Objectives and design: Aluminium (Al) is an environmental neurotoxin that affects cerebral functions and causes health complications. The antioxidant and memory enhancing effects of Centella Asiatica (CA) are well known in the past few decades. Therefore, the present study has been designed to investigate the therapeutic potential of CA against chronic Aluminium Chloride (AlCl3) exposure induced rats. Wistar albino rats were segregated into four groups: group 1-control rats, group 2-rats received AlCl3 (300 mg/kg body weight, every day orally) for 60 days, rats in group 3-received CA (500 mg/kg body weight, orally) and group 4 rats were initiated with both AlCl3 and CA treatment.
Material and methods: Neurotoxicity was assessed by measuring brain biogenic amines, Al concentration in blood and brain, the levels of pro-inflammatory cytokines and later the apoptotic related components. Significant depletion of neither brain Dopamine (DA), Epinephrine (E), Nor Epinephrine (NE) nor 5-Hydroxytryptamine (5-HT) levels was observed following AlCl3 exposure. Administration of AlCl3 raised the pro-inflammatory cytokines levels (tumor necrosis factor alpha, TNF-α and inducible nitric oxide synthase, iNOS) and modulated the levels of apoptotic proteins cytochrome c, caspases 3, with altered Bax/Bcl-2 ratio in favour of apoptosis in cortex that are determined by western blotting and immunohistochemistry.
Results: Administration of CA significantly increased biogenic amine levels, decreased Al concentration compared to Al-induced animals. CA ameliorates AlCl3-induced inflammatory niche. Further, treatment with CA ameliorated neuronal apoptosis by reducing cytochrome c, caspase- 3 expressions significantly and by modulating Bax/Bcl-2 ratio.
Conclusion: The findings of this study suggest that CA protects the cerebral cortex against AlCl3-induced neurotoxicity. This neuroprotective effect is partially mediated by its anti-oxidant, anti-inflammatory and anti-apoptotic activities as well as elevating biogenic amine levels.
Aluminium chloride; Apoptosis; Centella asiatica; iNOS; TNF-?
Aluminum (Al) is a potent environmental neurotoxin and has been associated with several disorders. Al and its salts are widely exposed to human body due to their presence in several products including cosmetics, drugs, food additives (salt, spices, yellow cheese and herbs) and a powerful flocculants in water purification. Al has the potential to be toxic for humans and its compounds have neurotoxic manifestations which further shown to contribute several age-related neurodegenerative diseases . Chronic exposure to Al causes pathogenesis of age-related illnesses such as the Alzheimer’s Disease (AD), the Parkinson’s disease, the Huntington’s disease, inflammation, Amyloid Beta (Aβ) deposition and plaque formation in the brain and suggested that this excess might be a possible contributing factor for the aging process by inflicting oxidative damage . Evidence suggests that inhaled Al not only causes neurologic signs, which mimic progressive neurodegeneration, but also results in neurofilamentous changes in the cerebral cortex focal region where more Al accumulation takes place [3,4]. Previous studies have shown that, the excessive concentration of Al in neurotic deposits, plaques and neurofibrillary tangles in the brain of Alzheimer’s patients .
Al is a well-documentedan experimental neurotoxicant that enhances neuroinflammatory events in the brain by multiple mechanisms. Accumulating evidence suggests that Aluminium Chloride (AlCl3) promote free radical generation by potentiating the pro-oxidant properties of transition metals (iron and copper) . In addition, AlCl3 exerts its own pro-oxidant effect by altering calcium flux and homeostasis and to facilitate peroxidation of membrane lipids and antioxidant enzymes . Beside this, various Al salt complexes can alter neuronal signal transduction pathways associated with glial cell activation, which further promote an uncontrolled inflammatory cascade in the brain. These activated glial cells secrete cytotoxic agents including Reactive Oxygen Species (ROS), Nitric Oxide (NO), inflammatory cytokines Like Tumor Necrosis Factor alpha (TNF-α), inducible Nitric Oxide Synthase (iNOS) which are toxic and inturn cause synaptic abnormalities ultimately related to memory dysfunction. Earlier study has evidenced that AlCl3 activates astrocytes and glial cells which releases different inflammatory mediators due to induction of apoptosis [8,9]. Therefore, overall effects are to reduce pro-inflammatory mediator’s production by activated microglia and astrocytes should be useful for prevention of neuroinflammation and eventually neuroprotection.
Apoptosis under mitochondrial control has been implicated in a progressive and selective loss of neurons, which involve the release of cytochrome c into the cytoplasm and initiation of the apoptosis cascade. AlCl3 is a potent cholinotoxin and causes aberrant apoptotic neuronal loss by stimulating ROS production, which is a characteristic symptom of neurodegeneration [10-12]. It induces apoptosis of neurons and astrocytes via involvement of caspases activation in rodent and in vitro models [13,14]. Reactivation of caspases has been implicated in mediating neural death during pathological processes causing neuronal loss in acute and chronic neurodegenerative diseases. Caspase-induced apoptosis has been documented in Amyloid-β-induced Alzheimer's and 6-hydroxydopamine-induced Parkinson's neuronal apoptosis [15,16]. Restriction of apoptosis and its associated factors during neurodegeneration is presumed to be beneficial for the survival of neurons. Thus clinical trials are in progress that anti caspase approach in neurodegeneration will be an exciting avenue for therapeutic neuroprotective interventions .
The antioxidant capabilities of Centella Asiatica (CA) have been documented previously in different experimental models. Centella Asiatica (CA), also known as gotu kola and Indian pennywort, is a perennial herbaceous creeper of the Apiaceae family. Recently, CA has been reported as a neuroprotectant against different degenerative disorders. CA modulate the immune system , act as antioxidant , prevent alleviation of oxidative stress , act as anti-inflammatory agent , and inhibit proliferation of cancer cells . Yet, the most widely reported health benefit of this herb is in improving the brain function, particularly related to learning and memory. In fact, CA has been reported to stimulate nerve regeneration in vitro , which could explain its brain protective effect. In humans, it has been reported to improve mental ability of the mentally retarded children and decrease the amyloid-β levels in the brain [24,25]. Previously, we have demonstrated that CA inhibited cognitive impairment and improves learning and memory in Al-induced rats by reducing oxidative stress and Acetyl Cholinesterase (Ach E) activity . Based on this background, the present study was carried out to investigate the protective effect of CA against AlCl3 induced inflammation and oxidative stress associated mitochondria apoptosis. Therefore in this study, we have investigated the efficacy of CA in terms of biogenic amines, expression of pro-inflammatory cytokines TNF-α, iNOS and apoptotic inducing factors in cortex of rat brain.
Figure 1: Effect of CA supplementation on Al concentration in serum and brain tissue of control and experimental rats subjected to AlCl3. Results were expressed as mean ± SD (n=6). Values are statistically significant at p<0.05; compared with aAlCl3 vs. control; bAlCl3+ CA vs. AlCl3.
Figure 2: Graph represents the effect of CA supplementation on the levels of brain monoamine neurotransmitters in the rats of control and experimental groups. Dopamine (DA, ng/g), epinephrine (E, ng/100g), norepinephrine (NE, ng/100g) and 5-hydroxytryptamine (5-HT, ng/g). Results were expressed as mean ± SD (n=6). Values are statistically significant at p<0.05; compared with aAlCl3 vs. control; bAlCl3+ CA vs. AlCl3.
Figure 3: Representative micrographs of immune stained specimens of cortex from control and experimental groups of rats. Brain specimens were probed with antibody specific for TNFα and iNOS. a) TNF-α expression in cortex of control group; b) TNF-α expression in cortex of AlCl3-induced group; c) CA alone (drug control) group; d) TNF-α expression in cortex of AlCl3-induced and CA treated group; e) iNOS expression in cortex of control group; f) iNOS expression in cortex of AlCl3-induced group; g)CA alone (drug control) group; h) iNOS expression in cortex of AlCl3-induced and CA treated group; Scale bar 100 ?m, magnification ×20. (i) Immunoreactivity was assessed and measured objectively by two independent observers. For quantitation, data expressing the respective protein expression were quantitated in ten fields/section and an average was used to denote the no. of positively stained cells and is represented in graph. Arrows indicate the positively stained cells. Hypothesis testing method included two-way Analysis of Variance (ANOVA) followed by Least Significant Difference (LSD). Results are given as statistically significant at p<0.05; compared with aAlCl3 vs. control; bAlCl3+ CA vs. AlCl3.
Figure 4: Representative immunoblots of TNF-α and iNOS protein expressions in the cortex of control and experimental groups of rats. Western blot analyses were performed using probes specific for TNF-α, iNOS, and β-actin as indicated. Lane 1 Control; Lane 2 AlCl3-induced; Lane 3 CA alone; Lane 4 AlCl3-induced and CA treated. Data expressing the corresponding protein levels were assessed using densitometry and is expressed in relative intensity (arbitrary units). Hypothesis testing method included two-way Analysis of Variance (ANOVA) followed by Least Significant Difference (LSD). Results are given as statistically significant at p<0.05; compared with aAlCl3 vs. control; bAlCl3+ CA vs. AlCl3.
Figure 5: (A) Representative micrographs of immune stained sections of cytochrome c expression in cortex from control and experimental groups of rats. Brain specimens were probed with antibodies specific for cytochrome c (Magnification ×40). Immunoreactivity was assessed and measured objectively by two independent observers. B) For quantitation, data expressing total number of positively stained cells quantification were performed by counting the positively stained cells in 10 fields/section by two independent observers in blinded fashion, and the average was used to denote the total number of positively stained cells. Hypothesis testing method included two-way Analysis of Variance (ANOVA) followed by Least Significant Difference (LSD). Results are given as statistically significant at p<0.05; compared with aAlCl3 vs. control; bAlCl3+ CA vs. AlCl3.
Al is a ubiquitous metal and implicated in the pathophysiology of neurodegenerative diseases. Reports suggest that AlCl3 alters cytoskeletal dynamics of neurons and astrocytes leading to pathogenesis of neurodegeneration. In mammals, Al accumulation in the brain has been linked to neurodegenerative diseases such as PD and AD [4, 35]. Understanding the etiology of neurodegeneration and identifying ways to achieve early prevention are being considered as important approaches in the management of neurological diseases. Counteracting attempts to prevent/inhibit neurodegeneration and enhance neuronal survival has been greatly summoned. Recently, the traditional medicinal plant CA and its extracts have gained much interest for their ability to modulate neuronal function and influence learning and memory processes [36,37]. Hence, the present study was an attempt to understand the reduction of neuroinflammatory signals and apoptotic mechanisms underlying neuroprotective efficacy of CA in a rat model of neurodegeneration.
Brain is considered a preferential site of Al accumulation mainly at some cortical regions and hippocampus via crossing the Blood-Brain Barrier (BBB) . The obtained data demonstrated that the serum Al level was elevated in Al exposed rats, indicated an occurrence of serum hyper-aluminemia which resulted in an elevation of brain tissue Al content. This elevation appears to be a consequence of an alteration in the permeability of the BBB, it has been shown that Al somehow interacts with the BBB via interfering with its function at the endothelial cell level, whereas positively charged Al ions neutralize the anionic sites of the endothelial cell surface and also may consequently altering the function of the BBB . Concomitant treatment with CA was associated with a significant reduction in serum and brain Al content this depletion may be due to CA’s free radical scavenging potential . This may preserve cell membrane function including ion transport and membrane fluidity.
In the current study, the effect of chronic Al exposure on brain monoaminergic systems as stress response indicators was assayed. Primarily, the data presented in this work elicited a marked depletion in the brain monoaminergic system (5-HT, DA, E, NE and 5-HT) levels, which negatively correlated with the brain Al level. 5-HT is an excitatory neurotransmitter synthesized from tryptophan via the intermediate 5-hydroxytryptophane. The decreases in the brain 5-HT levels are associated with a decrease in the synthesis of 5-HT and loss of serotonergic neurons . The decline in the levels of DA, E and NE neurotransmitters may be linked to their closely related synthetic route. These neurotransmitters are derived from tyrosine, which is converted to DA by the enzymes tyrosine hydroxylase and DOPA decarboxylase. DA is converted through the action of dopamine-β-hydroxylase to NE . Similar decreases were previously reported by Fernandez-Davila et al., . In the current study CA supplementation might be useful in maintaining brain neurotransmitters levels to some extent.
Al intoxication leads to marked astrocytes and microglial activation that contribute to the pathogenesis of neuroinflammation and neurodegeneration . Chronic administration of AlCl3 causes a greater inflammatory response over immunoreactivity of astrocytes and microglia associated with cognitive dysfunction and disease . Reactive gliosis and the associated proinflammatory cytokines/chemokines could be result of inflammation and neuronal injury in the rat brain . Inhibition of pro-inflammatory mediators would be a better approach in regulating the progression of neurodegenerative disorders. The pro-inflammatory cytokines, TNF-α and iNOS, contribute to neuronal damage and death in vivo and in vitro . Activation of iNOS in glial cells is a key step in neuroinflammation and is often related with TNF-α release. A potent proinflammatory cytokine TNF-α is synthesized by microglia, astrocytes and neurons . TNF-α together with other transcription factors (NF-?B, nuclear factor kappa B) involved in cell responses including inflammation, proliferation, cell migration and apoptosis . NO, the enzymatic product of iNOS, is associated with neuronal death by inhibiting mitochondrial respiration and excitotoxicity . iNOS is normally not expressed in the brain, but is induced by pro-inflammatory cytokines and pathogenic components like endotoxins. To explore the mechanism of CA on the attenuation of AlCl3-induced neuroinflammation, the expression of TNF-α and iNOS was detected in the rat brain. Reduced expressions of TNF-α and iNOS were observed in the cortex of rats treated with CA, which demonstrates that CA exerts anti-inflammatory effect. Hence, we assume that CA regulates focal microglia activation and proinflammatory cytokine production in cortex and this antioxidant is an effective experimental tool to reduce the brain lesions associated with inflammatory responses . It also influences memory performance and, hence, CA can be utilized as a protective agent against neurodegenerative disorders .
Apoptosis is one of the mechanisms contributing to neuronal loss in AlCl3 induced neurodegeneration. Aβ oligomers are majorly contributed to neuronal cell death by inducing apoptotic proteins during AlCl3 administration. Bcl-2 is a member of apoptotic regulator protein that plays a major role in the regulation of cell death under both the physiological as well as pathological conditions. Bcl-2 over expression is perceived as a survival factor to protect against neurons during brain injury [12,13]. In the present study, induction of AlCl3 resulted in a decrease in the activity of Bcl2 where as increased the activity of baxin cerebral cortex region of rat brain. The ratio of Bax/Bcl2 levels in cortex was turned in favour of apoptosis in AlCl3-administered rats that denotes that AlCl3 induces neuronal apoptosis in cortex region. Results of the present study are supported by documented report denoting the involvement of Bax, Bcl2 in AlCl3-induced apoptosis in rabbit [13,50]. Further, we demonstrate that treatment with CA reduces Bax/Bcl2 ratio thereby antagonizing apoptotic niche in cortex of AlCl3-administered rat brain. Mobilization of proapoptotic Bcl2 family signaling protein, Bax from cytosol to mitochondria causes mitochondrial outer membrane permeabilisation leading to cytochrome c expulsion from mitochondrial inter-membrane space leading to Caspase activation . In this study, protein levels of cytochrome c and expressions of caspase-3 were significantly increased upon AlCl3-exposure in the cortex denotes that AlCl3-induces neuronal apoptosis. Results of present study are in accordance with documented reports denoting the involvement of caspase activation in many neurodegenerative disorders . Treatment with CA attenuates AlCl3-induced cytochrome c expression and also its downstream effectors caspases-3. This indicates that CA exerts neuroprotective effect by inhibiting neuronal apoptosis and is further supported that dietary flavonoids are capable of inhibiting intrinsic apoptosis of neurons .
In summary, the present investigation sheds light on the potent neuroprotective potential of CA in AlCl3-induced neurotoxicity. Our observations suggest that CA helps to maintain brain neurotransmitters levels and further regulates apoptotic neuronal cell death, focal microglia activation and inflammation in the cortex. Therefore, it is sensible to suggest from the previous and the present studies that CA may be used as therapeutic agents in the treatment of cognitive dysfunction-associated disorders such as Alzheimer’s disease. Future research may be directed towards the use of CA in clinical trials to evaluate its neuroprotective effect on humans.
Citation: Amjad S, Umesalma S (2018) Centella Asiatica Extracts Regulates Aluminium Chloride-Induced Neurotoxicity in Rats: Impact on Inflammation, Apoptosis and Biogenic Amine Levels. J Pharmacol Pharmaceut Pharmacovigil 2: 007.
Copyright: © 2018 Shaik Amjad, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.