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

doi:10.12122/j.issn.1673-4254.2025.12.17

Abstract

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

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.

Figures/Tables 6

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

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.

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.

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.

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.

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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