Combination of vitamin E and Lactobacillius plantarum reverses mercuric chloride-induced neurotoxicity: Implication of BDNF, CREB and MAPK proteins expressions

1 Introduction

Mercury is an extensive metal in environments and there are unlimited populations that are currently exposed to this metal at low levels as a result of ever-present ecological factors (Eagles-Smith et al., 2018). It has been documented that low levels of mercury exposure caused immune system alterations (Nyland et al., 2011). Mercury toxicity is attributed to its capability to diminish free sulfhydryl groups of GSH and other antioxidants enzymes (Eagles-Smith et al., 2018).

Accumulating evidence illustrated that oxidative stress evokes numerous intracellular events, such as gene expression, cell-cycle arrest, and apoptosis (Wu et al., 2018). In addition, mercuric chloride (MC) has been found to damage function of many organelles (such as lysosomes that keep proton gradient through the membrane) and decline renal glutathione peroxidase activity (Buelna-Chontal et al., 2017). Some studies have found increased serious health effects among dental workers (Sahani et al., 2016). Mercury induces disruption of the cytochrome c oxidase system/ATP energy function (do Nascimento et al., 2008).

Numerous studies indicated that oxidative stress represents a dangerous event correlated to the neurotoxic effects of MC (Lohren et al., 2015). The levels of Reactive oxygen species (ROS) are dramatically increased upon MC exposure (Gökçe et al., 2018).

The central nervous system is one of the most susceptible organs affected by mercury toxicity. Both acute and chronic exposure to mercury is also known to cause a variety of neurological or psychiatric disorders (Moneim, 2015).

There is some evidence indicating that Mercury may be an etiological factor in Alzheimer’s disease (Siblerud et al., 2019). Thus, agents with the ability to suppress oxidative stress may be beneficial in reducing the risk of tissue injury and organ dysfunction during MC toxicity.

Brain-derived neurotrophic factor (BDNF) has emerged as a key neurotrophin regulating synaptic plasticity, neuronal differentiation and survival of new neurons and synapses. (Finkbeiner et al 1997). cAMP response element-binding (CREB) is a transcription activator that is implicated in the neuro-protection and survival-enhancing properties (Hatalski and Baram 1997; Conti et al 2002).

Mitogen-activated protein kinase (MAPKs) are a highly conserved family of structurally related serine/threonine protein kinases which is responsible for coordinating a variety of extracellular signaling pathways, regulating fundamental cellular processes involved in cell growth and survival (Johnson and Lapadat, 2002).

Alpha-tocopherol’s (vitamin E; VE) antioxidant activity plays a vital roles in the neuroprotection of mercury on the cerebellum and its movement-coordinating function (Owoeye et al., 2018).

The protective effect of VE on MC-induced hepatic and renal functions impairment and oxidative stress in male mice is well documented (Ali, 2018; Per, 2019; Owoeye et al., 2019).

Several studies have revealed that specific Lactobacillus strains alleviated heavy metal toxicity (Tian et al., 2017; Trinder et al., 2016; Yu et al., 2016; Alcantara et al., 2017). Recently, Majlesi et al., have proved the L. plantarum protected significantly against mercury toxicity in liver and kidney in rats by decreasing, creatinine, urea, bilirubin, ALT, and AST level, and preventing alterations in the levels of GPx and Superoxide dismutase (SOD) (Majlesi et al., 2017). It was previously reported that lactobacillus attenuates inflammation in mice by inhibiting NF-κB signaling pathway (Lim et al., 2017).

Although mercury is broadly sulfhydryl reactive, yet signaling cascade implicated in mediating MC-induced neurotoxicity is not fully investigated. This initiates the interest of the present study to investigate the new mechanistic role of MC neurotoxicity at the molecular level and to implement a trail for its amelioration using a combination of vitamin E (VE) and the probiotics Lactobacillus plantarum (LTB).

The current research highlights the potential impact of VE and/or LTB against brain injury induced by MC. The assessment of the efficacy of the antioxidants in question against brain injury was performed biochemically through measuring oxidative status biomarkers Malondialdehyde (MDA), Glutathione (GSH) levels and Superoxide dismutase (SOD) activity, inflammatory markers such as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), the apoptotic marker, cysteine-aspartic acid protease (caspase-3) and the expression of apoptosis regulator (Bax). At the molecular level protein expression of the MAPK and the neurotrophin BDNF and CREB were performed, DNA fragmentation was conducted as well. The previous parameters were supported by histopathological examination.

2 Materials and methods

3 Results

The outcomes of the current study confirmed MC-induced elevation in serum TNF-α, IL 6 levels and Caspase 3 activity compared to the control group (P ≤ 0.001). The intake of VE and/or LTB for fourteen consecutive days along with MC intoxication significantly reduced the increase in serum TNF-α, IL-6 levels and Caspase 3 activity comparad to the MC injected group (P ≤ 0.001) (Table 2). Similarly, MC injection produced a significant increase in MDA level and a decline in GSH level and SOD activity in the brain tissues (P ≤ 0.001) matched with the control group. Oral treatment of MC injected rats with VE and/or LTB significantly modulated the MDA and SOD activity (P ≤ 0.001),while the GSH level was significantly up regulated only in the combination group (P ≤ 0.001) and moderately increased in VE treated rats (P ≤ 0.05), as depicted in Fig. 1. MC exhibited a significant increase in MAPK (Fig. 2), while protein expression for BDNF, CREB (Fig. 3), were significantly down regulated (P ≤ 0.001). BAX expressions were unregulated (P ≤ 0.001) (Fig. 4) compared to the normal group. On the other hand, oral treatment with VE and/or LTB significantly down regulated MAPK and BAX expression (P ≤ 0.001), contrary protein expressions of BDNF as well as CREB were significantly over expressed (P ≤ 0.001) matched with MC – intoxicated rats. Fig. 5 reveals that DNA fragmentation significantly increased in MC injected rats compared to the control group (P ≤ 0.001), in contrast, there was a significant decrease in DNA fragmentation post VE and/or LTB treatments (P ≤ 0.001).

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Fig. 1

GSH, SOD and MDA in brain tissue in control, MC and in all treated groups. The animals were treated with either distilled water (control). Data are Mean ± SEM (n = 6). ⁺⁺⁺P ≤ 0.001 Vs control, *P ≤ 0.05, ^***P ≤ 0.001 Vs MC group, ^πππP < 0.001 Vs the combination group.

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Fig. 2

Protein expression of MAPK in control, MC and in all treated groups. Data are Mean ± SEM (n = 6). ⁺⁺⁺P ≤ 0.001 Vs control, ^***P ≤ 0.001 Vs MC group, ^πP ≤ 0.05 Vs the combination group.

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Fig. 3

Protein expression of BDNF and CREB in control, MC and in all treated groups. Data are Mean ± SEM (n = 6). ⁺⁺⁺P ≤ 0.001 Vs control, ^***P ≤ 0.001 Vs MC group, ^ππP < 0.01, ^πππP < 0.001 Vs the combination group.

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Fig. 4

mRNA gene expression of BAX in control, MC and all treated groups. The animals were treated with either distilled water (control). Data are Mean ± SEM (n = 6). ⁺⁺⁺P ≤ 0.001 Vs control, ^***P ≤ 0.001 Vs MC group, ^πππP ≤ 0.001 Vs the combination group.

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Fig. 5

DNA Fragmentation in control, MC and all treated groups. Data are Mean ± SEM (n = 6). ⁺⁺⁺P ≤ 0.001 Vs to control, ^***P ≤ 0.001 Vs to MC group.

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Histopathological examination of the H&E-stained brain sections illustrated that the cerebellum was characterized by a reduction in the cellular size of the molecular layer, distortion of granular cell layer and scattered, sparse cell distribution of Purkinje layers in MC injected rats compared to the normal brain tissues (Fig. 6B). On the other hand, examining sections of the brains treated with VE and LTB either alone or together; the cerebellum revealed almost normal histological features, illustrating well-defined molecular, granular and Purkinje layers (Fig. 6C–E). The combination of VE and LTB was the most effective regimen in the improvement of brain architecture in MC-induced brain injury.

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Fig. 6

Histopathology analysis of brain sections (A) Brain section of normal control group, showed normal histological features of the cerebellum (black arrow) with the presence of numerous closely packed small cells in the granular layer (red arrow) and Purkinje layers (large Purkinje cells) (yellow arrow) with normal structure of neuronal cells. (B) Brain section of MC group, showed the cerebellum with thin, reduction in cellular size of the molecular layer (black arrow), distortion of granular cell layer (red arrow) and scattered, sparse cell distribution of Purkinje layers (yellow arrow), (C) In brain section treated with VE, the cerebellum showed almost normal histological features, illustrating a well-defined molecular (green arrow), granular (presence of numerous closely packed small cells in the granular) (red arrow) (D) Brain section of treated with LTB, the cerebellum showed almost normal histological features, illustrating a well-defined molecular (yellow arrow), granular (presence of numerous closely packed small cells in the granular) (red arrow) and Purkinje layers (large Purkinje cells) (yellow arrow) with vacuolization and edema of neuronal cells of the frontal cortex (green arrow). (E) Brain section of treated with VE + LTB cerebellum showed almost normal histological features, illustrating a well-defined molecular (black arrow), granular (presence of numerous closely packed small cells in the granular) (red arrow) and Purkinje layers (large Purkinje cells) (yellow arrow) with normal structure of neuronal cells, (H&E stain, ×400).

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4 Discussion

The central nervous system is one of the most vulnerable organs affected by mercury toxicity, which causes a variety of neurological or psychiatric disorders (Moneim, 2015; Sakamoto et al., 2015, 2017). MC caused significant behavioral alteration and induced Purkinje neuron lesion in the cerebellum. VE has antioxidant roles in the reducing the toxic effects of mercury on the cerebellum and its movement-coordinating function. It is well known that LTB has antioxidative potential and able to decrease oxidative stress-mediated atherogenicity in humans, (Kullisaar et al., 2003). However, the mechanisms underlying the neuroprotective efficacy of VE or LTB are not fully understood.

In this study, the effect of VE and/or LTB on oxidative stress, inflammation and apoptosis in brain of MC-intoxicated rats was investigated. Rats received MC exhibited brain injury evidenced by the significant increase in brain LPO and diminished GSH level and SOD activity indicating oxidative stress. Moreover, MC showed cerebellum with reduction in cellular size of the molecular layer, distortion of granular cell layer and scattered, sparse cell distribution of Purkinje layers. In accordance, previous studies have demonstrated diminished the expression of SOD, reduced glutathione, and catalase and increased LPO in testes of MC -intoxicated rats (Muthu and Krishnamoorthy, 2012).

It was reported that the prime mechanism related to mercuric toxicity is the formation of ROS and nitrogen species that exhibit an imbalance between – oxidants/anti-oxidants in the body (Moneim, 2015).

ROS provoke injury through oxidizing lipids and proteins, inactivating antioxidant enzymes and triggering DNA damage (Martindale and Holbrook, 2002). In the current study, VE and/or LTB reduced LPO and boosted cellular antioxidants in brain of MC-intoxicated rats, demonstrating a potent antioxidant efficacy. Upon treatment with VE, the cerebellum showed almost normal histological features. Treatement with LTB revealed that the cerebellum showed almost normal histological features, large Purkinje cells with vacuolization and edema of neuronal cells of the frontal cortex, while brain sections of rats treated with VE & LTB cerebellum showed large Purkinje cells with normal structure of neuronal cells.

In accordance, Celikoglu et al. (2015) found that supplementation of VE alleviated MC-induced oxidative stress and increased the activities of SOD, catalase, glutathione peroxidase that were depleted as a result of the toxicity induced by mercury. Beside its antioxidant activity, VE plays a vital role in decreasing the absorption of mercury from the gastrointestinal tract (Su et al., 2008). Matched with the obtained results, it was documented that Lactobacillus brevis 23,017 relieves mercury toxicity in the colon by modulation of oxidative stress and inflammation through the interplay of MAPK and NF-κB Signaling Cascades (Jiang et al., 2018).

Besides oxidative stress, inflammation has also been implicated in the adverse toxic effects of MC (Rao and Purohit, 2011). In the current study, MC induced an inflammatory response illustrated by the production of inflammatory mediators, including TNF-α, IL-6 and increased DNA fragmentation and enhanced MAPK signaling pathways.

TNF-α is released by activated macrophages and participates in both local and systemic inflammation (Turner et al., 2014). Betti et al. (1993) showed that administration of MC impairs the cells, cell membrane, and DNA which leads to cell necrosis. The increased level of ROS production, inflammatory cytokines, and NO represent a main culprit behind MC toxicity in different tissues (Ahmad and Mahmood, 2019). In accordance, MC induces an autoimmune syndrome and necrotizing leucocytoclastic vasculitis in the Brown Norway rat (Qasim et al., 1994). Mercury stimulated the expression of TNFα, and increased LPS-induced TNFα and interleukin-6 mRNA expression, activated p38 mitogen-activated protein kinase (Kim et al., 2002). In addition, excessive release of TNF-α and IL −1β was recorded in response to MC intoxication (Almeer et al., 2019).

Long exposure of vascular smooth muscle cells (VSMC) to low doses of mercury activates MAPK signaling pathways resulting in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induces proliferation of VSMC and changes in cell size (Aguado et al., 2013). Other researches have revealed that oral mercury exposure affects the gut ecology, increases inflammation and susceptibility to colitis in mice (Stejskal, 2013) (Toomey et al., 2014; Eaton et al., 2017). In the present study, treatment with VE and/or LTB reduced TNF-α, interleukin (IL)-6 levels, decreased DNA fragmentation and down regulated MAPK signaling pathways. It was shown that VE ameliorated oxidative damage in vivo in systemic vasculitides. Numerous researches indicated that lactobacillus attenuates inflammation in mice by inhibiting NF-κB and p38 MAPK signaling pathways (Chon et al., 2010; Saito et al., 2012) that leads to suppress oxidative stress and inflammation (Lim et al., 2017). CREB and BDNF are the major proteins that play a vital role in brain function and regulation. BDNF has emerged as a key neurotrophin regulating synaptic plasticity, neuronal differentiation and survival of new neurons and synapses. CREB, is a transcription activator that is implicated in both stress- (Hatalski and Baram 1997) and antidepressant-induced transcriptional regulation (Conti et al 2002). The cAMP-CREB signalling cascade is critical to the generation of new neurons in the rodent hippocampus (Nakagawa et al 2002), and also facilitates their subsequent morphological maturation (Fujioka et al 2004). Thus, given its neuro-protective and survival-enhancing properties. Shared interactions between BDNF and CREB were well documented: BDNF promotes the phosphorylation of CREB, which, in turn, promotes the transcription of BDNF gene. BDNF activates CREB, in part, by increasing intracellular Ca²⁺ leading to the activation of CaMKIV, which ‘phosphorylates CREB (Finkbeiner et al 1997). In the present study, MC intoxication induces down regulation of protein expression of both CREB and BDNF in the brain tissues. Previous studies demonstrated that MC induced Purkinje neuron lesion in the cerebellum, thus caused significant behavioural alteration (Owoeye and Arinola, 2017; Owoeye et al., 2018).

VE supported its antioxidant roles in neuroprotection, thus reducing the toxic effects of mercury on the cerebellum and its movement-coordinating function.

The current study introduced new information that modulation of CREB and BDNF signaling plays a role in the protective mechanism of VE against MC neurototoxicity.

Treatment with the antioxidants in question alone or together mitigated the alteration in CREB and BDNF signaling pathways.

In the present study, MC intoxication induces up regulation of caspase-3 activity and mRNA expression of Bax in brain tissues. Previous studies demonstrated that pro-apoptotic proteins was favored following MC intoxication. Caspase-3 activity was higher with MC apoptosis, as indicated by the elevation of Bax/Bcl-2 ratio in brain tissues (Moneim, 2015).

It was reported that VE has an antioxidant defense with anti-inflammatory and anti-apoptotic activities against MC-induced neurotoxicity (Moneim, 2015).

In the present study, the intake of VE and/or LTB alleviated the increase in the studied apoptotic markers. Interestingly, the combination regimen exhibited the most admirable results in all the studied parameters

5 Conclusions

This study demonstrates that VE prevents MC neurotoxicity by attenuating oxidative stress, inflammation, DNA damage and cell death. The neuroprotective efficacy of VE was reinforced by the intake of LTB. The obtained results introduced new information that BDNF and CREB/MAPK signaling is implicated in MC -induced brain injury and dysfunction.