Neuroprotective Effect of Selected Phytocandidates against Neurotoxicity Induced Human Neuroblastoma PC-12 and SHSY5Y Cell Lines – An In Vitro Model for Brain Aging

Received: 31-Aug-2022, Manuscript No. jdar-22-76611;;Accepted Date: Sep 21, 2022; Editor assigned: 02-Sep-2022, Pre QC No. jdar-22-76611 (PQ); Reviewed: 16-Sep-2022, QC No. jdar-22-76611; Revised: 21-Sep-2022, Manuscript No. jdar-22-76611 (R); Published: 28-Sep-2022, DOI: 10.4303/jdar/236199

Abbreviations

(HRMS) High-Resolution Mass Spectrometry, (PC-12) Pheochromocytoma, (RPMI) Roswell Park Memorial Institute, (DMEM) Dulbecco’s Modified Eagle Medium, (FBS) Foetal Bovine Serum, (MTT) 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-2H-tetrazolium bromide, (KPD) Kaempferide, (NRG) Norbergenin, (Aβ) Amyloid-β protein, (6-OHDA) 6-hydroxy dopamine, (DMSO) Dimethyl sulfoxide, (ROS) Reactive Oxygen Species.

Introduction

Brain aging is a neurodegenerative disease, which is correlated with cognitive decline, dementia, and loss of locomotor activity due to changes in the pathophysiological process of the brain [1,2]. Neurodegeneration is mainly due to excess generation of Reactive oxygen species in the brain, which affects the organelles of Mitochondria formation of amyloid-β plaques due to the aggregation of amyloid-β proteins in the brain. Statistics state that almost 46.8 million people are affected by this disease at the age of ≥ 65 years [3,4]. It increases the implications on worldwide medical management. The outset of neurodegeneration was procrastinated and averted by the use of natural compounds which exerts anti-oxidant activity and neuroprotection [5]. Phytocandidates like Prunetin, Kaempferide, Piceatannol, Norbergenin, and Isookanin were exerting potent antioxidant activity. Dietary flavonoids are natural antioxidants mostly present in fruits and vegetables. Recent studies mainly concentrate on the development of natural immunity and antioxidant activity which can prevent oxidative stress. Natural products are promising therapy for age-related disorders like Brain aging, Alzheimer’s, Parkinson’s, and multiple sclerosis [6,7]. Prunetin is an O-methylated isoflavone, one group of flavonoids. It exhibits antioxidant, antihyperlipidemic, anti-inflammatory, and proteolytic activity [8,9]. Kaempferide is a mono methoxy flavone. KPD is an O-methyl derivative of Kaempferol. It is a natural compound present in Alpinia conchigera, chromolaena odorata, Syzygium aromaticum, Prunus domatica, Citrus X paradisi, and in several herbs and spices [10]. KPD has Anti-oxidant and Anti-hypertensive activity. Recent studies revealed that it shows action against SARS-Cov-2 Nucleocapsid Phosphoprotein[11]. Norbegenin is an O-methylated derivative of bergenin.It is found in Ardisia Sanguinolenta, Ficus racemosa, Mallotus japonicus etc.,. It shows anti-oxidant activity, acts against lung carcinoma, and inhibition of adrenal tyrosine hydroxylase in in vitro studies [12]. Isookanin is a biological Product present in Canarium album, Acacia melanoxylon, Bidens pilosa L, and other organisms. It shows anti-oxidant activity and anti-inflammatory and anti-viral activity [13]. Piceatannol is a natural stilbene. It is a polyhydroxylated stilbene. It is a potent bioactive stilbene that was found in berries, Vitis amurensis, smilax braceteata, and other organisms. It is an analog of resveratrol. Reported activities of piceatannol are anti-cancer activity with less cytotoxicity, hepatoprotective activity, anti-fibrotic activity, and hypoglycemic activity [14,15]. Natural products have been most widely used all over the world. In neurodegenerative diseases to enhance cognitive ability and memory, we use herb alternatives. Because it proved to get relief from the diseases with fewer side effects. Mainly, plant derivatives have more safety, potency, and acceptability with fewer side effects. Identification and preparation of medicinal plants are required for future therapy. The main scope of natural products is the antioxidant activity by decreasing oxidative stress in living cells. We speculate on the function of selected phytocandidates for neuroprotection because oxidative stress and amyloid beta generation are considered to be the major cause of neurodegeneration [16]. in vitro studies were performed to evaluate the antioxidant and neuroprotective activity of selected phytocandidates by using Pc12 and SHSY5Y human neuroblastoma cell lines.

Methodology

Structural confirmation using 1H-13C-NMR and HRMS analysis: All molecules Prunetin, Kaempferide, Piceatannol, Norbergenin, and Isookanin were structurally proved by 1H-13C-Nuclear Magnetic Resonance (NMR) (were represented in results section Figures 1 to 15) and High-resolution Mass Spectrometry (HRMS) analysis reports were represented in 3.1 methodology section.

Figure 1: Represents ¹H-NMR of Prunetin.

Figure 2: Represents ¹C-NMR of Prunetin.

Figure 3: Represents HRMS Spectra of Prunetin.

Figure 4: Represents ¹H-NMR of Kaempferide.

Figure 5: Represents ¹C-NMR of Kaempferide.

Figure 6: Represents HRMS Spectra of Kaempferide.

Figure 7: Represents ¹H-NMR of Piceatannol.

Figure 8: Represents ¹C-NMR of Piceatannol.

Figure 9: Represents HRMS Spectra of Piceatannol.

Figure 10: Represents ¹H-NMR of Norbergenin.

Figure 11: Represents ¹C-NMR of Norbergenin.

Figure 12: Represents HRMS Spectra of Norbergenin.

Figure 13: Represents ¹H-NMR of Isookanin.

Figure 14: Represents ¹C-NMR of Isookanin.

Figure 15: Represents HRMS Spectra of Isookanin.

Cell culture

The experiment was performed using PC-12 and SHSY5Y neuroblastoma cell lines, which were acquired from Sigma Aldrich, USA.PC-12cell lines were maintained in RPMI1640 medium and SHSY5Y were maintained in DMEM and both were supplemented with 10% Foetal Bovine Serum, 100 μg/mL streptomycin, and 100 IU/mL penicillin. Both the cells were kept at 37 ^°C in a 5% CO₂ humidified atmosphere. PC12 cells were cultured for 5 days after seeding and then treated with mouse nerve growth factor (mNGF) and medium (1:100) for differentiation into neuronal-type cells. The culture medium was changed every second day. The cells were treated with routine enzymatic digestion and passage. Cultured cells Figure 1 were then counted by using a haemocytometer.

Cell viability assay

To study the cytotoxicity effects of phytocandidates (Prunetin, Kaempferide, Piceatannol, Norbergenin, and Isookanin), PC12 and SHSY5Y cells were treated with concentrations ranging from 5-100 μM for 72 hrs exposed to the MTT assay. Cells were plated at a density of 3500 and 5000 cells per well in 96-well culture plates. After overnight maturation, the medium was restore with fresh media, and the compounds were added to attain desired concentrations. Following a 24 hr incubation, all groups were treated with the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to determine cell viability. MTT (5 mg/mL) was added at 20 μg per well to both cells and incubated for 4 hrs. Next, the growth medium was removed from the culture plates, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Multiwell Microtitre Spectrophotometer (Thermo ScientificTM, MultiskanTM FC Microplate Photometer 51119000, Massachusetts, USA) was used for quantitative analysis. The percentages of viable cells are indicated in Figures 16-18. based on the survival of the control group computed using absorbance intensities. Each experiment was replicated three times.

Figure 16: Culture of SHSY5Y (a) and PC-12 cells.

Figure 17: Cellular cytotoxicity of Phytocandidates against PC-12 cells.

Figure 18: Cellular cytotoxicity of Phytocandidates against SHSY5Y cells.

Neuroprotective effects of phytocandidates

To determine the neuroprotective effects of phytocandidates, against SHSY5Y cells (Figure 20), the cells were treated in the range of 0 to 50 μM for 24 h, and were further subjected to treatment with 2 μM β-amyloid (Aβ) for 48 h before the MTT assays. To analyze the neuroprotective effect, the PC-12 cells (Figure 19) were pre-treated with test samples for 24 h, followed by exposure to 6-hydroxydopamine (6-OHDA) for an additional 48 h, and the cells were analyzed using an MTT assay. After treatment, the cells were treated with 5 mg mL-1 MTT for 4 hrs at 37 ^°C. The neuroprotective effect of Phytocandidates against SHSY5Y cells and PC-12 cells was noted down. After the media were carefully removed, 100 μL DMSO (≥99%) was added to dissolve the formazan crystals formed. The absorbance was measured at 570 nm using a microplate reader (TECAN, Switzerland). Control consists of cells treated with 0.15% DMSO (vehicle control, VC) and cells treated with 2 μM Aβ without prior treatment with test compounds (Aβ control), and cells treated with 10 mM 6-OHDA without prior treatment with test compounds (OHDA control) was graphically represented in Figure 21. EC50 and EC100 of Phytocandidates against SHSY5Y and Pc-12 cells was represented in Figures 22 & 23.

Figure 19: Cell viability of various cell controls over the neurological conditions.

Figure 20: Neuroprotective effects of Phytocompounds against SHSY5Y cells.

Figure 21: Neuroprotective effects of Phytocompounds against PC-12 cells.

Figure 22: EC₅₀ of Phytocompounds against SHSY5Y and PC-12 cells.

Figure 23: EC₁₀₀ of Phytocompounds against SHSY5Y and PC-12 cells.

Anti-apoptotic study

To understand the anti-apoptotic effect of selected phytocandidates, the cells (2 mL) at a density of 5 x 10⁴ were grown in 40-mm Petri dishes and allowed to attach for 24 hrs. For SHSY5Y cells, cells were treated with the EC100 of KPD and NRG for 24 hrs and were further subjected to treatment with 2 μM β-amyloid (Aβ) for 48 hrs before the apoptotic analysis (Figures 24 & 25). The PC-12 cells were treated with KPD and NRG at EC100 for 24 hrs, followed by exposure to 6-hydroxydopamine (6-OHDA) for an additional 48 hrs, and the cells were examined using an MTT assay (Figures 26 & 27). Cells were taken after the various treatment intervals and centrifuged at 1000 g for 10 min. The supernatants were resuspended in Annexin V binding buffer after being washed in 1% PBS. The supernatants from the 1000 g for 10 min centrifugation of the cells were discarded. The cell extracts were mixed with 100 litres of annexin V binding buffer, 5 litres of annexin V Alexa Fluor 488, and left to incubate for 15 minutes in the dark. They were then given PI (4 L) diluted in 1x Annexin V binding buffer (1: 10) and allowed to incubate for 15 min at room temperature in the dark. The cells stained with Annexin/ PI were washed with 500 L of Annexin V binding buffer. The cells stained with Annexin/PI were washed with 500 L of Annexin V binding buffer. On a Becton Dickinson FAC Scan equipment (BD Biosciences Pharmingen, San Diego, CA, USA) outfitted with a 488 nm argon laser, annexin/PI was analysed in accordance with the procedure previously reported. Cell Quest Pro software was used to gather and analyse a minimum of 10,000 cells per sample.

Figure 24: Flow cytometric analysis of Apoptosis in SHSY5Y cell lines.

Figure 25: Apoptotic cellular distribution in SHSY5Y cells over the treatment.

Figure 26: Flow cytometric analysis of Apoptosis in PC12 cell lines.

Figure 27: Apoptotic cellular distribution of PC-12 cells over the treatment.

Scavenging activity

To examine the outcome of KPD and NRG on the production of cellular reactive oxygen species induced by Aβ in SH-SY5Y cells and 6-OHDA in PC-12 cells, were pre-treated with KPD and NRG at EC100 for 24 hrs before the addition of 2 μM Aβ in SHSY5Y cells and 10 mM of 6-OHDA in PC-12 cells for another 48 hrs. The effect of KPD or NRG on Aβ/6-OHDA induced production of reactive oxygen species was assessed using the DCFDA-cellular reactive oxygen species detection assay kit. The treated cells were stained with DCFDA solution for 45 min at 37 ^°C. The fluorescence at (Ex/Em = 485/535 nm was measured using a microplate reader. Percentage inhibition of reactive oxygen species over treatment of KPD and NRG in SHSY5Y and PC-12 cells were represented respectively in Figures 28 & 29.

Figure 28: % ROS content over treatment of KPD and NRG in SHSY5Y cells.

Figure 29: % ROS content over treatment of KPD and NRG in PC-12 cells.

Caspases

3/7 and 9 activities in SHSY5Y and PC-12 cells: The estimation of caspase-3/7 was carried out according to the manufacturer’s instructions. Briefly, 100 μL of 5 x 10⁴ cells/mL were seeded in white-walled 96-well microplates and incubated for 24 h. The cells were treated with the KPD and NRG at a concentration of EC100 for 24 hrs followed by treatment with 2μM Aβ and 10mM 6OHDA in SHSY5Y and PC-12 cells for 48 hrs respectively. After the treatment, an equal volume of Caspase-Glo 3/7 reagent was added and stirred for 30 seconds. After an hour of incubation, the luminescence signal was recorded using the GloMax-Multi Detection System (Promega, USA). The same procedure as above was used to treat the cells in order to evaluate caspase-9 (represented in Figure 30). Control cells included SHSY5Y and SHSY5Y A, PC-12 control, and PC-12 6OHDA. Interpretation: ¹H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 9.56 (s, 1H), 8.33 (s, 1H), 7.43 – 7.37 (m, 2H), 6.90 – 6.84 (m, 2H), 6.44 – 6.39 (m, 2H), 3.79 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 181.58, 165.03, 161.64, 157.63, 157.11, 152.67, 130.02, 129.94, 125.10, 122.51, 114.98, 104.89, 97.79, 92.23, 59.93, 55.31; HRMS: HRMS of C16H12O5 for the determination of (M+H)+ is 285.0757 (Found); 285.0751 (Calculated).

Figure 30: Caspases 3 and 9 expressional analysis over the treatment.

Kaempferide

1H-NMR of Kaempferide

Interpretation: ¹H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H), 12.24 (s, 1H), 11.69 (s, 1H), 7.74 – 7.67 (m, 2H), 7.02 – 6.96 (m, 2H), 6.27 (d, J = 2.1 Hz, 1H), 6.21 (d, J = 1.8 Hz, 1H), 3.76 (s, 3H); 13C NMR (125 MHz, DMSO- d6)) δ 177.04, 164.48, 160.34, 160.19, 157.12, 147.97, 136.09, 129.12, 129.05, 122.38, 113.71, 102.56, 98.93, 93.75, 54.70; HRMS: HRMS of C₁₆H₁₂O₆ for the determination of (M+H)+ is 301.0701 (Found); 301.712 (Calculated).

Interpretation: ¹H NMR (500 MHz, DMSO-d6) δ 9.27 (s, 2H), 7.10 (d, J = 15.6 Hz, 1H), 7.06 – 7.01 (m, 1H), 6.90 (d, J = 2.1 Hz, 1H), 6.86 – 6.78 (m, 3H), 6.44 (d, J = 2.1 Hz, 2H), 6.17 (t, J = 2.1 Hz, 1H), 4.94 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 158.16, 147.16, 146.40, 140.12, 130.85, 127.58, 127.41, 119.56, 115.73, 113.33, 105.29, 101.84; HRMS: HRMS of C₁₄H₁₂O₄ for the determination of (M+H)+ is 245.0813 (Found); 245.0808 (Calculated).

Interpretation: ¹H NMR (500 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.63 (s, 1H), 7.21 (s, 1H), 7.00 (s, 1H), 5.35 (d, J = 6.2 Hz, 1H), 5.33 – 5.26 (m, 1H), 5.02 – 4.96 (m, 1H), 4.34 (d, J = 4.8 Hz, 1H), 4.07 – 3.99 (m, 2H), 3.85 – 3.71 (m, 3H), 3.60 – 3.53 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 164.97, 146.31, 142.85, 139.94, 116.13, 113.79, 109.77, 81.72, 79.89, 74.17, 72.84, 70.85, 61.18; HRMS: HRMS of C₁₃H₁₄O₉ for the determination of (M+H)+ is 315.0715 (Found); 315.0710 (Calculated).

Interpretation: ¹H NMR (500 MHz, DMSO-d6) δ 8.21 (s, 1H), 8.01 (s, 1H), 7.60 (d, J = 9.4 Hz, 1H), 7.23 (s, 1H), 6.93 (d, J = 2.5 Hz, 1H), 6.91 – 6.86 (m, 1H), 6.82 (dd, J = 9.3, 6.8 Hz, 2H), 5.59 (dd, J = 6.4, 5.5 Hz, 1H), 5.39 (s, 1H), 3.24 (dd, J = 16.8, 5.8 Hz, 1H), 2.99 (dd, J = 16.8, 5.8 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 192.60, 150.78, 149.55, 145.15, 144.89, 134.40, 131.07, 120.01, 118.44, 115.65, 114.63, 114.10, 108.88, 79.74, 43.89; HRMS: HRMS of C₁₅H₁₂O₆ for the determination of (M+H)+ is 289.0712 (Found); 289.0633 (Calculated).

Statistics

All values are given as mean ± S.D.For the examination of variations between variously treated cells, GraphPad Prism 5.0 software was utilised after repeated measures ANOVA. A Student’s paired t-test was used to examine tests on caspase-3 and caspase-9 activation (GraphPad Prism).

Discussion

Brain aging is the most common neurodegenerative disease which declines cognitive ability. It affects the people in developing countries, including India [17,18]. Flavonoids are natural plant constituents, which are promising molecules for enhancing therapeutic potential [19]. In this study, we reveal that the Phyto candidates exert a protective effect against neurodegeneration induced by Aβ and 6-OHDA in cell lines. Phytocandidates Prunetin, Piceatannol, Kaempferide, Norbergenin, and isookanin were structurally confirmed by 1H-13C-NMR and HRMS analysis. Among the five molecules, four are flavonoids one is stilbene i.e., piceatannol. All these phytocandidates are soluble in DMSO. Cytotoxic effect was assessed for these molecules by cell viability test using MTT reagent [20]. Scientific evidence suggests a clear relation between oxidative stress and neurodegeneration [21]. Neurodegenerative diseases like Alzheimer’s, and Parkinson’s mainly affect cognition and memory [22,23]. Various in vivo studies were reported on stress-induced and streptozocin-induced neurodegenerative diseases that were attenuated by natural molecules with potent anti-oxidant activity [24]. Based on this evidence, select different natural flavonoids to study the protective activity of the neurons in the brain in in vitro studies. The current in vitro study spotlight the Neuroprotective effect of phytocandidates against the degenerative effect of Aβ and 6-OHDA in SHSY5Y and PC12 cell lines mediated through anti-apoptotic, free radical scavenging activity and caspase enzyme inhibition. SHSY5Y cell lines incubated with Aβ for 48 hrs lead to the accumulation of Aβ proteins and reactive oxygen species generation in vitro. PC12 cell lines exposed to 6-OHDA [25,26] for 48hrs lead to neurodegeneration by apoptosis and generation of free radicals in vitro. Many researchers proved that Aβ and 6-OHDA treatment causes oxidative stress and mitochondrial dysfunction which leads to apoptosis. Aβ causes neurotoxicity by the formation of neurofibrillary tangles of amyloid proteins causing degeneration [27,28] while 6-OHDA affects in two ways the inhibition of mitochondrial respiratory enzymes and excess generation of free radicals leads to cell death. Aβ and 6-OHDA induced neurodegenerative effect was diminished by selected phytocandidates in both the cell lines. Mainly Kaempferide (KPD) and Norbergenin (NRG) show a significant protective effect in the neuroprotective effect test performed in both SHSY5Y and PC12 cell lines. Different pharmacological activities of kaempferide include Potent anti-oxidant, acts against cancer, cardioprotective and anti-hypertensive. All these actions are mediated by modulating apoptosis and inflammatory response, angiogenesis, and excess free radical generation. Previous research reported on Kaempferide isolated from C.odorata acts against cervical cancer. Reported activities of NRG are anti-oxidant, anti-inflammatory, anti-viral, anti-microbial, and acts on the urinary system. In our research, KPD and NRG clearly show a protective effect on neurodegeneration caused by amyloid-β proteins and 6-OHDA in both cell lines. We found that Kaempferide exerts a more potent effect than Norbergenin. KPD as a natural compound elicits a neuroprotective effect on neurotoxic induced SHSY5Y and PC12 cell lines by anti-apoptotic and inhibitory action on caspase enzyme 3/7 and 9. Various doses of KPD and NRG decrease oxidative stress in 6-hydroxydopamine, and amyloid-β-induced neurotoxicity indicate in these in vitro models. This research additionally supports the in vivo studies of kaempferide and norbergenin in brain aging models.

Conclusion

It can be concluded that we explain the in vitro neuroprotective effect in the SHSY5Y and PC12 cell lines. This model imparts knowledge about the effect of phytocandidates against Aβ-induced neurodegeneration in SHSY5Y and 6-OHDA treated PC12 cell lines. In our in vitro studies, results revealed that flavonoids KPD and NRG significantly inhibit reactive oxygen species, apoptosis of cells, and caspase enzyme activity. Therefore, the present study focuses on the protective effect of KPD and NRG flavonoids. Consequently, in vivo neuroprotective activity studies of isolated flavonoids are ongoing in the lab to traverse the other possible mechanisms to control the neurodegeneration in the Alcl3-induced brain aging in rats.

Acknowledgement

We Acknowledge Dr.Appaji, DBT-center for DNA printing and diagnostics for technical help.

Conflict Of Interest

The Authors have no conflict of interest.

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