|Year : 2017 | Volume
| Issue : 2 | Page : 121-126
African mistletoe (Loranthaceae) ameliorates cholesterol-induced motor deficit and oxidative stress in adult BALB/c mice
Ademola A Oremosu1, Edem Ekpenyong Edem2, Olufunke O Dosumu1, AA Osuntoki3
1 Department of Anatomy, College of Medicine, University of Lagos, Lagos, Nigeria
2 Department of Anatomy, College of Medicine, University of Lagos, Lagos; Department of Anatomy, College of Medicine and Health Sciences, Afe Babalola University, Ado Ekiti, Nigeria
3 Department of Biochemistry, College of Medicine, University of Lagos, Lagos, Nigeria
|Date of Web Publication||4-Jun-2018|
Dr. Edem Ekpenyong Edem
Department of Anatomy, Afe Babalola University, Ado Ekiti, Ekiti
Source of Support: None, Conflict of Interest: None
INTRODUCTION: Disturbances in cholesterol homeostasis can influence neuronal cell membranes and induce oxidative stress, which can impact motor function. African mistletoe (Loranthaceae) is a hemiparasitic plant which has been employed in the treatment and management of several ailments including strokes and epilepsies.
AIM: This study was undertaken to determine the ameliorative effect of African mistletoe in cholesterol-induced motor deficit in BALB/c mice.
MATERIALS AND METHODS: Twenty mice were used for this study. They were divided into four groups, namely, control, experimental, extract, and treatment. The motor deficit was established by feeding the mice with a diet enriched with 2% cholesterol for 8 weeks. Mice were subsequently treated with a mistletoe methanolic extract preparation through oral administration (200 mg/kg daily for 2 weeks) or with normal saline (0.5 ml) as a control. Data were expressed as a mean ± standard error of the mean; P < 0.05 and P < 0.01 (level of significance).
RESULTS: The high cholesterol diet (HCD) induced a statistically significant motor deficit when compared to the other groups. After 8 weeks of HCD feeding, histological results showed remarkable structural disruptions in the corpus striatum and the cerebellar cortex of the BALB/c mice. Administration of 200 mg/kg of methanolic extract of mistletoe ameliorated histomorphological distortion produced by the chronic exposure to an HCD.
CONCLUSION: The study findings have revealed that chronic exposure to a HCD can impact the motor neural systems and their functions, and treatment with methanolic extract of African mistletoe improves motor function in BALB/c mice.
Keywords: African mistletoe, cholesterol, motor deficit, oxidative stress
|How to cite this article:|
Oremosu AA, Edem EE, Dosumu OO, Osuntoki A A. African mistletoe (Loranthaceae) ameliorates cholesterol-induced motor deficit and oxidative stress in adult BALB/c mice. J Exp Clin Anat 2017;16:121-6
|How to cite this URL:|
Oremosu AA, Edem EE, Dosumu OO, Osuntoki A A. African mistletoe (Loranthaceae) ameliorates cholesterol-induced motor deficit and oxidative stress in adult BALB/c mice. J Exp Clin Anat [serial online] 2017 [cited 2019 Jan 16];16:121-6. Available from: http://www.jecajournal.org/text.asp?2017/16/2/121/233675
| Introduction|| |
Mistletoes are hemiparasitic plants belonging to the families Loranthaceae (Troncoso et al., 2010). They have been recognized world over for the treatment of a myriad of medical conditions ranging from cancers to diabetes. In Africa, mistletoes are predominantly employed in the treatment of hypertension, malaria, epilepsy, and mistletoe infusions are used for stroke management (Chaulya et al., 2011). They have been reported to be effective for treating metabolic disorders (Ojewole and Adewole, 2007). It is immunomodulating (Onay-Ucar and Karagoz, 2006); hence, it being used as cancer adjuvant therapy. Recently, it has been shown to be neuroprotective and to enhance the brain-derived neurotrophic factor (BDNF) in a mouse model (Edem et al. 2016; Oremosu et al., 2016).
There are compelling reports and substantial evidence of an undeniable link between disturbed cholesterol homeostasis in the brain and its implication in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (Vance, 2012). Human studies have revealed a strong connection between elevated plasma and serum cholesterol levels and incidence of Parkinson's disease (Hu et al., 2006; Hu et al. 2008). In Parkinson's disease, a major motor disorder, hypercholesterolemia has been identified to disrupt the antioxidant system and mitochondrial functions in the brain, leading to loss of dopaminergic neurons in the substantia nigra (Paul et al., 2017). Impairments in the antioxidant balance trigger oxidative stress caused by the overproduction of reactive oxygen species. This pathophysiological phenomenon is implicated in the molecular and cellular mechanism of tissue damage in different human diseases (Valko, 2007). Several findings have shown the occurrence of pathologies in cellular neuronal models caused by chronic exposure to cholesterol or oxysterols which are equivalent to Parkinson's disease (Marwarha et al. 2011; Bar-On et al. 2008; Rantham et al., 2008). Thus, we sought to investigate whether a high cholesterol diet (HCD) feeding would deteriorate motor function and impact neurohistomorphology in the light of oxidative stress in BALB/c mice; and if methanolic extract of African mistletoe can ameliorate these effects.
| Materials and Methods|| |
Twenty male BALB/c mice (20–25 g) were procured from the Department of Pharmacology, College of Medicine of the University of Lagos, Nigeria. They were housed (5 per cage) in plastic cages (57 cm × 44 cm × 28 cm) bedded with wood shavings changed every 24 h and had free access to drinking water and food. The animals were maintained in compliance with the principles and guidelines of the Health Research Ethics Committee of the College of Medicine of the University of Lagos, Nigeria.
Preparation of experimental feed
The experimental feed was a high-cholesterol diet comprising 2% cholesterol and 5% peanut oil mixed with the powdered standard pellet diet (Kalaivanisailaja et al., 2003; Woodruff-Pak, 2008).
Plant material and extraction
Fresh, healthy leaves of African mistletoe (Loranthaceae) were collected in late June 2016 from orange trees in an orchard in Ikole Local Government Area of Ekiti State, Nigeria. They were shade dried and ground with the help of an electric blender to get a free-flowing powder. The powdered sample (1.5 kg) was defatted with n-hexane and extracted with methanol in a Soxhlet extractor, overnight. The crude methanolic extract was concentrated and evaporated to dryness with a rotary extractor under reduced pressure. The yield of the crude extract was 6.8%. It was set aside for dosing.
The animals were divided into four groups. Water was given ad libitum. Animals in groups 1 and 3 (control and extract groups) received standard pellet diet and animals in groups 2 and 4 (Experimental and Extract Groups) received a high-cholesterol diet during the initial period of 8 weeks. At the end of the initial period of 8 weeks, the animals were treated as follows for the next 15 days. Group 1 – Control mice continued on standard pellet diet. Group 2– HCD mice continued on the experimental diet comprising 2% cholesterol and 5% peanut oil mixed with the powdered standard pellet diet, for 15 days. Group 3 – Extract mice continued on standard pellet diet and given African mistletoe methanolic extract (200 mg/kg) (Oremosu et al., 2016), orally every alternate day for 15 days. Group 4 – Animals continued on HCD and given African mistletoe methanolic extract (200 mg/kg) orally every alternate day for 15 days. At the end of the total experimental period of 45 days after an overnight fast, the animals were anesthetized using ketamine in a desiccator and sacrificed by cervical dislocation. Biochemical analyses were performed to assay the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) antioxidant enzymes, and total protein level. Animals for histological studies were transcardially perfused with normal saline and paraformaldehyde, after which the brains were extracted and preserved in paraformaldehyde for further histological analyses. Brain tissue for total protein determination and oxidative stress markers were preserved in phosphate buffer solution and stored at 4°C while awaiting biochemical analyses.
After treatment, the animals were made to undergo motor function tests-the Rotarod and Parallel Bar tests.
This was done to assess motor performance in the mice after the experiments. The test involved three trials of 3 min each (T1, T2, and T3) separated by an inter-trial time of 60 min. The time spent on the Rotarod in T1, T2, and T3 were determined and averaged to determine the latency of fall (LOF) for each group (Ishola et al., 2015).
This test was conducted to measure the explorative activity of the animals. This was done using two raised 1 m long (1 mm) parallel bars (3 cm apart) mounted on the 60 cm high wooden frame. Each mouse was placed at the 0.5 m mark (center of the raised bars) and allowed to roam freely on the bar. The duration taken for the mice to make a 90° turn was recorded as the latency of turn (LOT) for a 3 min' trial (Ishola et al., 2015).
Oxidative stress markers
Biochemical analyses were performed to assay the activities of CAT, SOD, and GSH antioxidant enzymes, and total protein level.
Determination of total protein
Protein concentrations were determined using the Lowry's method as described by Lowry et al., (1951) bovine serum albumin (BSA) was used as standard. This method is based on the reaction between aromatic amino acids residues of the protein in the sample and phosphomolybdic-phosphotungstic acid present in the Folin–Ciocalteu reagent. The amount of protein in the sample is estimated by measuring absorbance at 750 nm. The standard curve of BSA is prepared.
Determination of catalase activity
CAT activity was determined according to the method of Sinha (1971) which measures the initial rate of H2O2 decomposition. This method is based on the reduction of dichromate in acetic acid to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The chromic acetate produced is measured colorimetrically at 570–610 nm and used as an index for estimating H2O2 decomposition and CAT activity.
Determination of superoxide dismutase activity
The SOD activity was determined using the method of Misra and Fridowich (1972). The SOD assay is determined based on the ability of SOD to inhibit the autooxidation of epinephrine at pH 10.2 with an increase in absorbance at 480 nm. The reaction serves as a rationale in assaying for SOD.
Determination of reduced glutathione concentration
The reduced GSH level was determined using Ellman's reagent, 5,5'-dithio-bis-2-nitrobenzoic acid as described by Sedlak and Lindsay (1968) and Jollow et al., (1974). This method is based on the development of a relatively stable yellow complex formed as a result of reaction between Ellman's reagent and free sulfhydryl groups. The chromophoric product, 2–nitro–5– thiobenzoic acid, resulting from the reaction of Ellman's reagent with GSH possesses a molar absorption at 412 nm. The absorbance of this complex at 412 nm is proportional to the level of GSH in the sample.
After postfixation, tissue processing began. Serial sections of the brain were made. They were stained with hematoxylin and eosin dyes using the method of Baker and Silverton (1985).
With the aid of GraphPad Prism V.5.0, California, United States of America; SPSS 20, IBM, Armonk, NY, United States of America applications, data were analyzed using one-way ANOVA followed by Tukey's multiple range test; P < 0.05 and P < 0.01 as the level of significance.
| Results|| |
Motor function tests
HCD feeding induced Parkinson's-like behavior, which was observable in the reduction of LOF when the HCD group was compared with the control. Posttreatment with 200 mg/kg of methanolic extract of African mistletoe improved motor function in the treatment group as the mice recorded an increase in LOF when compared to the control and the HCD groups. Although there was an increase in LOF in the extract group, it was not statistically significant when compared to the control (P < 0.05) [Figure 1]a. The number of times a mouse held on to the rotating bar and rotated together with the bar is termed passive rotation. In [Figure 1]b, the passive rotation count was statistically lower in the group exposed to an HCD, when compared to the control and treatment groups.
|Figure 1: (a) Rotarod test and latency of fall assessment. A significant difference (*P < 0.05) was obtained when the experimental (HCD) group recorded a reduced latency of fall compared to the control group. The mice in the experimental group spent lesser time on the rotarod wheel thereby having a latency of fall. Treatment with 200 mg/kg of African mistletoe produced a statistically significant increase in the latency of fall of mice in the treatment group as the animals were recorded to have spent more time on the wheel. (b) Passive rotation of the animals on the Rotarod test. Passive rotation decreased significantly in the experimental group compared to the control and treatment groups (*P < 0.05)|
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Parallel bar test
In this test, a significant increase or decrease in LOT scores was considered as abnormal motor coordination when the treatment groups were compared against the control. HCD feeding increased the LOT significantly versus the control (P < 0.05) as the mice showed a characteristic decline in motor coordination on the raised bars. In addition, there was a significant increase in the LOT values of the experimental group as against the extract group. After an intervention with 200 mg/kg of methanolic extract of African mistletoe, a decline in LOT was observed in the Treatment group when compared to the experimental group (P < 0.05). Similar to our observations in Rotarod test, induced Parkinson's-like behavior with a significant increase in LOT when compared to the control; and a reduction in LOT following treatment with 200 mg/kg of methanolic extract of African mistletoe (P < 0.05) [Figure 2]a. Time taken for animals to edge was significantly higher in the group of animals exposed to an HCD feeding versus the other groups (P < 0.05) [Figure 2]b.
|Figure 2: (a) Latency of turning of the animals on parallel bar test. Mice exposed to a high cholesterol diet feeding had the highest latency of turning compared to control, extract and treatment groups (P > 0.05). (b) Evaluation of the time taken by the animals to reach the edge of the parallel bar (time to edge). The experimental group mice spent more time to get to the edge of the bar compared to animals in the other groups (P < 0.05)|
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Determination of total protein levels
Total protein level after HCD feeding in BALB/c mice was significantly lower compared to the control mice [Table 1]a.
Oxidative stress markers
Oxidative stress markers in the brain of the control mice, mice fed HCD (experimental group) for 8 weeks, mice administered 200 mg/kg of methanolic extract of African mistletoe (extract group) for 2 weeks, and mice treated with 200 mg/kg of methanolic extract of African mistletoe for 2 weeks after 8 weeks of HCD (treatment group) are shown in [Table 1]b. The antioxidant enzyme activities of CAT, SOD, and reduced GSH level in brains of the experimental group mice were significantly (P < 0.01) reduced compared to the control mice.
At the end of the experiment, the mice were sacrificed by cervical dislocation. After perfusion, brain tissue for histological studies was collected and fixed in 10% formal saline. The brain tissue was passed through a process of tissue processing. The routine stains used were hematoxylin and eosin (H and E). Results are as shown in [Figure 3] and [Figure 4].
|Figure 3: Photomicrographs of the striatum. H and E staining observed at x100 magnification. Control group (a) reveal a normal striatal histomorphology; Experimental group mice (b) which were exposed to a high cholesterol diet for 8 weeks show different stages of reactive gliosis, pyknosis and neurodegeneration. Mice that received 200 mg/kg of methanolic extract of African mistletoe in the extract group (c) no significant histological changes. Treatment group (d) mice that were posttreated with 200 mg/kg of methanolic extract of African mistletoe for 2 weeks after 8 weeks of feeding on a high cholesterol diet, reveal mild structural recovery|
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|Figure 4: Photomicrographs of the Cerebellar Cortex. H and E staining observed at x100 magnification. Control group (a) reveal a normal cerebellar cortex histoarchitecture of molecular layer, Purkinje cell layer and granular layer; Experimental group mice (b) which were exposed to a high cholesterol diet for 8 weeks show different stages of reactive gliosis, pyknosis, and remarkable degeneration of the Purkinje cells. There are also vascular degeneration and reduction of the granule cells in the granular layer. Mice that received 200 mg/kg of methanolic extract of African mistletoe in the Extract group (c) shows an intact histomorphology. Treatment group (d) mice that were posttreated with 200 mg/kg of methanolic extract of African mistletoe for 2 weeks after 8 weeks of feeding on a high cholesterol diet, reveal significant structural recovery compared with the eontrol and experimental groups|
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| Discussion|| |
Plant-based therapies have over the years proven to be effective against a variety of health conditions. They have been employed as potent, noninvasive and inexpensive alternatives (Akhtar et al., 2011). There is a strong connection between the etiology of motor disorders like PD and environmental factors such as diet lifestyle (Tanner et al., 1996). Clinical studies have suggested that high total cholesterol at baseline is associated with an increased risk of PD (Hu et al., 2008). It would be important to find mechanisms behind the association between a high cholesterol level and PD risk (Hu, 2010). Thus, the present study investigated the effect of a HCD on motor function. Behavioral results after 8 weeks of a HCD showed a decline in motor coordination as seen in the reduction in the LoF [Rotarod; [Figure 1]a and an increase in the LoT [Parallel Bar; [Figure 1]b. The recorded motor deficit could be attributed to changes in extracellular calcium hyperpolarization in the corpus striatum after chronic exposure to a HCD. The anatomical integrity of the corpus striatum and cerebellar were significantly altered after 8 weeks of HCD feeding. The histoarchitectural disruptions are attributable to oxidative stress. Neuronal cholesterol accumulation induces oxidative stress (Gezginci-Oktayoglu et al., 2009), which aggravates neurodegeneration; thus, the varying degrees of neurodegeneration revealed in the corpus striatum and cerebellar cortex of mice exposed to a HCD for 8 weeks. In our oxidative stress marker findings, there was a statistically significant decrease in the level of antioxidant enzymes– SOD, GSH, and CAT. There was equally a decrease in the total protein level in the brains of mice fed a HCD for 8 weeks. These findings agree with Yang et al.(2008).
African mistletoe has been reported to possess antioxidant properties (Turkkan et al., 2016). It has also been shown to be neuroprotective (Oremosu et al., 2016), and to enhance the BDNF (Edem et al., 2016). In the present study, 200 mg/kg of methanolic extract of African mistletoe significantly increased the levels of both total protein and antioxidant enzymes (SOD, GSH, and CAT) in the brains of the mice after 8 weeks of HCD feeding. This corresponds with the findings of Turkkan et al.(2016). Methanolic extract of African mistletoe from our behavioral studies was shown to have significantly improved motor function after 8 weeks of HCD-induced motor function in mice.
| Conclusion|| |
Our novel study has revealed that chronic exposure of HCD induces oxidative stress and motor deficit in mice. Treatment with methanolic extract of African mistletoe improves motor function and ameliorates the harsh effects of oxidative stress on brain tissue by regulating neuronal cholesterol accumulation and antioxidant enzymes homeostasis in the BALB/c mice brains.
Financial support and sponsorship
This work was supported by a grant (CRC 2016/13) from the Central Research Committee of the University of Lagos, Nigeria.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]