Noscapine Prevents Rotenone-Induced Neurotoxicity: Involvement of Oxidative Stress, Neuroinflammation and Autophagy Pathways

Abstract

Parkinson's disease is characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the resultant loss of dopamine in the striatum. Various studies have shown that oxidative stress and neuroinflammation plays a major role in PD progression. In addition, the autophagy lysosome pathway (ALP) plays an important role in the degradation of aggregated proteins, abnormal cytoplasmic organelles and proteins for intracellular homeostasis. Dysfunction of ALP results in the accumulation of α-synuclein and the loss of dopaminergic neurons in PD. Thus, modulating ALP is becoming an appealing therapeutic intervention. In our current study, we wanted to evaluate the neuroprotective potency of noscapine in a rotenone-induced PD rat model. Rats were administered rotenone injections (2.5 mg/kg, i.p.,) daily followed by noscapine (10 mg/kg, i.p.,) for four weeks. Noscapine, an iso-qinulinin alkaloid found naturally in the Papaveraceae family, has traditionally been used in the treatment of cancer, stroke and fibrosis. However, the neuroprotective potency of noscapine has not been analyzed. Our study showed that administration of noscapine decreased the upregulation of pro-inflammatory factors, oxidative stress, and α-synuclein expression with a significant increase in antioxidant enzymes. In addition, noscapine prevented rotenone-induced activation of microglia and astrocytes. These neuroprotective mechanisms resulted in a decrease in dopaminergic neuron loss in SNpc and neuronal fibers in the striatum. Further, noscapine administration enhanced the mTOR-mediated p70S6K pathway as well as inhibited apoptosis. In addition to these mechanisms, noscapine prevented a rotenone-mediated increase in lysosomal degradation, resulting in a decrease in α-synuclein aggregation. However, further studies are needed to further develop noscapine as a potential therapeutic candidate for PD treatment.

Keywords: Parkinson’s disease; autophagy; inflammation; noscapine; oxidative stress; rotenone.

Figure 1

Noscapine prevented rotenone-induced oxidative stress and enhanced antioxidant enzymes in rats. Administration of rotenone increased (p < 0.05) lipid peroxidation, which is represented by increase in malonaldehyde levels with significant reduction in vital antioxidant levels in the midbrain (n = 5). However, treatment with noscapine prevented lipid peroxidation with a significant decrease in MDA levels and reverted brains’ vital antioxidant (CAT, GSH and SOD) levels. Data are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group.

Figure 2

Noscapine inhibited MMP-9 production and modulated pro-inflammatory factors in experimental animals. Rotenone administration caused a significant increase in expression of pro-inflammatory cytokines (A) and enhanced production of MMP-9 (B). Western blotting analysis of midbrain protein samples probed with Cox-2 and iNOS (C). Quantitative analysis of western blots was performed using Image J and corresponding results were represented as bar diagram (D). However, noscapine treatment decreased expression and production of pro-inflammatory factors in rotenone-treated animals. Data are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group (n = 5).

Figure 3

Noscapine prevented activation of microglia and astroglia. Enhanced expression of ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) is an indicator of reactive microgliosis and reactive astrocytes, respectively. Our results (n = 5) showed that activation of microglia by rotenone is evident from larger cell bodies and fewer processes (A). Similarly, activation of astrocytes is evident from enhanced expression of GFAP positive cells (C). Conversely, administration of noscapine to rotenone-treated rats significantly decreased the expression of Iba-1 and GFAP in the striatum of experimental animals. These results were concordant with the decrease in production of pro-inflammatory factors in experimental animals. Quantification of activated microglia and astrocytes is represented as percentage of control and depicted as histogram in (B,D) respectively. Data are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group.

Figure 4

Noscapine protected dopaminergic neurons against rotenone toxicity. Parkinson’s disease is histologically characterized by significant loss of tyrosine hydrolase (TH)-positive dopaminergic neurons in the substantia nigra pars compacta (SNpc) with profound decrease in TH expression in the striatum. Therefore, we quantified TH-positive dopaminergic neurons in SNpc and expression of TH in the striatum (n = 5). Rotenone administration caused a significant decrease in number of TH positive neurons (A,B) which in turn caused significant decrease in the intensity of TH-positive striatal fibers (C,D). However, administration of noscapine prevented neuronal loss and increased TH expression in striatal fibers. Data are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group.

Figure 5

Noscapine modulates the expression of α-synuclein in rotenone-treated animals. Representative immunoblots of alpha-synuclein expression in experimental animals (n = 5) (A). Image J was used for quantitative analysis (B). Data are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group.

Figure 6

Noscapine diminished rotenone-induced neuronal apoptosis by restoring mTOR pathway. Western blotting of midbrain samples (n = 5) with mTOR pathway (A) proteins and apoptotic markers (C). Immunoblots of phosphor mTOR and p70S6K (B), Bax and Bcl-2 (D) were quantified using Image J and represented as fold of control. Results are expressed as mean ± SEM. ^a p < 0.05 compared to control, ^b p < 0.05 compared to rotenone-treated group.

Figure 7

Noscapine prevented dopaminergic degeneration through regulating autophagy. Immunoblotting of midbrain samples (n = 5) with p62 (A). Quantitative analysis of immunoblotting of p62 (B). Values are represented as mean ± SEM. ^a p < 0.05 compared to control.