Protective Effects And Mechanisms Of Procyanidins On Parkinson’s Disease In Vivo And In Vitro Part 2

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2.7.Effects of PCs on Oxidative Stress of Zebrafish Larae Treated with MPTP

Treatment of zebrafish larvae with PCs decreased MPTP-induced increased intracellular ROS formation (Figure 7a,b). Furthermore, the lipid peroxidation assay results showed that PC exposure （16 μg/mL）blocked the MPTP-induced MDA levels in zebrafish larvae (Figure 7c). Additionally, the MPTP-induced reduction in GSH-Px activity was reversed by PC（16 μg/mL） treatment （Figure 7d）， and PCs（4，8， and 16 ug/mL） increased MPTP-induced decreases in CAT and SOD activity (Figure 7e,f).

Figure 7. Effects of PCs on oxidative stress of zebrafish larvae treated with MPTP. (a)ROS levels were measured by fluorescent microscopy and imaging analysis;(b)ROS levels were measured via imageJ software;(c) MDA levels;(d) GSH-Pxactivity;(e)CATactivity;(f) SOD activity. F and p values of the one-way analysis of variance are presented above each chart.The results of Tukey's post hoc test are presented for selected comparisons: ns, p>0.05;*,p<0.05;*,p<0.01;***, p <0.001; the error bars are SD.

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2.8. Effects of PCs on Nr2/ARE Patlhroay in MPTP-Induced Zebrafish Laroa

Treatment of zebrafish larvae with PCs underscored the premise that activation of Nr2/ARE pathways was involved in PC-mediated protective properties. The expression of Nrf2, HO-1, NQO1, GCLC and GCLM markers was upregulated by PCs treatment compared with MPTP exposure alone (Figure 8a-e).

Figure 8. Effects of PCs on Nrf2/ARE pathway in MPTP-induced zebrafish larvae. (a) Nrf2 levels;(b)HO-1 levels;(c)NQO1 levels;(d) GCLClevels;(e) GCLM levels. F and p values of the one-way analysis of variance are presented above each chart. The results of Tukey's post hoc test are presented for selected comparisons: ns,p>0.05;*,p<0.05;**,p<001;***,p<0.001:the error bars are SD.

3. Discussion

PCs exhibit strong radical scavenging abilities and antioxidant activity, and oxidative stress, caused by the excessive generation of ROS or/and the impaired antioxidant defense system, plays a critical role in PD[37-39]. However, whether PCs can play a neuroprotective role through antioxidant effects remains unclear. This research aimed to investigate the molecular mechanism of PCs against MPP+/MPTP-induced PD models.

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In this study, compared with the MPP+/MPTP-alone group, PCs markedly raised the activity level of antioxidant enzymes (including GSH-Px, CAT, and SOD) and decreased levels of ROS and MDA. The current findings are consistent with a previous study that suggests that a low PC supplement via diet improves the activities of GSH-Px, CAT, and SOD in weaned piglets [40]. Another study also suggests that PCs significantly increase CAT and SOD activities and decrease MDA content, thus improving the quality of goat sperm[41]. These findings suggest that PCs have a protective effect on oxidative damage of nerve cells. Additionally, PCs improved cell viability compared with the MPP+-alone group in MPP+-induced PC12 cells, markedly increased total distance moved, and decreased TH+cell density relative to the MPTP group in MPTP-induced zebrafish larvae. These data indicate that PCs have a protective effect on nerve cells.

We further observed that PCs significantly increased nuclear Nrf2 accumulation compared with that of MPP+ alone in PC12 cells. Indeed, PCs markedly upregulated the expression levels of NQO1, HO-1, GCLM, and GCLC[42]. Furthermore, Nfr2gene silencing via Nrf2 siRNA was used to investigate the role of Nrf2/ARE activation in PCs-mediated neuroprotection against MPP+-induced oxidative damage: Nrf2-siRNA-transfected cells indicated a marked decrease in Nrf2 expression. This current study also found that Nrf2 knockout abolished both PCs-mediated protection against MPP+-treated impairments in cell viability and the antioxidant effects of PCs. These results are consistent with a previous study suggesting that improving activation of the Nrf2/ARE pathway contributes to neuroprotection [43].

The Nrf2/ARE pathway is a significant antioxidant pathway [44-46]. Normally, Nrf2 resides in the cytoplasm, where it is bound to the inhibitory protein, Keapl[47,48]. When cells undergo oxidative stress, Nrf2 dissociates from Keap1, initiates the endogenous antioxidant defense system, and subsequently translocates into the nucleus [46,49,50]. It then interacts with ARE to activate a series of cell-protective and antioxidant genes, including GCLC, GCLM, NQO1, and HO-1 [51-54]. In response to oxidative stress, Nrf2 dissociates from Keap1 in the cytosol and is then translocated into the nucleus, binding to the ARE sequence to activate transcription of cryoprotective genes [53-58]. The current results indicate that PCs can activate the Nrf2/ARE pathway, transfer Nrf2 from the cytoplasm to the nucleus, accumulate in the nucleus, upregulate the expression of GCLC, GCLM, NOO1, and HO-1 and improve the ability of cells to resist oxidative stress. Indeed, the Nrf2/ARE pathway may represent a pharmacological target of PCs for the prevention of PD.

4. Materials and Methods

4.1. Chemical Compounds and Reagents

MPP+ and MPTP agents were purchased from Sigma (St. Louis, MO, USA), and the Cell Counting Kit-8 was obtained from Beyotime (Shanghai, China). The PCs,2',7/-Dichlorofluorescin diacetate, MDA, GSH-Px, SOD, and CAT diagnostic kits were all obtained from Solarbio (Beijing, China). The RNAiso Plus, PrimeScriptTMRT Reagent Kit with gDNA Eraser, and SYBRPremix Ex TaqTMⅡI were purchased from Takara(Shiga, Japan); the Nrf2-siRNA, control-siRNA, and Lipofectamine 2000 were obtained from GenePharma (Shanghai, China). Finally, the primary antibodies, Nrf2, HO-1, GCLC, GCLM, NAD(P)H: NQO1, Lamin B, GAPDH, Antityrosine Hydroxylase (TH), and corresponding secondary antibodies were obtained from Proteintech (Wuhan, China).

4.2.Cell Culture

PC12 cells were obtained from the National Collection of Authenticated Cell Cultures. The cells were maintained in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U/mL; 100 ug/mL) in a humidified atmosphere incubator at 37 °C with 5% CO2 [59].

4.3. Cell Viability Assay

PC12cells(1×104 cells/well)were incubated with 1,2or4ug/mLof PCsor deprenvl （30 μM） for 24 h， and then incubated with 1.5 mM MPP+ for 24 h. Next， 10 μL of Cell Counting Kit-8 solution was added to each well and incubated for 1 h. The absorbance was measured at 450 nm [60].

4.4. Fish Maintenance

Ethical approval for animal use was granted by the animal conservation and use committee of the experimental animal center, Zhejiang University (ZJU20200125). Adult zebrafish(AB strain) were obtained from the laboratory animal center of Zhejiang University (Hang Zhou, China) and maintained at 28±1°Cunder 14h light/10h dark cycles. The fish were fed Artemia nauplii twice daily [61]. To produce embryos, adult zebrafish were placed in breeding tanks overnight at a 1:1 male: female ratio. Spawning was triggered after the lights were turned on the next morning and completed in 2 h. Embryos were raised in embryo water（13.7 mM NaCl，540 μM KCl，25 uM Na2HPOA44 uM KHPOA， 300 uM CaCl2， 100 μM MgSO， 420 μM NaHCO3， pH 7.4） at 28°C【62，63】.

4.5. ROS Measurement

To measure ROS production, PC12 cells(2×104 cells/well) were exposed to 1,2 or 4 μg/mL of PCs or deprenyl （30 μM） for 24 h and then incubated with 1.5 mM MPPt for 24 h 【64】. The cells were exposed to 10 μM 2'，7'-Dichlorofluorescin diacetate solution in dark conditions for 30 min; the dye solution was then removed, and the cells were washed with phosphate-buffered saline (PBS)3 times. The images of the cells were observed using an Olympus laser scanning confocal microscope(Olympus, Tokyo capital, Japan). The fluorescence intensity of the cells was quantified using Image J software v1.8.0(Olympus, Tokyo capital, Japan). The results are expressed as a percentage of the area of the ROS regions in the control group.

To measure ROS production, the zebrafish larvae at 3 days post fertilization (dpf) were exposed to deprenyl（40 μM） or 4，8 or 16 ug/mL of PCs with or without 400 μM MPTP for 4 days. Zebrafish larvae at 7 dpf were transferred to a 24-well plate(10 larvae per group), treated with 20 μM 2'，7'-Dichlorofluorescin diacetate solution, and incubated for 60 min in the dark at 28.5°C [65]. The larvae were then washed three times with embryo medium to remove excess 2',7'-Dichlorofluorescin diacetate. The images of the stained larvae were observed using an Olympus laser scanning confocal microscope. The fluorescence intensity of the individual larva was quantified using Image J software. The results are expressed as a percentage of the area of the ROS regions in the control group. 4.6.Assessment of MDA, GSH-Px, SOD, and CAT

PC12 cells(2×104 cells/well) were exposed to 1,2 or 4 ug/mL of PCs or deprenyl （30 μM） for 24h， and then incubated with 1.5 mM MPP+ for 24h. Then，1 mL of extract was added to 4× 10° cells, and cells were broken by ultrasonic centrifuging at 8000 rpm at 4°C for 10 min. The supernatant was then put on ice for testing, and reagents were added for the determination of MDA, GSH-Px, SOD, and CAT, respectively. Each index was repeated in triplicate.

The zebrafish larvae at 3dpf were incubated with deprenyl（40 μM）or4，8 or 16 μg/mL of PCs with or without 400 μM MPTP for 4 days. Then，0.05 g of zebrafish larvae tissue and 0.5 mL of extract were homogenized in an ice bath and then centrifuged at 4°C for 10 min. The supernatant was put on ice for testing. Reagents were subsequently added for the determination of MDA, GSH-Px, SOD, and CAT, respectively. Each index was repeated in triplicate.

4.7. Preparation of Whole-Cell, Cytoplasmic, and Nuclear Protein

For whole-cell protein extraction, cells were collected and incubated with RIPA lysis buffer containing 1% PMSF and 1% protease inhibitor cocktail for 30 min on ice. Cell lysates were centrifuged, and the supernatant was collected and stored. For subcellular fractionation preparation, cell samples were processed using the nuclear and cytoplasmic protein extraction kit. The protein content was assayed using the BCA(Beyotime, Shanghai, China) assay.

4.8. Nrf2 siRNA Transfection

Nrf2-siRNA was used to knockdown Nrf2. PC12 cells were transfected with Nrf2-siRNA (80 nM) or control-siRNA using Lipofectamine 2000, according to the manual. 4.9.Western Blotting

Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene di-fluoride(PVDF) membranes. The blots were exposed to appropriate primary antibodies: Nrf2, Keap1, HO-1, NQO1, GCLC, GCLM, Lamin B, GAPDH and peroxidase-conjugated secondary antibodies. Protein bands were visualized using ECL(Beyotime, Shanghai, China) plus Western blotting detection reagents [36,43].

4.10.Locomotor Behaioral Test

The zebrafish larvae at 3dpf were incubated with deprenyl（40 μM）or4，8or16 ug/mL of PCs with or without 400uM MPTP for 4 days. The 7 dpf zebrafish were then placed into 24-well plates(1 fish per well and 12 larvae per group), and the total distance each fish swam over 10 min was recorded. Zebrafish behavior was monitored and analyzed using an automated video tracking system(Any-maze 4.73, Stoelting, Wood Dale, IL, USA)[66].

4.11.Total RNA Extraction, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction

RNA was extracted using RNAiso Plus following the manufacturer's instructions. RNA was reverse transcribed using a PrimeScriptTMRT Reagent Kit with gDNA Eraser, following the manufacturer's instructions. Quantitative real-time PCR analysis was performed on an Applied Biosystems ViATM 7 Real-Time PCR system using SYBRPremix Ex TaqTM II(Takara, Shiga, Japan).β-actin was used as a reference gene and relative gene expression was calculated using the 2-△Ct method. The primer sequences utilized in the research are listed in Table 1.

4.12. Zebrafish Antityrosine Hydroxylase(TH)Whole-Mount Immunostaining

Zebrafish larvae at 1 dpf were incubated with deprenyl （40 μM） or 4，8 or 16 ug/mL of PCs with or without 400 uM MPTP for 2 days(10 fish/group batches). Larvae were fixed with 4% paraformaldehyde in PBS for 30 min. After fixation, they were treated for whole-mount immunostaining of TH [67]. At room temperature, 2%(v/o) lamb serum and 0.1%（w/ø）bovine serum albumin （BSA）were blocked in phosphate-buffered saline Tween-20(PBST) for 1 h. They were then exposed in the blocking buffer to anti tyrosine hydroxylase antibody(1:200 diluted, Proteintech) for 2 h, and then rinsed 6 times with PBST. The final whole-mount immunostaining step was performed in staining buffer with 488 goat antirabbit(1:500) for 1 h and washed again with PBST. After sufficient color development, the zebrafish were flat-mounted with 3.5% methylcellulose and imaged using an Olympus laser scanning confocal microscope.

4.13. Statistical Analysis

One-way analysis of variance(ANOVA) followed by Tukey's multiple comparison test was used to compare the means of different groups. Graphpad Prism v6.01(GraphPad Software, San Diego, CA, USA) was used for statistical analysis and plotting the graphs.

5. Conclusions

In conclusion, our findings indicate that PCs exert neuroprotective effects via activation of the Nrf2/ARE pathway and its downstream detoxification and antioxidant enzymes. Taken together, these insights suggest that PCs may be useful for treating PD.

This article is extracted from Molecules 2021, 26, 5558. https://doi.org/10.3390/molecules26185558 https://www.mdpi.com/journal/molecules