The ability to sense oxidative and proteotoxic insults and to coordinate defensive stress response are basic elements for cellular adaptation and survival [ 23 24 ]. With regard to this, medicinal mushrooms, including HE may have beneficial protective effects in low doses [ 25 27 ]. Consistently, hormesis dose response is characterized by low dose stimulation and a high dose inhibition. The biphasic dose-response phenomenon is characterized by a U-shaped or inverse dose response curve, depending on the different measured endpoints [ 28 30 ]. The hormetic dose response results from either a direct stimulation or through an overcompensation stimulatory response following disruption in homeostasis [ 31 ]. Such hormetic dose responses provide a quantitative description of the bounds of biological plasticity, and a measure of the extent to which adaptive processes may be upregulated, which is especially relevant to the comprehension of protective effects induced by plant and fungal species [ 32 36 ]. It has been known that mushrooms activate the heat shock protein (Hsp) pathway in different brain regions of rats, which plays a crucial role in the cellular stress response [ 22 38 ], and hence,preparations can have neuroprotective effects through modulation of inflammatory processes associated with the neuropathology, as well as through regulation of brain stress response plasticity mechanisms [ 39 ]. In addition, antioxidant activity of HE has also been demonstrated in other tissues, including liver [ 40 ]. In light of the above-mentioned evidence in the present study, we have investigated in vitro the neuroprotective role of HE biomass preparation against DEHP-induced neurotoxicity.

Moreover, mushroom-derived polysaccharides are complex molecules that cannot be synthesized, as the mass production of these compounds would require timely and costly extraction processes. As a result, many research efforts have focused on low molecular weight compounds, such as cordycepin, which is a cytotoxic nucleoside analog inhibitor of cell proliferation. Of the mushroom-derived therapeutics, polysaccharopeptides obtained from(HE) are commercially the best established. HE is known to have a neuroprotective property evidenced by the regulation of inflammation in relation to the pathology of Alzheimer’s disease. In another modality of protection against Alzheimer’s disease, HE has been shown to stimulate the synthesis of nerve growth factor (NGF) in cultured astrocytes [ 21 ]. This growth factor, acting on cholinergic neurons by modulating the activity of two enzymes: cholineacetyltransferase and acetylcholinesterase in Alzheimer’s disease, the activity of these two enzymes is inhibited since the dysfunction of cholinergic neurons is an initial event in Alzheimer’s disease [ 22 ].

Apoptosis is the most common and well-defined form of programmed cell death which is a genetically directed process of cell self-destruction that is essential for embryonic development, immune-system function, and the maintenance of tissue homeostasis in multi-cellular organisms [ 11 ]. It is marked by different changes in the mitochondria such as the release of caspase activators, changes in electron transport, loss of mitochondrial membrane potential (MMP), and involvement of pro and anti-apoptotic Bcl-2 family proteins [ 12 ]. In recent years, considerable research has been carried out on identifying naturally occurring substances endowed with neuroprotective properties, impacting on apoptotic processes, and thus capable to prevent or delay neurodegenerative processes. Mushrooms, which have been used in traditional medicine for thousands of years [ 13 14 ], have been reported to possess various biological actions, including antitumor, immunomodulatory, antioxidant, antiviral, antibacterial, and hepatoprotective effects [ 15 16 ]. Some of the most potent immunostimulatory molecules derived from mushrooms are β glucans, which activate many types of immune cells and stimulate cytokine responses [ 17 20 ]. Administration of complex mixtures of molecules to unknown concentrations is difficult to reconcile with current pharmaceutical practices involving highly purified compounds, and hence, as the active ingredients may be unknown, to patent mushroom extracts is a very difficult task.

Recently, an increasing number of studies have provided evidence of a significant association between DEHP exposure and neuronal disruption. The toxicity of DEHP in mammalian cells has been investigated, and it has been demonstrated that this phthalate is cytotoxic and induces apoptosis in Neuro-2-a cells, a neuroblastome cell line [ 5 ]. Other investigators have studied the in vivo administration of DEHP and demonstrated that this plasticizer causes neurodegeneration in the brain of rats [ 6 ]. Other lines of evidence indicate that prenatal and postnatal exposure of DEHP affect CNS [ 7 ]. Indeed, in utero exposure, DEHP caused disruption of rat brain development [ 8 ], while a reduction in the number of mid brain dopaminergic neurons and a motor hyperactivity have been demonstrated after postnatal exposure to this phthalate [ 9 ].

Phthalates are common plasticizers, used in a large variety of household and medical products to confer flexibility to many polyvinyl chlorides (PVC)–based plastics [ 1 ]. Di(2- ethylhexyl)phthalate (DEHP) is one of the most extensively used phthalates, which have a variety of applications including food packages, cosmetics, clothing, children’s toys, and medical devices such as blood storage bags [ 2 ]. DEHP is highly hydrophobic compound and it is well absorbed after oral exposure [ 3 ], being estimated that human absorption of DEHP could be as high as 25%. Due to its liposolubility, DEHP leaches from plastics following lipophilic fluids [ 4 ], making it possible that DEHP crosses the blood-brain barrier into the central nervous system (CNS) tissue, which could lead to neural toxic effects.

The results in Figure 7 show that the expression of apoptosis biomarker, p53, increased after DEHP treatment ( Figure 7 a,b). This induction was associated with the overexpression of proapoptotic protein, Bax ( Figure 7 a,b), and with a decrease in antiapoptotic protein, Bcl2 ( Figure 7 a,b). All these effects were prevented by pretreatment with HE ( Figure 7 a,b). We next examined involvement of caspase 3 in apoptosis pathways. Western blotting analysis showed that DEHP treatment resulted in increased level of cleaved active caspase 3 ( Figure 7 a,b). In addition, caspase-3 activity was measured by fluorimetric assay. As shown in Figure 7 c, DEHP treatment significantly increased caspase-3 activity. HE pretreatment was found to be effective to prevent DEHP-induced activation of caspase-3 ( Figure 7 a–c). Our results indicate the involvement of the caspase-mediated apoptosis pathway.

It has been reported that electron transport chain (ETC) complexes are important sources of mitochondrial reactive oxygen species, and their inhibition has been associated with elevated levels of ROS [ 41 ]. Given the observed induction of ROS after DEHP treatment, we hypothesized that this excess of ROS may be mediated by mitochondrial dysfunction. To assess this, we analyzed the activity of respiratory chain complexes in PC12 cells treated with DEHP and the effect of HE pretreatment. The data presented in Table 1 shows that the enzymatic activities of complexes I, II-III, IV, as well as ATP synthase drastically decreased after DEHP treatment. However, pretreatment with HE reduces DEHP-induced alterations in mitochondrial respiratory complex activities and significantly restored activity of complex I and II+III (< 0.05).

Indeed, the DEHP treatment results in 51.16 % cell viability, but pretreatment for 2 h with HE at 0.5 mg/mL increase cell viability at 78.73 % ( Figure 1 c). Thus, we asked whether DEHP exposure affects mitochondrial ROS formation. Therefore, ROS production in PC12 cells was measured using fluorescent dye DCFDA. The levels of intracellular ROS markedly increased after treatment with DEHP. However, pretreatment with HE (0.5 mg/mL) significantly decreased the intracellular ROS generated by DEHP in PC12 cells ( Figure 2 ).

We first evaluated whether DEHP alone or HE alone treatment was toxic to pheochromocytoma 12 (PC12) cell line. Cells were treated for 24 h with increasing concentrations of DEHP (20, 40, 60, 80, and 100 µM) and HE (0.5, 1 1.5, and 2 mg/mL) ( Figure 1 a) and cell survival was determined by 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( Figure 1 a,b). DEHP treatment induced a dose-dependent reduction in cell viability with approximately IC50 observed at 85 µM ( Figure 1 a). Consequently, cytotoxic induction with 85 µM DEHP for 24 h was used in the subsequent experiments. Besides, while HE alone exhibited no toxicity towards PC12 cells ( Figure 1 b), pretreatment with this mushroom at 0.5 mg/mL, significantly decreased DEHP-mediated cytotoxicity ( Figure 1 c).

3. Discussion

Although DEHP is extensively used as a plasticizer, few studies have focused on its brain tissue toxicity. In the present study, we investigated the protective effect of HE to counteract oxidative stress, mitochondrial energy deficit and cell death in PC12 cells. Our results showed that HE significantly increased PC12 cells survival against DEHP insult, an effect associated with a decrease in the level of ROS generation, and with modulation of mitochondrial respiratory complex activities, as well as reduction of apoptosis. The present data shows that DEHP induces oxidative stress as demonstrated by robust increase in ROS generation. ROS excessively produced during oxidative stress cause cell damage leading to cell death [ 33 ].

Pre-treatment of neuronal cells with HE at the dose of 0.5 mg/mL inhibits intracellular ROS formation and protects cells against DEHP-induced oxidative stress. Protective effects of HE against oxidative stress-induced injuries have been reported in various in vitro and in vivo studies [ 39 44 ]. So, we proposed that this anti-oxidative effect of HE may be partly though the possibility of direct elimination of ROS or by rescuing the efficiency of complex I given that the inhibition of this complex is related to ROS overproduction. In fact, it is known that overproduction of ROS might occur through energy dependent mechanisms, mainly a consequence of an inhibition of complex I which generally is associated with cell death [ 45 46 ].

To assess the possible alterations in energy metabolism following exposure to DEHP, we measured mitochondrial respiratory complex activities, i.e., NADH-UQ oxidoreductase (complex I), Succinate-cytochrome c oxidoreductase (complex II-III), Cytochrome c oxidase (complex IV) and ATP synthase (complex V). Our results clearly show that DEHP inhibits the mitochondrial respiratory complex I, II-III, IV and V. The mechanism by which DEHP inhibits these complexes is still unclear, however, we hypothesize that inhibition might result from an imbalance in redox status associated with alteration in mitochondrial membrane potential, which can eventually lead to structural disorganization and dysfunction of critical enzymes. Further studies are necessary, however, to determine whether DEHP induces structural and/or post-translational modifications of these enzymes. On the other hand, our results show for the first time that HE treatment could restore the activity of respiratory complexes I, II-III, IV, and V, suggesting that HE may play a direct role in preserving complex activities and eventually prevent ROS production, thus renewing the capacity of neurons to produce energy. Such alterations in respiratory complex activities could be associated with a disturbance of the oxidant/antioxidant balance equilibrium which induces cell degeneration.

42,+ as a cofactor, catalyze the deacetylation reaction of histone substrates and transcriptional regulators. Sirtuins regulate different biological processes, such as apoptosis, cell differentiation, energy transduction, and glucose homeostasis [ Cells are endowed with cellular pathways involved in the maintenance of cellular homeostasis to recovery mitochondrial function and confer protection against oxidative stress [ 39 ]. Among these pathways, there is a complex network of the so-called longevity assurance processes, composed of several genes termed vitagenes [ 27 ], which include members of the HSP family, such as HO-1, Hsp72, but also sirtuins and the thioredoxin/thioredoxin reductase system [ 27 47 ]. Molecular chaperones have been known to protect cells against a wide variety of toxic conditions as well as oxidative stress, extreme temperatures or heavy metals exposure. Chaperones play also an important role in the preservation and repair of the correct conformation of the cellular macromolecules, such as proteins, RNAs, and DNA [ 48 ]. Indeed, HO-1 catalyzes the degradation of heme and produces carbon monoxide and bilirubin, which can directly scavenge free radicals and repair DNA damage caused by oxidative or nitrosative stresses [ 39 ]. Sirtuins are histone deacetylases which, in the presence of NADas a cofactor, catalyze the deacetylation reaction of histone substrates and transcriptional regulators. Sirtuins regulate different biological processes, such as apoptosis, cell differentiation, energy transduction, and glucose homeostasis [ 42 ]. Furthermore, Trx, is a major redox control system, consisting of a 12 kDa redox active protein Trx, and a homodimeric seleno-protein called thioredoxin reductase (TrxR1). TrxR1 is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized thioredoxin protein. It is usually located in the cytosol, but it translocates into the nucleus in response to various stimuli associated with oxidative stress, thereby playing a central role in protecting against oxidative stress [ 27 ].

In this work we provide experimental evidence that PC12 cells treatment with DEHP for 24 h results in upregulation of vitagenes, in particular Hsp70, HO-1, and Trx and in down regulation of SIRT1. However, our study shows for the first time that HE pretreatment modulates vitagenes expression in PC12 cells. Our results are consistent with evidence obtained in mice, showing neuroprotection by HE on Aβ25–35 peptide-induced cognitive dysfunction [ 49 ].

We also provide experimental evidence that upregulation of HO-1 might involve the transcription factor Nrf2, which was highly expressed in the nuclear fraction of cells exposed to DEHP. Nrf2 is a transcription factor that regulates the expression of genes involved in protection against oxidative stress. Nrf2-dependent transcription is under control of the amount of ROS present in cells. Indeed, under basal conditions, Nrf2 is localized in the cytoplasm in its inactive form where it is bound to its inhibitor Keap1 which promotes its degradation by the proteasome via an E3 ubiquitin ligase complex. However, under oxidative stress, Nrf2 dissociates from Keap1, moves into the nucleus, and activates AREs present in promoter regions of a set of genes [ 50 ]. Upregulation of Nrf2 observed in our study might be due to oxidative stress generated by this phthalate, which on the other hand, was modulated by HE pretreatment. In addition to this, we found inhibition of the mitochondrial enzyme complexes activities, which can be considered an event preceding the reduction of the mitochondrial membrane potential. Consistent to this finding, excess ROS production by decreasing mitochondrial complex activities and promoting the decline of mitochondrial membrane potential, can induce, as a potent mediator, cell pathway death [ 39 ].