豆腐果苷通过NCALD/sGC/cGMP/PKG轴缓解慢性不可预见性温和应激大鼠的抑郁样行为

doi:10.12122/j.issn.1673-4254.2025.12.17

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (12): 2699-2707.doi: 10.12122/j.issn.1673-4254.2025.12.17

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豆腐果苷通过NCALD/sGC/cGMP/PKG轴缓解慢性不可预见性温和应激大鼠的抑郁样行为

杜鑫¹^,²(), 张晓彤⁴, 芦娟¹^,², 葛鹏¹^,², 胡海涛¹^,², 付梦雪¹^,², 张袁祥¹^,⁵^,⁶, 王国栋¹^,⁵^,⁶^,⁷(), 童九翠¹^,²^,³()

^1.皖南医学院药学院，安徽芜湖 241002
^2.皖南医学院安徽高等院校非编码RNA转化研究重点实验室，安徽芜湖 241001
^3.皖南医学院第一附属医院（弋矶山医院）药学部安徽芜湖 241001
^4.皖南医学院第一附属医院（弋矶山医院）临床药学科，安徽芜湖 241001
^5.安徽省多糖药物工程技术研究中心，安徽芜湖 241002
^6.安徽省皖南地区植物药活性物质筛选与再评价工程实验室，安徽芜湖 241002
^7.安徽省代谢性疾病药物基础研究创新中心，安徽芜湖 241002

收稿日期:2025-06-30 出版日期:2025-12-20 发布日期:2025-12-22
通讯作者: 王国栋,童九翠 E-mail:20239150@stu.wnmc.edu.cn;wangguodong@wnmc.edu.cn;tongjiucui@wnmc.edu.cn
作者简介:杜鑫，在读硕士研究生，E-mail: 20239150@stu.wnmc.edu.cn
基金资助:
安徽省卫生健康科研重点项目(AHWJ2022a027);安徽省高校科研计划项目-省高校重大科研项目(2024AH040248)

Helicid alleviates depression-like behavior in rats with chronic unpredictable mild stress through the NCALD/sGC/cGMP/PKG axis

Xin DU¹^,²(), Xiaotong ZHANG⁴, Juan LU¹^,², Peng GE¹^,², Haitao HU¹^,², Mengxue FU¹^,², Yuanxiang ZHANG¹^,⁵^,⁶, Guodong WANG¹^,⁵^,⁶^,⁷(), Jiucui TONG¹^,²^,³()

^1.School of Pharmacy, Wannan Medical College, Wuhu 241002, China
^2.Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution, Wannan Medical College, Wuhu 241001, China
^3.Department of Pharmacy, Yijishan Hospital, Wannan Medical College, Wuhu 241001, China,
^4.Department of Clinical Pharmacy, Yijishan Hospital, Wannan Medical College, Wuhu 241001, China
^5.Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wuhu 241002, China
^6.Anhui Provincial Engineering Laboratory for Screening and Re-evaluation of Active Compounds of Herbal Medicines in Southern Anhui, Wuhu 241002, China
^7.Anhui Innovative Center for Drug Basic Research of Metabolic Diseases, Wuhu 241002, China

Received:2025-06-30 Online:2025-12-20 Published:2025-12-22
Contact: Guodong WANG, Jiucui TONG E-mail:20239150@stu.wnmc.edu.cn;wangguodong@wnmc.edu.cn;tongjiucui@wnmc.edu.cn

摘要/Abstract

摘要：

目的探讨豆腐果苷（Helicid）通过神经钙调素δ（NCALD）改善慢性不可预见性温和应激（ CUMS）大鼠抑郁样行为的功能及其分子机制。方法 SD大鼠进行单笼适应性饲养1周后，随机分成正常对照组（n=20）、CUMS组大鼠（n=70）。正常对照组大鼠始终不进行任何刺激，CUMS组大鼠连续给予慢性不可预知温和刺激6周，建立大鼠抑郁模型。成功建模后，根据旷场实验和体质量结果，将CUMS组大鼠进一步分组，分别开展两部分实验：侧脑室注射腺相关病毒沉默NCALD，分为CUMS+SAL组、CUMS+AAV组和CUMS+AAV-si-NCALD组，10只/组；豆腐果苷灌胃治疗的基础上侧脑室注射腺相关病毒过表达NCALD，分为CUMS+SAL组、CUMS+Helicid+SAL组、CUMS+Helicid+AAV组和CUMS+Helicid+AAV-oe-NCALD组，10只/组。通过体质量、蔗糖偏好实验和旷场实验评估大鼠抑郁状态，Western blotting法测定海马组织中NCALD、sGCα1、sGCβ1、PKG1/2和cleaved-caspase-3蛋白水平，ELISA法测定cGMP蛋白水平。结果与正常对照组比较，CUMS大鼠海马中NCALD表达上调（P<0.05）、cleaved-caspase-3水平增加（P<0.05）及sGC/cGMP/PKG信号通路异常激活（P<0.05）；而腺相关病毒沉默NCALD后，下调了cleaved-caspase-3水平，抑制了sGC/cGMP/PKG信号通路的异常激活，改善了CUMS大鼠抑郁样行为（P<0.05）。豆腐果苷治疗同样下调了上述指标（P<0.05），减轻抑郁样行为（P<0.05），但在过表达NCALD条件下，豆腐果苷的保护作用被逆转（P<0.05）。结论豆腐果苷通过下调NCALD表达，抑制了sGC/cGMP/PKG通路的异常活化，改善CUMS大鼠的海马组织凋亡，从而发挥抗抑郁作用。

关键词: 豆腐果苷, 神经钙素, sGC/cGMP/PKG信号通路, 慢性温和不可预知应激模型

Abstract:

Objective To investigate the molecular mechanism of helicid for improving depressive-like behaviors in rats exposed to chronic unpredictable mild stress (CUMS). Methods SD rats were randomly divided into normal control group (n=20) and CUMS group (n=70) to receive no stimulation and mild unpredictable stress for 6 weeks, respectively. After successful modeling, CUMS rats were further divided into 7 subgroups for intracerebroventricular injection with saline, adeno-associated virus (AAV) vector, or AAV carrying si-NCALD (NCALD silencing experiment, n=10); or intracerebroventricular injection with saline, saline with daily helicid gavage, AAV vector with helicid gavage, or NCALD-overexpressing AAV with helicid gavage (NCALD overexpresison experiment, n=10). The depressive state of the rats was evaluated by assessing changes in body weight, sucrose preference, and open field test. The expressions of NCALD, sGCα1, sGCβ1, PKG1/2, and cleaved-caspase 3 in the hippocampus of the rats were detected by Western blotting, and hippocampal cGMP level was determined with ELISA. Results Compared with the normal control rats, CUMS rats showed significantly increased hippocampal expressions of NCALD and cleaved caspase-3 and abnormal activation of the sGC/cGMP/PKG pathway. Silencing NCALD by intracerebroventricular injection of AAV-si-NCALD significantly reduced cleaved caspase-3 and inhibited sGC/cGMP/PKG pathway activation in the hippocampus, and improved depressive-like behaviors of the rats. Helicid treatment produced similar effects, but its effect was abolished by intracerebroventricular injection of NCALD-overex-pressing AAV. Conclusion Helicid relieves depressive-like behaviors in CUMS rats by downregulating NCALD, inhibiting abnormal sGC/cGMP/PKG activation, and reducing hippocampal apoptosis.

Key words: helicid, neurocalcin δ, sGC/cGMP/PKG signaling pathway, chronic mild unpredictable stress

杜鑫, 张晓彤, 芦娟, 葛鹏, 胡海涛, 付梦雪, 张袁祥, 王国栋, 童九翠. 豆腐果苷通过NCALD/sGC/cGMP/PKG轴缓解慢性不可预见性温和应激大鼠的抑郁样行为[J]. 南方医科大学学报, 2025, 45(12): 2699-2707.

Xin DU, Xiaotong ZHANG, Juan LU, Peng GE, Haitao HU, Mengxue FU, Yuanxiang ZHANG, Guodong WANG, Jiucui TONG. Helicid alleviates depression-like behavior in rats with chronic unpredictable mild stress through the NCALD/sGC/cGMP/PKG axis[J]. Journal of Southern Medical University, 2025, 45(12): 2699-2707.

图/表 6

图1 动物实验分组情况及流程图

Fig.1 Grouping of the rats and flow chart of the animal experiment.

图2 CUMS刺激6周后大鼠的体质量及行为学变化

Fig.2 Body Weight and behavioral changes of the rats after 6 weeks of CUMS stimulation. A: Changes of body weight of rats during CUMS. B: Effect of CUMS on total distance traveled of the rats in open field test. C: Effect of CUMS on rearing times of the rats in open field test. D: Effect of CUMS on the number of zone crossing in open field test. E: Effect of CUMS on sucrose preference of the rats in each group. F: Effect of CUMS on immovability time of the rats during forced swimming test. *P<0.05, **P<0.01, ***P<0.001 vs Control.

图3 沉默NCALD缓解CUMS诱导的大鼠抑郁样行为

Fig.3 NCALD silencing attenuates CUMS-induced depression-like behaviors in rats. A: Changes of body weight of the rats after NCALD silencing. B: Changes of total distance of the rats in open field test after NCALD silencing. C: Changes of rearing times in open field test after NCALD silencing. D: Changes of the number of zone crossing in open field experiment after NCALD silencing. E: Changes of sucrose preference in rats after NCALD silencing. F: Changes of immovability time of the rats after NCALD silencing. ###P<0.001 vs CON+SAL; *P<0.05, **P<0.01, ***P<0.001 vs CUMS+AAV.

图4 沉默NCALD对sGC/cGMP/PKG信号通路和凋亡细胞因子cleaved-caspase 3的调节作用

Fig.4 Regulatory effect of AAV-si-NCALD on the sGC/cGMP/PKG signaling pathway and hippocampal expression of caspase-3 in CUMS rats. A, B: Protein levels of NCALD in the hippocampus detected using Western blotting. C, D: Protein levels of cleaved caspase-3 in the hippocampus detected using Western blotting. E-H: Protein levels of sGCα, sGCβ and PKG in the hippocampus detected using Western blotting. I: Protein levels of cGMP in the hippocampus detected using ELISA.**P<0.01, ***P<0.001 vs CON+SAL; #P<0.05, ##P<0.01 vs CUMS+AAV.

图5 过表达NCALD逆转了豆腐果苷的治疗作用

Fig.5 Overexpression of NCALD attenuates the therapeutic effect of helicid. A: Changes of body weight of the rats after helicid treatment and NCALD overexpression. B: Changes of total distance traveled of the rats in open field test in different groups. C: Changes of rearing times in open field test of the rats in different groups. D: Changes of the number of zone crossing in open field test of the rats in different groups. E: Changes of sucrose preference of the rats in different groups. F: Changes of immovability time of the rats in different groups. ***P<0.001 vs CON+SAL; &&P<0.01, &&&P<0.001 vs CUMS+SAL; #P<0.05, ##P<0.01, ###P<0.01 vs CUMS+helicid+AAV.

图6 豆腐果苷通过NCALD对sGC/cGMP/PKG信号通路和凋亡细胞因子cleaved-caspase 3的调节作用

Fig.6 Helicid modulates the sGC/cGMP/PKG signaling pathway and hippocampal caspase-3 expression by regulating NCALD expression. A, B: Protein levels of NCALD in the hippocampus detected using Western blotting. C, D: Protein levels of cleaved caspase-3 in the hippocampus detected using Western blotting. E-H: Protein levels of sGCα, sGCβ and PKG in the hippocampus detected using Western blotting. I: Protein levels of cGMP in the hippocampus detected using ELISA.**P<0.01, ***P<0.001 vs CON+SAL; &P<0.05, &&P<0.01, &&&P<0.001 vs CUMS+SAL; #P<0.05, # #P<0.01, # # #P<0.001 vs CUMS+helicid+AVV.

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豆腐果苷通过NCALD/sGC/cGMP/PKG轴缓解慢性不可预见性温和应激大鼠的抑郁样行为

Helicid alleviates depression-like behavior in rats with chronic unpredictable mild stress through the NCALD/sGC/cGMP/PKG axis

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