Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway

Abstract

Background: Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway.

Methods: We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways.

Results: METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on mitochondrial membrane integrity was also confirmed by flow cytometry and immunofluorescence staining.

Conclusions: Based on the literatures and our findings, AA is a promising candidate for an anti-neurotoxic agent, and it can potentially be used for the prevention and treatment of various neurological disorders.

Keywords: Asiatic acid; ERK; Methamphetamine; NF-κB; Neuroinflammation; Neurotoxicity; STAT3.

Fig. 1

AA inhibits METH-induced TNF-alpha and IL-6 production and mRNA expression levels. SH-SY5Y cells were incubated in the presence or absence of AA (1, 10, and 20 μM) for 1 h and then treated with 1 mM METH for 24 h. a TNFR overexpression was significantly inhibited in METH-stimulated SH-SY5Y cells by AA administration. AA strongly suppressed METH-induced TNFα and IL-6 production both in extracellular (b) and mRNA levels (c, d). β-actin was used to confirm equal sample loading. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 4 to 9). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment

Fig. 2

AA suppresses the translocation of NF-κB and STAT3 in METH-stimulated SH-SY5Y cells. a Immunoblot analysis shows the effects of 20 μM AA on the inhibition of translocation of NF-κB/STAT3 and phosphorylation of JAK2 (an upstream activator of STAT3) in 1 mM METH-stimulated SH-SY5Y cells. β-actin and lamin B were used to confirm equal sample loading. DNA- and Ab-binding activity of NF-κB (b) and STAT3 (c) in nuclear extracts was measured by EMSA and supershift assay. d Immunofluorescence double staining for p-NF-κB p65 (green) and p-STAT3 (red) localization. Cells were counterstained with Hoechst 33342 (blue). Magnifications ×200. Immunoblotting, EMSA, and supershift assay were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 3 to 4). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment

Fig. 3

AA inhibits pro-inflammatory cytokine secretion through suppression of NF-κB, STAT3, and ERK pathway in METH-stimulated SH-SY5Y cells. SH-SY5Y cells were pretreated with 20 μM AA or various inhibitors for 1 h and then stimulated with 1 mM METH for 24 h. a Phosphorylation of ERK was strongly inhibited in METH-stimulated SH-SY5Y cells by AA administration. Inhibitors of NF-κB (Bay11-7085), STAT3 (S3I-201), ERK (PD98059), or AA strongly suppressed METH-induced TNFα and IL-6 production both in extracellular (b) and mRNA levels (c, d). METH-induced translocation of p65 and STAT3 was inhibited when the ERK pathway of these cells were downregulated. β-actin and lamin B were used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 4 to 6). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment

Fig. 4

AA suppresses METH-stimulated TNFα and IL-6 production through inhibition of NF-κB/STAT3 and MAPK-ERK signaling pathways in microglia BV2 cells. BV2 cells were pretreated with 20 μM AA or various inhibitors for 1 h and then stimulated with 1 mM METH for 24 h. a 20 μM AA suppresses 1 mM METH-induced translocation of NF-κB/STAT3 and phosphorylation of JAK2/ERK in BV2 cells. β-actin and lamin B were used to confirm equal sample loading. b Inhibitors of NF-κB (20 μM Bay11-7085), STAT3 (20 μM S3I-201), ERK (20 μM PD98059), or 20 μM AA strongly suppressed METH-induced TNFα and IL-6 production in extracellular levels. DNA-binding activity of NF-κB (c) and STAT3 (d) in nuclear extracts was measured by EMSA. Immunoblotting and EMSA were quantified by densitometric analysis. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 4 to 6). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment

Fig. 5

Protective effects of AA on METH-stimulated mesencephalic neurons. a Along with the presence or absence of AA, the morphological changes (upper) and TH expression (lower) in dopaminergic neurons after exposure to METH. Immunofluorescence staining for TH (green) localization. Cells were counterstained with Hoechst 33342 (blue). Magnifications ×400. b AA strongly reverses TH expression and inhibits phosphorylation of ERK and translocation of NF-κB/STAT3. β-actin and lamin B were used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. c Inhibitors of NF-κB (Bay11-7085), STAT3 (S3I-201), ERK (PD98059), or AA significantly suppresses TNFα and IL-6 secretion in METH-stimulated mesencephalic neurons. d Immunofluorescence double staining for TH (green) and p-NF-κB p65/p-STAT3 (red) localization. Cells were counterstained with Hoechst 33342 (blue). Magnifications ×200. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 4 to 6). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment

Fig. 6

AA protects METH-induced apoptosis in SH-SY5Y cells. a Annexin V/PI staining by flow cytometry. AA inhibits cleavages of caspase-3 and PARP (b) and regulates apoptotic proteins (c). VDAC was used as mitochondrial loading control, and β-actin was used to confirm equal sample loading. Immunoblotting was quantified by densitometric analysis. SH-SY5Y cells were evaluated by morphological criteria after JC-1 mitochondria staining (red and green) by using fluorescent microscope (upper) and flow cytometry (lower). Magnifications ×200. The data are representative of three independent experiments and quantified as mean values ± SEM (n = 3 to 9). Tukey’s multiple comparison test, *p < 0.05 compared to normal control, ^† p < 0.05 compared to METH treatment