Introduction
Lead (Pb) is a toxic heavy metal widely distributed across environments all around the world with no beneficial roles in biological systems [
1]. Exposure to Pb has been a problem of global concern for decades especially in children [
2]. Pb poisoning is known to alter the body function as it can affect the hematologic, hepatic, cardiovascular, reproductive, gastrointestinal and neurological systems in the body [
3]. As such, Pb has been reported to affect speech, nerve conduction and hearing, as well as cause weight loss, hyperactivity, intestinal discomfort, vomiting, constipation, and muscle aches. Pb poisoning can equally lead to anemia, paralysis, nephropathy, convulsions or death [
4]. In pregnancy, Pb can affect the foetus causing intrauterine deaths, miscarriages and/or low birth weight [
5].
Neurological damage induced by Pb toxicity is reported to be a base for multiple disorders such as Parkinson’s and Alzheimer’s diseases [
6]. Pb has the ability to pass through the blood-brain barrier due to its calcium ion-substituting ability to damage the prefrontal cortex, cerebellum and hippocampus [
7]. The disruption of the brain barrier by Pb causes albumin to enter the tissues of the central nervous system (CNS), resulting in increased intracranial pressure, edema, and encephalopathy [
8]. Pb exposure in the brain affects neurotransmitters including cholinergic, dopaminergic and glutaminergic systems [
9]. One of the characteristic features of Pb intoxication in the brain is impairment of the action of acetylcholinesterase (AChE) in cholinergic nerve cells transmission [
10]. The central cholinergic system is essential for the regulation of cognitive functions. As such, cholinergic receptor agonists and inhibitors of AChE are used to control endogenous acetylcholine levels to overcome cognitive deficits [
11].
Pb neurotoxicity also promotes pro-inflammatory effects in the brain. Pb is known to induce increase gene expression and secretion of interleukin 6 (IL-6), tumour necrotic factor-α (TNF-α) and transforming growth factor β1 (TGF-β1) leading to increased inflammation in the brain [
11]. Inflammatory processes have been observed in the pathogenesis of Parkinson’s, Alzheimer’s diseases and in multiple sclerosis [
12]. Also, Pb toxicity is known to cause apoptotic neurodegeneration [
13]. Studies have shown Pb to increase mRNA and protein levels of apoptotic factors including caspase and Bcl-2 Associated X Protein (Bax), evidence that Pb increases neurodegeneration [
14].
Pb toxicity induces oxidative stress by generating reactive oxygen species (ROS) such as superoxide (O
2−), hydroperoxides (HO
2•) and hydrogen peroxide (H
2O
2), as well as by depleting the antioxidant defence systems [
15]. Glutathione (GSH) is an important antioxidant that directly or indirectly scavenges ROS. Also, antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) are important ROS scavengers. However, these enzymes can be rendered inactive or reduced by Pb [
15]. Failure of the body to meet the homeostatic requirement due to overwhelming ROS level results in the occurrence of oxidative stress characterised by oxidative damage of proteins, nucleic acid and membrane lipids (lipid peroxidation) [
16]. Cellular by-products of oxidative damage also stimulate inflammation and cell death.
In recent years, studies conducted on the use of antioxidants as an intervention for Pb-induced neurotoxicity and oxidative stress has shown success. Gallic acid, a phenolic compound of natural origin attenuated locomotor damage and brain oxidative stress induced by Pb exposure [
17]. Also, geinstein was shown to alleviate Pb-induced neurotoxicity through the involvement of multiple signalling pathways in vitro and in vivo [
18]. Plants rich in phenolic compounds are known to play an important role as antioxidants which enhances their medicinal properties. One of such plant is
Lippia javanica (Burm.f.) Spreng. which has been shown to possess antioxidant activities in in-vitro studies [
19‐
21]. Traditionally, parts of
L. javanica have been used medicinally for the treatment of respiratory ailments such as colds, coughs, asthma and tuberculosis [
22]. It has also been reported in the treatment of gastrointestinal tract disorders such as abdominal pain [
23] which is a common symptom of Pb poisoning. Although
L. javanica has been shown to possess higher antioxidant capacity as well as better free radical scavenging activity than a known antioxidant, rooibos (
Aspalathus linearis) in vitro [
24], it remains unknown whether the in vitro antioxidant properties of
L. javanica can translate to in vivo neuroprotective effects of Pb-induced toxicity against oxidative stress, inflammation, apoptosis and acetylcholinesterase activity. Therefore, the aim of this study was to investigate the effectiveness of
L. javanica in attenuating Pb-induced toxicity in rat’s brain.
Discussion
Pb-induced toxicity, particularly neurotoxicity is known to be mediated through impairment of cholinesterase activity, generation of ROS as well as pro-inflammatory cytokines which can also generate ROS.
L. javanica has been reported to possess antioxidant activity to scavenge ROS [
24]. However, it remains unknown whether the
L. javanica can promote neuroprotective effects through its antioxidant activity against Pb-induced oxidative stress, inflammation, apoptosis and cholinesterase activity. This study was conducted to investigate the in vivo effects of
Lippia javanica (Burm.f.) Spreng on Pb-induced brain oxidative stress and associated neurodegenerative effects.
Pb has the ability to cross the blood-brain barrier by competing with Ca
2+ during neuronal firing and has been associated with neurodegenerative disorders [
37]. It is well established that the neurodegenerative effects of Pb are linked to its role in causing oxidative stress [
1,
6,
27]. As such, studies have looked at combating brain oxidative stress and neurodegeneration with antioxidants [
38].
Lippia javanica, a herbal tea plant whose antioxidant activity has been studied almost exclusively in vitro [
24,
39]
, has not been investigated for antioxidant effects in vivo especially in the brain. To our knowledge, this is the first study to investigate effects of
L. javanica on Pb-induced brain damage. To achieve this, we exposed rats to Pb to induce damage and observed the effect on the animal’s organs, paying attention to the brain which was our organ of interest. Brain weight was reduced as well as toxic effects on liver, heart and spleen were observed, and consistent with observations in previous studies attributed to Pb-induced oxidative stress, inflammation, fibrosis and cell death [
40].
The brain is highly susceptible to oxidative damage and lipid peroxidation due to its high oxygen requirements and high lipid content. The levels of glutathione and α-tocopherol, both non-enzymatic antioxidants, are relatively low in the brain compared to the rest of the organs in the body [
41]. Ingestion of plant-based antioxidants in the form of polyphenols may increase the brain’s antioxidant capacity. Faria and colleagues [
42] demonstrated that flavonoids are able to cross the blood-brain barrier and showed intact anthocyanins within various brain regions. In our study, the TEAC assay was used to measure the overall non-enzymatic antioxidant status in the brain, which included hydrophilic and lipophilic antioxidants as well as –sulfhydryl-containing antioxidants. Pb exposure had no effect on overall antioxidant status in the brain, nor did
L. javanica. However, glutathione was significantly increased in the
L. javanica-treated Pb exposed animals compared to animals exposed to Pb only. These results suggest that though the total antioxidant level was same,
L. javanica specifically increased glutathione level. Khalaf and colleagues [
43] reported that Pb exposure at 100 mg/kg BW resulted in lowered antioxidant status in the brain while green tea consumption at 5 g/L ad libitum increased overall non-enzymatic antioxidant status in the brain, also using the TEAC assay.
SOD is an enzyme family that protects cells from the harmful effects of O
2− radical. In light of the brain’s low, non-enzymatic antioxidant status, enzymatic antioxidants such as SOD are extremely important in protecting neurons against free radicals and oxidative stress. The SOD status in the brain was increased with Pb exposure,
L. javanica consumption as well as with
L. javanica-treated Pb exposure. This increased SOD activity may have a protective role in the cells of the brain which, in the case of Pb exposure, may be a protective response while the increase of SOD status may be due to upregulation by
L. javanica treatment. Again, in parallel with our results, Reckziegel and colleagues [
17] reported increased SOD status with Pb exposure as well as with gallic acid treatment.
Lipid peroxidation (LPO) can be defined as a free radical oxidation of polyunsaturated fatty acids [
44]. LPO, as determined by MDA concentration, was increased in the Pb exposed group indicating that Pb exposure led to LPO in the brain as has previously been demonstrated [
45]. On the other hand, though Pb-exposed
L. javanica treatment group had increased LPO, it showed a trend towards a decrease compared to Pb exposure alone group (
p = 0.1) indicating that
L. javanica treatment prevented LPO to an extent. This was confirmed as treatment with
L. javanica had no effect on LPO as the MDA level was similar to controls. Similarly, Reckziegel and colleagues reported that MDA concentration was increased with Pb exposure, but treatment of Pb exposure with gallic acid, a known flavonoid, reduced Pb-induced LPO in the brain [
17]. Also, curcumin has been shown to inhibit Pb-induced oxidative stress and chelating activity [
46].
Pb exposure has been suggested to promote inflammation. Generally, inflammatory cytokine expression is very low or undetectable in the brain under physiological conditions but tends to increase in conditions such as infections, trauma, autoimmune diseases or exposure to toxic agents [
47]. Pb toxicity has been shown to induce increase level of pro-inflammatory cytokines such as IL-6, TGF-β1 [
11] and TNF-α [
48]. Findings from this study showed that Pb induced inflammation as increased level of TNF-α was observed in Pb exposed animals but was reduced by
L. javanica as the Pb exposed animals treated with
L. javanica had a significantly lower TNF-α level. This finding confirms previous studies which have also shown some plants to prevent Pb-induced inflammation [
49]. Pb toxicity is known to promote apoptosis as it has been shown to increase the expression and synthesis of some pro-apoptotic proteins such as bcl-2 [
14] and Bax proteins [
50]. Pb induce the synthesis of Bax protein as it significantly increased in Pb exposed animals but was reduced by
L. javanica treatment suggesting that
L. javanica can prevent Pb-induced apoptosis. Pb exposure has also been observed to affect neurotransmission. Pb has been shown to affect cholinergic systems in the hippocampus and septum impairing cholinergic nervous transmission [
10]. Acetylcholinesterase regulates cholinergic neurotransmitters. Increased acetylcholinesterase activity impairs nervous transmission [
11]. In this study, Pb exposed animals which were treated with
L. javanica had a significantly reduced acetylcholinesterase level which was high in Pb exposed animals. Therefore,
L. javanica treatment reduced cholinesterase activity which was induced by Pb. This finding is consistent with previous studies which have also shown
Thunbergia laurifolia (Linn.) [
51] and propolis [
52] to attenuate Pb-induced cholinesterase activities in animal models.
The hippocampus is a bilateral brain structure located in the temporal lobe on which learning and memory are critically dependant [
53]. Episodic memory impairment is a hallmark of neurodegenerative diseases, such as Alzheimer’s disease, which are neurobiologically linked to the hippocampus [
54]. Liu and colleagues [
48] reported that Pb exposure induced significant microgliosis and astrogliosis in hippocampus of young mice. In Pb treated rats, vacuolization and oedema was observed in the hippocampus area [
43]. In our current study, Pb exposure caused severe hippocampal cellular damage which was similarly manifested as vacuolization and oedema. The hippocampal structure remained normal with
L. javanica treatment, consistent with controls. The effects of Pb-induced hippocampal damage were decreased with
L. javanica, similar to previously published studies using green tea [
43]. Thus,
L. javanica protects against Pb-induced neuronal damage.
To ascertain the phytochemicals in
L. javanica that were responsible for the neuroprotective effects of Pb-induced brain damage, LC-MS analysis was done
. L. javanica was shown to be rich in phenolic acids among which vanillic acid, caffeic acid,
p-coumaric acid, protocatechuic acid and syringic acid were high in abundance accounting for about 88% of the total phenolics while gallic acid, syringaldehyde, trans-cinnanic acid and ferulic acid were in small amounts. This finding confirms previous reports which have shown
L. javanica to be rich in polyphenols such as flavonols flavonoids, and proanthocyanidin [
55,
56]. Most of these abundant phenolics compounds (syringic,
p-coumaric, caffeic and vanillic acids) found in
L. javanica have been reported in experimental models to possess anti-inflammatory and antioxidant activities [
57‐
61] as well as anticholinesterase activity [
62,
63] and in apoptosis in reducing the expression of Bcl [
64]. This may suggest that the observed effects of
L. javanica in this study may be accounted for by these phenolic compounds.
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