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. 2023 Mar 9;24(6):5222.
doi: 10.3390/ijms24065222.

Limonene, a Monoterpene, Mitigates Rotenone-Induced Dopaminergic Neurodegeneration by Modulating Neuroinflammation, Hippo Signaling and Apoptosis in Rats

Lujain Bader Eddin  1 , Sheikh Azimullah  1 , Niraj Kumar Jha  2 , Mohamed Fizur Nagoor Meeran  1 , Rami Beiram  1 , Shreesh Ojha  1   3
Affiliations

Limonene, a Monoterpene, Mitigates Rotenone-Induced Dopaminergic Neurodegeneration by Modulating Neuroinflammation, Hippo Signaling and Apoptosis in Rats

Lujain Bader Eddin et al. Int J Mol Sci. .

Abstract

Rotenone (ROT) is a naturally derived pesticide and a well-known environmental neurotoxin associated with induction of Parkinson's disease (PD). Limonene (LMN), a naturally occurring monoterpene, is found ubiquitously in citrus fruits and peels. There is enormous interest in finding novel therapeutic agents that can cure or halt the progressive degeneration in PD; therefore, the main aim of this study is to investigate the potential neuroprotective effects of LMN employing a rodent model of PD measuring parameters of oxidative stress, neuro-inflammation, and apoptosis to elucidate the underlying mechanisms. PD in experimental rats was induced by intraperitoneal injection of ROT (2.5 mg/kg) five days a week for a total of 28 days. The rats were treated with LMN (50 mg/kg, orally) along with intraperitoneal injection of ROT (2.5 mg/kg) for the same duration as in ROT-administered rats. ROT injections induced a significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and DA striatal fibers following activation of glial cells (astrocytes and microglia). ROT treatment enhanced oxidative stress, altered NF-κB/MAPK signaling and motor dysfunction, and enhanced the levels/expressions of inflammatory mediators and proinflammatory cytokines in the brain. There was a concomitant mitochondrial dysfunction followed by the activation of the Hippo signaling and intrinsic pathway of apoptosis as well as altered mTOR signaling in the brain of ROT-injected rats. Oral treatment with LMN corrected the majority of the biochemical, pathological, and molecular parameters altered following ROT injections. Our study findings demonstrate the efficacy of LMN in providing protection against ROT-induced neurodegeneration.

Keywords: Parkinson’s disease; dopaminergic neurons; limonene; monoterpenes; neurodegeneration; neuroinflammation; rotenone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rotarod test performance of rats. The values are presented as mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 2
Figure 2
TH-ir neurons and TH-ir fibers in the substantia nigra (SN) and striatum are presented, respectively (a,b) (scale bar is 20 µm). (c) Quantification of the TH-ir neurons in SNc and the density of TH-ir fibers is also shown. Each group contains three rats, and the data are represented as percent mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 3
Figure 3
Representative images of Western blots and densitometric analysis for BDNF and α-synuclein expressions in the striatal tissues (a,b). Immunoblotting was performed in duplicates and the data are expressed as the mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 4
Figure 4
Effect of limonene on the levels of MDA, SOD, catalase (CAT), and GSH in the midbrain of rats (a–d). The values are presented as mean ± SEM (n = 6–8); * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 5
Figure 5
Effect of limonene on the concentration of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the midbrain of rats (a–c). The values are presented as mean ± SEM (n = 6–7); * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 6
Figure 6
The activation of GFAP and Iba-1 in striatum was explored by immunofluorescence staining of striatum. A remarkable expression of activated astrocytes (GFAP-positive) (a), and microglia (Iba-1-positive) (b), was indicated in the fluorescent images taken from ROT-injected rats when compared to the control rats. (c), The quantitative analysis of the number of activated astrocytes and microglia is shown. Each group consists of three rats and the values are presented as mean ± SEM; * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 7
Figure 7
Immunoblotting analysis and quantification of iNOS, COX-2, and p-NF-κB in the striatal tissues of rats (a,b). Immunoblotting was performed in duplicate and the results are presented as mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 8
Figure 8
Effect of limonene on the protein expressions of P38, JNK, m-TOR, and p-m-TOR densitometric analysis in the striatal tissues (a,b). Immunoblotting was performed in duplicate and the results are presented as mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 9
Figure 9
Western immunoblotting and densitometric analysis on the protein expression of mitochondrial complex-I in the striatal tissues of rats (a,b). Immunoblotting was performed in duplicate and the values are presented as mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 10
Figure 10
Immunoblotting and densitometric analysis of Bcl2. Antiapoptotic protein and pro-apoptotic proteins, including Bax, cytochrome-C, cleaved caspase-3, cleaved caspase-9, CHOP, p-MST were evaluated in the striatum (a,b). Immunoblotting was performed in duplicate and the data are presented as mean ± SEM. * p < 0.05 CON vs. ROT; # p < 0.05 ROT vs. LMN + ROT (one-way ANOVA followed by DMRT). CON: normal control, ROT: rotenone, LMN: limonene, LMN + ROT: limonene and rotenone treatment.
Figure 11
Figure 11
Summarized mechanisms of neuroprotective effect of limonene in experimental model of Parkinson’s disease.
See this image and copyright information in PMC

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