药学学报

Gene	Primer sequence forward (5'-3')	Primer sequence reverse (5'-3')
GAPDH	AGCGAGACCCCACTAACATC	GGTTCACACCCATCACAAAC
CAT	AGCGACCAGATGAAGCAGTG	TCCGCTCTCTGTCAAAGTGTG
Mn-SOD	CAGACCTGCCTTACGACTATGG	CTCGGTGGCGTTGAGATTGTT
Klotho	GGCTTTCCTCCTTTACCTGAAAA	CACATCCCACAGATAGACATTCG

Gene

Primer sequence forward (5'-3')

Primer sequence reverse (5'-3')

GAPDH

AGCGAGACCCCACTAACATC

GGTTCACACCCATCACAAAC

CAT

AGCGACCAGATGAAGCAGTG

TCCGCTCTCTGTCAAAGTGTG

Mn-SOD

CAGACCTGCCTTACGACTATGG

CTCGGTGGCGTTGAGATTGTT

Klotho

GGCTTTCCTCCTTTACCTGAAAA

CACATCCCACAGATAGACATTCG

Gene	Primer sequence forward (5'-3')	Primer sequence reverse (5'-3')
GAPDH	AGCGAGACCCCACTAACATC	GGTTCACACCCATCACAAAC
CAT	AGCGACCAGATGAAGCAGTG	TCCGCTCTCTGTCAAAGTGTG
Mn-SOD	CAGACCTGCCTTACGACTATGG	CTCGGTGGCGTTGAGATTGTT
Klotho	GGCTTTCCTCCTTTACCTGAAAA	CACATCCCACAGATAGACATTCG

Gene

Primer sequence forward (5'-3')

Primer sequence reverse (5'-3')

GAPDH

AGCGAGACCCCACTAACATC

GGTTCACACCCATCACAAAC

CAT

AGCGACCAGATGAAGCAGTG

TCCGCTCTCTGTCAAAGTGTG

Mn-SOD

CAGACCTGCCTTACGACTATGG

CTCGGTGGCGTTGAGATTGTT

Klotho

GGCTTTCCTCCTTTACCTGAAAA

CACATCCCACAGATAGACATTCG

Task	Best performance score	Deficit score
Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)	0 (present at all paws)	4 (1 point for each missing reflex)
Stop at the edge of a table	0 (stopping at the end of table)	1 (no stopping behavior)
Turning the head (touching the ear from behind with a little rod)	0 (turning towards both directions)	2 (1 point for each failure to turn towards the stimulated ear)
Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)	0 (extension of both front paws)	1 (not reaching down towards the ground with both front paws)
Spontaneous motility (moving behavior on a flat surface)	0 (immediate, spontaneous activity)	1 (activity after 30-60 s) or 2 (no activity within 60 s)
Circling behavior (moving behavior on a flat surface)	0 (no circling, moving in all directions)	2 (spontaneous circling, turning only to one direction)
Pelt property (appearance of the coat)	0 (clean, well kept)	1 (coarse, scrubby)
Flight reaction (spontaneous behavior on a flat surface)	0 (normal behavior)	1 (anxious, jumpy behavior)
Maximum total score		14

Task

Best performance score

Deficit score

Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)

0 (present at all paws)

4 (1 point for each missing reflex)

Stop at the edge of a table

0 (stopping at the end of table)

1 (no stopping behavior)

Turning the head (touching the ear from behind with a little rod)

0 (turning towards both directions)

2 (1 point for each failure to turn towards the stimulated ear)

Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)

0 (extension of both front paws)

1 (not reaching down towards the ground with both front paws)

Spontaneous motility (moving behavior on a flat surface)

0 (immediate, spontaneous activity)

1 (activity after 30-60 s) or 2 (no activity within 60 s)

Circling behavior (moving behavior on a flat surface)

0 (no circling, moving in all directions)

2 (spontaneous circling, turning only to one direction)

Pelt property (appearance of the coat)

0 (clean, well kept)

1 (coarse, scrubby)

Flight reaction (spontaneous behavior on a flat surface)

0 (normal behavior)

1 (anxious, jumpy behavior)

Maximum total score

Task	Best performance score	Deficit score
Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)	0 (present at all paws)	4 (1 point for each missing reflex)
Stop at the edge of a table	0 (stopping at the end of table)	1 (no stopping behavior)
Turning the head (touching the ear from behind with a little rod)	0 (turning towards both directions)	2 (1 point for each failure to turn towards the stimulated ear)
Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)	0 (extension of both front paws)	1 (not reaching down towards the ground with both front paws)
Spontaneous motility (moving behavior on a flat surface)	0 (immediate, spontaneous activity)	1 (activity after 30-60 s) or 2 (no activity within 60 s)
Circling behavior (moving behavior on a flat surface)	0 (no circling, moving in all directions)	2 (spontaneous circling, turning only to one direction)
Pelt property (appearance of the coat)	0 (clean, well kept)	1 (coarse, scrubby)
Flight reaction (spontaneous behavior on a flat surface)	0 (normal behavior)	1 (anxious, jumpy behavior)
Maximum total score		14

Task

Best performance score

Deficit score

Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)

0 (present at all paws)

4 (1 point for each missing reflex)

Stop at the edge of a table

0 (stopping at the end of table)

1 (no stopping behavior)

Turning the head (touching the ear from behind with a little rod)

0 (turning towards both directions)

2 (1 point for each failure to turn towards the stimulated ear)

Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)

0 (extension of both front paws)

1 (not reaching down towards the ground with both front paws)

Spontaneous motility (moving behavior on a flat surface)

0 (immediate, spontaneous activity)

1 (activity after 30-60 s) or 2 (no activity within 60 s)

Circling behavior (moving behavior on a flat surface)

0 (no circling, moving in all directions)

2 (spontaneous circling, turning only to one direction)

Pelt property (appearance of the coat)

0 (clean, well kept)

1 (coarse, scrubby)

Flight reaction (spontaneous behavior on a flat surface)

0 (normal behavior)

1 (anxious, jumpy behavior)

Maximum total score

Klotho对缺血性脑损伤的抗氧化保护作用及机制研究

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李欣 , 杨雪琴 , 史梦琪 , 杜俊蓉 ^*

药学学报 | 研究论文 2018,53(8): 1331-1336

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药学学报 | 研究论文 2018, 53(8): 1331-1336

Klotho对缺血性脑损伤的抗氧化保护作用及机制研究

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李欣, 杨雪琴, 史梦琪, 杜俊蓉^*

作者信息

四川大学华西药学院, 四川成都 610041

通讯作者:

* 杜俊蓉, Tel: 86-28-85503938, Fax: 86-28-85501371, E-mail: dujr_1@163.com

Anti-oxidative protective effect and mechanism of klotho on ischemic brain injury

Xin LI, Xue-qin YANG, Meng-qi SHI, Jun-rong DU^*

Affiliations

West China School of Pharmacy, Sichuan University, Chengdu 610041, China

出版时间: 2018-08-12 doi: 10.16438/j.0513-4870.2018-0328

文章导航

摘要

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本实验通过侧脑室注射慢病毒载体编码的小鼠klotho基因（LV-KL），研究klotho过表达对缺血再灌损伤的抗氧化保护作用及潜在机制。C57Balc/6J小鼠双侧侧脑室注射慢病毒4周后，通过双侧颈总动脉结扎法（2VO）建立小鼠全脑缺血再灌注模型。应用神经行为学评分、Nissl染色、qPCR及Western blot等指标研究klotho对脑缺血的作用及相关抗氧化机制。实验结果表明，klotho过表达明显改善了2VO小鼠的神经行为学障碍，改善海马CA1区和纹状体区（CPu）神经元损伤。同时，LV-KL增加了脑内锰超氧化物歧化酶（Mn-SOD）和过氧化氢酶（CAT）的表达，减少了脑内丙二醛（MDA）含量。Western blot结果显示，klotho过表达抑制了Akt和FoxO1的磷酸化，表明klotho的抗氧化作用可能与抑制Akt/FoxO1信号通路有关。由此可见，利用klotho或klotho增强剂可以减缓衰老相关的klotho表达下降，而本实验也为治疗老年急性缺血性脑卒中提供了一种新的研究方向。

关键词

klotho / 脑缺血 / 氧化应激 / Akt/FoxO1信号通路 / 衰老

Abstract

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Present study was designed to investigate the effects and underlying antioxidant mechanism of klotho overexpression through an intracerebroventricular injection of a lentiviral vector that encoded murine klotho (LV-KL) on cerebral ischemia injury. Four weeks after the injection of lentivirus into the lateral ventricle of C57Balc/6J mice, a mouse model of global cerebral ischemia-reperfusion was established by bilateral common carotid artery occlusion (2VO). Klotho overexpression significantly improved neurobehavioral deficits and increased the number of survival neurons in the hippocampal CA1 and caudate putamen subregions. The overexpression also decreased malondialdehyde (MDA) content in brain, while mitochondrial manganese- superoxide dismutase (Mn-SOD) and catalase (CAT) expression in brain were increased. Moreover, klotho overexpression decreased Akt and forkhead box class O1 phosphorylation. These findings suggest that klotho may compensate for its aging-related decline to provide a promising therapeutic approach for the acute ischemic stroke during aging.

Key words

klotho / ischemic stroke / oxidative stress / Akt/FoxO1 signaling / aging

引用本文

李欣, 杨雪琴, 史梦琪, 杜俊蓉. Klotho对缺血性脑损伤的抗氧化保护作用及机制研究. 药学学报, 2018 , 53 (8) : 1331 -1336 . DOI: 10.16438/j.0513-4870.2018-0328

Xin LI, Xue-qin YANG, Meng-qi SHI, Jun-rong DU. Anti-oxidative protective effect and mechanism of klotho on ischemic brain injury[J]. Acta Pharmaceutica Sinica, 2018 , 53 (8) : 1331 -1336 . DOI: 10.16438/j.0513-4870.2018-0328

正文

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缺血性脑卒中是由于脑血管供血不足而引起脑损伤的一种疾病, 死亡率高, 约占全球死亡总数的11.3%^[1]。研究表明, 衰老是脑卒中发生及预后不良的最大风险因素^[2]。与衰老相关的脑部病理生理学变化非常复杂, 其中氧化应激与衰老大脑中的缺血性损伤呈现出高度正相关^[3], 然而, 氧化应激与脑损伤相关的潜在分子机制目前仍不清楚。

Klotho是一种具有多种生物学活性的抗衰老基因, 该基因缺陷小鼠在出生4周后会出现器官功能衰竭、认知障碍和寿命缩短等一系列与人类衰老相似的表型变化, 而上调klotho的表达则能使小鼠的寿命延长20%~30%^[4]。最新研究表明, klotho可能通过激活由insulin/IGF-1信号通路负调节的FoxO转录因子, 诱导Mn-SOD和CAT等抗氧化酶的转录, 清除体内产生的过多的ROS, 从而减轻氧化应激^[5]。

临床前基础研究和临床数据表明, 动物和人脑内klotho表达随年龄增长而下降。老年猕猴脑内klotho水平低于青年猕猴, 而血清klotho水平与受试者年龄呈现负相关^{[6, 7]}。以往的研究也表明, 衰老小鼠脉络丛中klotho蛋白和mRNA水平均显著降低^[5]。据估计, 到2050年, 脑卒中发病率有望翻倍, 其中75岁以上的老年人口则是脑卒中发病的主要人群^[8]。通过对韩国女性缺血性脑卒中患者进行基因分型发现, klotho是缺血性脑卒中一个重要基因风险因子^[9]。由此推测, 随年龄增长, 内源性klotho减少, 氧化应激反应增强, 最终导致脑细胞对缺血的敏感性增加和老年卒中患者的神经障碍加重。同时, Zeldich等^[10]的相关研究也已表明, klotho蛋白对谷氨酸诱导产生的神经氧化损伤具有直接性的保护作用, Zhou等^[11]研究发现上调klotho的表达能够抑制缺血后的炎症反应, 证实了klotho在缺血脑损伤中的保护作用, 这些都为本实验提供了研究基础与依据。本实验采用侧脑室注射慢病毒载体编码的小鼠klotho基因(LV-KL)上调klotho的表达, 研究klotho过表达对缺血再灌损伤的氧化保护作用及潜在机制, 以期为老年急性缺血性脑卒中治疗提供新的靶点。

材料与方法

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实验动物 SPF级C57Balc/6J小鼠, 雄性, 6周龄, 20~22 g左右, 购自四川省成都市达硕生物科技有限公司, 合格证号: SCXK (川) 2013-15。饲养环境温度维持在25±1 ℃, 相对湿度50%~70%, 12 h/12 h昼夜节律, 自由摄食饮水。

材料和试剂 HEK293细胞(American Type Culture Collection); Klotho重组质粒、对照载体质粒(美国OriGene Technologies公司); Lipofectamine 3000转染试剂盒(美国Invitrogen公司); GM easy^TM慢病毒包装试剂盒、GML-PC^TM慢病毒浓缩试剂盒、Polybrene (上海吉满生物科技有限公司); DMEM高糖培养基购自(美国Gibco公司); RevertAid First Strand cDNA Synthesis Kit、SsoFast^TM EvaGreen^® Supermix (Bio-Rad公司); SDS-PAGE蛋白上样缓冲液、SDS-PAGE凝胶配制试剂盒、BCA蛋白浓度测定试剂盒(碧云天生物科技有限公司)。Akt、pAkt (Ser473)、FoxO1、pFoxO1 (Ser256)抗体购自(美国Cell Signaling Technology公司)。实验中用到的所有引物序列见表 1。

实验动物分组 用于研究LV-KL对正常小鼠klotho表达影响的C57Balc/6J小鼠分为3组:空白对照组(Sham)、对照病毒组(LV-GFP)、上调病毒组(LV-KL), 每组4只, 仅注射慢病毒, 不进行双侧颈总动脉结扎(2VO); 用于研究LV-KL对2VO小鼠klotho表达影响的C57Balc/6J小鼠分为3组:空白对照组(Sham+LV-GFP)、对照病毒组(2VO+LV-GFP)、上调病毒组(2VO+LV-KL), 每组10只。

慢病毒生产及双侧侧脑室注射 HEK293细胞汇合达90%~100%时, 将GM easy^TM lentiviral mix、lipofectamine 3000和慢病毒编码的klotho或GFP基因加到无血清、无抗生素的DMEM中混合均匀, 于CO₂培养箱中培养, 72 h后, 连续稀释病毒液转染HEK293细胞, 计算病毒滴度^[11]。小鼠脑立体定位仪进行注射位点的定位(前卤后0.2 mm, 中线旁开1.0 mm, 硬脑膜下2.5 mm)并用颅骨钻打孔, 使用微量注射器向小鼠双侧侧脑室分别注射3 μL慢病毒溶液(2.1×10⁷ TU·mL^-1), 骨蜡封闭^[12]。实验中仅注射1次病毒。

2VO模型建立 慢病毒注射4周后, 进行2VO手术。4%水合氯醛腹腔麻醉后, 暴露双侧颈总动脉鞘, 剥离迷走神经, 动脉夹夹闭20 min后松开, 并确保再灌成功, Sham+LV-GFP组仅暴露双侧颈总动脉, 不进行夹闭^[13]。

标本收集 慢病毒注射4周后, 部分C57Balc/6J小鼠直接处死取脑, 用于检测正常小鼠脑内和脉络丛klotho的表达(n = 4);部分C57Balc/6J小鼠于2VO手术再灌72 h后, 处死取脑, 用于组织病理学和生化检查(n = 10)。

神经行为学评分 分别在术后24、48、72 h进行神经行为学评分^[14], 评分方法见表 2。

Nissl染色 脑石蜡切片于0.5%焦油紫水溶液染色10 min, 镜下分色晾干后, 封片。光镜下(×400)盲法计数海马CA1区和CPu区3个微观区域神经元活细胞数^[13]。

Real-time PCR 用Trizol从小鼠脑组织和脉络丛中分离总RNA, 并逆转录为cDNA, 随后进行qPCR反应。特异性引物序列见表 1。Klotho mRNA水平归一化为GAPDH。结果表示为使用2^-ΔΔC_t方法相对于空白对照组的阈循环(C_t)值的倍数变化。

MDA测定 MDA试剂盒用于检测脑内脂质过氧化代表性产物MDA含量, 将脑匀浆与三氯乙酸混合, 离心取上清。将TBA加入到上清液中, 然后采用分光光度法在532 nm处进行测定。

Western blot检测 脑组织提取总蛋白, BCA法测定蛋白浓度。然后用10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离等量的脑组织蛋白, 并转移至聚偏氟乙烯膜上。将膜与一抗在4 ℃孵育过夜, 第二天加入辣根过氧化物酶标记的相应二抗溶液在37 ℃下孵育1 h, 使用ECL化学发光试剂盒进行显影分析, 并用Gel Pro Analyzer 6.0测定每条带的OD值。对免疫印迹结果进行定量分析, 并表示为各组相对于空白对照组的比值。

统计学处理 SPSS 19.0统计软件用于数据分析, 实验数据以mean±SEM表示, 组间比较采用单因素方差分析, 神经行为学评分采用多因素方差分析, P < 0.05表示有显著性差异。

结果

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1 LV-KL上调正常小鼠klotho表达

如图 1所示, 与Sham组相比(1.07±0.14和0.98±0.07), LV-GFP组脑内和脉络丛klotho mRNA水平(1.13±0.13和1.01±0.08)均无显著变化(P > 0.05), 而LV-KL显著上调正常小鼠脑内和脉络丛klotho表达(1.71±0.16和1.82±0.12, P < 0.01)。

2 LV-KL上调缺血小鼠klotho表达

如图 2所示, 与Sham+LV-GFP组相比(1.0±0.07和1.01±0.11), 2VO手术对2VO+LV-GFP组脑内和脉络丛klotho mRNA水平(1.03±0.09和1.05±0.07)均无显著影响(P > 0.05), 而LV-KL显著上调2VO+LV-KL组小鼠脑内和脉络丛klotho表达(1.53±0.05和1.71±0.12, P < 0.01)。

3 Klotho过表达减轻2VO小鼠神经行为学障碍

如图 3所示, Sham+LV-GFP组没有表现出神经行为学障碍, 神经行为学评分为0。20 min的脑缺血引起了神经行为学障碍, 表现为2VO+LV-GFP组缺血后神经行为学评分增加(24 h: 4.9±0.63, 48 h: 4.4±0.52, 72 h: 4.2±0.79)。LV-KL有效改善了2VO导致的神经损伤(F_{2, 27} = 11.459, P < 0.001), 表现为在相应时间点, 2VO+LV-KL组的神经行为学评分显著低于2VO+LV-GFP组(24 h: 3.9±0.63, 48 h: 3.5±0.53, 72 h: 3.2±0.42)。

4 Klotho过表达改善2VO小鼠神经元损伤

如图 4所示, 2VO手术72 h后, 2VO+LV-GFP组海马CA1神经元活细胞总数和CPu神经元活细胞均数(158±18和29±3)明显少于Sham+LV-GFP组(397±10, P < 0.01; 117±6, P < 0.01)。而klotho过表达有效地改善了缺血后海马CA1和CPu神经元损伤(266±21和54±8; P < 0.05)。

5 Klotho过表达抑制2VO小鼠脑内氧化应激

如图 5所示, 与Sham+LV-GFP组相比(1.02±0.11和1.01±0.08), 2VO+LV-GFP组Mn-SOD mRNA和CAT mRNA表达水平均明显下降(0.2±0.06, P < 0.05, 图 5A; 0.36±0.06, P < 0.05, 图 5B)。而klotho过表达有效抑制2VO诱导的脑内氧化应激的增加, 表现为2VO+LV-KL组Mn-SOD和CAT mRNA水平明显升高(2±0.48, P < 0.01; 1.17±0.08, P < 0.01)。另外, 与Sham+LV-GFP组相比, 2VO增加了2VO+LV-GFP组的MDA水平(2.6±0.07, P < 0.01, 图 5C), 而LV-KL有效抑制脂质磷酸化, 降低了缺血脑中MDA含量(2.14±0.03, P < 0.05)。

6 Klotho过表达抑制2VO小鼠脑内Akt/FoxO1磷酸化

如图 6所示, 与Sham+LV-GFP组相比(1.0±0.03和1.0±0.14), 2VO+LV-GFP组Akt和FoxO1磷酸化水平增加(1.21±0.09和1.23±0.09), 提示全脑缺血/再灌注促进了Akt/FoxO1磷酸化, 可能导致2VO小鼠脑内抗氧化酶表达减少, 脑内氧化应激水平增加。而klotho过表达明显逆转了2VO手术引起的脑内Akt和FoxO1磷酸化的增加(0.46±0.11和0.53±0.05, P < 0.01)。这些结果表明, 抑制Akt/FoxO1磷酸化可能是klotho过表达发挥抗氧化保护作用的潜在靶标。

讨论

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缺血性脑卒中是脑卒中最普遍的疾病亚型, 1/3缺血性脑卒中发生在老年人群^[15]。此外, 老龄化增加了静脉溶栓给药后出血性转化的风险, 大大限制了溶栓治疗的效果^[2]。Klotho作为一种抗衰老基因, 其表达随年龄增长而下降, 表明了其在衰老相关疾病中的潜在作用。

短暂性2VO诱导的全脑缺血作为一种缺血耐受小鼠模型, 很好地模拟了由晚期动脉老化引发的进行性结构和功能改变引起的脑灌注不足^[16]。本实验采用此小鼠模型发现, 2VO手术引起了明显的神经功能障碍, 海马CA1区和纹状体神经元大量死亡, 表明2VO手术造成了明显的缺血再灌损伤。

2VO+LV-KL组实验结果表明, 慢病毒介导的klotho过表达显著改善了神经功能障碍, 逆转了2VO手术引起的神经元死亡, 并抑制了脑内氧化应激水平, 表现为降低MDA含量和增加Mn-SOD和CAT mRNA表达, 抑制缺血脑内Akt和FoxO1的磷酸化。综上所述, klotho过表达可能是通过抑制Akt/FoxO1信号通路来发挥抗脑缺血的作用。

FoxOx蛋白由一个核转录因子家族组成, 其中FoxO1被Akt磷酸化后, 从胞核转移至胞质, 失去转录活性, 这可能介导缺血预处理后的缺血耐受。同时, FoxOs蛋白磷酸化后, 其转录减少, Mn-SOD和CAT等下游抗氧化酶的表达降低, 导致ROS的大量累积, 引起严重的氧化损伤^[5]。有研究表明, Akt/FoxO1通路可能在藁本内酯(ligustilide, LIG)抗衰老相关的氧化损伤的神经保护作用中发挥了重要作用, 而对脑缺血的作用少有报道。通过实验结果推测, klotho过表达之所以能够减轻脑内的氧化损伤, 可能是因为其明显抑制了2VO小鼠缺血脑中FoxO1磷酸化, 上调了抗氧化酶的活性及其表达, 有助于O₂^-、过氧化氢等ROS的清除。由此可见, klotho可作为脑卒中等Akt/FoxO1信号通路相关疾病的潜在治疗手段。

基金

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国家自然科学基金资助项目(81473219)

参考文献

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排序方式：

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Pedersen L, Pedersen SM, Brasen CL, et al. Soluble serum klotho levels in healthy subjects. Comparison of two different immunoassays [J]. Clin Biochem, 2013, 46:1079-1083.

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Zeldich E, Chen CD, Colvin TA, et al. The neuroprotective effect of klotho is mediated via regulation of members of the redox system [J]. J Biol Chem, 2014, 289:24700-24715.

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Zhou HJ, Li H, Shi MQ, et al. Protective effect of klotho against ischemic brain injury is associated with inhibition of RIG-I/NF-κB signaling[J]. Front Pharmacol, 2018, 8:950.

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2018年第53卷第8期

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doi: 10.16438/j.0513-4870.2018-0328

接收时间：2018-04-11
首发时间：2026-01-15
出版时间：2018-08-12

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收稿日期：2018-04-11
修回日期：2018-05-07

基金

国家自然科学基金资助项目(81473219)

作者信息

四川大学华西药学院, 四川成都 610041

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* 杜俊蓉, Tel: 86-28-85503938, Fax: 86-28-85501371, E-mail: dujr_1@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Gene	Primer sequence forward (5'-3')	Primer sequence reverse (5'-3')
GAPDH	AGCGAGACCCCACTAACATC	GGTTCACACCCATCACAAAC
CAT	AGCGACCAGATGAAGCAGTG	TCCGCTCTCTGTCAAAGTGTG
Mn-SOD	CAGACCTGCCTTACGACTATGG	CTCGGTGGCGTTGAGATTGTT
Klotho	GGCTTTCCTCCTTTACCTGAAAA	CACATCCCACAGATAGACATTCG

Gene

Primer sequence forward (5'-3')

Primer sequence reverse (5'-3')

GAPDH

AGCGAGACCCCACTAACATC

GGTTCACACCCATCACAAAC

CAT

AGCGACCAGATGAAGCAGTG

TCCGCTCTCTGTCAAAGTGTG

Mn-SOD

CAGACCTGCCTTACGACTATGG

CTCGGTGGCGTTGAGATTGTT

Klotho

GGCTTTCCTCCTTTACCTGAAAA

CACATCCCACAGATAGACATTCG

Task	Best performance score	Deficit score
Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)	0 (present at all paws)	4 (1 point for each missing reflex)
Stop at the edge of a table	0 (stopping at the end of table)	1 (no stopping behavior)
Turning the head (touching the ear from behind with a little rod)	0 (turning towards both directions)	2 (1 point for each failure to turn towards the stimulated ear)
Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)	0 (extension of both front paws)	1 (not reaching down towards the ground with both front paws)
Spontaneous motility (moving behavior on a flat surface)	0 (immediate, spontaneous activity)	1 (activity after 30-60 s) or 2 (no activity within 60 s)
Circling behavior (moving behavior on a flat surface)	0 (no circling, moving in all directions)	2 (spontaneous circling, turning only to one direction)
Pelt property (appearance of the coat)	0 (clean, well kept)	1 (coarse, scrubby)
Flight reaction (spontaneous behavior on a flat surface)	0 (normal behavior)	1 (anxious, jumpy behavior)
Maximum total score		14

Task

Best performance score

Deficit score

Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw)

0 (present at all paws)

4 (1 point for each missing reflex)

Stop at the edge of a table

0 (stopping at the end of table)

1 (no stopping behavior)

Turning the head (touching the ear from behind with a little rod)

0 (turning towards both directions)

2 (1 point for each failure to turn towards the stimulated ear)

Falling reflex (lifting the mouse at the tail and lowering with the front legs towards the ground)

0 (extension of both front paws)

1 (not reaching down towards the ground with both front paws)

Spontaneous motility (moving behavior on a flat surface)

0 (immediate, spontaneous activity)

1 (activity after 30-60 s) or 2 (no activity within 60 s)

Circling behavior (moving behavior on a flat surface)

0 (no circling, moving in all directions)

2 (spontaneous circling, turning only to one direction)

Pelt property (appearance of the coat)

0 (clean, well kept)

1 (coarse, scrubby)

Flight reaction (spontaneous behavior on a flat surface)

0 (normal behavior)

1 (anxious, jumpy behavior)

Maximum total score