Rutin Attenuates Oxidative Stress Via PHB2-Mediated Mitophagy in MPP+-Induced SH-SY5Y Cells

Oxidative stress plays a crucial role in the occurrence and development of Parkinson’s disease (PD). Rutin, a natural botanical ingredient, has been shown to have antioxidant properties. Therefore, the aim of this study was to investigate the neuroprotective effects of rutin on PD and the underlying mechanisms. MPP+(1-methyl-4-phenylpyridinium ions)-treated SH-SY5Y cells were used as an in vitro model of PD. Human PHB2-shRNA lentiviral particles were transfected into SH-SY5Y cells to interfere with the expression of Prohibitin2 (PHB2). The oxidative damage of cells was analyzed by detecting intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial membrane potential (MMP). Western blotting was used to detect the protein expression of antioxidant factors such as nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO-1), and mitophagy factors PHB2, translocase of outer mitochondrial membrane 20 (TOM20), and LC3II/LC3I (microtubule-associated protein II light chain 3 (LC3II) to microtubule-associated protein I light chain 3 (LC3I)). In addition, we also examined the expression of PHB2 and LC3II/LC3I by immunofluorescence staining. MPP+ treatment significantly increased the generation of ROS and MDA and the level of MMP depolarization and decreased the protein expression of Nrf2, HO-1, NQO1, TOM20, PHB2, and LC3II/LC3I. In MPP+-treated SH-SY5Y cells, rutin significantly decreased the generation of ROS and MDA and the level of MMP depolarization and increased the protein expression of Nrf2, HO-1, NQO-1, TOM20, PHB2, and LC3II/LC3I. However, the protective role of rutin was inhibited in PHB2-silenced cells. Rutin attenuates oxidative damage which may be associated with PHB2-mediated mitophagy in MPP+-induced SH-SY5Y cells. Rutin might be used as a potential drug for the prevention and treatment of PD.


Introduction
Parkinson's disease (PD) is the world's second most common neurodegenerative disease. The main pathological change in PD is the degeneration and death of substantia nigra dopaminergic (DA) neurons, which eventually leads to a series of clinical symptoms. Studies have shown that oxidative stress is one of the main pathogenesis of PD (Archibald et al. 2009;Schapira et al. 2014;Trist et al. 2019), and mitochondrial dysfunction is a major source of ROS generation (Peoples et al. 2019;Subramaniam and Chesselet 2013).
Mitophagy can selectively remove damaged mitochondria, which are eventually degraded by lysosomes. Studies have found that PD is closely related to mitophagy (Clark et al. 2021;Malpartida et al. 2021); activation of mitophagy can protect DA neurons from oxidative stress damage (Di Rita and Strappazzon 2019;Shefa et al. 2019), but the mechanism remains unclear. PHB2 is a mitochondrial inner membrane protein that plays an important role in maintaining mitochondrial morphology and function . Since the discovery that PHB2 is a key mitochondrial inner membrane autophagy receptor that can promote mitophagy (Wei et al. 2017), the research on PHB2 and mitophagy has received more and more attention. Xu et al. reported that PHB2-mediated mitophagy could attenuate renal tubular epithelial cell injury by regulating mitochondrial dysfunction (Xu et al. 2019). Zhang et al. found that mitoquinone could activate PHB2-mediated mitophagy and thus inhibit oxidative stress-related neuronal death (Zhang et al. 2019).
Rutin is a flavonoid widely distributed in nature that has various pharmacological effects such as antioxidant, anti-inflammatory, antiviral, and antitumor (Ganeshpurkar and Saluja 2017). In recent years, the research on rutin in PD has received more and more attention (Christmann et al. 2022;Sharma et al. 2016), and both in vivo and in vitro studies have confirmed that rutin can prevent and reverse PD. In a 6-OHDAinduced PD cell model, rutin treatment activated antioxidant enzymes, decreased lipid peroxidation levels, and attenuated 6-OHDA-induced neurotoxicity in PC12 cells (Magalingam et al. 2013(Magalingam et al. , 2016. PARK et al. found that rutin treatment inhibited ROS generation and calcium elevation, attenuated MMP depolarization levels, and ultimately prevented rotenone-induced oxidative damage in SH-SY5Y cells (Park et al. 2014). KHAN et al. found that rutin could increase the level of antioxidants, protect substantia nigra DA neurons, and significantly improve the motor deficit caused by 6-OHDA in rats (Khan et al. 2012). The above studies suggest that rutin may be a promising neuroprotective drug for the treatment of PD. Therefore, the present study aimed to investigate whether rutin could alleviate MPP + -induced oxidative damage in SH-SY5Y cells and whether its protective effect was related to PHB2-mediated mitophagy.

Cell Culture and Drug Treatment
Human neuroblastoma cell line SH-SY5Y was obtained from Sun Yat-sen University (Guangzhou, China) and cultured in DMEM/H medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in a humidified incubator with 5% CO 2 at 37 °C. The medium was changed every 3 days, and the cells were subcultured when the density reached 80%. Cells were seeded in culture plates at a certain density. The next day, a drug-containing medium was added, and the cells were cultured for another 24 h for subsequent experiments. MPP + iodide was purchased from Sigma-Aldrich (D048, St Louis, USA) and was dissolved in sterile water. Rutin was purchased from Selleck.CN (S235002, Shanghai, China) and was dissolved in DMSO. CCK-8 kit (Solarbio, CA1210, Beijing, China) was used to detect cell viability. Following the manufacturer's instructions, SH-SY5Y cells were seeded at 5 × 10 3 cells/well in a 96-well plate and incubated overnight. The next day, the medium containing the drug was added, and a control group was set at the same time. After 24 h, aspirate the medium in each well, and add the medium-containing CCK-8. After 2 h, the absorbance was measured at 450 nm with a microplate reader (EnSpire, PerkinElmer, Singapore), and the cell viability was calculated. The cell viability of the control group was set as 100%, and the cell viability of the other groups was compared with that of the control group.

LDH Release Assay
LDH release rate was detected with a lactate dehydrogenase cytotoxicity assay kit (Beyotime Biotechnology, C0017, Shanghai, China). Briefly, cells were seeded at 5 × 10 3 cells/ well in 96-well plates and grouped according to the instructions. The next day, drugs were added to the drug-treated groups and continued to culture for 24 h. One hour before detection, add the LDH release reagent into the "sample maximum enzyme activity control well", and mix it by pipetting repeatedly. One hour later, the 96-well plate was centrifuged at 400 × g for 5 min in a multi-well plate centrifuge. Take 120 μl of the supernatant from each well to a new 96-well plate, and add 60 μl of LDH assay solution. After incubating at room temperature for 30 min in the dark, the absorbances at 490 nm and 600 nm (reference wavelength) were measured with a microplate reader (EnSpire, PerkinElmer, Singapore), and the LDH release rates were calculated according to the instructions.

Reactive Oxygen Species (ROS) Assay
ROS levels can directly reflect the oxidative stress of cells. We used a ROS assay kit (Beyotime Biotechnology, S0033M, Shanghai, China) to detect the ROS levels. Following the manufacturer's instructions, cells were harvested and suspended in serum-free medium containing 10 μM DCFH-DA. After incubating at 37 °C for 20 min, the cells were washed three times with serum-free medium. Finally, the cells were suspended in serum-free medium, and the fluorescence intensity was measured by flow cytometry (BD Accuri C6, NJ).

MDA Assay
MDA content not only directly reflects the degree of lipid peroxidation but also indirectly reflects the ability of cells to scavenge free radicals. We used a lipid oxidation (MDA) detection kit (Beyotime Biotechnology, S0131M, Shanghai, China) to detect the MDA content. Following the manufacturer's instructions, cells were lysed and centrifuged at 10,000 × g for 10 min. Collect the supernatant, and measure the absorbance at 532 nm with a microplate reader (EnSpire, PerkinElmer, Singapore). The concentration of MDA was calculated from absorbance. MDA content = (concentration of MDA in the sample * sample volume)/protein mass.

Mitochondrial Membrane Potential (MMP) Assay
The level of MMP depolarization can directly reflect the degree of mitochondrial damage. We used an MMP assay kit (JC-1) (Beyotime Biotechnology, C2006, shanghai, China) to detect changes in MMP. Following the manufacturer's instructions, aspirate the medium and wash the cells once with PBS. Second, cells were incubated with JC-1(1 ×) staining buffer for 20 min at 37 ℃. Then, remove the JC-1 staining buffer, and wash the cells twice. Finally, add an appropriate amount of cell culture to each well, and observe the fluorescence with an inverted fluorescence microscope (Nikon, ECLIPSE Ti, Tokyo, Japan); the green fluorescence was observed with the GFP channel, and the red fluorescence was observed with the TRITC channel.

Establishment of PHB2-Silencing (PHB2-shRNA) Cell Line
According to the manufacturer's instructions, the medium containing PHB2-shRNA lentiviral particles (Santa Cruz, sc-45849-V, Dallas, USA) and polybrene cation was added to the culture plate when the cell density reached 50%, and the control group was transfected with scramble-shRNA lentiviral particles. After 8 h, the old medium was removed, and the new medium without polybrene cation was added. To select cells stably expressing PHB2-shRNA, the cells were subcultured with medium containing puromycin (final concentration 10 μg/ml) when the cell density reached 80%. The old medium was replaced with new medium containing puromycin every 3 days thereafter until all untransfected cells were killed. Puromycin-resistant cells were expanded, and the transfection efficiency was detected by Western blot. Finally, a cell line stably expressing PHB2-shRNA was established and used for subsequent studies.

Statistical Analysis
GraphPad Prism 7.0 was used to analyze the data. The results were expressed as the mean ± standard error of the mean (SEM). One-way ANOVA was used to compare multiple groups, followed by Tukey's post hoc test. A difference of p < 0.05 was considered statistically significant. Results are expressed as the relative expression of β-actin. Data are expressed as mean ± SEM; *p < 0.05 vs. control group, ***p < 0.001 vs. control group; # p < 0.05 vs. MPP + group, ## p < 0.01 vs. MPP. + group, n = 3
The MMP staining results are shown in Fig. 2d, e. Green fluorescence represents JC-1 monomers, indicating the depolarization of MMP; red fluorescence represents JC-1 aggregates, indicating the normal state of MMP; the ratio of red fluorescence to green fluorescence is used to measure the level of MMP depolarization. As shown in Fig. 2d, compared with the control group and the rutin group, JC-1 staining shows weaker red fluorescence and brighter green fluorescence in the MPP + group, and the ratio of red fluorescence to green fluorescence is significantly decreased (***p < 0.001, n = 6), suggesting the level of MMP depolarization was significantly increased. Compared with the MPP + group, JC-1 staining showed brighter red fluorescence and weaker green fluorescence in the Rutin + MPP + group, and the ratio of red fluorescence to green fluorescence was significantly increased ( ## p < 0.01, n = 6), suggesting the level of MMP depolarization was significantly decreased. Figure 2e shows the ratio of red fluorescence intensity to green fluorescence intensity.

Effect of Rutin on ROS, MDA Generation, and the Level of MMP Depolarization After Silencing PHB2
Silencing PHB2 had no significant effect on cell viability (Fig. S1). Then, we used PHB2-silenced cells to investigate the effect of rutin on ROS, MDA production, and MMP depolarization levels after MPP + treatment to explore whether the protective effect of rutin was related to PHB2.
As shown in Fig. 5a, b, the ROS generation in the MPP + group is significantly increased than that in the control group (***p < 0.001, n = 6), and the ROS generation in the MPP + + Rutin group is significantly decreased than that in the MPP + group ( ### p < 0.01, n = 6). The ROS generation in MPP + + Rutin + PHB2 shRNA group was significantly increased than that in the MPP + + Rutin group ( && p < 0.001, n = 6). However, there was no significant difference in ROS generation between MPP + + Rutin + PHB2 shRNA group and MPP + + PHB2 shRNA group (p > 0.05, n = 6).
As shown in Fig. 5c, compared with the control group, the MDA generation in the MPP + group is significantly increased (***p < 0.001, n = 6); compared with the MPP + group, the MDA generation in the MPP + + Rutin group is significantly decreased ( ### p < 0.001, n = 6). The MDA generation in the MPP + + Rutin + PHB2 shRNA group was significantly increased than that in the MPP + + Rutin group ( &&& p < 0.001, n = 6). However, there was no significant difference in MDA generation between MPP + + Rutin + PHB2 shRNA group and MPP + + PHB2 shRNA group (p > 0.05, n = 6).
As shown in Fig. 5d, e, compared with the control group, the level of MMP depolarization in the MPP + group is significantly increased (***p < 0.001, n = 6); compared with the MPP + group, the level of MMP depolarization in the MPP + + Rutin group is significantly decreased ( ### p < 0.001, n = 6). Compared with the MPP + + Rutin group, the level of MMP depolarization in the MPP + + Rutin + PHB2 shRNA group was significantly increased ( &&& p < 0.001, n = 6). However, there was no significant difference in the level of MMP depolarization between MPP + + Rutin + PHB2 shRNA group and MPP + + PHB2 shRNA group (p > 0.05, n = 6).

Effect of Rutin on the Protein Expression of Nrf2, HO-1, and NQO-1 After Silencing PHB2
We transfected SH-SY5Y cells with PHB2 shRNA lentiviral particles and scramble-shRNA lentiviral particles. The transfection efficiency is shown in the Fig. 6a, b; the expression of PHB2 is significantly decreased in the PHB2 shRNA group compared with the control group (***p < 0.01, n = 3).

Discussion
DA neurons in PD are particularly sensitive to oxidative stress (He et al. 2020); therefore, antioxidants play an important role in the prevention and treatment of PD. MPP + is a neurotoxin that can selectively destroy substantia nigra DA neurons (Sun et al. 2018). MPP + can easily accumulate in mitochondria and inhibit the activity of mitochondrial complex I, eventually leading to oxidative stress damage in cells (Risiglione et al. 2020). Therefore, it is commonly used to replicate the cellular model of PD. As a natural antioxidant, rutin has been reported to have neuroprotective effects (Budzynska et al. 2019;Enogieru et al. 2018;Pan et al. 2019). In this study, we found that the generation of ROS and MDA and the level of MMP depolarization increased after MPP + treatment. However, rutin significantly decreased MPP + -induced ROS and MDA generation and the level of MMP depolarization. Our study showed that rutin could protect DA neurons from oxidative damage, which was consistent with previous studies (Khan et al. 2012;Magalingam et al. 2013). However, the effect of rutin on ameliorating MPP + -induced oxidative damage was abolished by silencing PHB2, suggesting that the protective effect of rutin might be related to PHB2.
Nrf2 is a member of the transcription factor family and is a key nuclear transcription factor regulating oxidative stress (Jung and Kwak 2010). When cells are stimulated by oxidative stress, Nrf2 is translocated into the nucleus and combined with antioxidant response elements (AREs) to mediate the activation of its important downstream antioxidant enzyme genes such as HO-1 and NQO-1 (Ke et al. 2020;Waz et al. 2021). However, Nrf2/HO-1/NQO-1 signaling is considered to be an important pathway to mitigate ROS damage (Zhao et al. 2019). In this study, we found that MPP + treatment significantly decreased the expression of Nrf2, HO-1, and NQO-1, while rutin treatment increased the protein expression of Nrf2, HO-1, and NQO-1. These results suggested that the antioxidant effect of rutin was related to the activation of the Nrf2/HO-1/NQO-1 pathway. After silencing PHB2, the effect of rutin on increasing the expression of Nrf2, HO-1, and NQO-1 in MPP + -treated cells was abolished which suggested that rutin might activate the Nrf2/HO-1/NQO-1 pathway through PHB2. Fig. 9 The effect of rutin on mitophagy in MPP + -induced SH-SY5Y cells. Rutin alleviates MPP + -induced oxidative damage in SH-SY5Y cells by inhibiting PHB2-mediated mitophagy It has been confirmed that PHB2 is involved in neuronal protection in neurodegenerative diseases (De Rasmo et al. 2020;Guyot et al. 2020;Iridoy et al. 2018), but the mechanism remains unclear. Yan et al. reported that PHB2 could promote PINK1/Parkin-dependent mitophagy through the PARL-PGAM5-PINK1 axis (Yan et al. 2020). In a Drosophila model of Parkinson's disease, PHB2 plays an important role in mitochondrial health and biosynthesis, and azaflavonoids can increase motor coordination and tyrosine hydroxylase expression by increasing PHB2 expression (Pant et al. 2021). PHB2 has a LC3 interaction region domain and can bind to LC3 to promote mitophagy upon the mitochondrial outer membrane rupture (Wei et al. 2017). In our study, we found that the protein expression of PHB2, TOM20, and LC3II/LC3I decreased after MPP + treatment, and rutin could significantly increase the protein expression of PHB2, TOM20, and LC3II/LC3I. However, the effect of rutin on increasing the protein expression of TOM20 and LC3II/LC3I in MPP + -treated cells was abolished by silencing PHB2. Our results suggest that rutin can activate mitophagy by increasing the expression of PHB2 in MPP + -induced cells.
This study confirms that rutin can be used as a potential agent for the prevention and treatment of PD. However, there are still some shortcomings: (1) Although we have demonstrated for the first time that rutin attenuates MPP + -induced oxidative damage in DA neurons through PHB2-dependent mitophagy, further studies are needed to elucidate the specific mechanism.
(2) This study provides a basis for the treatment of PD with rutin through in vitro experiments, but it still needs to be verified in animal experiments.
In conclusion, our study suggests that PHB2 may be involved in PD as a key molecule and rutin could attenuate oxidative damage through PHB2-mediated mitophagy in MPP + -induced SH-SY5Y cells (Fig. 9). Rutin may be used as a potential drug for the prevention and treatment of PD.