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. 2022 Nov 3;8(11):e11434.
doi: 10.1016/j.heliyon.2022.e11434. eCollection 2022 Nov.

Effect of citronellol on oxidative stress, neuroinflammation and autophagy pathways in an in vivo model of Parkinson's disease

Richard L Jayaraj  1 , Sheikh Azimullah  1 , Khatija A Parekh  1 , Shreesh K Ojha  1 , Rami Beiram  1
Affiliations

Effect of citronellol on oxidative stress, neuroinflammation and autophagy pathways in an in vivo model of Parkinson's disease

Richard L Jayaraj et al. Heliyon. .

Abstract

Citronellol, a monoterpene found in the essential oils of Cymbopogo plants has been reported to possess various biological properties. In the present study, we investigated the neuroprotective mechanisms of citronellol against rotenone induced neurodegeneration by using rat model of Parkinson's disease (PD). Our results demonstrated that oral administration of citronellol prevented rotenone induced reactive oxygen species production, lipid peroxidation and enhanced Nrf2 expression, catalase, glutathione peroxidase and superoxide dismutase levels in the brain. Enzyme-linked immunosorbent assays showed that citronellol reduced secretion of TNF-α, IL-1β, IL-6 and decreased MMP-9 expression levels. Further, citronellol prevented rotenone induced microglia (Iba-1 staining) and astrocyte (GFAP staining) activation. Western blot analysis showed that citronellol significantly decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase-2 that are key markers of neuroinflammation. We further evaluated the effect of citronellol on dopaminergic neurons in substantia nigra pars compacta (SNpc) and striatum (ST) which are key anatomical structures in PD. Tyrosine hydroxylase (TH) immunoreactivity showed that citronellol preserved Tyrosine hydroxylase (TH) positive dopaminergic neurons and enhanced TH striatal expression levels significantly compared to rotenone alone group. Further, to understand the effect of citronellol on apoptosis and proteotoxicity, we evaluated apoptotic markers (Bax, Bcl-2), growth regulator (mTOR) and α-synuclein expression. Citronellol attenuated rotenone induced expression of pro-apoptotic protein Bax, reduced α-synuclein expression and enhanced Bcl-2 and mTOR levels. In addition, citronellol modulated autophagy pathway by decreasing LC-3 (Microtubule-associated proteins) and p62 levels. Taken together, our results demonstrate that citronellol protected dopaminergic neurons through its antioxidant, anti-inflammatory, anti-apoptotic and autophagy modulating properties.

Keywords: Apoptosis; Citronellol; Dopaminergic neurons; Neuroprotection; Rotenone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Chemical structure of Rotenone.
Figure 2
Figure 2
Chemical structure of citronellol.
Figure 3
Figure 3
Citronellol prevented rotenone induced oxidative stress. Effect of citronellol on Nrf2 expression and antioxidant enzymes have been measured in midbrain of experimental animals. Antioxidant activities in experimental samples (midbrain) were measured using commercially available kits. Rotenone administration (2.5 mg/kg, i.p) significantly increased lipid peroxidation and decreased Nrf2 expression (a) and antioxidant levels (b) in the midbrain. However, oral administration of citronellol (25 mg/kg) reduced lipid peroxidation levels and significantly reverted antioxidant (CAT, GSH and SOD) levels. Quantitative evaluations of blots were done using ImageJ and their results were represented in histogram (c). Data are represented as mean ± SD. ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group.
Figure 4
Figure 4
Citronellol prevented production of pro-inflammatory factors in experimental animals. ELISA and western blotting were performed to analyze the effect of citronellol on pro-inflammatory markers in the midbrain of experimental animals. Administration of rotenone (2.5 mg/kg, i. p) for four weeks significantly enhanced secretion of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and MMP-9. (a). Further, rotenone enhanced the expression of COX-2 and iNOS in midbrain of experimental animals (b). Quantitative evaluations of blots were done using ImageJ and their results were represented in histogram (c). However, administration of citronellol (25 mg/kg, oral) for four weeks significantly decreased the expression of pro-inflammatory factors. Data are represented as mean ± SD. ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group.
Figure 5
Figure 5
Citronellol reduced microglia and astrocytes activation. Immunofluorescent staining for microglia and astrocyte was performed to understand the anti-inflammatory property of citronellol. Over expression of Iba-1 and GFAP is an indicator of activation of microglia and astrocyte respectively. Our studies showed that rotenone administration (2.5 mg/kg, i.p) activated microglia as evident from larger cell bodies and higher fluorescence intensity (a). Similarly, higher expression of GFAP represents activation of astrocytes (c). However, citronellol administration (25 mg/kg, oral) decreased the activation of microglia and astrocytes. ImageJ was used to quantify the activation of microglia and astrocytes. Results are represented as percentage of control in the corresponding histograms (b and d). Data are expressed as mean ± SD. ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group.
Figure 6
Figure 6
Citronellol diminished rotenone induced dopaminergic neuronal loss. Post-mortem studies clearly demonstrated loss of tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNpc with significant decrease in the expression of tyrosine hydroxylase in the striatum. Cryoprotected animal brains were serially sectioned (20 μm) using a cryostat and tyrosine hydroxylase immunohistochemistry was performed. Our results demonstrated significant decrease in number of TH positive dopaminergic neurons (a, b) with concomitant decrease in the intensity of TH + ve striatal fibers (c, d) in rotenone intoxicated animals. However, citronellol administration reduced dopaminergic neuronal loss and preserved the expression of tyrosine hydroxylase in striatal fibers. Data are expressed as mean ± SD. ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group.
Figure 7
Figure 7
Citronellol prevents α-synuclein over-expression in rotenone animals. Immunoblots representing alpha-synuclein expression in midbrain of experimental animals (a). Blots were quantified using ImageJ and the results are represented as histogram (b). Results are represented as mean ± SD. ∗p < 0.001 compared to control, #p < 0.001 compared to rotenone treated group.
Figure 8
Figure 8
Citronellol prevented apoptosis and enhanced mTOR expression. Western blot analysis was done to examine the expression of Bax, Bcl-2 and mTOR in the midbrain of experimental samples (a). Results were quantified using ImageJ and represented as fold of control (b). Results are expressed as mean ± SD. mTOR: ∗p = 0.037 compared to control, Bcl-2: ∗p = 0.001 compared to control, Bax: ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group.
Figure 9
Figure 9
Citronellol protected dopaminergic neurons by restoring autophagy pathway. Midbrain levels of autophagy markers LC3 and p62 were evaluated by western blotting (a). Quantitative evaluation of LC3 and p62 levels were done using ImageJ (b). Values are represented as mean ± SD. ∗p < 0.05 compared to control, #p < 0.05 compared to rotenone alone treated group, $p = 0.05 compared to control.
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