Protein Kinase C Involvement in Neuroprotective Effects of Thymol and Carvacrol Against Toxicity Induced by Amyloid-ß in Rat Hippocampal Neurons.

Introduction: We have reported that thymol and carvacrol can improve cognitive abilities in Alzheimer Disease (AD) rat models. However, the mechanism of their action is not yet fully understood. Recently, our in vitro results suggested that PC12 cell death induced by Aß25-35 can be protected by thymol and carvacrol via Protein Kinase C (PKC) and Reactive Oxygen Species (ROS) pathways. So, we hypothesize that the mechanisms of thymol and carvacrol in improving the learning impairment in the AD rat model may be related to their effects on PKC. So, the activity of PKC and protein expression levels of PKCα were examined in the hippocampal cells of the AD rat model. Methods: To examine the thymol and carvacrol effects, we performed a behavioral test in AD rat models induced by Aß25-35 neurotoxicity. To access the underlying mechanism of the protective effects, western blotting was performed with antibodies against PKCα. We also measured the PKC activity assay by Elisa. Histopathological studies were carried out in the hippocampus with Hematoxylin and Eosin (H&E) staining. Results: The escape latency increased in Aß-received rats compared to the control group, and thymol and carvacrol reversed this deficit. Furthermore, these compounds could enhance the PKC activity and increase the PKCα expression ratio. Moreover, H&E staining showed that Aß caused shrinkage of the CA1 pyramidal neurons. However, thymol and carvacrol treatments could prevent this effect of Aß peptides. Conclusion: This study suggests that Amyloid-Beta (Aß) results in memory decline and histochemical disturbances in the hippocampus. Moreover, these results revealed that thymol and carvacrol could have protective effects on cognition in AD-like models via PKC activation. Highlights: Rat's ability to find the invisible platform in the Morris Water Maze (MWM) was impaired by Amyloid-Beta (Aß) infusion in the hippocampus, while this effect was reversed by thymol or carvacrol administration.Aß significantly downregulated the Protein Kinase C (PKC) activity in rats' hippocampus.Western blot analysis demonstrated that Aß significantly reduced PKCα protein expression in AD rat model hippocampal cells.The expression ratio of PKCα was upregulated following the injection of thymol and carvacrol in rats.Injection of Aß in the hippocampus resulted in histochemical disturbances in CA1 pyramidal neurons.Carvacrol and thymol can prevent several histological changes induced by Aß. Plain Language Summary: Alzheimer's disease is one of the most important brain diseases in which the learning and memory are impaired. One of the main causes of Alzheimer's disease is the presence of amyloid beta plaques in the neurons. Protein kinase C enzyme reduces amyloid production and accumulation in the brain. In the present study, we tested the possible effects of carvacrol and thymol in a rat model of Alzheimer's disease. Memory impairment was induced in adult rats by intra-cerebral infusion of amyloid ß. One week later, the memory-impaired animals were treated with carvacrol and thymol. Finally, we tested their memory in a Morris water maze apparatus. Furthermore, their hippocampus was dissected and PKC activity and the neuronal injury was evaluated. Our findings exhibited that thymol and carvacrol improved rats' memory performance. In addition, thymol and carvacrol significantly increased PKC activity and prevented neuronal cell loss in the rat hippocampus. This study shows that thymol and carvacrol have beneficial effects on memory and cognitive function via PKC activation.

Neuroprotective effects of carvacrol against Alzheimer's disease and other neurodegenerative diseases: A review.

Objective: Neurodegenerative diseases are considered an important cause of cognitive deficit and morbidity in old ages. Alzheimer's disease (AD) is one of these disorders affecting about 40 million people in the world at the present time. Available drug therapy is mostly symptomatic and does not modify or stop disease progression. Recently, biologically active chemicals from herbs have been studied to develop new therapeutic drugs. Carvacrol has shown positive properties on many neurological diseases. This compound is expected to have the ability to affect AD pathogenesis and therefore, it is considered an anti-AD agent. Materials and Methods: This review was conducted using PubMed, Google Scholar and Science Direct bibliographic databases until November 2021. For data collection, the following keywords were used: carvacrol, neuroprotective, cognition, anti-inflammatory, antioxidant, Acetylcolinesterase inhibitor (AChEI), Alzheimer's, Parkinson's, epilepsy, stroke, ischemic brain injury, and neurodegenerative diseases. Results: This review summarizes in vitro and in vivo studies on protective potential of carvacrol in neurodegenerative disorders and various underlying mechanisms, such as anti-inflammatory, antioxidant, and anticholinesterase effects. Conclusion: We gave an overview of available literature concerning neuroprotective effects of carvacrol in ameliorating the neurodegenerative diseases symptoms in vivo and in vitro. Particular attention is given to AD. Several neuro-pharmacological actions of carvacrol have been summarized in the current review article including anti-inflammatory, antioxidant, and AChEI properties.

Cognitive-enhancing activity of thymol and carvacrol in two rat models of dementia.

This study evaluated the efficacy of thymol and carvacrol against cognitive deficits induced by amyloid ß (Aß) or scopolamine. Rats received bilateral intrahippocampal injections of Aß(25-35) or intraperitoneal injections of scopolamine, and the effect of different doses of thymol, or carvacrol (0.5, 1, or 2 mg/kg) on cognitive function was determined. Animals were subjected to 5 days of training in the Morris water maze: 4 days with an invisible platform to test spatial learning and the 5th day with a visible platform to test motivation and sensorimotor coordination. The acute toxicities of thymol and carvacrol were also studied. The results showed increases in escape latency and decreases in target quadrant entries in Aß or scopolamine-treated groups. These impairments were reversed by pretraining administration of either thymol or carvacrol. The calculated LD50's of thymol (565.7 mg/kg) and carvacrol (471.2 mg/kg) were found to be much higher than their therapeutic doses (thymol 0.5 mg/kg, carvacrol 1 mg/kg). These findings provide preliminary positive evidence for the effectiveness and safety of thymol and carvacrol in alleviating cognitive impairments caused by increased Aß levels or cholinergic hypofunction. Anticholinesterase, antioxidant, and anti-inflammatory activities may be the mechanisms contributing toward their beneficial effects in these models.

Amelioration of amyloid ß-induced cognitive deficits by Zataria multiflora Boiss. essential oil in a rat model of Alzheimer's disease.

INTRODUCTION: The limitations of current Alzheimer's disease (AD) therapeutics have prompted investigation into innovative therapeutics focused on antiinflammatory, antioxidant, and neuroprotective agents including those from medicinal plants. Numerous plants have been tested for their potential for alleviating symptoms of AD. AIMS: Zataria multiflora Boiss. (ZM) a member of Lamiaceae family has been used in Iranian traditional medicine for its beneficial effects on mental abilities. Therefore, the effect of its essential oil was evaluated in a rat model of AD. METHODS: Amyloid ß-protein (Aß) fragment 25-35 was injected bilaterally in the CA1 region of rats hippocampus and the effect of different doses of ZM essential oil (50, 100, or 200 µL/kg) on cognitive function was investigated in the Morris water maze. Acute toxicity of the essential oil was also studied. RESULTS: The results showed increases in escape latency, traveled distance, heading angle, and decreases in target quadrant entries in Aß-received groups as compared to the control group. This impairment was reversed by ZM essential oil. The results of acute toxicity testing revealed that the calculated LD50 (1264.9 µL/kg) is much higher than the therapeutic dose (100 µL/kg). CONCLUSIONS: It seems that antioxidant, antiinflammatory, and anticholinesterase activities of ZM or its main constituents might contribute to its beneficial effects in this model. Our findings suggest that ZM may be a potentially valuable source of natural therapeutic agents for the treatment of AD. However, further investigations are necessary to establish its clinical efficacy and potential toxicity, before any recommendations concerning its use as a medication in the treatment of AD.

Association between NOS3 gene G894T polymorphism and late-onset Alzheimer disease in a sample from Iran.

Alzheimer disease (AD) is the most common age-associated neurodegenerative disease caused by complicated interactions between genetic and environmental factors. The presence of the apolipoprotein epsilon4 allele, the only confirmed genetic risk factor for late-onset AD (LOAD), is neither sufficient nor necessary to explain all occurrences of the disease. Aberrant expression of the endothelial nitric oxide synthase (NOS3) gene has been demonstrated in degenerating neurons and glial cells in brains with AD. Molecular epidemiologic studies have presented contradictory results concerning a potential role of NOS3 gene G894T polymorphism in AD. To define a possible association of this polymorphism with LOAD in an Iranian population, we conducted a case-control study including a clinically well-defined group of 100 LOAD patients and 100 age-matched controls. G894T polymorphism in NOS3 gene was determined by polymerase chain reaction-restriction fragment length polymorphisms assay. Chi-square analysis showed a significantly increased number of individuals with the G/G genotype in AD patients compared with controls (P<0.05). These results demonstrate an association between G894T polymorphism and LOAD in an Iranian sample and the G/G genotype seems to have some effects in the development of AD either alone or through interaction with other risk factors.

Involvement of hippocampal nitric oxide in spatial learning in the rat.

Nitric oxide (NO) is thought to be involved in synaptic plasticity contributing to learning and memory in several brain areas including the hippocampus. The hippocampus is believed to have a critical role in the processing of spatial information. But, data on the role of hippocampal NO in spatial learning are not consistent. So the effect of NO synthase (NOS) inhibition in the CA1 region of rat hippocampus on spatial localization was investigated in the Morris water maze (MWM). Male albino Wistar rats cannulated in their CA1 region received bilateral injections of vehicle (saline) or N(omega)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (50, 100 and 200 microg/0.5 microl) through the cannulae 30 min before training each day. Animals were subjected to 5 days of training in the MWM; 4 days with the invisible platform to test spatial learning and the 5th day with the visible platform to test motivation and sensorimotor coordination. The results showed dose-dependent increases (p<0.001) in escape latency, traveled distance, heading angle, and dose-dependent decreases (p<0.01) in target quadrant entries in L-NAME-received groups as compared to the control group. This impairment was reversed by co-administration of mole-equivalent doses of L-arginine (L-Arg), the NO precursor. L-Arg alone at the dose of 129.2 microg, increased heading angle (p<0.01) with no effect on other parameters. On the basis of the present data, it is concluded that processes mediated by NO synthesis in the hippocampus are essentially involved in spatial learning.

The effect of intrahippocampal injection of testosterone enanthate (an androgen receptor agonist) and anisomycin (protein synthesis inhibitor) on spatial learning and memory in adult, male rats.

In most mammals, the hippocampus has a well-documented role in spatial memory acquisition. High concentration of androgen receptors in fundamental centers of learning and memory in brain such as hippocampus shows that there may be some relationships between androgen receptors and cognitive aspects of brain. Previous studies, which have shown sex-dependent differences in hippocampal morphology and physiology, suggest a modulatory role for sex steroids in hippocampal function. Androgens have been shown to modulate some hippocampal-mediated behaviors including learning and memory. To study the mechanism of action of androgens in processes underlying learning and memory, anisomycin, a protein synthesis inhibitor was used to prevent the genomic effects of testosterone. Therefore, the effects of anisomycin and testosterone together were assessed on rat's performance in MWM. Rats received anisomycin (2.5 microg/0.5 microl), testosterone (80 microg/0.5 microl) or both anisomycin (2.5 microg/0.5 microl) and testosterone (80 microg/0.5 microl) through the connulas in the CA1 region. Anisomycin was injected 20 min and testosterone was injected 35 min before training each day. The results showed that anisomycin (2.5 microg/0.5 microl) and testosterone (80 microg/0.5 microl) increased latencies to find the invisible platform. But the group that received testosterone and anisomycin together was decrease in latency and traveled distance to find the invisible platform.

The effects of anisomycin (a protein synthesis inhibitor) on spatial learning and memory in CA1 region of rats hippocampus.

Inhibition of protein synthesis has been shown to affect long-term memory in a wide variety of animal species. But little is known regarding the neuroanatomical location of protein synthesis in different memory tasks. In this study, the effect of intrahippocampal injection of anisomycin, an inhibitor of brain protein synthesis on spatial memory was examined in Morris Water Maze. At first, rats were connulated bilaterally into the CA1 region and then different doses of anisomycin (1.25-2.5 micro g/0.5 micro l) on its vehicle (saline) were injected bilaterally into the CA1 region of rats hippocampus 20 min before training each day. The results showed dose-dependent increases in latencies to find the invisible platform and traveled distances in anisomycin received group compared to the control group. Therefore, it appears that protein synthesis inhibition in the CA1 region of hippocampus impair spatial learning in Morris Water Maze.

Serotonin depletion in rat hippocampus attenuates L-NAME-induced spatial learning deficits.

Inhibition of nitric oxide (NO) synthesis has been found to produce learning deficits in spatial tasks. Recent studies also suggest a regulatory effect of endogenous NO on hippocampal serotonin (5-HT) release and have shown that NO-synthase (NOS) inhibitors increased extracellular levels of serotonin (5-HT) in the rat hippocampus. To clarify possible interactions between NO and 5-HT in the hippocampus on learning processes, the effect of selective hippocampal 5-HT depletion on NOS inhibition-induced spatial learning deficits was investigated. Rats received bilateral injections of 5,7-dihydroxytryptamine (5,7-DHT), a 5-HT neurotoxin, or its vehicle in the CA1 region of hippocampus following pretreatment with desipramine. Rats were subjected to 5 days of training in the Morris water maze (MWM); 4 days with the invisible platform to test spatial learning and the 5th day with the visible platform to test motivation and sensorimotor coordination. Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, was administered to either sham-operated or 5,7-DHT-lesioned groups 30 min before training each day. Results showed that L-NAME significantly impaired the ability of rats to locate the hidden platform. This impairment was reversed by co-administration of mole equivalent dose of L-arginine, the NO precursor. Although the 5,7-DHT-induced lesion had no effect by itself on rat performance in the MWM, it attenuated the memory impairment caused by L-NAME. The observed effect suggests an interaction between NO and 5-HT in the hippocampus on spatial memory formation; however, the mechanism of interaction is still unclear and requires further investigation.