|Year : 2018 | Volume
| Issue : 53 | Page : 9-16
Demethoxycurcumin, a natural derivative of curcumin abrogates rotenone-induced dopamine depletion and motor deficits by its antioxidative and anti-inflammatory properties in Parkinsonian rats
Muthu Ramkumar1, Srinivasagam Rajasankar2, Veerapan Venkatesh Gobi1, Udaiyappan Janakiraman3, Thamilarasan Manivasagam3, Arokiasamy Justin Thenmozhi3, Musthafa Mohamed Essa4, Ranganathan Chidambaram5, Saravana Babu Chidambaram6, Giles J Guillemin7
1 Department of Anatomy, Bharath University, Selaiyur, Chennai, India
2 Department of Anatomy, Velammal Medical College and Hospital, Madurai, India
3 Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Tamil Nadu, India
4 Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat; Ageing and Dementia Research Group, Sultan Qaboos University, Muscat, Oman; Food and Brain Research Foundation, Chennai, Tamil Nadu, India
5 Department of Radiology, Sri Lakshminarayana Institute of Medical Sciences, Puducherry, India
6 Department of Pharmacology, JSS College of Pharmacy, JSS University, SS Nagar, Mysore, Karnataka, India
7 Neuroinflammation Group, Department of Biomedical Research, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia
|Date of Submission||20-Mar-2017|
|Date of Acceptance||27-Apr-2017|
|Date of Web Publication||20-Feb-2018|
Department of Anatomy, Velammal Medical College and Hospital, Madurai - 625 009, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder (NDD) associated with the loss of dopaminergic neurons in the substantia nigra and subsequently has an effect on motor function and coordination. The pathology of PD is multifactorial, in which neuroinflammation and oxidative damage are the two of the main protagonists. Objectives: The present study aims to assess the potential antioxidant and anti-inflammatory effects of demethoxycurcumin (DMC), a natural derivative of curcumin, against rotenone-induced PD in rats. Materials and Methods: Rats were randomized and divided into six groups: control, rotenone (0.5 mg/kg/day, intraperitoneal in sunflower oil) treated for 7 days, rotenone and DMC (5, 10, and 20 mg/kg b.w) cotreated, and DMC (20 mg/kg b.w) alone treated groups. Results: Based on the dopamine concentration and biochemical estimations, the effective dose of DMC was selected and the chronic study was performed. At the end of the experimental period, behavioral studies and protein expression patterns of inflammatory markers were analyzed. Rotenone treatment led to motor dysfunctions, neurochemical deficits, and oxidative stress and enhanced expressions of inflammatory markers, whereas oral administration of DMC attenuated all the above. Conclusion: Even though further research is needed to prove its efficacy in clinical trial, the results of our study showed that DMC may offer a promising and new therapeutic lead for the treatment of NDDs including PD.
Abbreviations used: COX-2: Cyclooxygenase-2; DA: Dopamine; DMC: Demethoxycurcumin; DMRT: Duncan's multiple range test; GSH: Reduced glutathione; GPx: Glutathione peroxidase; IL-1 β: Interleukin-1 β; IL-6: Interleukin-6; iNOS: Inducible nitric oxide synthase; PD: Parkinson's disease; SN: Substantia nigra; SOD: Superoxide dismutase; TBARS: Thiobarbituric acid reactive substances; TNF-α: Tumor necrosis factor-α.
Keywords: Demethoxycurcumin, dopamine, neurodegenerative disease, neuroinflammation, oxidative stress
|How to cite this article:|
Ramkumar M, Rajasankar S, Gobi VV, Janakiraman U, Manivasagam T, Thenmozhi AJ, Essa MM, Chidambaram R, Chidambaram SB, Guillemin GJ. Demethoxycurcumin, a natural derivative of curcumin abrogates rotenone-induced dopamine depletion and motor deficits by its antioxidative and anti-inflammatory properties in Parkinsonian rats. Phcog Mag 2018;14:9-16
|How to cite this URL:|
Ramkumar M, Rajasankar S, Gobi VV, Janakiraman U, Manivasagam T, Thenmozhi AJ, Essa MM, Chidambaram R, Chidambaram SB, Guillemin GJ. Demethoxycurcumin, a natural derivative of curcumin abrogates rotenone-induced dopamine depletion and motor deficits by its antioxidative and anti-inflammatory properties in Parkinsonian rats. Phcog Mag [serial online] 2018 [cited 2019 Nov 22];14:9-16. Available from: http://www.phcog.com/text.asp?2018/14/53/9/225655
- Curcumin and their derivatives have been shown to be potent neuroprotective effect
- Demethoxycurcumin (DMC) amolerated the rotenone induced behavioural alterations
- DMC abrogated the rotenone induced dopamine deficits
- DMC attenuated the rotenone induced oxidative stress
- DMC diminished the rotenone mediated inflammation.
| Introduction|| |
The disabling symptoms of Parkinson's disease (PD) are related to the movement abnormalities such as tetrad of tremor at rest, rigidity, akinesia (difficulty in initiating movement), bradykinesia (slowness of voluntary movements), gait, and posture abnormalities. These symptoms are mainly due to the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the consequent degeneration of their projecting nerve fibers in the striatum (ST), which leads to shortage in striatal dopamine (DA) content. Although the exact cause of PD remains unclear, enhanced oxidative stress and mitochondrial dysfunction are reported to be associated with the pathological processes underlying PD. Other prominent neuropathological features of PD include gliosis  and the presence of intraneuronal proteinaceous inclusions called Lewy bodies (LBs) that further induce the loss of neurons in the substantia nigra (SN).
Using the animal models, the reliability and internal validity mimicking human PD can be achieved. The striatal DA deficiency associated with motor symptoms of PD is evaluated through the use of an animal model as this is not possible in a cellular model. Two types of animal models (neurotoxin and transgenic) resembling the familial and sporadic forms of PD have been used., Toxin models represent the classic experimental PD models, which are able to replicate most of the phenotypic and/or pathological features of PD. The neurotoxin agents have been used to induce PD in a broad range of organisms from small single cell yeast to large, nonhuman primates including 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), rotenone, paraquat, and maneb.
Rotenone is considered as one of the best neurotoxins for induction of in vitro and in vivo models of PD., Previous studies indicated that the administration of rotenone to animals induces biochemical, anatomical, and behavioral symptoms similar to those observed in PD patients., It is a potent inhibitor of mitochondrial electron transport chain complex-I  that leads to production of free radicals resulting in oxidative damage. It also has the ability to activate both the microglial and astroglial cells and trigger the neuroinflammatory responses. Activated microglia might cause dopaminergic cell death by releasing cytotoxic inflammatory cytokines, such as interleukin-1 β, 6 (IL-1 β, 6) and tumor necrosis factor-α (TNF-α). TNF-α damages dopaminergic neurons by activating intracellular death signaling pathways (necrosis factor kappa B [NF-κB], JNK, and p38 pathways) and inducing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions, which further amplify inflammatory cascades.
Even today, L-DOPA therapy remains the most effective treatment for PD. Substitution treatment of PD by L-DOPA initially reduces motor symptoms. However, long-term administration typically leads to motor complications, including involuntary choreoathetoid movements and motor fluctuations, collectively known as L-DOPA-induced dyskinesia (LIDS), due to excessive response of DA. Therefore, the long-term L-DOPA treatment worsens the neurodegeneration and produces LIDS. The current pharmacological therapies for the PD are inadequate. These are only able to provide symptomatic relief and after long use produce stern side effects and worsen the condition. An alternative therapeutic approach would be the use of natural plant extracts or their associated phytochemicals that are reported to have access to the brain and offer neuroprotection by virtue of their radical scavenging, iron chelating, anti-inflammatory, and anti-apoptotic activities.,
Curcuma longa (turmeric) has a long history of use in Ayurvedic medicine. The pharmacological properties of turmeric may be due to the presence of curcumin, the active polyphenol which offers anti- Parkinsonism More Details effect attributed to its antioxidant, mitochondrial protective, signal modulating, anti-inflammatory,, and antiapoptotic properties. Commercially available preparations of curcumin contain at least three curcumin compounds including curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). The ratio of curcumin: DMC: BDMC in commercially available preparations is about 66:23:11. DMC and BDMC are also used in the food industry, for domestic cooking and folk medicines.
Curcumin analogs such as DMC and BDMC have also been reported to possess considerable antioxidant, anti-inflammatory, and antiproliferative activities.,, DMC is reported to have better anticancer and anti-inflammatory activity compared with curcumin., Few researchers have indicated that the methoxy groups on the phenyl rings in curcumin have important health effects. The curcuminoids, curcumin, DMC, and BDMC strongly inhibited βA-fibril formation in Alzheimer's disease. Based on the above literature, the present study focused on the neuroprotective role of DMC against rotenone-induced motor deficits, neurochemical alteration, and oxidative stress and the expression of inflammatory markers in rats.
| Materials and Methods|| |
Animals and drug treatment
Male Wistar rats (225–250 g) were procured from the Biogen Laboratory, Bangalore, India. They were kept under the ambient conditions and fed with standard pellet and water ad libitum. All the experimental protocols met with the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000) and were approved by the Animal Ethics Committee (IAEC/KMPC/230/2015–2016).
About 36 rats were randomized and divided into the following groups (n = 6): control rats (0.5 ml of sunflower oil intraperitoneal [i.p.] for 3 weeks), rotenone (2.5 mg/kg/day, i.p in sunflower oil for 7 days) alone treated, rotenone (as group II) and low dose of DMC (5 mg/kg/b.w. was administered orally after 1 h after rotenone treatment) treated, rotenone and middle dose of DMC (10 mg/kg b.w.) treated, rotenone and high dose of DMC (20 mg/kg b.w.) treated, and DMC (20 mg/kg/day) alone treated. After the end of the experimental period, a behavior test (narrow beam walking) was carried out and the ST and SN were procured for the estimation of DA and oxidative stress-related indices, respectively.
Based on the DA level, we considered 10 mg/kg of DMC treatment as the effective dose and used it for further experiments. Twenty-four animals were randomized and divided into four groups (n = 6): control group (0.5 ml of sunflower oil i.p. for 45 days), rotenone-treated group (2.5 mg/kg/day i.p. in sunflower oil for 45 days) (as group II), DMC (10 mg/kg b.w. p.o. for 45 days) + rotenone (2.5 mg/kg/day i.p. in sunflower oil for 45 days) treated group, and DMC (10 mg/kg) alone treated. After the end of the experimental period, behavior tests (Rotarod and Hang test) were carried out. Then, the animals were sacrificed by cervical dislocation and the SN was dissected, rinsed in ice-cold saline, and stored at −80°C for protein expression studies of inflammatory markers.
Narrow beam walking
Rats were pretrained to cross an elevated and narrow beam to reach an enclosed escape platform in the beam-walking test. After the experimental period, the animals were allowed to transverse the narrow, flat wooden beam (L100 cm × W1 cm), which was placed at a height of 100 cm from the floor. The time taken to cross the starting area to the finishing area was measured.
The rotarod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration an animal stays on the drum in relation to the drum speed. Rats were allowed to adjust their posture in order to maintain their balance on a rotating rod at speeds of 5, 10, 15 and 20 rpm. The average retention time on the rod was calculated.
Rats were placed on a horizontal grid and were supported until they grabbed the grid with their fore and hind paws. Then, the grid was inverted so that the rats were allowed to hang upside down. The falling time was measured.
High-performance liquid chromatographic analysis of dopamine
Striatal DA levels were determined by the high-performance liquid chromatography (HPLC) with an electrochemical detector by the method of Muralikrishnan and Mohanakumar. Briefly, the procured ST region was immediately sonicated in 0.1 M perchloric acid (ice-cold condition) containing 0.01% ethylenediaminetetraacetic acid (EDTA). Samples were centrifuged (10,000 g for 10 min) and the supernatant was collected and injected (10 μl) into the HPLC system using a rheodyne 7725i injector valve with a 20 μl injection loop. The mobile phase consisting of 7% acetonitrile, 3% methanol, and 90% 20 mM citric acid, 10 mM monobasic phosphate sodium, 3.25 mM octane sulfonic acid, 0.1 mM EDTA, 3 mM heptane sulfonic acid, 2 mM KCL, 6 mL/L o-phosphoric acid, and 2 mL/L diethylamine (pH 3) was allowed to flow at a rate of 0.3 mL/min with a Gold 118 system (Beckman, Fullerton, CA, USA). Results were expressed in ng/mg weight of brain tissue.
Estimation of the thiobarbituric acid reactive substances
Briefly, the tissue was homogenized in chilled 0.1 M phosphate buffer. The assay mixture contained 0.67% thiobarbituric acid, 10% chilled trichloroacetic acid, and homogenate (10%) in a total volume of 3 ml. The reaction mixture was centrifuged at 4000 rpm for 15 min, and the supernatant was boiled for 10 min. After cooling, the samples were read at 535 nm. The rate of lipid peroxidation was expressed as l mol of thiobarbituric acid reactive substances (TBARSs) formed/mg protein.
Estimation of the levels of reduced glutathione
Reduced glutathione (GSH) levels were determined by the method of Jollow et al. Postmitochondrial supernatant (PMS) (1.0 ml) was precipitated with 1.0 ml of sulfosalicylic acid (4.0%). The samples were kept at 4°C for 1 h and then subjected to centrifugation at 1200 g for 15 min at 4°C. The assay mixture consists of 0.5 ml of aliquot, 2.3 ml of sodium phosphate buffer (0.1 M, pH 7.4), and 0.2 ml of DTNB. The yellow color thus developed was read immediately at 412 nm on a spectrophotometer.
Determination of glutathione peroxidase activity
To 0.1 ml of PMS, 1.44 ml of sodium phosphate buffer (0.1 M, pH 7.4), 0.1 ml of EDTA (1 mM), 0.1 ml of sodium azide (1 mM), 0.05 ml of glutathione reductase (1 IU/ml), 0.1 ml GSH (1 mM), 0.1 ml NADPH (0.02 mM), and 0.01 ml of hydrogen peroxide (H2O2) (0.25 mM) were added. Glutathione peroxidase (GPx) activity was calculated as nmol NADPH oxidized min/mg protein at 340 nm.
Determination of catalase activity
To 0.05 ml of PMS, 1.95 ml of phosphate buffer (0.1 M, pH 7.4) and 1 ml of H2O2 (0.09 M) were added. Catalase activity was calculated in terms of nmol H2O2 consumed min/mg protein at 240 nm.
Determination of superoxide dismutase activity
Superoxide dismutase (SOD) activity was assayed using an indirect inhibition assay, in which xanthine and xanthine oxidase serve as a superoxide generator and nitro blue tetrazolium (NBT) is used as a superoxide indicator. To 20 μl of PMS, 960 μl of 50 mM sodium carbonate buffer (pH 10.2) containing 0.1 mM xanthine, 0.025 mM NBT, and 0.1 mM EDTA and 20 μl of xanthine oxidase were added. Changes in absorbance were observed spectrophotometrically at 560 nm. The activity was expressed as units/min/mg protein.
Nigral tissues were homogenized in an ice-cold RIPA buffer and the homogenate was centrifuged (12,000 rpm/min for 15 min at 4°C) to remove debris. Protein level was measured by the method of Lowry et al. 50 μg of total cellular protein samples was loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, the gel was transferred on to a nitrocellulose membrane (PALL Corporation, Biotrace). The membranes were incubated with the blocking buffer consisting of 5% nonfat dry milk powder for 2 h to reduce nonspecific binding sites and then the blots were probed with various antibodies: IL-1 β, IL-6, NF-κB, TNF-α, iNOS, COX-2, brain-derived neurotrophic factor (BDNF), and β-actin (1:2000) with gentle shaking overnight at 4°C. After this, membranes were incubated with their corresponding secondary antibodies (anti-rabbit IgG conjugated to horseradish peroxidase) for 2 h at room temperature. The membrane was washed thrice with TBST for 30 min. Immunoreactive protein was visualized by the chemiluminescence protocol (GenScript ECL kit, USA), scanned bands were quantitated by TotalLab Quant 1.11 software, and the control was set to 100.
Statistical analysis was executed by one-way analysis of variance followed by Duncan's multiple range test using Statistical Package for the Social Science (SPSS) software package version 15.0 (GraphPad Software, Inc., USA). All data are expressed as mean ± standard error of mean for six rats from each group. Results were considered statistically significant at P < 0.05.
| Results and Discussion|| |
Behavioral indices and dopamine levels
Rats treated with rotenone displayed a significantly increased beam-crossing duration and declined rotarod performance and neuromuscucular strength than the control group [Figure 1], [Figure 2], [Figure 3]. Previous studies indicated the impaired motor functions such as diminished walking balance, vestibular integrity, and muscular coordination in narrow beam walking test, lack of coordination during walking, bradykinesia, and rigidity in the rotarod test  and lowered neuromuscular strength in the hang test  in various PD models. Administration of rotenone at low doses causes DA neurodegeneration in the nigro-striatal pathway. Striatal DA depletion is the pathological hallmark of PD. The ST harbors a population of dopaminergic neurons that is thought to act as a local source of DA and this neurotransmitter is responsible for balance, control of movements, and walking. [Figure 4] reveals a significant decline in the levels of striatal DA in the rotenone alone treated rats as compared with control rats, whereas administration of DMC dose dependently enhanced the levels of dopamine in rotenone-induced PD rats. However, it was observed that both the 10 and 20 mg/kg b.w showed similar induction but more significant than 5 mg/kg b.w. Hence, we have chosen the 10 mg/kg b.w of DMC as the optimum dose for further studies. The decreased levels of DA cause the neurons of the ST to fire uncontrollably, preventing the PD patient from directing motor function. In the present study, rotenone administration caused a significant reduction in levels of striatal DA, whereas cotreatment of DMC ameliorated the rotenone-induced dopamine depletion. Drugs that are able to ameliorate DA depletion are considered to be neuroprotective. In this context, the cotreatment of DMC improved the behavioral status in the animals intoxicated with rotenone, which may be due to its neuroprotective effect on enhancing the depleted dopamine levels.
|Figure 1: Measurement of balance and muscular coordination by narrow beam walking test. Rotenone-treated rats took more time to cross the narrow beam, whereas administration of demethoxycurcumin significantly attenuated motor deficts induced by rotenone. Results given are mean ± standard error of mean (n = 6), values not sharing common superscript are significant with each other P < 0.05, one-way analysis of variance followed by Duncan's multiple range test|
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|Figure 2: Rotarod performance of control and experimental rats: Rotenone-induced rats illustrate significantly reduced rotarod performance compared to the control rats (different rpm such as 5, 10, 15, and 20). Treatment of demethoxycurcumin to rotenone-induced rats significantly improved the retention time in rotarod as compared to the demethoxycurcumin-untreated rotenone rats. Values are given as mean ± standard error of mean for six rats in each group. *P < 0.05 compared to the control, #P < 0.05 compared to the rotenone control|
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|Figure 3: Determination of neuromuscular strength by hang test. Hanging time of rotenone induced rats were drastically decreased, meanwhile treatment with demethoxycurcumin significantly increased hanging time of rotenone induced rats. Results given are mean ± standard error of mean (n = 6), values not sharing common superscript are significant with each other P < 0.05, one-way analysis of variance followed by Duncan's multiple range test|
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|Figure 4: Rotenone-induced depletion of dopamine can be conserved by demethoxycurcumin treatment. Administration of rotenone depleted the levels of dopamine, which is attenuated dose-dependently by treatment of demethoxycurcumin. Results given are mean ± standard error of mean (n = 6), values not sharing common superscript are significant with each other P < 0.05, one-way analysis of variance followed by Duncan's multiple range test|
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Levels and activities of lipid peroxidative products and antioxidants
Oxidative stress is the main trigger of neurodegeneration in PD. The levels of TBARS and GSH and the activities of SOD, catalase, and GPx were assayed in the SN of control and experimental animals to examine the antioxidative role of DMC. The levels of TBARS were significantly elevated, whereas the levels of GSH and activities of SOD, catalase, and GPx were significantly diminished in the rats treated with rotenone. Treatment of DMC significantly and dose dependently attenuated rotenone-induced oxidative stress by diminishing the levels of TBARS and elevating the levels and activities of antioxidants. On its own, DMC (20 mg/kg) treatment did not show any significant change in the levels and activities of oxidative and antioxidative indices as compared to control rats [Figure 5]. Compared to other tissues, the brain is more vulnerable to oxidative stress because of (i) high metabolic activity and utility of more oxygen, (ii) auto-oxidation of DA, its metabolites, and precursors to form semiquinones and quinones able of adducting sulfhydryl groups of protein, including GSH, and (iii) presence of enhanced iron levels in the SN leading to the reduction of H2O2 to form the highly reactive hydroxyl radical (OH) (Fenton chemistry).,, Postmortem analysis of the brains from PD patients confirmed the enhanced level of oxidative stress in the SN,, diminished level of GSH,, and enhanced lipid peroxidation., The extent of lipid peroxidation processes was measured by quantifying the level of TBARS, collective products of lipid peroxidation. Bashkatova et al. showed increased NO and TBARS level in the ST and cortex, following the chronic rotenone treatment, which verifies our study. Increased production of reactive oxygen species (ROS) during neurodegenerative disorders is an indication of the oxidative stress and leads to a rapid consumption of endogenous scavenging antioxidants. Rotenone-administered PD rats enhanced the level of TBARS, a marker of lipid peroxidation processes, and reduced the levels and activities of enzymatic antioxidants which are consistent with the previous studies.,, Previous studies demonstrated decreased GSH level, SOD activity, and oxidative DNA  and enhanced levels of MDA  in rotenone models of PD.
|Figure 5: Rotenone induced oxidative stress can be attenuated by demethoxycurcumin. The levels of thiobarbituric acid reactive substances (a), and activities of superoxide dismutase (b) and catalase (c) were increased along with decreased levels of reduced levels of reduced glutathione (d) and activities of GPx (e) in the substania nigra of rotenone treated rats--oxidative stress|
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However, cotreatment of DMC-reduced, rotenone-induced TBARS levels ameliorates lipid peroxidation. The methoxy group present on the phenyl ring is one of the functional groups responsible for the antioxidant property of curcumin. As DMC has one methoxy group, it is also considered as a potent antioxidant. A fault in individual or more endogenous antioxidant molecules, particularly GSH, is an important factor in etiology of PD. Dairam et al. demonstrated that the administration of DMC increased the levels of GSH in lead-treated animals, which is consistent with our work. In the present study, diminished activity of SOD in the rotenone-treated rats indicated activation of SOD by ROS  and leads to the scenario of increased superoxide radical production. Enhanced superoxide radicals spontaneously dismutated to form H2O2. Elevated levels of H2O2 could lead to depletion of their metabolizing enzymatic antioxidants such as catalase and GPx. DMC has antioxidant effects and improved the activities of SOD, catalase, and GPx against the 6-OHDA-induced rat model of PD, middle cerebral artery occlusion-induced experimental stroke, and other in vitro studies.,
Protein expressions of interleukin-1 β, interleukin-6, necrosis factor kappa B, tumor necrosis factor-α, inducible nitric oxide synthase, cyclooxygenase-2, and brain-derived neurotrophic factor
Western blot analysis was performed in the SN regions of experimental rats. Rotenone treatment markedly increased the expressions in IL-1 β, IL-6, NF-κB, TNF-α, iNOS, and COX-2 and reduced the expression of BDNF. Oral administration of DMC significantly attenuated the rotenone-induced inflammation. No significant changes were observed between control and DMC alone treated groups [Figure 6]. Inflammation is one among the several assumed factors that play an important role in PD pathogenesis. Inflammatory mediators modulated the immune and neuronal cells, thereby contributing to the cascade of events leading to neuronal degeneration. These mechanisms comprise microglial activation, astrogliosis, and lymphocytic infiltration. Activation of microglia is related with dopaminergic neuronal loss and α-synuclein that have been reported to play an important role in the initiation and progression of inflammation in PD. Microglia in PD have been observed to grow densely in the ST and SN with increased expression of pro-inflammatory mediators, including TNF-α, IL-1 β, IL-2, IL-4, IL-6, COX-2, and iNOS. In addition, levels of inflammatory cytokines such as TNF-α, IL-6, and IL-10 are elevated in different brain regions of patients with PD.
|Figure 6: Rotenone persuaded inflammation can be amolierated by demethoxycurcumin administration. Expressions of interleukin-1β, interleukin-6, tumor necrosis factor-α, Necrosis Factor kappa B, inducible nitric oxide synthase, cyclooxygenase-2, and brain-derived neurotrophic factor in the substantia nigra were analyzed by Western blot (a), lane 1: Control, saline control, lane 2: Rotenone, Rotenone-treated only, lane 3: Rotenone - demethoxycurcumin, Rotenone with demethoxycurcumin, lane 4: Demethoxycurcumin, demethoxycurcumin alone administrated. The band density was quantified by scanning densitometry (b and c). Results given are mean ± standard error of mean, (n = 6), values not sharing common superscript are significant with each other P < 0.05, one-way analysis of variance followed by Duncan's multiple range test|
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The pro-inflammatory cytokines TNF-α and IL-1 can also trigger direct toxicity in neurons , and can potentiate an ongoing inflammatory response by enhancing microglial NO production., Upregulation of inflammatory mediators involved in apoptotic cell death through TNF-α-induced signaling pathway, including caspase-1, caspase-3, and TNF receptor R1 (TNF-R1 or p55), were identified in the SN from parkinsonian patients, indicating the occurrence of a proapoptotic environment in PD. Curcuminoids attenuated H2O2-enhanced production of pro-inflammatory molecules such as IL-6, TNF-α, monocyte chemoattractant protein-1, and NF-κB.
Recently, Zhang et al. reported that DMC exhibited a stronger inhibitory activity on nitric oxide and TNF-α production compared to curcumin in lipopolysaccharide-activated rat primary microglia. NF-κB may be activated by oxidative stress, which in turn regulates the expressions of many genes including pro-inflammatory cytokines, particularly, TNF-α, IL-1 β, iNOS, IL-6, and IL-10. Pro-inflammatory cytokines degraded the inhibitor kappa B by phosphorylation and activated the NF-κB signaling cascade. Activated NF-κB promotes the induction of iNOS from activated microglia. NO derived from iNOS-expressing microglia partially accounted for the loss of dopaminergic neurons in PD patients.
Enhanced expressions of inflammatory indices including NF-κB and iNOS were demonstrated in rotenone-injected rats, and on the other hand, treatment with DMC not only diminished expression of iNOS and NF-κB significantly but also diminished IL-6, IL-1 β, and TNF-α expression. This indicated that microglial activation has been alleviated by DMC administration. Activated microglia upregulated the levels of COX-2, a key enzyme responsible for the synthesis of inflammation-related prostaglandins. The prostaglandins can be directly toxic to neurons through activation of caspase 3 or indirectly through the release of glutamate by astrocytes leading to excitotoxicity. Subsequently, the expression of its products such as prostaglandin E2, which is cytotoxic, is also increased during times of microglial activation. COX-2 expression is normally limited to the forebrain; however, under pathological conditions involving inflammation, COX-2 expression is significantly increased throughout the brain. It is unsurprising, therefore, that increased COX-2 expression has been found to be localized to the SNpc in the postmortem brains of PD patients. Moreover, the inhibition of COX-2 has been shown to attenuate the toxic effects of MPTP in mice, further suggesting that microglial-mediated COX-2 expression is harmful to DA neurons of the SNpc.
Our study found significant enhancement in COX-2 expression after rotenone administration, which is concordant with previous studies. Inhibition of COX-2 activity improves behavioral impairment and shields dopaminergic neuronal loss against various PD inducing neurotoxins., Astrocytes have diverse and critical functions in the central nervous system that includes providing energetic, antioxidant, and other trophic support essential for the survival and function of neurons. Several reports have suggested that the activation of astrocytes is due to secretion of inflammatory cytokines, such as TNF-α and IFN-γ, by the surrounding microglial cells.,
The astrocytic reaction is another well-known neuropathological characteristic of the SN in PD. Damier et al. showed that astrocytes are heterogeneously distributed within the mesencephalon in healthy individuals and their density of astrocytes is low in the SNpc, which is severely affected in PD. One of the earlier studies reporting neuroinflammation in PD involved a quantitative confirmation of the astroglial reaction using glial fibrillary acidic protein (GFAP) immune-staining in the SN of patients with PD. Reactive astrocytes have been demonstrated in regions of dopaminergic nerve cell loss in PD. Guo et al. reported that DMC and BDMC inhibited NO production and COX-2 and iNOS expression and suppressed LPS-induced IB-phosphorylation and degradation in a dose-dependent manner. Astrogliosis induced by cytokines such as IL-1 β and IL-6 could accelerate GFAP release, which favored neuroinflammatory response and neuronal loss. In contrast, it is observed that treatment of DMC significantly diminished GFAP expression, which indicates that astrogliosis activation has been mitigated.
| Conclusion|| |
Our study demonstrated that DMC treatment could be able to provide protection against the neurotoxic effects of rotenone in rats and the mechanisms of protection induced by this agent possibly due to its anti-inflammatory and antioxidant activities.
Red ferm communication for language assistance and Prof Guillemin is supported by the National Health and Medical Research Council, the Australian Research Council and Macquarie University. Dr. Mohamed Essa is supported by an internal grant from Sultan Qaboos University (IG/AGR/FOOD/17/02).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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