3-N-butylphthalide attenuates neuroinflammation in rotenone-induced Parkinson's disease models via the cGAS-STING pathway

Abstract

Neuroinflammation is crucial in the onset and progression of dopaminergic neuron loss in Parkinson's disease (PD). We aimed to determine whether 3-N-Butylphthalide (NBP) can protect against PD by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway and the inflammatory response of microglia. MitoSOX/MitoTracker/Hoechst staining was used to detect the levels of mitochondrial reactive oxygen species (ROS) in BV2 cells. Quantitative Real-Time Polymerase Chain Reaction was used to measure the levels of free cytoplasmic mitochondrial DNA (mtDNA) in BV2 cells and mouse brain tissues. Behavioral impairments were assessed using rotarod, T-maze, and balance beam tests. Dopaminergic neurons and microglia were observed using immunohistochemical staining. Expression levels of cGAS, STING, nuclear factor kappa-B (NfκB), phospho- NfκB (p-NfκB), inhibitor of NfκBα (IκBα), and phospho-IκBα (p-IκBα) proteins in the substantia nigra and striatum were detected using Western Blot. NBP decreased mitochondrial ROS levels in rotenone-treated BV2 cells. NBP alleviated behavioral impairments and protected against rotenone-induced microgliosis and damage to dopaminergic neurons in the substantia nigra and striatum of rotenone-induced PD mice. NBP decreased rotenone-induced mtDNA leakage and mitigated neuroinflammation by inhibiting cGAS-STING pathway activation. NBP exhibited a protective effect in rotenone-induced PD models by significantly inhibiting the cGAS-STING pathway. Moreover, NBP can alleviate neuroinflammation, and is a potential therapeutic drug for alleviating clinical symptoms and delaying the progression of PD. This study provided insights for the potential role of NBP in PD therapy, potentially mitigating neurodegeneration, and consequently improving the quality of life and lifespan of patients with PD. The limitations are that we have not confirmed the exact mechanism by which NBP decreases mtDNA leakage, and this study was unable to observe the actual clinical therapeutic effect, so further cohort studies are required for validation.

Keywords: 3-N-butylphthalide; Parkinson’s disease; cyclic GMP-AMP synthase-stimulator of interferon genes pathway; microglia; neuroinflammation.

Figure 1.

Effects of the STING inhibitor (C-176) on the inflammatory response of rotenone-induced BV2 cells. (a)-(e) Expression levels of cGAS, STING, NfκB, p-NfκB, IκBα, and p-IκBα proteins in BV2 cells were detected using Western Blot. β-Actin was used as the control. (f)-(j) The mRNA expression of IL-1β, IL-6, TNF-α, cGAS, STING in BV2 cells was measured using qRT-PCR. GAPDH was used as the control. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 2.

NBP inhibits rotenone-induced cell viability injury, mitochondrial ROS levels and mtDNA leakage. (a) MitoSOX (red)/MitoTracker (green)/Hoechst (blue) staining was performed to detect the level of mitochondrial ROS in BV2 cells. (b) Cell viability was measured using a CCK8 assay in BV2 cells. (c) The levels of cytoplasmic free mtDNA in BV2 cells were detected using qRT-PCR. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 3.

NBP inhibits the activation of the cGAS-STING signaling and inflammation induced by rotenone in BV2 cells. (a)-(e) Western Blot for cGAS, STING, NfκB, p-NfκB, IκBα, and p-IκBα to detect protein levels. β-Actin was used as the control. (f)-(j) mRNA expression levels of IL-1β, IL-6, TNF-a, cGAS, STING in BV2 cells were measured by qRT-PCR. GAPDH was used as the control. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 4.

NBP alleviates the behavioral impairments in rotenone-induced PD mice. (a) Design of the study and scheme about drug administration. (b) Latency to fall off in the rotarod test. (c) Correct rate in the T-maze test. (d) Time to cross the balance beam. Data are expressed as the means ± SEM (n = 12). p value is provided in all histograms.

Figure 5.

NBP protects dopaminergic neurons in rotenone-induced PD mice. (a) Typical immunohistochemical staining showing TH positive cells in the substantia nigra. (b) Typical immunohistochemical staining showing TH positive nerve fibers in the striatum. (c) Numbers of TH positive cells in substantia nigra. (d) The relative optical density of TH staining in striatum. (e)-(h) Western Blot for TH protein levels in tshe striatum and substantia nigra. GAPDH was used as an internal control. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 6.

NBP inhibits microglial activation and mtDNA leakage in the striatum and substantia nigra induced by rotenone. (a)-(b) Immunohistochemical staining showing IBA-1 positive cells in the striatum and substantia nigra. (c)-(d) Numbers of IBA-1 positive cells in substantia nigra and striatum. (e)-(f) The levels of free cytoplasmic mtDNA in the substantia nigra and striatum were analyzed by qRT-PCR. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 7.

NBP reduces cGAS-STING associated neuroinflammation in the substantia nigra and striatum. (a), (f) Expression levels of cGAS, STING, NfκB, p-NfκB, IκBα, and p-IκBα proteins in the striatum and substantia nigra were measured using Western Blot. (b)-(e) Statistical analysis of cGAS, STING, NfκB, p-NfκB, IκBα, and p-IκBα protein levels in substantia nigra. (g)-(j) Statistical analysis of cGAS, STING, NfκB, p-NfκB, IκBα, and p-IκBα protein levels in striatum. Data are expressed as the means ± SEM (n = 3). p value is provided in all histograms.

Figure 8.

Schematic diagram of the protective effect of NBP in rotenone-induced PD models. Rotenone increases mitochondrial ROS levels and mtDNA leakage. The free cytoplasmic mtDNA activates the cGAS-STING pathway and leads to neuroinflammation, which is neurotoxic to dopaminergic neurons. NBP decreases rotenone-induced mitochondrial ROS levels and mtDNA leakage. NBP inhibits the activation of the cGAS-STING pathway and neuroinflammation, which is helpful to delay the progression of degeneration of dopaminergic neuron.