鼠尾草酸对慢性不可预见性轻度应激诱导的抑郁大鼠的抗抑郁作用及其SIRT1/ERS调控机制

doi:10.13481/j.1671-587X.20230212

摘要/Abstract

摘要：

目的探讨鼠尾草酸（CA）对慢性不可预见性轻度应激（CUMS）诱导的抑郁大鼠内质网应激（ERS）的影响，并阐明其抗抑郁机制。方法采用随机数字表法从66只SD大鼠中随机选取12只为对照组，剩余54只大鼠采用CUMS联合孤养诱导建立抑郁模型，取48只建模成功的大鼠随机分为模型组、氟西汀（3.17 mg·kg^－1）组、CA（40 mg·kg^－1）组和CA（40 mg·kg^－1）+EX527沉默信息调节因子1（SIRT1）抑制剂（5 mg·kg^－1）组，每组12只。给予相应的药物干预后，旷场实验（OFT）检测大鼠运动总距离和中间停留时间；强迫游泳实验（FST）检测大鼠强迫游泳时的不动时间；糖水偏好实验（SPT）检测大鼠糖水偏好度；TUNEL法检测各组大鼠海马组织中TUNEL阳性细胞百分率；免疫荧光法检测各组大鼠海马组织中C/EBP同源蛋白（CHOP）和葡萄糖调节蛋白78（GRP78）阳性表达强度；实时荧光定量PCR（RT-qPCR）法检测各组大鼠海马组织中SIRT1、GRP78、CHOP和Caspase-12 mRNA表达水平；Western blotting法检测各组大鼠大脑海马组织CA1区中SIRT1、GRP78、CHOP、Caspase-12和裂解的Caspase-12（cleaved Caspase-12）蛋白表达。结果与对照组比较，模型组大鼠OFT中的总距离、中间停留时间、糖水偏好百分率及海马组织CA1区中SIRT1 mRNA和蛋白表达水平均明显降低（P<0.05）；强迫游泳时的不动时间，TUNEL阳性细胞百分率，GRP78、CHOP、Caspase-12和cleaved Caspase-12蛋白表达水平均明显升高（P<0.05）。与模型组比较，氟西汀组和CA组大鼠OFT中的运动总距离、中间停留时间、糖水偏好度及海马组织CA1区中SIRT1 mRNA和蛋白表达水平明显升高（P<0.05），强迫游泳时的不动时间，TUNEL阳性细胞百分率，GRP78、CHOP、Caspase-12和cleaved Caspase-12蛋白表达水平明显降低（P<0.05）；与CA组比较，CA+EX527组大鼠OFT中的总距离，中间停留时间，糖水偏好度，大脑海马组织CA1区中SIRT1 mRNA和蛋白表达水平均明显降低（P<0.05）；强迫游泳时的不动时间，TUNEL阳性细胞百分率，GRP78、CHOP、Caspase-12和cleaved Caspase-12蛋白表达水平均明显升高（P<0.05）。结论 CA可减轻CUMS引起的大鼠抑郁样行为，其抗抑郁机制可能与SIRT1介导的ERS抑制有关。

关键词: 鼠尾草酸, 抑郁症, 内质网应激, 沉默信息调节因子1

Abstract:

Objective To investigate the effect of carnosic acid （CA） on the endoplasmic reticulum stress （ERS） in the depression rats induced by chronic unpredictable mild stress （CUMS）， and to elucidate its antidepressant mechanism. Methods Twelve SD rats were randomly selected as control group from 66 SD rats by random number table method， and the remaining 54 rats were induced to establish depression model by CUMS combined with solitary feeding， and 48 rats were randomly divided into model group， fluoxetine（3.17 mg·kg^－1） group， CA （40 mg·kg^－1） group， CA （40 mg·kg^－1）+ EX527 silence information regulator 1 （SIRT1） inhibitor （5 mg·kg^－1） group， and there were 12 rats in each group. After corresponding drug intervention， the total exercise distance and intermediate residence time of the rats were measured by open field test （OFT）； the immobility time of the rats during forced swimming was observed by forced swimming test （FST）； the surge water perference of the rats in various groups were observed by sugar water preference test （SPT）； the percentages of the TUNEL positive cells in hippocampus tissue of the rats in various groups were detected by TUNEL method； immunofluorescence method was used to detect the positive expression intensities of C/EBP-homologous protein （CHOP） and glucose regulated protein 78 （GRP78） in hippocampus tissue of the rats in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of SIRT1， GRP78， CHOP， and Caspase-12 mRNA in hippocampus tissue of the rats in various groups； Western blotting method was used to detect the expression levels of SIRT1， GRP78， CHOP Caspase-12， and cleaved Caspase-12 （cleaved Caspase-12） proteins in CA1 region in hippocampus tissue in brain of the rats in various groups. Results Compared with control group， the total exercise distance， intermediate residence time， sugar water preference in OFT， and expression levels of SIRT1 protein and mRNA in CA1 region in hippocampus tissue in brain of the rats in model group were significantly decreased （P<0.05）， the immobility time during forced swimming， percentage of TUNEL positive cells，expression levels of GRP78， CHOP，Caspase-12， and cleaved Caspase-12 proteins were significantly increased （P<0.05）.Compared with model group， the total exercise distance， intermediate residence time，sugar water preference in OFT，and expression levels of SIRT1 protein and mRNA in hippocampus tissue of the rats in fluoxetine group and CA group were significantly increased （P<0.05）， the immobility time during forced swimming， percentage of TUNEL positive cells， expression levels of GRP78， CHOP，Caspase-12， and cleaved Caspase-12 proteins were significantly decreased （P<0.05）；compared with CA group， the total exercise distance， intermediate residence time，sugar water preference in OFT， and expression levels of SIRT1 protein and mRNA in CA1 region in hippocampus tissue in brain of the rats in CA+EX527 group were significantly decreased （P<0.05）， the immobility time during forced swimming， percentage of TUNEL positive cells， expression levels of GRP78， CHOP，Caspase-12， and cleaved Caspase-12 proteins were significantly increased （P<0.05）. Conclusion CA can alleviate the depression-like behaviors of the rats caused by CUMS， and its antidepressant mechanism may be related to the inhibition of ERS mediated by SIRT1.

Key words: Carnosic acid, Depression, Endoplasmic reticulum stress, Silent information regulator 1

中图分类号:

R966

谭东明,尹红英,邓祥敏,丁旭. 鼠尾草酸对慢性不可预见性轻度应激诱导的抑郁大鼠的抗抑郁作用及其SIRT1/ERS调控机制[J]. 吉林大学学报(医学版), 2023, 49(2): 360-368.

Dongming TAN,Hongying YIN,Xiangmin DENG,Xu DING. Antidepressant effect of carnosic acid on chronic unpredictable mild stress-induced depression in rats and its SIRT1/ERS regulatory mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(2): 360-368.

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Gene	Forward primer (5'－3')	Reverse primer (5'－3')
SIRT1	GTTGTGTGCCTTCGTTTTGGA	AGGCCGGTTTGGGCTTATACA
GRP78	ACACTTGGTATTGAAACTGTGGGAG	CTTGATTGTTACGGTGGGCTG
CHOP	TCTTCATACACCACCACACCTG	CTTCGTTTCCTGGGGATGAGA
Caspase-12	TTGGATACTCAGTGGTGATAAAGGA	GGATGCCGTGGGACATAAAGA
β-actin	GTTGTCTCCTGCGACTTCA	GCCCCTCCTGTTATTATGG

Group	Total exercisedistance (l/m)	Intermediate residence time (t/s)	Immobility time during forced swimming (t/s)	Sugar water preference (η/%)
Control	10.45±1.37	68.39±9.26	20.30±3.48	67.80±7.24
Model	5.12±0.86^*	32.27±5.40^*	76.74±5.19^*	25.31±4.69^*
CA	7.33±0.91^*△	49.58±6.35^*△	45.29±4.62^*△	43.54±5.81^*△
Fluoxetine	7.84±1.15^*△	56.42±8.16^*△	39.61±5.07^*△	52.72±6.35^*△
CA+EX527	5.50±0.76^*#	35.07±6.24^*#	70.43±6.25^*△#	27.43±4.06^*#

Group	Percentage of TUNEL positive cells
Control	7.25±1.10
Model	21.34±2.06^*
Fluoxetine	10.39±1.28^*△
CA	14.60±1.75^*△
CA+EX527	18.52±1.91^*△#

Group	CHOP	GRP78
Control	7.25±2.07	2.30±0.26
Model	42.81±4.13^*	16.17±2.09^*
Fluoxetine	19.40±2.65^*△	5.09±0.72^*△
CA	23.36±3.29^*△	8.28±1.35^*△
CA+EX527	35.78±4.02^*△#	12.75±1.46^*△#

Group	SIRT1 mRNA	GRP78 mRNA	CHOP mRNA	Caspase-12 mRNA
Control	1.01±0.12	0.98±0.10	1.02±0.14	1.00±0.09
Model	0.35±0.05^*	2.43±0.28^*	2.18±0.25^*	2.05±0.22^*
Fluoxetine	0.79±0.08^*△	1.57±0.21^*△	1.45±0.18^*△	1.33±0.15^*△
CA	0.64±0.07^*△	1.81±0.25^*△	1.59±0.20^*△	1.46±0.19^*△
CA+ EX527	0.45±0.06^*#	2.26±0.29^*#	1.92±0.23^*#	1.80±0.20^*#