Evaluation of Newly Synthesized Chalcone Derivatives effect on PC12 Cells in in vitro model of the Parkinson's disease as a potential Treatment

: Chalcone is a common simple scaffold found in many naturally occurring compounds. Many Chalcone derivatives have also been prepared due to their convenient synthesis. These natural products and synthetic compounds have shown numerous interesting biological activities, such as antioxidant, anti-inflammatory, induction of apoptosis, and angiogenesis. As the second most common neurodegenerative disease after Alzheimer's disease, Parkinson’s disease is most common motor function disorder. Even though this disease is not fully understood, processes such as oxidative stress and neuronal apoptosis are largely involved in its progress. As such, antioxidants are significant agents in slowing down the process through running interference in ROS production and apoptosis. Here, we present the effect of three newly synthesized Chalcone compounds on 6-OHDA-induced cytotoxicity on the PC12 cells in Parkinson's disease model by integrating several experimental (MTT assay, ROS assay, Annexin & PI assay, Western blotting P53, Bax, Bcl2) data and validating the results based on the interactional contribution equations of these compounds obtained from previous experimental and theoretical study carried out on the molecular resonance and interactional behavior of these compounds via Linear solvation energy relationship (LSER) model and time-dependent density functional theory and conﬁguration interaction calculations. We conclude that all three Chalcones have neuroprotective activity, and presented a reduction in ROS production and an increment in cell viability in the groups treated with 6-OHDA. This effect was observed at lower concentrations for all Chalcone compounds. At higher concentrations Chalcones 1 and 2 showed cytotoxicity. However, Chalcone 3 did not show any cytotoxicity, even for high doses, which points out the therapeutic potential of this Chalcone in reducing the dopaminergic cell destruction. on the toxicity of mesencephalic cells in rats with 6-OHDA. The results


Introduction
In the year 1817, James Parkinson, presented the detailed description of the Parkinson's disease symptoms and named the disease "Shaking palsy". Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, which is resulted from the death of cells in the substantia nigra, a region of the midbrain, leading to a dopamine deficit [1], [2]. The cause of this cell death is poorly understood but the most obvious symptoms are resting tremor, bradykinesia, postural instability, and also cognitive and behavioral problems [3], [4].
The prevalence rate of Parkinson's in people over 50 years old has been reported at approximately 2%. The reactive oxygen species, such as anion superoxide, hydroxyl radicals, and hydrogen peroxide, created as the by-products during the metabolism process of oxygen, are some of the most important causes of neuronal damage. Oxidative stress-induced apoptosis is a prominent feature of age-related neurodegenerative diseases [5]- [7].
To study the molecular mechanism and the behavioral and pathological variations of this heterogeneity a variety of animal models is required, to better understand the different aspects of the disease. Three main approaches are used to model PD in experimental animals: genetics [8], viral models [9], and neurotoxins [10]- [12]. The latter, as the easiest and the less expensive method, is the mostly used technique for researchers. 6-hydroxydopamine (6-OHDA) is one of the most widely used neurotoxic compounds that can regenerate oxidative stress, neurodegeneration, neuroinflammation, and cell death; as some of PD's main cellular processes in animal models [13], [14]. To this day, there are three reported mechanisms to explain the cytotoxicity of 6-OHDA: 1) intra-or extracellular auto-oxidation of 6-OHDA that mostly leads to the generation of hydrogen peroxide and superoxide and hydroxyl radicals [15]; 2) Action of monoamine oxidase, which results in the formation of hydrogen peroxide [16], and 3) Direct inhibition in the respiratory chain complex I of mitochondrial [17]. Generation of reactive oxygen species (ROS) may be the result of these mechanisms, independently or together [18], [19]. For this reason, a vast range of studies is focused on oxidative stress and antioxidants.
Flavonoids are strong antioxidants due to the virtue of their phenolic structure as chelators and free radical scavengers [20]. The physiological effects of flavonoids are largely due to their antioxidant properties in plasma, which serve as an enzyme cleaner for ornithine carboxylase, protein kinase and calmodulin enzymes [19]. Epidemiological studies show that flavonoid intake will reduce the risk of coronary heart disease [21], stroke [22], diabetes [23], and cancer; including breast cancer [24], prostate cancer [25], lung cancer [26], colon cancer [27], and stomach cancer [28], [29].
Chalcones are considered as the precursors of flavonoids and isoflavonoids [30]. Due to the presence of unsaturated α-β, (Enone) group between the two phenyl rings, chalcones possess many biological and pharmacological properties [31]. Therapeutic applications of these materials go back to the thousand-year history of using herbs and plants for medicinal purposes [32]. The most challenging aspect of the use of chalcones is its pharmacokinetic properties and metabolic instability. Though, it appears that changes in their structure can overcome this obstacle.
Alterations of chalcones substitutes can produce different properties.
In 2008, Nobre-Junior et al. investigated the neuroprotective effects of Chalcone, isolated from myracroduonu-randeuvao, on the toxicity of mesencephalic cells in rats with 6-OHDA. The results of this study showed the neuroprotective effects of chalcones, which can reduce the oxidative stress and apoptotic damage induced by 6-OHDA. These results also showed that Chalcones has the potential to be a therapeutic aid in the treatment of neurodegenerative disorders such as Parkinson's [43].
So far, many therapeutic approaches have been suggested for Parkinson's disease, including the administration of various drugs and surgeries. Since the administration of medicines only helps to improve the quality of life and increase the functional capacity of patients, the need to employ new compounds in people with PD is sensed.
Due to the chalcones various properties, such as anti-inflammatory, anti-leishmania, antimalarial, antivirus, anti-fungal, anti-metastatic, apoptotic, anti-parasitic, anti-angiogenic, antiappetite, anti-diabetic, antioxidant, etc. effects, this study aims to investigate the effects of new Chalcone derivatives, with different properties, on the PC12 cells, as an in vitro model of the Parkinson's disease. The results from this study is evaluated based on the previous experimental and theoretical report on the molecular structure, interactional behavior, and molecular resonance of these Chalcones, using Linear solvation energy relationship (LSER) model and time-dependent density functional theory and configuration interaction calculations [44].

Results and Discussion
It is well known that interactions of chemical materials depend on many parameters, such as chemical structure, active groups and solvent media. Solvent media plays a significant role especially in biological systems due to their complex nature. In such systems, polarizability/polarity, acidity and basicity are major players, simultaneously, which add to the complexity of an already complicated behavior in these media [45]- [47].
According to our previous experimental and theoretical study carried out on the molecular resonance and interactional behavior of these newly synthetic Chalcones via Linear solvation energy relationship (LSER) model and time-dependent density functional theory and configuration interaction calculations, the interactional behavior of these Chalcone compounds are governed by specific and non-specific interactions [31], [44]. Specific interactions include hydrogen bond donor ability (acidity) and hydrogen bond acceptor ability (basicity) whereas the non-specific interactions include polarity and polarizability interactions [48].
Results of the multiple linear analyses are presented, in detail, in Ref [44], (one can refer to The media parameters representing these interactions are as follows: acidity -α; basicity -β; dipolarity/polarizability -π* (R 2 : values of the regression coefficient). These parameters are usually are values between 0 and 1, for example for DMSO, these parameters are α=0, β =0.76 and π * =1.
In a healthy biological system the solvent parameters of the whole system is in balance. However, presence of a harmful foreign compound in a biological system, like 6-OHDA in the present study, disturbs this balance and alters the interactional environment, ensuing complications, such as ROS production, P53 expression and etc. In this regard, using chemical compounds, like Chalcones, as treatments in such biological systems is also affected and controlled by these parameters (α, β, and π * ). As such, contributions of these parameters in the interaction of these Chalcone compounds defines the obtained behavioral results, which are presented in the following.

Intracellular ROS levels with all three chalcones.
Introduction of 6-OHDA to the PC12 cells results in the production of free radicals, which, as mentioned, were measured via ROS assay. This assay was done considering the effective time of 24 hours and low effective doses. All selected doses of Chalcone 1, Chalcone 2, and Chalcone 3 had significant effects with the positive control group. According to Figure 1, Chalcone 3 showed a greater ability to reduce the ROS production remarkably.
As said before, the 6-OHDA act to produce ROS through generation of hydrogen peroxide and superoxide and hydroxyl radicals, causing cell death. Treated with Chalcones 1, 2 and 3, the number of produced ROS is reduced. According to the interactional behaviour of the Chalcones, investigated before (ref 44), the dominant media parameter for Chalcones 1 and 2 is the nonspecific interactions -polarity/polarizability -and for the Chalcone 3 the main effective factor is the specific interactions -formation of complexes between hydrogen-bond donors (HBDs) and hydrogen-bond acceptors (HBAs).
Due to the high activity of Chalcones 1 and 2 in the presence of media with high polarity, a degree of interaction is observed between the 6-OHDA and Chalcones 1 and 2. These interactions not only reduce the effect of 6-OHDA and diminish its products, but also hinder the effect of 6-OHDA on the cell due to the formation of complexes and blocks between the Chalcones and the 6-OHDA that decreases the effect of 6-OHDA on the production of ROS in the cells. As seen in Figure 1 ((a) and (b)), using Chalcones 1 and 2 in cell treatment results in diminishing the ROS.
The higher the dosage of Chalcones in cells, the more interactions are occurred between the molecules and the less ROS is produced.
In the case of Chalcone 3, as mentioned before (see ref 44) the main factor in the inter/intra molecular interactions is the specific interactions -hydrogen bonding -, meaning that Chalcone 3 is significantly active in the presence of material with the capacity for specific interactions, like 6-OHDA. This leads to a high degree of interactions between these two materials that concludes in preventing 6-OHDA from generating ROS. As can be seen from Figure 1 (c), there is a significant reduction in the production of ROS as a result of treatment with Chalcone 3. This decrement is higher for Chalcone 3, in comparison to Chalcones 1 and 2 ( Figure 1(d)).

Western Blotting.
Results of the expression of P53 protein in all three Chalcones showed a decrease in this protein for all concentrations, compared to the positive control group, and as shown in Figure 2, the reduction of the expression of this protein for Chalcone 3 is higher in comparison.
For Chalcone 1, there was no change in the expression of Bax and Bcl-2 protein, whereas for Chalcone 2 an increase is observed in the expression of Bcl-2 protein at the dose of 10 μm/ml, and a reduction of the expression for the dose of 40 μm/ml. Chalcone 3 showed an increment in the expression levels of these proteins by increasing the dose.
It has been reported that 6-OHDA destroys catecholaminergic structures by the combined effect of reactive oxygen species (ROS) and quinones. It is assumed that the ROS initiate cellular oxidative stress and p-quinone mediates 6-OHDA-induced cell death. To cope with and manage various types of stress a large number of tightly regulated stress response pathways have evolved.
The P53 pathway is the primary response to stress [49]. P53, activated by external and internal stress signals, promote its nuclear accumulation in an active form. In response to stress, P53 selectively regulates the expression of its target genes, which results in cell cycle arrest, apoptosis, or senescence [50].
This effect of 6-OHDA can be seen from Figure  The interactional behavior of Chalcone 2 is similar to Chalcone 1, however, beside the polarity/polarizability, the roll of hydrogen bond acceptor ability is stronger for Chalcone 2, in comparison to Chalcone 1. As a result, in treatment with this Chalcone, there is a major interaction between the 6-OHDA and the Chalcone 2 inside the cell. Thus, the effect of 6-OHDA and Chalcone 2 in triggering the P53 is reduced, and as seen from Figure 1 the expression of P53 is significantly diminished.
For Chalcone 3, the main factor in the inter/intra-molecule interactions is the specific interactions. Treating the samples with Chalcone 3 results in a notable reduction in the expression of P53. Upon treatment with Chalcone 3, there is high interactions between Chalcone 3 and 6-OHDA, since 6-OHDA possesses a high ability for specific interactions due to its molecular structure.

Apoptosis Assessment.
To further study the effect of Chalcones on the PC12 cells and the apoptosis of cells, an apoptosis assessment was carried out. The results of this evaluation via Annexin & PI assay are shown in

Viability assay.
Evaluation of the viability of the Parkinson's cells treated with 5-10-20-40 µg/ml doses of As shown before, upon treatment with Chalcone 1, ROS assay showed a decrement in the generation of ROS (Figure 1(a)) and Western Blotting confirmed an increase in the expression level of P53 (Figure 2(c)). Consequently, the sum of paths to cell death comes down to an increment in the number of live cells for low doses. However, according to apoptosis assessment This high activity of Chalcone 3 makes this material a superb candidate as a new treatment drug for Parkinson's disease.

Materials and methods
Materials. The PC12 cell line was purchased from the Pasteur Institute of Iran. 6-Hydroxy dopamine (Sigma), Fetal bovine serum (FBS) (Gibco), dimethyl sulfoxide (Sigma) and 2-7dichlorofluorescent diacetate (DCF_DA) and Annexin & PI kit were procured from Sigma-Aldrich, and Bax, P53, Bcl-2 antibodies were purchased from Abcam. Chalcones 1, 2 and 3 were synthesized with the chemical structure as shown in Figure 8. The details of synthesis are reported elsewhere [51].

Cell culture.
Cells were cultured in flasks containing RPMI and 10% FBS medium, Penicillin/Streptomycin antibiotics, and placed in CO2 incubator with 95% humidity, at 37°C. After forming a layer on the bottom of the flask, cells were passaged thrice using Trypsin and used for experiments.

Experimental groups.
Negative control group: The group that did not receive any treatment.
Sham group: received DMSO, the solvent of 6-HODA, as well as Chalcones' solvent.
Positive control group: received a dose of 75μM, 6-HODA.

MTT assay.
MTT assay was performed to determine the effects of three new Chalcone derivatives on 6-OHDA treated PC12 cells. Cells were cultured in a 96-well plate. One day after the cells adhered to the bottom of the plate, 6-OHDA with a concentration of 75µM was added to the cells and then treated with different doses of Chalcones, at 24h and 48h. Afterward, the final volume of the columns, with said treatments, was brought up to 100µL. After the incubation was complete, 100μL of MTT was added to the wells and incubated for 2h to 4h. Following, 100 µL of DMSO was added to the wells and the plate was placed in an Elisa reader, with the wavelength of 570nm.
The optical absorption rate was calculated using the following formula.

Annexin & PI assay.
Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. Annexin & PI assay was done according to the method mentioned in the Roche catalog. Cells were cultured and treated, and after 24 hours, they were treated with Chalcone, and evaluated under an inverted microscope, then collected in separate micro-tubes on ice. Afterward, the cells were centrifuged at 2,000 rpm for 5 minutes, and the cell plaque was collected. The Annexin V and PI were added in order and in a specific time, and the apoptotic cells were detected and counted in a flow cytometer.

Western blotting P53, Bax, Bcl2.
The SDS PAGE gel was used to purify proteins of U87MG cells. The protein was extracted from the cell, and was placed on the SDS-PAGE gel. After the sample thawed, the electroplating step was performed and the gel-protein samples were transferred to the PVDF membrane, and eventually the protein samples, which were revealed on the PVDF membrane, were detected using the ECL kit. The Bcl-2 antibodies were added to the membrane. Within 24 hours, a secondary antibody was added so that the entire surface of the membrane was covered. The membrane container and secondary antibodies were placed on a slow-moving pad at ambient temperature for an hour. After the incubation time, the secondary antibodies were removed, and then washed by Tris-buffered saline (TBS) for 3 to 10 minutes.
Statistical test. To compare the mean value of data between the groups, the results were analyzed using the SPSS software via one way ANOVA, for all groups, using Tukey's test with three repeats for each test (significance level is considered to be P <0.05).

Conclusion
Chalcones; derivatives of (trans-1 and 3-diaryl-2-propene-1-one), widely found in edible plants, possess biological activities, such as; antioxidant, cytotoxicity, anticancer, antibacterial, antihistamine, and anti-inflammatory properties. With an increase in pharmaceutical applications, Chalcones and its derivatives have attracted much attention. New Chalcone compounds exhibit better and stronger biological effects, so this study aimed to investigate the effect of three new Chalcone derivatives on the cell death and oxidative stress in PC12 Parkinson model cells.
To do so, MTT assay, ROS, apoptosis, Bax, Bcl2, and P53 proteins using Western blotting were carried out. Results were analyzed via one-way ANOVA and the Duncan test less than 0.05 was considered significant (P˂0.05).
The results showed that the doses of 5-10-20-40 µg/ml of Chalcone 1 and 2, after 24 h, showed a significant increase in cell viability, compared to the positive control group (the group that received 75μM 6-OHDA), but doses of 80 and 160 µg/ml were toxic and presented a noticeable decrease in viability. There was also a significant difference in vitality gained at two-time points of 24 and 48 hours. Plus, this study showed that Chalcone 3, which according to Figure 8

Author Contributions
These authors contributed equally to this work.