key: cord-0950499-drrlez63
authors: Pattarachotanant, Nattaporn; Prasansuklab, Anchalee; Tencomnao, Tewin
title: Momordica charantia L. Extract Protects Hippocampal Neuronal Cells against PAHs-Induced Neurotoxicity: Possible Active Constituents Include Stigmasterol and Vitamin E
date: 2021-07-10
journal: Nutrients
DOI: 10.3390/nu13072368
sha: c5cf56301a91b289b54f18132a07f1bc3b5bf843
doc_id: 950499
cord_uid: drrlez63

Polycyclic aromatic hydrocarbons (PAHs) have been recognized to cause neurobehavioral dysfunctions and disorder of cognition and behavioral patterns in childhood. Momordica charantia L. (MC) has been widely known for its nutraceutical and health-promoting properties. To date, the effect of MC for the prevention and handling of PAHs-induced neurotoxicity has not been reported. In the current study, the neuroprotective effects of MC and its underlying mechanisms were investigated in mouse hippocampal neuronal cell line (HT22); moreover, in silico analysis was performed with the phytochemicals MC to decipher their potential function as neuroprotectants. MC was demonstrated to possess neuroprotective effect by reducing reactive oxygen species’ (ROS’) production and down-regulating cyclin D1, p53, and p38 mitogen-activated protein kinase (MAPK) protein expressions, resulting in the inhibition of cell apoptosis and the normalization of cell cycle progression. Additionally, 28 phytochemicals of MC and their competence on inhibiting cytochrome P450 (CYP: CYP1A1, CYP1A2, and CYP1B1) functions were resolved. In silico analysis of vitamin E and stigmasterol revealed that their binding to either CYP1A1 or CYP1A2 was more efficient than the binding of each positive control (alizarin or purpurin). Together, MC is potentially an interesting neuroprotectant including vitamin E and stigmasterol as probable active components for the prevention for PAHs-induced neurotoxicity.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, persistent environmental pollutants that result from the half-finished burning of coal, oil, tobacco, and garbage. They are integrated into every compartment of daily life as contamination in dietary ingredients, harvested agricultural production, food preparation processes, human activities, and industrial processing. Humans are exposed to the intake of PAHs through several ways including air, water, skin contact, food, and occupational settings. PAHs have been described as causes of toxicity, mutagenicity, and cancer in human [1, 2] . Normally, they contain more than two fused aromatic rings, with partial water solubility and high lipophilicity. Their high affinity for lipid-rich tissues might induce several pathological processes in the brain. The acquaintance of prenatal PAHs is related with disorders in childhood including cognition and behavioral patterns [3, 4] . PAHs can cause neurotoxicity by inducing apoptosis and cell cycle arrest, the processes requiring many proteins.

Cytochrome P450 (CYP), the enzyme found in the membrane of mitochondria and endoplasmic reticulum, plays critical functions including metabolism of vitamin D and All chemical structures of the phytochemicals were obtained from PubChem database. We minimized energy using Discovery Studio Visualizer (BIOVIA, San Diego, CA, USA) and converted the file format to the protein databank, partial charge (Q), and atom type (T) or PDBQT using AutoDockTools-1.5.6 software (The Scripps Research Institute, San Diego, CA, USA).

The X-ray crystallographic structures of CYP1A1 (PDB ID: 4I8V) [17] , CYP1A2 (PDB ID: 2HI4) [18] , and CYP1B1 (PDB ID: 3PM0) [19] were retrieved from RCSB Protein Data Bank. Protein structures were processed using the Prepare Protein Set Up in AutoDock tools and converted to PDBQT file format as the inputs for the docking study [20] .

The docking analyses were performed according to the previous report [20] . In brief, AutoDock 4.2 software package supported by Autodock tools 1.5.6 was used. Lamarckian Genetic Algorithm with default parameters was used to perform the protein-ligand interaction studies and the results were further visualized using the Discovery Studio Visualizer (BIOVIA, San Diego, CA, USA).

The data were represented as mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by a post hoc Tukey test (p value < 0.05).

The MC extract showed a reduction in cell viability in a dose-dependent manner. At 48 h, MC showed 91.44 ± 6.32% and 84.99 ± 6.33% viability for 50 and 100 μg/mL, respectively. The lowest concentrations of MC (5, 10, and 25 μg/mL), with no observed cytotoxicity, were subsequently used for further experiments.

In the case of Polycyclic Aromatic Hydrocarbons (PAHs) (Figure 1b) , the result indicated that the concentration of 5 μg/mL of phenanthrene (Phe) caused significant cytotoxicity (73.75 ± 8.81% viability) was used for further analysis. 

To evaluate the protective effects of MC on PAHs-induced cytotoxicity, cells were treated with 5 µg/mL Phe alone or in combination with different concentrations of MC (5, 10, and 25 µg/mL).

It was observed that at all the tested concentrations, MC extract was able to protect HT-22 cells from Phe-induced neurotoxicity. Co-treatment with MC showed 82.63% ± 9.68, 83.47% ± 2.76, and 81.34% ± 4.32 cell viability for 5, 10, and 25 µg/mL, respectively, against Phe ( Figure 2 ). To evaluate the protective effects of MC on PAHs-induced cytotoxicity, cells were treated with 5 μg/mL Phe alone or in combination with different concentrations of MC (5, 10, and 25 μg/mL).

It was observed that at all the tested concentrations, MC extract was able to protect HT-22 cells from Phe-induced neurotoxicity. Co-treatment with MC showed 82.63% ± 9.68, 83.47% ± 2.76, and 81.34% ± 4.32 cell viability for 5, 10, and 25 μg/mL, respectively, against Phe ( Figure 2 ). (n = 3; * p < 0.05 vs. control (0 μg/mL Phe); ** p < 0.05 vs. Phe alone; * p value was 0.000 in 5 μg/mL Phe alone and ** p values were 0.002, 0.001, and 0.006 in Phe combined with 5, 10, and 25 μg/mL MC groups, respectively; ### p value was 0.000, 0.001, and 0.000 in Phe group combined with 5, 10, and 25 μg/mL MC compared with control group, respectively.

Based on the results, 25 μg/mL MC was used as the working concentration against Phe-induced neurotoxicity models.

To examine the protective effect of MC extract on Phe-induced ROS generation, HT-22 cells were treated with 5 μg/mL Phe alone or in combination with 25 μg/mL MC. The results showed that the percentage of intracellular ROS (of control) was 130.30 ± 4.40 and 87.70 ± 4.86 in groups treated with Phe alone and combination with 25 μg/mL MC, respectively ( Figure 3 ), indicating MC extract could significantly reduce ROS' formation. ✱ ✱✱ Figure 2 . Cytoprotective effect of MC against PAHs-induced neurotoxicity. Cell viability of HT-22 cells treated with 5 µg/mL Phe alone or co-treatment with different concentrations of MC for 48 h; (n = 3; * p < 0.05 vs. control (0 µg/mL Phe); ** p < 0.05 vs. Phe alone; * p value was 0.000 in 5 µg/mL Phe alone and ** p values were 0.002, 0.001, and 0.006 in Phe combined with 5, 10, and 25 µg/mL MC groups, respectively; ### p value was 0.000, 0.001, and 0.000 in Phe group combined with 5, 10, and 25 µg/mL MC compared with control group, respectively.

Based on the results, 25 µg/mL MC was used as the working concentration against Phe-induced neurotoxicity models.

To examine the protective effect of MC extract on Phe-induced ROS generation, HT-22 cells were treated with 5 µg/mL Phe alone or in combination with 25 µg/mL MC. The results showed that the percentage of intracellular ROS (of control) was 130. 30 

To understand the ability of MC on Phe-induced neurotoxicity, the effect on apoptosis and cell cycle analysis was studied using flow cytometer. The results revealed that the percentage of apoptotic cells treated with 5 μg/mL Phe was significantly higher than in the control group. When co-treated with 25 μg/mL MC, the percentage of apoptotic cells 

To understand the ability of MC on Phe-induced neurotoxicity, the effect on apoptosis and cell cycle analysis was studied using flow cytometer. The results revealed that the percentage of apoptotic cells treated with 5 µg/mL Phe was significantly higher than in the control group. When co-treated with 25 µg/mL MC, the percentage of apoptotic cells was significantly decreased compared with 5 µg/mL Phe alone ( Figure 4) .

Furthermore, Phe could induce neurotoxicity by interfering with cell cycle distribution causing cell cycle arrest. Phe (5 µg/mL) could significantly arrest cells at G1 phase and reduce cell distribution at both S and G2/M phases compared to the control group (p < 0.05). However, MC treatment could improve cell distribution at all cell cycle phases ( Figure 5 ). Population of apoptotic cells in each group is shown as bar graphs (b). * p < 0.05 vs. control (0 μg/mL Phe); ** p < 0.05 vs. Phe alone. * p value was 0.000 in 5 μg/mL Phe alone, ** p value was 0.034 in Phe group combined with 25 μg/mL MC, and ### p value was 0.037 in Phe group combined with 25 μg/mL MC compared with control group; n = 3. ✱ Figure 4 . The anti-apoptotic effect of MC against PAHs in HT-22 cells. Apoptotic histograms from each experimental group showed (a). Population of apoptotic cells in each group is shown as bar graphs (b). * p < 0.05 vs. control (0 µg/mL Phe); ** p < 0.05 vs. Phe alone. * p value was 0.000 in 5 µg/mL Phe alone, ** p value was 0.034 in Phe group combined with 25 µg/mL MC, and ### p value was 0.037 in Phe group combined with 25 µg/mL MC compared with control group; n = 3. (b) The percentage of cell number 

Regulation of cell cycle plays a crucial role in the control of neurotoxicity. From flow cytometry analysis, it can be found that Phe causes neurotoxicity by inducing cell apoptosis and affecting cell cycle progression. However, MC extract could reverse the effect of Phe. In addition, the role of p38 in Phe-induced neurotoxicity was assessed with trolox (p38 inhibitor [21] ) being used as positive control. Trolox is a well-known, water-soluble vitamin E analogue with reported antioxidant properties against ischemia damage [22, 23] . Furthermore, stigmasterol was also used as another positive control because it was one of three major phytochemicals identified by GC-MS/MS. To approve all of these, the expression of four proteins, p38, phospho-p38, p53, and cyclin D1, was studied. The expression of cyclin D1, p53, and phospho-p38/p38 ratio was significantly increased upon treatment with 5 μg/mL Phe alone (* p < 0.05 vs. control) ( Figure 6 ). However, after treatment with 

Regulation of cell cycle plays a crucial role in the control of neurotoxicity. From flow cytometry analysis, it can be found that Phe causes neurotoxicity by inducing cell apoptosis and affecting cell cycle progression. However, MC extract could reverse the effect of Phe. In addition, the role of p38 in Phe-induced neurotoxicity was assessed with trolox (p38 inhibitor [21] ) being used as positive control. Trolox is a well-known, water-soluble vitamin E analogue with reported antioxidant properties against ischemia damage [22, 23] . Furthermore, stigmasterol was also used as another positive control because it was one of three major phytochemicals identified by GC-MS/MS. To approve all of these, the expression of four proteins, p38, phospho-p38, p53, and cyclin D1, was studied. The expression of cyclin D1, p53, and phospho-p38/p38 ratio was significantly increased upon treatment with 5 µg/mL Phe alone (* p < 0.05 vs. control) ( Figure 6 ). However, after treatment with 25 µg/mL MC, 0.1 mM stigmasterol, or 0.5 mM trolox, both p53 and phospho-p38/p38 ratios were significantly decreased (** p < 0.05 vs. 5 µg/mL Phe alone).

Nutrients 2021, 13, 2368 9 of 20 25 μg/mL MC, 0.1 mM stigmasterol, or 0.5 mM trolox, both p53 and phospho-p38/p38 ratios were significantly decreased (** p < 0.05 vs. 5 μg/mL Phe alone).

(a) 

The GC-MS/MS analysis showed the presence of 28 phytochemicals. Peaks of the phytochemical compounds in MC were shown in Figure 7 with three major constituents being n-Hexadecanoic acid (palmitic acid, 24.13%), stigmasterol (6.88%), and (Z,Z,Z)-9,12,15-octadecatrienoic acid (linolenic acid, 4.60%). Additionally, the other phytochemical constituents present were classified as (1) organic acid (34.4%), mainly composed of fatty acids including myristic acid, palmitic acid, linolenic acid, and stearic acid; (2) phytosterols (11.19%) such as stigmasterol; (3) esters of organic acid (5.04%); (4) phenol (3.5%) including salicylic acid and vitamin E; and (5) other compounds such as coumarins, terpenoid, ketones, amides, aldehydes, and alcohol. , p53 (c), and p38 and phospho-p38 (d) against GAPDH. * p < 0.05 vs. control; ** p < 0.05 vs. 5 µg/mL Phe alone. For Cyclin D1, * p = 0.002. For p53, * p = 0.002; ** p = 0.024, 0.003, and 0.001. For phospho-p38/p38 ratio, * p = 0.000; ** p = 0.000, 0.001, and 0.000, respectively. For p53 expression, there was no significant difference in all MC-, stigmasterol-, and trolox-treated groups compared with control groups. For cyclin D1 expression, ### p value was 0.007, 0.003, and 0.001 in Phe group combined with 25 µg/mL MC, 0.1 mM stigmasterol, and 0.5 mM trolox, respectively, compared with control group. For phospho-p38/p38 ratio, ### p value was 0.046 in Phe group combined with 0.1 mM stigmasterol compared with control group (n = 3).

The GC-MS/MS analysis showed the presence of 28 phytochemicals. Peaks of the phytochemical compounds in MC were shown in Figure 7 with three major constituents being n-Hexadecanoic acid (palmitic acid, 24.13%), stigmasterol (6.88%), and (Z,Z,Z)-9,12,15-octadecatrienoic acid (linolenic acid, 4.60%). Additionally, the other phytochemical constituents present were classified as (1) organic acid (34.4%), mainly composed of fatty acids including myristic acid, palmitic acid, linolenic acid, and stearic acid; (2) phytosterols (11.19%) such as stigmasterol; (3) esters of organic acid (5.04%); (4) phenol (3.5%) including salicylic acid and vitamin E; and (5) other compounds such as coumarins, terpenoid, ketones, amides, aldehydes, and alcohol. Retention time (RT), molecular formula (MF), molecular weight (MW), nature of compound, and relative concentrations (peak areas %) of these phytochemicals are given in Table 1 . Retention time (RT), molecular formula (MF), molecular weight (MW), nature of compound, and relative concentrations (peak areas %) of these phytochemicals are given in Table 1 . 

In the present-day experiment, alizarin and purpurin previously described as strong inhibitors of the activity of all the three CYP isomers were used as the positive control in the molecular docking study [24] .

Alizarin exerted the binding energy of −8.63, −8.09, and −8.17 kcal/mol for CYP1A1, CYP1A2, and CYP1B1, respectively (Tables 2-4 ). Furthermore, the binding energy of purpurin to CYP1A1, CYP1A2, and CYP1B1 was −8.84, −7.84, and −8.45 kcal/mol, respectively.

Based on the docking results (Tables 2-4) , two phytochemicals including vitamin E and stigmasterol showed outstanding inhibition against all of CYP isomers with higher binding energy compared to others.

Noticeably, the energy of both vitamin E binding to CYP1A1 (−8.91 kcal/mol) and CYP1A2 (−8.65 kcal/mol) and stigmasterol binding to CYP1A1 (−8.97 kcal/mol) was higher than both positive controls. The 3D and 2D diagrams of three phytochemicals with higher binding energy are shown in Figures 8-10 for CYP1A1, CYP1A2, and CYP1B1, respectively. Table 2 . Docking results of the phytochemical constituents with 1 CYP1A1 ( 2 PDB ID: 4I8V). Table 3 . Docking results of the phytochemical constituents with 1 CYP1A2 ( 2 PDB ID: 2HI4). Table 3 . Docking results of the phytochemical constituents with 1 CYP1A2 ( 2 PDB ID: 2HI4). 

Momordica charantia L. (MC) is a nutraceutical used in traditional medicine to relieve various ailments and inflammatory diseases including diabetes, cancer, gastric ulcer, fever, high blood pressure, worm infections, malaria, dysentery, and rheumatism therapy [25] [26] [27] . The metabolite profiling indicated that MC extract contained many functional 

Momordica charantia L. (MC) is a nutraceutical used in traditional medicine to relieve various ailments and inflammatory diseases including diabetes, cancer, gastric ulcer, fever, high blood pressure, worm infections, malaria, dysentery, and rheumatism therapy [25] [26] [27] . The metabolite profiling indicated that MC extract contained many functional components, which is consistent with the previous report [15] . It was shown that the bioactive components of MC possess various pharmacological effects including antidiabetic, immunomodulation, neuroprotection, and antitumor properties. This is the first report for MC extract showing the reduction of ROS, anti-apoptotic activity, induction of cell cycle progression, and the expression of apoptosis and cell cycle-checkpoint proteins phospho p38/p38 MAPK, p53, and cyclin D1 against Phe-induced toxicity in HT-22 cells.

Phe is a persistent environmental contaminant belonging to PAHs with applications in the making of resins and pesticides. Phe and PAHs are ubiquitously dispersed in air, landforms, water bodies and enter humans via both occupational and non-occupational exposure by breathing contaminated air, consuming PAHs-contaminated foods, dermal contact, and exposure to smokes from cigarette and open fireplaces [28] .

In response to various extracellular stimuli, the p38 MAPK kinase pathway is activated and plays a key role in learning and memory and acts as a vital factor in brain functions [29] . Consequently, deregulation of this pathway may cause neurological disorders.

Interestingly, p38 MAPK responds to various types of cellular stress such as ROS stress and cellular senescence via a series of checkpoints. In addition, p38 mediates G1/S checkpoint through cyclin D1. Normally, binding of cyclin D1 to cyclin-dependent kinase 4 and 6 (Cdk4/6) activates the complex, which is required for the switch to the S phase and cell proliferation. The p38 MAPK keeps a check on cyclin D1 function via phosphorylation, resulting in subsequent degradation. Further, p38 also activate p53 to control cell survival and induce cell apoptosis.

Treatment of Phe significantly increased ROS' generation and phosphorylation of p38 in HT-22 cells followed by upregulation of p53 and cyclin D1, causing apoptosis and cell cycle arrest.

However, MC significantly decreased p53 and the ratio of phospho-p38/p38 in extracttreated groups except for cyclin D1. Further, the p38 inhibitor trolox was used to confirm that p38 MAPK is a crucial pathway for MC treatment against Phe-induced neurotoxicity. As seen in Figure 6 , the expression of all proteins in the MC-treated group was consistent with the expression in trolox-treated group. Noticeably, cyclin D1 expression in both trolox-treated and stigmasterol-treated groups was almost similar to the Phe-treated group, which is in agreement with the previous study reporting that phytosterols could play important roles in anti-cancer activity by increasing cyclin D1 expression causing G1/S phase arrest [30] . The results indicate that the combinatorial effect of stigmasterol and trolox with other phytochemicals in MC extract could probably be more effective than in the form of individual phytochemicals.

The binding affinity of the constituents of MC extract against CYP was analyzed by in silico analysis. For favorable reaction, Gibbs free energy was found to be negative and lessened the binding energy better than the interaction between ligand and protein [31] .

These results indicate that both vitamin E and stigmasterol are more potent inhibitors of cytochrome P450 than alizarin and purpurin. In addition, several other phytoconstituents of MC extract also showed interaction indicating the combinatorial activity. The requirement of additional studies is required to verify and confirm the neuroprotective effectiveness of those constituents.

Taken together, our results showed that MC extract could provide a neuroprotective effect against Phe-induced toxicity through p38 MAPK pathway, with vitamin E and stigmasterol as the effective phytochemical constituents.

Our results reveal that MC is a medicinal plant with antioxidant properties and biologically active constituents. MC could protect cultured neuronal cells (HT22) from PAHs-induced neurotoxicity. The neuroprotective effect of MC is mediated by regulating p38/cyclin D1 checkpoint proteins-dependent cell cycle progression and inhibiting apoptosis. In addition, vitamin E and stigmasterol may be the prominent phytochemicals that, through binding with CYP enzymes, prevent CYP-induced PAHs' metabolism into toxic metabolites. Nevertheless, the bioactivities of the MC extract need to be additionally studied in a higher-model system, which may provide insight on the mechanism of MC extract against PAHs-induced neuronal cytotoxicity and possibility for the development of novel agents against environmental pollutants.

Author Contributions: Conceptualization, A.P. and T.T.; methodology, N.P. and A.P.; software, N.P.; validation, N.P. and A.P.; formal analysis, N.P.; investigation, N.P.; data curation, N.P.; writingoriginal draft preparation, N.P.; supervision, A.P. and T.T.; project administration, A.P.; funding acquisition, A.P. and T.T. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University (CU_GR_63_30_53_02), and also by the grant from Natural Products for Neuroprotection and Anti-ageing Research Unit. N.P. was supported by the Second Century Fund (C2F), Chulalongkorn University.

Institutional Review Board Statement: Not applicable.

Data Availability Statement: Not applicable.

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The authors would like to acknowledge the staff of the Division of Allergy and Clinical Immunology, Faculty of Medicine, Chulalongkorn University, for allowing the authors to access the flow cytometry facility. We would like to express our thankfulness to Panthakarn Rangsinth and Chanin Sillapachaiyaporn (Faculty of Allied Health Sciences, Chulalongkorn University) for their help in molecular docking analysis (in silico approach).

The authors declare no conflict of interest.