Amyloid Beta Exposure on Olfactory Ensheathing Cells Induces Different Expression Pattern of Tissue Transglutaminase and Its Isoforms: Modulatory Effect of Indicaxanthin


 Background

Alzhèimer Disease (AD) is characterized by intracellular and extracellular protein aggregates in the brain, including amyloid-beta (Aβ). Aβ is a substrate for tissue transglutaminase (TG2), an ubiquitarian calcium-dependent protein that induces Aβ oligomerization and aggregation. We assessed the effect of full native peptide of Aβ(1–42), the fragments (25–35 and 35–25) on TG2 expression levels and its isoforms (Long and Short) on Olfactory Ensheathing Cells (OECs). The levels of cytoskeletal proteins, Vimentin and Glial Fibrillary Acid Protein (GFAP), were also studied. The effect of the pre-treatment with Indicaxanthin on cell viability, total Reactive Oxygen Species (ROS), superoxide anion (O2−) and apoptotic pathway activation was assessed. Since Nestin is co-expressed in pluripotent stem cells with cyclin D1, their levels were evaluated.
Methods

Mouse primary OECs were exposed to 10 µM Aβ(1–42) or Aβ(25–35) or Aβ(35–25) for 24 h both in the absence and in the presence of Indicaxanthin. TG2 and its isoforms were evaluated through Confocal Laser Scanning Microscopy and Western Blot analysis. Data were statistically analysed using one-way analysis of variance followed by post hoc Holm–Sidak.
Results

Our findings highlight that OEC exposure to Aβ(1–42) and its fragments induced an increase of TG2 levels and the different expression pattern of its isoforms. Indicaxanthin pre-treatment reduced TG2 over-expression differently modulating its isoforms, following Aβ exposure of the cells. It was also able to prevent total ROS and O2−production, to reduce GFAP and Vimentin levels, inhibiting apoptotic pathway activation. It also leaded to an increase of Nestin and cyclin D1 expression levels.
Conclusions

Our results show that Aβ exposure on OECs induces an increase of TG2 levels and a different expression pattern of its isoforms and that Indicaxanthin pre-treatment stimulates OEC self-renewal through the reparative activity played by TG2. They also suggest that Aβ in OECs, both in the absence and in the presence of Indicaxanthin, might differently induce the transition of TG2 between “closed” and “open” conformation, providing a new mechanism involved in the signal pathways activated by the protein in Aβ injury important for neural regeneration of AD.

Several ndings reported that in AD patients an early sign of neurodegeneration is represent by a reduced function of olfactory performance [12]. In particular, a peculiar olfactory glial cell is represented by Olfactory Ensheathing Cells (OECs). This cellular type, showing a bipolar or multipolar morphology, surrounds the olfactory nerves, is able to secrete different growth factors, neurotrophins, adhesion molecules and numerous markers, which promote neuron survival and axonal growth, supporting also injured Central Nervous System [13][14][15][16]. OECs are able to stimulate angiogenesis and remyelination, therefore they play an important role in transplants in spinal cord injury [17]. In addition, OECs exhibit stem cell properties, expressing Nestin, a marker of precursor neural stem cells [17]. In previous researches we demonstrated that TG2 was overexpressed in OECs exposed to full native peptide Aβ  and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fragment and that some growth factors were able to down-regulate the expression levels of the protein [18].
In the recent years, growing attention rose on neuro-nutraceutics, such as Indicaxanthin, a phytochemical produced by cactus pear fruit from Opuntia cus-indica, L. Mill. [19,20]. Indicaxanthin possesses signi cant anti-proliferative, antitumor and anti-in ammatory effects both in vivo and in vitro [21,22]. In addition, it modulates reactive oxygen species (ROS) production, prevents mitochondrial damage, regulates cell redox balance and calcium homeostasis in a number of experimental in vitro models [23]. Interestingly enough, and in contrast with the majority of phytochemicals, Indicaxanthin is able to cross the blood brain barrier and to modulate the bioelectric neuronal activity in the hippocampus [24].

OEC cultures
Olfactory bulbs were removed from decapitated pups and placed in cold (+ 4 °C) Leibowitz L-15 medium [27]. Successively, pellets were digested in MEM-H, containing collagenase and trypsin mixture. Trypsinization was stopped by adding DMEM supplemented with 10% FBS (DMEM/FBS). Cells were resuspended and plated in asks fed with complete DMEM/FBS. Cytosine arabinoside (10 − 5 M), an antimitotic agent, was added 24 h after initial plating, in order to reduce the number of dividing broblasts. OECs were then processed to an additional step transferring from one ask to a new one, in order to reduce contaminating cells, following the method by Chuah and Au [28]. When OECs were con uent, they were removed by trypsin, transferred on 25 cm 2 asks and cultured in DMEM/FBS. Cells were then incubated at 37 °C in complete medium and fed twice a week. Puri ed OECs were grown in DMEM/FBS on 14 mm diameter glass coverslips and 96 multiwells at bottomed at a nal density of 1×10 4 cells/coverslip and on 25 cm 2 asks at a nal density of 1×10 6 . Cells were then incubated at 37 °C a humidi ed 5% CO 2 -95 % air mixture.

Indicaxanthin puri cation
Indicaxanthin was isolated from Opuntia cus indica L. Mill fruit pulp (yellow cultivar) and purity (97%) of the pigment was assessed by HPLC according to a previous described method [24].

Isolation of total protein and Western blot analysis
Untreated and treated OECs cells were harvested in cold PBS, collected by centrifugation, resuspended in cell lysis buffer containing 50 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 10 µg/mL of aprotinin, leupeptin, pepstatin, incubated for 30 min at 4 °C, centrifuged at 12,000 x g for 10 min at 4 °C and the supernatants containing total cell proteins were collected [5,18,30,31]. Brie y, extracted proteins were stored at − 80 °C, and protein quantitation was performed by bicinconinic acid method, according to to manufacturer's instruction. 40 µg of total proteins were separated through 4-15% precast SDSpolyacrylamide gels and transferred to nitrocellulose membranes. Filters obtained were then incubated with the following 1:1000 diluted antibodies: mouse monoclonal antiboby against TG2, rabbit monoclonal antibody against Cyclin D 1 , mouse monoclonal antibody against β-tubulin. Anti-rabbit IgG horseradish peroxidase-conjugated and anti-mouse IgG horseradish peroxidase-conjugated were then used. The expression of each protein was visualized through Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate after autoradiography lter exposure. Blots were then scanned and quanti ed through ChemiDoc Imaging System (ChemiDoc™ Imaging System, Bio-Rad, Milan). Densitometric analysis was performed through the integrated software and data obtained were normalized with β-tubulin.

Statistical analysis
Data were statistically analysed using one-way analysis of variance (one-way ANOVA) followed by post hoc Holm-Sidak test to calculate signi cant differences among groups. Data were reported represent the mean ± S.D. of ve separated experiments in triplicate, and differences among groups were considered to be signi cant at *p < 0.05.

Total ROS/O 2 generation
To monitor the intracellular oxidative status, the staining of total intracellular ROS levels (Fig. 4A, green) and O 2 − (Fig. 4B, red) generation in OECs exposed for 24 h to the full native peptide Aβ   Total TG2 expression through immunocytochemistry The Fig. 5 reports TG2 positivity and its localization performed on single cell through immunocytochemical procedures and CLSM analysis. In the control cells a low staining for TG2 was found and the protein was prevalently localized in the cytosol. A more intense staining for TG2 both in Aβ(1-42) and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) treated OECs was observed widely in the cytosol, when compared with the controls. In particular, the positivity of the cells for TG2 appeared more evident in Aβ(25-35) treated ones, when compared with the controls and Aβ(1-42) treated OECs. The exposure to the fragment Aβ  produced a light increase for TG2 cell positivity, when compared with the controls and the protein appeared prevalently localized in the cytosol. In 100 µM Indicaxanthin-treated cells, TG2 staining was slightly higher than controls and it was widely localized in the cytosol. In contrast, in the cells exposed to Aβ(1-42) and Aβ(25-35), Indicaxanthin pre-treatment was able to decrease the number of positive cells for TG2, when compared with the controls. Speci cally, in Indicaxanthin treated OECs and subsequently exposed to Aβ(1-42), TG2 was localized into the nucleus. Differently in Aβ(25-35)-Indicaxanthin pretreated cells, the protein was prevalently localized in the cytosol, even if some cells showed a low positivity for the protein in the nucleus and nucleoli. A low staining for TG2 in Indicaxanthin pre-treated OECs and then exposed to the fragment Aβ  was found, when compared both with the controls and the fragment Aβ  alone. In addition, the protein was prevalently localized in the cytosol. No speci c staining of OECs was observed in control incubation in which the primary antibody was omitted.

TG2 and its isoforms expression and effect of Indicaxanthin
To assess and better clarify CLMS analysis performed on single cell relative to the different intracellular localization of TG2, the expression levels of the total TG2 and its isoforms (TG2-S and TG2-L) induced by the different treatment types, were evaluated through Western blot analysis on total cellular lysates. Immunoblots (Fig. 6A) and densitometric analysis (Fig. 6B) show an signi cant increase of total TG2 expression levels both in Aβ(1-42) and Aβ(25-35) exposed OECs, when compared with the controls. The effect was more evident in Aβ(25-35)-treated cells. Slight but no signi cant changes in total TG2 expression levels in cultures exposed to Aβ  were found. Indicaxanthin pre-treatment did not induce signi cant modi cations in total TG2 expression levels, when compared with the controls. When Indicaxanthin was added to OECs following exposed to Aβ(1-42) or Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) or Aβ(35-25), a signi cant reduction of total TG2 expression levels was observed, when compared with the controls. The effect of the pre-treatment of the cells with Indicaxanthin was more evident in OECs exposed to Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). In Indicaxanthin pre-treated OECs and then exposed to the fragment Aβ(35-25), a signi cant reduction of total TG2 expression levels was relieved, when compared with Aβ , Indicaxanthin alone treated cells and with the controls.
To better elucidate the effect of OEC exposure to Aβ(1-42) or Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) or Aβ  both in the absence and in the presence of Indicaxanthin on the role played by TG2, the expression levels of its isoforms were detected. Figure 7 reports the immunoblots (Fig. 7A) and densitometric analysis (Fig. 7B, C) performed on all experimental conditions. On the basis of the treatment type, different expression patterns of the isoforms were found. In controls (PBS and DMSO) both TG2 isoforms were expressed at very low levels, even if TG2-S levels were higher than TG2-L. Aβ(1-42) treatment induced a signi cant increase of both isoform expression levels, when compared with the controls. The exposure of OECs to Aβ(25-35) caused a signi cant increase of TG2-S and TG2-L, even if TG2-S levels were higher than TG2-L ones, when compared with Aβ(1-42) exposed cells and with the controls. No signi cant change in TG2-L and TG2-S expression levels in cultures exposed to Aβ(35-25) was found. When OECs were exposed to 100 µM of Indicaxanthin, a signi cant increase of the both isoforms was found, when compared with the controls. The pre-treatment of the cells with Indicaxanthin and Aβ(1-42) exposure induced a signi cant increase of TG2-L, when compared with TG2-S and with the controls. In contrast, Indicaxanthin pretreatment in Aβ(25-35) exposed cells caused a signi cant enhancement of TG2-S expression levels, when compared with TG2-L ones. Furthermore, no signi cant changes in TG2-L expression levels in Aβ(35-25) exposed OECs were observed. Surprisingly, Indicaxanthin pre-treatment caused a signi cant increase of the TG2-S expression levels, when compared with Aβ(35-25), Indicaxanthin alone treated cells and with the controls. Densitometric analysis performed for each experimental conditions, after normalization with β-tubulin, con rmed all the results. These data highlighted that Aβ treatment both in the absence and in the presence of Indicaxanthin differently modulates TG2 isoforms acting or on apoptotic pathway activation or on the cell self-renewal ability.

Caspase-3 cleavage immunolabeling
To verify the TG2-mediated apoptotic pathway in OECs exposed to Aβ(1-42) or Aβ(25-35), we evaluated the caspase-3 cleavage through immunocytochemical techniques. Figure 8 highlights caspase-3-positive OECs exposed to different conditions. In controls (PBS and DMSO) the positivity of cells for caspase-3 was almost absent. When the cells were exposed to Aβ(1-42) or Aβ(25-35), a strong activation of positive cells for caspase-3 was found, which appeared mainly localized in the cytoplasm. The effect was particularly evident in Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), that is highly toxic for the cells. In Aβ(35-25)-treated cells a light positivity for caspase-3, when compared with the controls, was found. The treatment of OECs with 100 µM of Indicaxanthin did not produce any positivity for caspase-3, when compared with the controls. In Indicaxanthin pre-treated cells and subsequently exposed to Aβ(1-42) or Aβ(25-35) a decrease for caspase-3-positive cells, which appeared at similar expression levels of the controls, was observed. A light increase of positive OECs was found in Indicaxanthin pre-treated cells and then exposed to the fragment Aβ , when compared with Indicaxanthin alone and with the controls. No speci c staining of OECs was observed in control incubation in which the primary antibody was omitted. These ndings revealed the role played by TG2 in the control of apoptotic pathway activation both in Aβ exposed OECs and Indicaxanthin pre-treated ones.
This set of experiments demonstrated that Indicaxanthin pre-treatment stimulated TG2 repair activity in OEC exposed to Aβ, activating also the stem self-renewal through the increase of cyclin D 1 expression levels and the cell positivity for Nestin.

Discussion
The aim of this study was to assess TG2 and its isoform expression levels in both OECs exposed to full native peptide of Aβ(1-42) and its toxic fragment Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Epidemiological evidences report that the effects of Mediterranean Diet "MeDi" could be an alternative prophylaxis treatment for AD [32]. In particular, it has been identi ed in Sicily an increased frequency of centenarians, a reduced occurrence of mental and cognitive diseases, when compared with other Italian or European regions [19,20]. One of the factors that could contribute to this phenomenon is the large availability of some rare speci c nutrients, largely present in some area of Sicily, as well as Indicaxanthin from Opuntia cus-indica fruit. Therefore, for the rst time, we tested the effect of Indicaxanthin pre-treatment on OECs exposed to Aβ. Since cytoskeleton plays an important role in the pathogenesis of neurodegenerative diseases, including AD [33], particular attention was focused on the effect of Indicaxanthin on some cytoskeletal proteins, such as Vimentin, GFAP, that have an important role in astrogliosis, a typical sign of AD [34]. Furthermore, the expression levels of cyclin D 1 , which is induced in stem cell reprogramming and is co-expressed with Nestin, marker of neural stem cells [26], were assessed. In addition, the effect of Aβ(1-42), Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and Aβ (35 − 25) in the absence and in the presence of Indicaxanthin was tested on cellular viability and on the activation of apoptotic pathway. Intracellular total ROS and O 2 − production was also evaluated.
The experiments were performed on OECs because they represent a glial population of olfactory system that is also involved in AD [12]. It is note that olfactory dysfunction, as well as hyposmia and olfactory memory loss, represent the early symptoms of AD [18,35,36]. Furthermore, it has been demonstrated that anterior olfactory nucleus (AON) projects to hippocampus [37] and that it is the earliest site involved in AD, associated with cell loss, the neuro brillary tangles and senile plaques [12].
Previous our researches demonstrated that TG2 is overexpressed in OECs exposed to Aβ(1-42) and its toxic fragment Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and that the treatment with some Growth Factors (GFs) was able to restore its levels to control values [18]. In particular, TG2, a calcium-dependent protein with transamidanting activity, is involved in AD, inducing the formation of insoluble amyloid aggregates that can alter the properties of several proteins [2]. TG2 activity is down-regulated in response to oxidative stress [29,30] and this effect could be related to the increase of the intracellular Ca 2+ levels due to Aβ toxicity [18]. In fact, the accumulation of extracellular protein aggregates prevalently constituted by polymeric Aβ, caused by the aberrant transamidanting activity of TG2, are also related to a dysregulation of autophagy process [38].
These conditions contribute to oxidative stress and neural cell death, in which TG2 plays a key role [30]. It has been reported that hippocampal neurons are more responsive to Indicaxanthin [39]. In particular, it has an important role in several metabolic functions both in vitro and in vivo, reducing in ammation and enhancing immune response [22,23,40].
In this study, for the rst time, we highlight that the OEC exposure to Aβ(1-42), its fragments Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and Aβ(35 − 25) induces a different expression pattern of TG2-L and TG2-S, demonstrating the opposite role played by TG2. Furthermore, we show the protective effect exerted by Indicaxanthin pre-treatment on total TG2 and its isoforms expression levels. In particular, we found that in Aβ(1-42) treated cells the two isoforms appeared at same expression levels, whereas in Aβ(25-35) exposed ones TG2-S was at higher levels than TG2-L, when compared with Aβ(1-42) exposed cells and with the controls. In OECs exposed to Aβ(35 − 25), a light modi cation between TG2-L and TG2-S expression levels was observed. The pretreatment with Indicaxanthin was able to counteract the oxidative damage following the exposure of the cells to full native peptide of Aβ(1-42) and its toxic fragment Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), restoring the expression levels of total TG2 to control values. Furthermore, CLSM analysis performed on single cell showed that TG2 in OECs pre-treated with Indicaxanthin alone was localized in the cytosol. In contrast, when cells were pretreated with Indicaxanthin and then exposed to Aβ(1-42), the protein appeared prevalently localized into the nuclear compartment. In the cells pre-treated with Indicaxanthin and then stressed with Aβ(25-35), TG2 was localized both in the cytosol and in the nucleus. Western blot analysis showed a signi cant increase in TG2-L in Indicaxanthin alone treated cells and in those then exposed cells to Aβ . This effect might be correlated to the role played by TG2 when it is localized into the nuclear compartment, in which it acts on the control of cell proliferation, regulating gene expression, cell survival and differentiation, exerting an anti-apoptotic function [10,18,41]. In OECs treated with Indicaxanthin alone and in those subsequently exposed to Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), an increase of TG2-S expression levels was observed. The effect appeared more evident in the cells pre-treated with Indicaxanthin. TG2-S, even if reduced respect to that found in Aβ(25-35) treated cells, exerts transamidanting activity and acts as apoptotic factor [6,18]. Surprisingly, Indicaxanthin pre-treatment in Aβ(35 − 25) exposed cells, induced a signi cant increase of TG2-S expression levels, when compared with Aβ(35 − 25) alone and controls. This nding might be due to the strong protective effect of Indicaxanthin, since we hypothesize that Aβ(35 − 25) fragment, even if it was reported that is not toxic [25], was able to induce a low toxicity in OECs, as relieved by a very signi cant increase of TG2-S expression levels when compared with exposed cells to Aβ (35 − 25) alone. Thus, we suppose that this effect may be due to the protective role played by TG2, which stimulates its pro-apoptotic activity, in order to remove damaged cells and to induce cellular repair.
We also found that Indicaxanthin counteracted the oxidative stress induced by Aβ, as relieved by the reduction of total ROS and O 2 − production, that appeared similar to those observed in the controls. Thus, Indicaxanthin pre-treatment, for its antioxidant properties, was able to reduce the Aβ-toxicity, oxidative stress-dependent and mitochondrial damage. In addition, Indicaxanthin, with its anti-in ammatory proprieties, decreased GFAP and Vimentin expression levels, that were enhanced in Aβ exposed OECs. These results highlighted that Indicaxanthin exerted a protective effect on reactive astrogliosis induced by Aβ responsive of cytoskeleton modi cations. Furthermore, to clarify the protective role played by TG2 in the absence and in the presence of Indicaxanthin, the levels of Nestin, marker of neural stem selfrenewal, co-expressed with cyclin D 1 , marker of cellular proliferation [26], were assessed. These results show an increase of positive cells for Nestin and cyclin D 1 expression levels, demonstrating that Indicaxanthin pre-treatment, stimulating the activity played by nuclear TG2 on stem self-renewal OEC reprogramming, that stimulates cell proliferation repairing the damage induced by Aβ. We also observed that Indicaxanthin counteracted the TG2-aberrant cross-linking activity induced by Aβ-exposure on the cells, evaluating caspase-3 cleavage, that appeared reduced following to its treatment. This effect might be correlated to the function that TG2 exerts on the apoptotic pathway, as revealed by the increase of TG2-S expression levels observed in our experimental conditions, when cells were treated with Aβ  and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) in the absence of Indicaxathin. In contrast, total TG2 did not show its opposite role on the basis of cellular localization and did not evidence the effect of Aβ both in the absence and in the presence of Indicaxanthin.
Taken together, our ndings demonstrate that Aβ stress is responsible of TG2 up-regulation [18] and its structural modi cations in two distinct conformational states with different functions [10]. In fact, when the levels of Ca 2+ are low and those of guanosin triphosphate (GTP) or guanosin diphosphate (GDP) are high, TG2-L acts as a GTPase, is involved in signaling pathway, is inactive and is present in "closed" conformation, promoting cell growth and survival (Fig. 11A). Aβ exposure of OECs, increasing intracellular Ca 2+ and decreasing GTP or GDP levels, might cause a change of TG2-L from "closed" to "open" conformation, catalytically active. In addition, Aβ treatment induced an increase of the levels of TG2-S, an alternative splice variant of TG2 lacking of the portion of the carboxyl terminal essential for the maintenance of the protein in the "closed" conformation, that is responsible of apoptotic activation and cell death. The effect is more evident when the cells were exposed to the major toxic Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fragment, that strongly enhanced intracellular Ca 2+ levels (Fig. 11B). Indicaxanthin pre-treatment prevented total TG2 over-expression induced by the OEC exposure to full native peptide Aβ(1-42) and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fragment, probably binding to Ca 2+ [40]. The signi cant increase of TG2-L isoform expression levels induced by Aβ (1-42), accompanied by the decrease of TG2-S ones, is related to the role that the protein plays into the nucleus, in which it might stimulate OEC self-renewal and the reparative effect against Aβ toxicity (Fig. 11C). Furthermore, in Aβ(25-35) exposed OECs Indicaxanthin is able to signi cantly decrease TG2-S isoform expression levels enhancing at the same time those of TG2-L. The different expression pattern of TG2 isoforms in Aβ(25-35) exposed cells in the presence of Indicaxanthin might be due to the major toxicity of the fragment that induces a major enhancement of Ca 2+ . Thus, in this conditions, the protein was able to stimulate both apoptosis and self-renewal (Fig. 11D).

Conclusions
Our ndings clearly highlighted that Aβ exposure on OECs induced an increase of TG2 and a different expression pattern of its isoforms. Furthermore, the pre-treatment of the cells with Indicaxanthin was able to decrease total TG2 expression levels, inducing a different pattern of TG2 isoforms that might be due to change of TG2 state conformation. It also reduced total ROS and O 2 − production and the expression levels of GFAP and Vimentin, inhibiting glial reactivity and the activation of apoptotic pathway induced by Aβ. Furthermore, it leads to an increase of Nestin and cyclin D 1 expression levels, stimulating OECs selfrenewal and TG2 reparative role. In addition, our data suggest that in OECs exposed to Aβ both in the absence and in the presence of Indicaxanthin might differently induce the transition of TG2 between "closed" and "open" conformation providing a new mechanism involved in the signal pathways activated by the protein in Aβ injury. Therefore, further studies need to better clarify whether Indicaxanthin plays an important role for adopting the TG2 open conformation, that has a key role in self-renewal ability of OECs, which are cells capable of expressing and releasing neurotrophic receptors, and might represent a promising tool for neural regeneration in AD.

Consent for publication
All authors have given their consent for publication.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The Authors declare that they have no con icts of interest.
Funding represent the mean ± S.D. of ve separated experiments in triplicate. *p< 0.05 signi cant differences vs controls.