注射建立帕金森病模型。采用阿扑吗啡诱导的旋转实验、转棒实验评估小鼠造模后第21日的神经功能损伤情况；末次行为学实验后，取小鼠脑组织，通过蛋白质印迹法检测脑组织纹状体及黑质区域酪氨酸羟化酶（tyrosine hydroxylase，TH）蛋白含量；免疫荧光染色观察纹状体及黑质区域衰老标志物p16^Ink4a、p21的表达变化以及胶质细胞、衰老标志物双阳性的细胞数量变化；采用实时荧光定量PCR（RT-qPCR）检测纹状体及黑质区域的细胞周期蛋白依赖性激酶抑制剂p16^Ink4a、p15^Ink4b、p19^Ink4d、p21和p27^Kip1，以及衰老相关分泌表型（senescence-associated secretory phenotype， SASP）包括Cxcl10、Ccl2、肿瘤坏死因子α（tumor necrosis factor-α，Tnf-α）、白介素1α（interleukin-1 α，Il-1α）、Il-1β、Il-6、基质金属蛋白酶3（matrix metalloproteinase 3，Mmp3）的表达及变化。结果·转棒实验结果显示，与Sham组相比， 6-OHDA组小鼠在转棒上的停留时间较短（P=0.000）；阿朴吗啡诱导的旋转实验结果显示，与Sham组相比，6-OHDA组小鼠总旋转次数较多（P=0.000）；蛋白质印迹法检测结果显示，6-OHDA组纹状体及黑质区域TH蛋白表达量均显著低于Sham组（均P=0.000）。以上结果证实6-OHDA单侧纹状体小鼠帕金森病模型成功建立。免疫荧光染色结果显示：6-OHDA组纹状体和黑质区域p16^Ink4a阳性细胞数量增多；p21在2组均无明显表达；6-OHDA组同时伴有衰老的星形胶质细胞、小胶质细胞、少突胶质细胞产生，且星形胶质细胞数量大于少突胶质细胞和小胶质细胞（均P<0.05）。RT-qPCR检测结果显示，与Sham组相比：6-OHDA组造模侧纹状体和黑质区域p16^Ink4a、p15^Ink4b和p19^Ink4d的表达上调，差异具有统计学意义（均P<0.05）；p21轻微上调，但差异均无统计学意义（均P?0.05）；p27^Kip1轻微上调，但仅在黑质区域的差异有统计学意义（P=0.016）。与Sham组相比，6-OHDA组造模侧纹状体区域的Cxcl10、Ccl2、Tnf-α、Il-1α和Il-6显著上调，Il-1β显著下调（均P<0.05），黑质区域的Ccl2、Tnf-α、Il-1α和Il-6显著上调（均P<0.05），Mmp3和Il-1β均显著下调（均P<0.05）。结论·6-OHDA诱导的帕金森病小鼠模型表现出以p16上调和星形胶质细胞衰老为特征的衰老表型。

关键词: 帕金森病, 衰老, 6-羟基多巴胺, 动物模型, 星形胶质细胞

Abstract:

Objective·To explore whether 6-hydroxydopamine(6-OHDA)-induced Parkinson disease (PD) mouse models have senescent phenotypes and explore the changes of senescence-related glial cells.

Methods·Thirty 9?10 months old C57BL/6J male mice were divided into Sham-operated group (n=15) and 6-OHDA group (n=15). PD mouse models were established by stereotactic injection of 6-OHDA into striatum. The neurological function deficit was evaluated by rotarod test and apomorphine (APO)-induced rotational behavior on the 21st day after modeling. The brain was taken after the last behavioral experiment. Western blotting was used to detect the protein content of tyrosine hydroxylase (TH) in both caudate putamen (CPu) and substantia nigra (SN) regions. Immunofluorescence was used to observe the expression of glial cells and aging marker p16^Ink4a / p21 in both CPu and SN regions. RT-qPCR was also used to detect the expression and changes of cyclin-dependent kinase inhibitor including p16^Ink4a, p15^Ink4b, p19^Ink4d, p21 and p27^Kip1, and senescence-associated secretory phenotype including Cxcl10, Ccl2, tumor necrosis factor-α (Tnf-α), interleukin-1α (Il-1α), Il-1β, Il-6 and matrix metalloproteinase 3 (MMP3).

Results·The decreased falling latency in rotarod test (P=0.000), the increased number of rotations induced by APO (P=0.000) and the loss of TH protein measured by Western blotting (P=0.000), suggested that 6-OHDA unilateral striatum mouse model of PD was successfully established. Immunofluorescence staining showed that the upregulation of an aging marker p16^Ink4a in both CPu and SN regions of the 6-OHDA group compared with the Sham group, but p21 was not significantly expressed in both groups; senescent astrocytes, microglia and oligodendrocytes were accumulated in CPu and SN regions as well, while the number of astrocytes was larger than the other two types of glia cells (P<0.05). RT-qPCR results showed that, compared with those of the Sham group, p15^Ink4b, p16^Ink4a and p19^Ink4d in CPu and SN regions of the 6-OHDA group were up-regulated (P<0.05); p21 was slightly up-regulated, but the difference was not statistically significant (P?0.05); p27^kip1 was slightly up-regulated, but statistically significant difference only presented in SN region (P=0.016). RT-qPCR also showed that, compared with those of the Sham group, the levels of Cxcl10, Ccl2, Il-1α and Il-6 were significantly up-regulated while Il-1β was significantly decreased in CPu region of the 6-OHDA group (P<0.05); the levels of Ccl2, Tnf-α, Il-1α and Il-6 were significantly increased while Mmp3 and Il-1β were significantly decreased in SN region of the 6-OHDA group (P<0.05).

Conclusion·6-OHDA-induced PD mice exhibit senescent phenotypes characterized by upregulation of p16^Ink4a and astrocyte senescence.

Key words: Parkinson disease (PD), senescence, 6-hydroxydopamine (6-OHDA), animal model, astrocyte

中图分类号:

R742.5

袁笑, 田野野, 薛峥. 6-OHDA诱导的帕金森病小鼠表现出以p16^Ink4a上调和星形胶质细胞衰老为特征的衰老表型[J]. 上海交通大学学报(医学版), 2021, 41(7): 876-883.

Xiao YUAN, Ye-ye TIAN, Zheng XUE. 6-OHDA-induced Parkinson disease mice exhibit senescent phenotypes characterized by upregulation of p16^Ink4a and astrocyte senescence[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 876-883.

图/表 5

表 1 RT-qPCR引物

Tab 1 Primers for RT-qPCR

Gene	Forward primer （5′→3′）	Reverse primer （5′→3′）
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
p15	CCCTGCCACCCTTACCAGA	CAGATACCTCGCAATGTCACG
p16	CGCAGGTTCTTGGTCACTGT	TGTTCACGAAAGCCAGAGCG
p19	CTGAACCGCTTTGGCAAGAC	GCCCTCTCTTATCGCCAGAT
p21	CCTGGTGATGTCCGACCTG	CCATGAGCGCATCGCAATC
p27	TCAAACGTGAGAGTGTCTAACG	CCGGGCCGAAGAGATTTCTG
Ccl2	TTAAAAACCTGGATCGGAACCAA	GCATTAGCTTCAGATTTACGGGT
Cxcl10	CCAAGTGCTGCCGTCATTTTC	GGCTCGCAGGGATGATTTCAA
Il-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
Il-8	CAAGGCTGGTCCATGCTCC	TGCTATCACTTCCTTTCTGTTGC
Tnf-α	GACGTGGAACTGGCAGAAGAG	TTGGTGGTTTGTGAGTGTGAG
Il-1α	GCACCTTACACCTACCAGAGT	AAACTTCTGCCTGACGAGCTT
Il-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
Mmp3	ACATGGAGACTTTGTCCCTTTTG	TTGGCTGAGTGGTAGAGTCCC

图1 6-OHDA诱导的小鼠PD模型评估Note： A. Fall latency of mice recorded in rotarod test. B. Number of full rotations in 10 min in apomorphine-induced rotation test. C.TH protein levels in the CPu and SN regions detected by Western blotting. D. Comparison of TH protein levels between the Sham group and 6-OHDA group.

Fig 1 Evaluation of 6-OHDA-induced PD model

图2 细胞衰老标志物的检测（n=5）Note： A. Representative immunofluorescent staining images of p16Ink4a and p21 positive cells in 6-OHDA lesioned brains. Scale bar=40 μm. B. Statistical analysis of the p16Ink4a positive or p21 positive cells in the lesioned-side CPu regions. C. Statistic analysis of the p16Ink4a positive or p21 positive cells in the lesioned-side SN regions. D. mRNA expression of CDKi in the lesioned side of CPu and SN regions.

Fig 2 Detection of cell senescence markers (n=5)

图3 造模后第21日SASP表达水平的变化

Fig 3 SASP levels on day 21 post modelling

图4 CPu和SN区胶质细胞的衰老表型(n=5)Note： A. Representative immunofluorescent staining of Iba-1 (green), Olig2 (green), GFAP (green), p16Ink4a (red) and DAPI (blue) in CPu regions of both lesioned side and intact side of mice in the 6-OHDA group at day 21 after modelling (Scale bar=40 μm). B. Representative immunofluorescent staining of GFAP (green), p16Ink4a (red) and DAPI (blue) in SN regions of both lesioned side and intact side of mice in the 6-OHDA group at day 21 after modelling (Scale bar=40 μm).C. Statistical analysis of Olig2+p16Ink4a+, GFAP+p16Ink4a+ and Iba1+p16Ink4a+ cell numbers in CPu region of lesioned side in the 6-OHDA group. D.Statistical analysis of Olig2+p16Ink4a+, GFAP+p16Ink4a+ and Iba1+p16Ink4a+ cell numbers in SN region of lesioned side in the 6-OHDA group. E. Quantification of oligodendrocyte, astrocyte and microglia numbers in CPu region of the 6-OHDA group.

Fig 4 Senescent phenotype of glial cells in CPu and SN regions (n=5)

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