Structural changes of tubulin by interacting with Δ⁹-tetrahydrocannabinol: in-vitro and theoretical studies

Abstract

Background

There is growing evidence of the contribution of microtubule dynamics to dendritic spine changes, synaptic plasticity, axonal transportation, and cell polarity. Besides, one of the well-studied effects of Cannabis on human behavior is memory disability. As Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the most pivotal chemical of Cannabis, we investigated the effect of Δ⁹-THC on microtubule dynamicity and the structural study of tubulin (microtubule monomer).

Results

Our results show that Δ⁹-THC changes microtubule dynamicity compared to the control group. The turbidity assay results demonstrated that Δ⁹-THC reduces microtubule polymerization in a concentration-dependent manner. Circular Dichroism spectroscopy also studied the structural changes of the purified tubulin, which revealed significant changes in the secondary structure of the tubulin. Furthermore, Silico studies predicted one binding site for Δ⁹-THC on β-tubulin.

Conclusions

We concluded that Δ⁹-THC could reduce the microtubule’s stability, which may conversely affect brain function by microtubule dynamic changes caused by secondary structural changes of tubulin and preventing tubulin-tubulin interaction.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12868-025-00957-5.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12868-025-00957-5.

Introduction

Microtubules are cytoskeletal filaments in eukaryotic cells, made up of α and β subunits [1], and are polarized polymeric tubes with dynamic ends [2]. Microtubules are essential for numerous aspects of the cell’s function. Cell morphogenesis, chromosome segregation, and intracellular vesicle transport are critical roles of microtubules [3, 4].

Furthermore, microtubules are critical structural and functional connectivity structures across the neural network and are involved in signal transportation along the axons [5, 6]. Dynamic microtubules are involved in dendritic spine changes and synaptic plasticity [7]. Microtubules’ motor proteins (dynein) are involved in the trafficking of intracellular subunits [8–10]. Different research groups have studied the structural changes that lead to the functional alteration of microtubules [11–13].

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD), two major cannabinoids from Cannabis sativa, influence the cytoskeleton, particularly microtubules. Research on the direct interaction between the two ingredients and tubulin is limited. However, some studies have explored the effects of cannabinoids on the cytoskeleton, particularly microtubules composed of tubulin proteins. CBD has been observed to disrupt the organization of microtubules and microfilaments in PC12 cells, leading to a reduction in cytoskeletal integrity in a dose-dependent manner [14]. cannabinoid agonist affect acetylation of α-tubulin and gene expression in the prefrontal cortex of adult mice [15].

Δ⁹-THC is one of the most critical and psychoactive ingredients of Cannabis. It also impairs several aspects of cognitive functions, with the most robust effect on humans’ short-term, episodic, and working memory [16, 17].

The negative effect of Δ⁹-THC on synaptic plasticity is well-accepted [18, 19], and numerous studies have focused on this impairment’s signaling pathways, which are poorly understood [20–23]. Daniel Jimenez et al. reported that the activation of mouse astroglia type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) disturbs glucose metabolism and lactate production in the brain. This leads to an alteration in neuronal functions [24]. Other results show that Δ⁹-THC-induced apoptosis in glioma cells may be independent of CB1 receptors and due to the THC-stimulated hydrolysis of sphingomyelin in glioma cells [25]. It has been shown that the long-term administration of Δ⁹-THC in laboratory animals impairs microtubule dynamicity [26].

According to previous studies on the direct role of microtubules in synaptic plasticity, cell structure, and the proven effects of Δ⁹-THC on the cell function, it can be presumed that Δ⁹-THC could exert its effect through changes in microtubule dynamicity along with other CB1 receptor-related mechanisms signaling pathway. To study this assumption, following our previous study of the systemic administration of Δ⁹-THC to rats [26], we examined the interaction between Δ⁹-THC and microtubule protein kinetically using a set of spectrophotometric techniques. Our results indicated that Δ⁹-THC has a binding site on tubulin and changes the dynamic behavior of the protein by inducing structural changes in the tubulin dimers.

Materials and methods

Chemicals

Δ⁹-THC (Cat# T4764) in methanol, prepared in a vehicle containing 4% DMSO and 96% saline (sterile saline 9%). Guanosine 5′-triphosphate (GTP, Cat# G0635), Piperazine-N, N′-bis (2-ethane sulfonic acid) (PIPES, Cat# P6757), EGTA (Cat# E3889), MgSO₄ (Cat# E3889), Dimethyl sulfoxide (DMSO, Cat# D2650), and other chemicals were purchased from Merck.

Animals

Twenty-five adult Wistar rats, weighing 200–250 g, were purchased from the Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. The animals were kept under 12 h light/dark periods with access to standard food and water at the Neuroscience Research Center at the Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. The animals were anesthetized with an intraperitoneal injection of ketamine/xylazine (80 mg/kg and 10 mg/kg, respectively) as previously described [27]. The brain was quickly removed, and all brain tissue was fixed in a -80 °C for the next experiments, and the remaining carcasses were disposed of at the IBB animal care center.

Rat brain tubulin extraction and purification

Tubulin was purified from the rat brain in two cycles of polymerization and depolymerization [28, 29]. For further purification, we applied cellulose phosphate column chromatography to remove MAPs for structural studies of the purified tubulins. The purity of the obtained fractions was determined by Coomassie blue staining of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein kinetics via turbidity measurement

The tubulin assembly assay was performed at 37 °C in the presence of a final concentration of 1 mM GTP solution in double-distilled water with a UV/visible spectrophotometer (at 350 nm wavelength), which was an adjustable temperature device [30]. To examine the effect of Δ⁹-THC on microtubule and tubulin assembly, the protein was pre-incubated with different concentrations of Δ⁹-THC for 2 min, and GTP was added to the solution to start the assay.

Kinetic parameters of the tubulin polymerization

We followed the polymerization assay at 350 nm, as mentioned above. Information about the nucleation, lag phase, and elongation obtained from the polymerization curve [31] was assessed. The nucleation phase was studied with two different parameters, including (i) the tenth time or “t_1/10” which refers to the necessary time to produce 1/10 of the final amount of the polymer, and (ii) parameter p, known as the number of successive steps in the nucleation process. The parameter p was used for estimating the nucleus size, which can be characterized by plotting log A_(t)/A_max against log(t) where A_(t) is the absorbance at a given time and calculating the slope of the plot [32].

We also studied the elongation phase by plotting (1- A_t /A_max) against the time, which provides a first-order rate constant of elongation (k_obs). To analyze the tubulin assembly, we incubated the tubulin purified by cellulose phosphate column chromatography. Purified tubulin (30 µM) was incubated with different concentrations of Δ⁹-THC (0–1 µM) to test the direct interaction of Δ⁹-THC with tubulin in the absence of MAPs.

Intrinsic fluorescence spectroscopy

The tendency of Δ⁹-THC to tubulins was determined by the intrinsic fluorescence of tubulins, which was detected in the presence of different concentrations (0–1 µM) of Δ⁹- THC. The emission of tubulins was detected at 295 nm excitation in the range of 300–500 nm [33]. The abovementioned experiments were carried out using the Cary Eclipse fluorescence spectrophotometer (Varian, Australia).

Circular dichroism spectroscopy (CD)

The CD spectroscopy assay was performed by a model 215 circular Dichroism (Aviv Biomedical, USA). In this procedure, the tubulin dimers were pre-incubated with different concentrations of Δ⁹-THC (0–1 µM). The far UV CD spectra were recorded from 190 to 260 nm using a 1 mm path length quartz cuvette. Moreover, the secondary structural changes in tubulin were analyzed by the CDNN program.

Molecular Docking studies

Molecular docking was performed using AutoDock tools version 1.5.6 with standard parameters. The crystal structure of tubulin was obtained from the Brookhaven Protein Data Bank (PDB entry 1JFF), and the ligand-free structure was used as the initial protein structure [34]. The ligand structure was obtained from Pub Chem (Pub Chem CID 16078). The grid box was defined (100_ 86 _74 Å) for a general pre-calculation of the interaction of Δ⁹-THC over the whole macromolecule, grid maps with a spacing of 0.375 Å in each direction, and the center of the grid was set to 19.17,0 and 0 Å. After recognizing the approximate binding site of Δ⁹-THC on tubulin, flexible docking of Δ⁹-THC over the β-tubulin binding site was performed. The grid box was defined as 40_40_40 Å, and the center of the grid was set to x = 3.641, y = -16.032, and z = 19.64. Images were created using Python Molecule Viewer (PMV), PyMol molecular viewer v.1.1, and the program Lig Plot v.1.0, which generates schematic 2-D representations of protein-ligand complexes from the PDB file input [35].

Statistical analysis

The analysis of the data obtained was performed using SPSS software version 16. One-way analysis of variance (ANOVA) was performed, and Tukey post hoc analysis was performed to compare the means of the experimental groups. All results are expressed as mean ± SEM, and differences were considered significant at P < 0.05, P < 0.01, and P < 0.001.

Results

Rat brain tubulin extraction and purification

Tubulin dimers were obtained from two cycles of assembly and disassembly from the animals’ brain extracts, followed by a purification step with a phosphocellulose column (Fig. 1). MAP-free tubulin was successfully obtained, and a high level of purity was observed by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) stained by Coomassie Brilliant blue. As shown in Fig. 1, α-tubulin and β-tubulin are two separate narrow lines in lane 2.

Fig. 1.

Coomassie brilliant blue-stained SDS-PAGE of the extracted and purified tubulin. Protein ladder (lane 1), tubulin dimers prepared from one cycle of polymerization and depolymerization of brain extract (lane 2), and purified tubulin (α-tubulin and β-tubulin) by phosphocellulose column chromatography (lane 3). The cropped ladder is used to improve the figure clarity

Δ⁹-THC significantly decreased tubulin polymerization with a significant effect on nucleation and elongation

Turbidity measurements were conducted at 350 nm to characterize the effects of Δ⁹-THC on tubulin dynamicity (Fig. 2).

Fig. 2.

Increasing the concentrations of Δ⁹-THC significantly decreased the tubulin polymerization rate and maximum turbidity at the steady state

The kinetic parameters calculated from the polymerization curve are presented in Table 1. The maximum absorbance (A_max( at the steady state decreased significantly in a dose-dependent manner in the drug-treated samples as compared to the control (df = 11, F (3,8) = 119.1, p < 0.001, Fig. 3A). Δ⁹-THC significantly increased the tenth time of polymerization in all drug-treated tubulins (df = 11, F (3,8) = 114.56, p < 0.01 in 0.2 µM and p < 0.001 in 0.6, 1 µM Δ⁹-THC samples, Fig. 3B). Parameter p increased significantly in 0.6 and 1 Δ⁹-THC (df = 11, F (3, 8) = 61.3, p < 0.001, Fig. 3C), while k_obs decreased in a dose-dependent manner in Δ⁹-THC samples (df = 11, F (3, 8) = 207, p < 0.001, Fig. 3D).

Table 1.

The polymerization and purification of tubulin depict the influence of different concentrations of Δ⁹-THC (0.2 ΜM, 0.6 ΜM, and 1 µM) on four relevant parameters: maximum absorbance (Amax), half-life (t1/10), parameter P, and the observed rate constant (k_obs). It is noteworthy that Amax is reduced by increasing concentrations of Δ⁹-THC, whereas both t_1/10 and parameter P exhibit dose-dependent enhancement. In contrast, k_obs fell off steeply with increasing Δ⁹-THC concentrations, reflecting a concentration-dependent Inhibition of the observed rate constant. This table gives a quantitative summary of the dose-response relation of Δ⁹-THC with the parameters measured, supplying information about its biochemical and kinetic action. *P < 0.05, **p < 0.01, ***p < 0.001; values represent the mean ± SEM

Δ9-THC concentration	Amax	t1/10 (minutes)	parameter p	kobs
Control	0.071 ± 0.004	0.147 ± 0.016	0.68 ± 0.03	0.94 ± 0.04
0.2 µM Δ 9 -THC	0.048 ± 0.004**	0.37 ± 0.052*	0.77 ± 0.04	0.69 ± 0.055**
0.6 µM Δ 9 -THC	0.037 ± 0.002**	1.97 ± 0.03**	1.74 ± 0.100***	0.506 ± 0.037**
1 µM Δ 9 -THC	0.020 ± 0.001***	5.65 ± 0.529**	2.99 ± 0.11***	0.15 ± 0.016**

Fig. 3.

Kinetic parameters change significantly in purified tubulin interacting with different concentrations of Δ⁹-THC. (A) The maximum absorbance decreased with increasing concentrations of Δ⁹-THC. (B) The parameter t_1/10 increased with higher concentrations of Δ⁹-THC, with the highest t_1/10 observed at a concentration of 1 µM. Significant differences in t1/10 were observed between 0.6 µM and 0.2 µM concentrations (p < 0.001). (C) Δ⁹-THC significantly increased parameter p in a dose-dependent manner. The highest parameter p was observed significantly at a concentration of 1 µM (p < 0.001). (D) The observed rate constant (k_obs) for purified tubulin was significantly reduced in Δ⁹-THC-treated tubulin compared with the control (p < 0.001). The lowest k_obs value was observed at a concentration of 1 µM (p < 0.05). The values represent the mean ± SEM. ***p < 0.001

Δ⁹-THC interacts with the tubulin dimer through one binding site

Our results from the fluorescence titration indicated that Δ⁹-THC has a quenching effect on tubulin. The fluorescence emission intensity of tubulin decreased rapidly in the presence of increasing concentrations of Δ⁹-THC, an interaction between tubulin and Δ⁹-THC as shown in Fig. 4A. The number of binding sites and the binding constant were calculated by plotting log [ (F0–F)/F] against log [Δ⁹-THC] (Eq. 1), where F0 and F are the fluorescence intensities before and after the addition of the ligand, K_a is the binding constant, and [Q] is the concentration of the quencher.

Fig. 4.

Intrinsic fluorescence binding of Δ⁹-THC to tubulin provides insights into the interaction mechanism between Δ⁹-THC and tubulin dimers. (A) Fluorescence quenching spectra of tubulins in the presence of various concentrations of Δ⁹-THC. The data obtained from the fluorescence intensity show that Δ⁹-THC interacted with tubulin dimers using a quenching mechanism, suggesting a direct binding interaction. (B) The numbers of binding sites (n = 1) of tubulin were obtained from the graph slope, and log Ka was derived from the plot of log [F0- F /F] against log [THC] (Ka = 3.16 × 10⁴). This confirms a specific and measurable binding affinity between Δ⁹-THC and tubulin. (C) Plotting F0/F against [THC] suggests a static interaction between tubulin and Δ⁹-THC with a quenching constant of 2.59 × 10¹⁴ L.s.mol^− 1, further supporting the presence of a stable complex formation

This plot demonstrated that one binding site exists for Δ⁹-THC on the tubulin dimer and the binding constant is equal to 3.16 × 10⁴ L. mol^− 1, indicating the tubulin’s high affinity for Δ⁹-THC as shown in Fig. 4B.

1

The Stern-Volmer equation (Eq. 2) was used to define the quenching constant. Kq, Ksv, and τ₀ are the biomolecular quenching rate constants, the Stern–Volmer quenching constant, and the average lifetime of the biomolecule without the quencher (τ0 = 10^− 8 s), respectively.

This plot indicated that the quenching constant was equal to 2.59 × 10¹⁴ L. s. mol^− 1, suggesting a static interaction between Δ⁹-THC and tubulin (Fig. 4C). The fluorescence quenching mechanism is dynamic or static [36]. According to our data, the plot of Stern − Volmer is linear and the obtained K_q (2.59 × 10¹⁴ M^− 1 S^− 1) is larger than 2 × 10¹⁰ M^− 1 S^− 1 verifying that the main fluorescence quenching mechanism is likely static (Table 2).

Table 2.

Biophysical parameters of Δ⁹-THC interaction with tubulin

Ka (M− 1)	Ksv(M− 1)	Kq (M− 1 S− 1)	n
3.16 × 104	2.59 × 106	2.59 × 1014	1.053

2

CD spectroscopy demonstrated a notably decreased α-helix in proportion to other secondary structures

The CD data showed alterations in the secondary structure of tubulin (Fig. 5; Table 3). Decreasing intensity and broadening of both the positive and the negative CD bands led to a shift in the x-axis toward higher wavelengths and exhibited a slight red shift (Fig. 5).

Fig. 5.

Effect of Δ⁹-THC on the secondary structure of tubulin. The secondary structural change of the tubulin dimers in the presence of various concentrations of Δ⁹-THC shows a reduction in the negative ellipticity at 220 nm

Table 3.

Alterations of the tubulin secondary structures in the presence of different concentrations of Δ⁹-THC. The decrease in α-helix structure was significantly more in the drug-treated groups than in the control group. The increase in β-turn, β-antiparallel, β-parallel, and the random coil was significantly observed in the drug-treated groups compared with the control group. *p < 0.05, **p < 0.01, ***p < 0.001; Values represent the mean ± SEM

Δ9-THC concentration	α-helix (%)	β-turn (%)	β-antiparallel (%)	β-parallel (%)	Random coil (%)
Control	52.6 ± 1.447	12.27 ± 0.688	3.93 ± 0.497	5.2 ± 0.251	21.73 ± 0.933
0.2µM	42.75 ± 1.824*	14.77 ± 0.95	6.1 ± 0.264**	6.76 ± 0.145**	28.53 ± 0.78**
0.6µM	30.47 ± 0.913***	15.20 ± 1.035**	8.13 ± 0.185***	8.76 ± 0.12***	31.97 ± 1.126**
1µM	24.02 ± 2.170***	19.07 ± 0.548**	10.93 ± 0.348***	10.67 ± 0.176***	36.73 ± 1.146**

The increase in β-sheet structure and decrease in α-helix coils were significantly observed in the drug-treated groups compared with the control sample (Table 3). α-helix secondary structure decreased significantly in Δ⁹-THC treated groups compared with the control (df = 11, F (3,8) = 58.9, p < 0.001). On the contrary, β-turn (df = 11, F (3,8) = 11.48, p = 0.0029), β-antiparallel (df = 11, F (3,8) = 75.12, p < 0.001), β-parallel (df = 11, F (3,8) = 174.1, p < 0.001), and random coil (df = 11, F (3,8) = 39.11, p < 0.001) increased significantly compared with the control group (Table 3).

Docking studies

Molecular docking provides insight into the binding of Δ⁹-THC to tubulin. The conformer with the lowest energy in the cluster, with 20 members of 400 conformers, was selected. According to AutoDock calculations, the binding energy of this structure was − 7 kcal/mol. A hydrogen bond between the side chain of His 227 and the OH group of the cyclohexane ring of Δ⁹-THC is orienting the ligand molecule to occupy the binding site. The results show that van der Waals interactions, hydrogen bonds, and hydrophobic interactions play a significant role in the binding of Δ⁹-THC to a hydrophobic pocket in β-tubulin. Based on the modeling studies, the dominant interaction is hydrophobic. As shown in Figs. 6A and B, the hydrophobic site of β-tubulin interacts with Δ⁹-THC via Arg359, Pro358, leu228, cys211, leu215, leu225, leu273, Thr 274, Arg 282, leu 361, leu207, and Gly360. According to the first docking over the entire tubulin, there is no binding site on α-tubulin, and all the full binding sites are on β-tubulin (Fig. 7). Δ⁹-THC mainly binds close to the GTP binding site on β-tubulin.

Fig. 6.

View of the tubulin-THC interaction. (A) The diagram plotted by the Lig plot shows the interaction between Δ⁹-THC and tubulin. Hydrophobic interactions play a significant role; one hydrogen bond (His227, with THC) is shown in green in the diagram. (B) The image was obtained using PyMol V1.1

Fig. 7.

Stereo image presentation of the binding site of tubulin with Δ⁹-THC. Critical residues are shown in the zoom box, and THC is shown in green

Discussion

In this study, we proposed that Δ⁹-THC induces significant changes in the microtubule dynamicity resulting from the structural changes in the tubulin dimes.

Dynamic microtubules are involved in synaptic plasticity in the brain [7, 37]. Therefore, microtubule dysfunction may affect synaptic plasticity and spatial memory. Uv-visible spectroscopy represented that Δ⁹-THC exerts its effect through changes in microtubule dynamicity as well as, and microtubule dysfunction may affect synaptic plasticity as one, among others, possible mechanisms.

Studying the kinetics of the polymerization assay provided interesting data, increasing the tenth times (t_1/10) demonstrates a more extended lag phase in Δ⁹-THC incubated samples compared to the control group. This suggests that the necessary time for tubulin dimer assembly in nucleation increased by Δ⁹-THC. In addition, the p parameter increased by increasing the Δ⁹-THC concentration, implying that Δ⁹-THC disturbs the lag phase and nucleation, while the k_obs value decreased strongly in a dose-dependent manner, showing that increasing Δ⁹-THC concentration has a critical role in reducing the elongation process of polymerization. Therefore, Δ⁹-THC suppresses elongation. It seems that Δ⁹-THC decelerates the nucleation and elongation process and reduces the final amount of the protein polymer.

The fluorescence intensity of the tubulin dimers decreased in the presence of Δ⁹-THC. As the Δ⁹-THC did not have any intrinsic fluorescence intensity, the fluorescence quenching seems to be the interactions between Δ⁹-THC and tubulin dimers, which is confirmed by previous studies [26]. The fluorescent studies and Stern-Volmer plot in this experiment show that there is a single stable site of Δ⁹-THC- tubulin interaction. The reduction of the fluorescence intensity originated from one of the static quenching mechanisms. Furthermore, the number of tubulin-binding sites (n) was calculated to be one. Three significant classes of tubulin-binding drugs have been identified: I; Colchicine binding ligands; ii, the Vinca domain drugs; and iii; Texans and Epothilone family. Vinca domain inhibitors, next to the nucleotide-binding region, inhibit polymerization by preventing nucleic acid exchange [38, 39]. Our computer-based study demonstrated one binding site for Δ⁹-THC on tubulin, which is around the Vinca region and makes a hydrogen bond with His 227 and electrostatic and hydrophobic interactions with hydrophobic amino acids on β-tubulin and might inhibit the GDP-GTP exchange and prevent microtubule polymerization.

Δ⁹-THC is a hydrophobic structure that contains three cyclic structures: a phenol ring, a pyran ring, and a cyclohexane ring, causing rigidity in the tubulin’s structure and the alkyl moiety [40]. Adding Δ⁹-THC to the tubulin solution might induce the exposure of hydrophobic amino acids normally buried inside α- helix secondary structures.

Our CD spectroscopy results elucidated that the proportion of α-helix structures was reduced in Δ⁹-THC treated compared to the control. Additionally, the proportion of β-sheets and other structures was increased in Δ⁹-THC treated in comparison with the control. This result is notably similar to previous in vitro studies [26]. The addition of Δ⁹-THC to the protein solution induces the secondary structures of the protein to transit to β- sheet and random coil structures, aiming to expose more hydrophobic amino acids. Thus, we predict that Δ⁹-THC can bind to tubulin dimers through hydrophobic interactions.

Conclusions

In conclusion, our study reveals that Δ⁹-THC significantly disrupts microtubule dynamics by altering the structural properties of tubulin dimers, which may impair brain function. Δ⁹-THC extends the lag phase of tubulin assembly, reduces polymerization, and interacts with tubulin near the Vinca domain. Additionally, Δ⁹-THC induces a shift from α-helix to β-sheet and random coil structures, exposing the hydrophobic amino acids. These findings highlight the molecular mechanisms through which Δ⁹-THC affects neuronal functions. While this study provides insights into the direct interaction of Δ⁹-THC with tubulin, in vitro binding assays may not fully replicate cellular conditions. Our future studies will focus on cell culture studies to validate these findings.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

M.M., Gh.D., J.M., H.R., R.Gh., and R.H. designed the research. M.M. collected the experimental materials. M.M., Gh.D., and R.Gh conducted experiments. M.M. and Gh.D. wrote the manuscript. All authors have read and approved the final manuscript submission.

Funding

This research was supported by the Institute of Biochemistry and Biophysics, University of Tehran, and a research grant from the Amol University of Special Modern Technologies, Amol, Iran. The funding body did not participate in the design of the study, data collection, analysis, interpretation, or writing of the manuscript.

Data availability

The data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

We have complied with all relevant ethical regulations. In this research, all experiments were performed according to the Bioethical Committee of the Amol University of Special Modern Technologies with the code IR.ASMT.REC.1403.011 (https://ethics.research.ac.ir/EthicsProposalView.php?&code=IR.ASMT.REC.1403.011).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.