Andrographolide Exerts Significant Antidepressant-Like Effects Involving the Hippocampal BDNF System in Mice

Abstract

Background: Major depressive disorder is a worldwide neuropsychiatric disorder associated with various symptoms, but current antidepressants used in clinical practice have various side effects and high failure rates. Andrographolide is the main bioactive ingredient of Andrographis paniculata and exhibits numerous pharmacological actions. This study aimed to evaluate the antidepressant-like effects of andrographolide in male C57BL/6J mice.

Methods: The antidepressant-like effects of andrographolide in mice were explored in a forced swim test, tail suspension test, and chronic unpredictable mild stress model of depression. Western blotting and immunofluorescence were further performed to assess the effects of chronic unpredictable mild stress and andrographolide on the brain-derived neurotrophic factor signalling cascade and hippocampal neurogenesis. Moreover, a pharmacological inhibitor (K252a) and a lentiviral-short hairpin RNA (LV-TrkB-shRNA) were used to clarify the antidepressant-like mechanism of andrographolide.

Results: Andrographolide exhibited antidepressant-like potential in the forced swim test and tail suspension test without influencing the locomotor activity of mice. Repeated andrographolide treatment not only produced significant antidepressant-like effects in the chronic unpredictable mild stress model but also prevented the decreasing effects of chronic unpredictable mild stress on hippocampal brain-derived neurotrophic factor signalling and neurogenesis in mice. Importantly, blockade of the hippocampal brain-derived neurotrophic factor system by K252a and TrkB-shRNA fully abolished the antidepressant-like effects of andrographolide in mice.

Conclusions: Andrographolide exerts antidepressant-like effects in mice via promoting the hippocampal brain-derived neurotrophic factor signalling cascade.

Keywords: andrographolide; brain-derived neurotrophic factor; depression; hippocampus.

Figure 1.

A single andrographolide (Andro) injection produced antidepressant-like activity in naive mice, as seen in the forced swim test (FST) (A) and tail suspension test (TST) (B) without affecting their locomotor activity (C and D). All results are expressed as the means ± SEMs (n = 10); *P < .05, **P < .01 vs Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 2.

Repeated andrographolide (Andro) administration induced significant antidepressant-like effects in the chronic unpredictable mild stress (CUMS) model of depression, as revealed by the forced swim test (FST) (B), tail suspension test (TST) (C), and sucrose preference test (SPT) (D). A schematic of the procedural timeline is shown in A. All results are represented as the means ± SEMs (n = 10); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 3.

Repeated andrographolide (Andro) treatment fully reversed the effects of chronic unpredictable mild stress (CUMS) on the hippocampal brain-derived neurotrophic factor (BDNF) signalling cascade in mice. Representative western-blot images and the quantitative analyses are shown in A and B, respectively. All results are represented as the means ± SEMs (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 4.

Repeated andrographolide (Andro) treatment fully reversed the effects of chronic unpredictable mild stress (CUMS) on hippocampal neurogenesis in mice. (A) Representative confocal microscopy images showing the localization of doublecortin (DCX; green) and 4’,6-diamidino-2-phenylindole (DAPI; blue) in the dentate gyrus (DG). The scale bar represents 150 µm for the representative images and 50 µm for the enlarged images. (B) Representative microscopic images showing the co-staining (yellow) of neuronal nuclei (NeuN; green) and 5-Bromo-2-deoxyUridine (BrdU; red) in the DG. The scale bar represents 150 µm for the representative images and 75 µm for the enlarged images. The quantitative analyses are shown in (C). All results are represented as the means ± SEMs (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 5.

The usage of K252a attenuated the antidepressant-like effects of andrographolide (Andro) both in naive mice (B) and in the chronic unpredictable mild stress (CUMS) model of depression (C–E). Schematics showing the timelines of the procedures are shown in A. All results are represented as means ± SEM (n = 10); *P < .05, **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The comparisons were made by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 6.

K252a blocked the promoting effects of andrographolide (Andro) on hippocampal brain-derived neurotrophic factor (BDNF) signalling in chronic unpredictable mild stress (CUMS)-treated mice. Representative western-blot images and the quantitative analyses are shown in A and B, respectively. All results are represented as the means ± SEMs (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 7.

K252a blocked the enhancing effects of andrographolide (Andro) on hippocampal neurogenesis in chronic unpredictable mild stress (CUMS)-treated mice. (A) Representative confocal microscopy images showing the localization of doublecortin (DCX; green) and 4’,6-diamidino-2-phenylindole (DAPI; blue) in the dentate gyrus (DG). The scale bar represents 150 µm for the representative images and 50 µm for the enlarged images. (B) Representative microscopic images showing the co-staining (yellow) of neuronal nuclei (NeuN; green) and 5-Bromo-2-deoxyUridine (BrdU; red) in the DG. The scale bar represents 150 µm for the representative images and 75 µm for the enlarged images. The quantitative analyses are shown in (C). All results are represented as the means ± SEMs (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 8.

The usage of TrkB-shRNA fully abolished the antidepressant-like effects of andrographolide (Andro) both in naive mice (B) and in the chronic unpredictable mild stress (CUMS) model of depression (C–E). Schematics showing the timelines of the procedures are shown in (A). All results are represented as the means ± SEMs (n = 10); *P < .05, **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.

Figure 9.

TrkB-shRNA completely abolished the promoting effects of andrographolide (Andro) on hippocampal brain-derived neurotrophic factor (BDNF) signalling in chronic unpredictable mild stress (CUMS)-treated mice. Representative western-blot images and the quantitative analyses are shown in (A) and (B), respectively. All results are represented as the means ± SEM (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the (LSD) test.

Figure 10.

Tyrosine receptor kinase B- short hairpin RNA (TrkB-shRNA) completely abolished the enhancing effects of andrographolide (Andro) on hippocampal neurogenesis in chronic unpredictable mild stress (CUMS)-treated mice. (A) Representative confocal microscopy images showing the localization of doublecortin (DCX; green) and 4’,6-diamidino-2-phenylindole (DAPI; blue) in the dentate gyrus (DG). The scale bar represents 150 µm for the representative images and 50 µm for the enlarged images. (B) Representative microscopic images showing the co-staining (yellow) of neuronal nuclei (NeuN; green) and 5-Bromo-2-deoxyUridine (BrdU; red) in the DG. The scale bar represents 150 µm for the representative images and 75 µm for the enlarged images. The quantitative analyses are shown in C. All results are represented as the means ± SEMs (n = 5); **P < .01 vs Vehicle; ^##P < .01 vs CUMS + Vehicle. The data were compared by 1-way ANOVA followed by the least significant difference (LSD) test.