Protective Effects and Mechanisms of Pectolinarin against H2O2-Induced Oxidative Stress in SH-SY5Y Neuronal Cells

This study aims to investigate the protective effects and mechanisms of pectolinarin against oxidative stress-induced cell damage in SH-SY5Y cells. Neurodegenerative diseases—such as Alzheimer’s disease—are potentially associated with oxidative stress, which causes excessive production of reactive oxygen species (ROS) that damage DNA and proteins in neuronal cells. The results of this study demonstrate that pectolinarin can scavenge hydroxyl and nitric oxide radicals in a concentration-dependent manner. Moreover, pectolinarin significantly increased cell viability while reducing ROS production and LDH release in the hydrogen peroxide (H2O2)-induced control group. Additionally, Pectolinarin recovered protein expression from H2O2-altered levels back to close-to-normal SH-SY5Y cell levels for components of the oxidative stress, inflammation, and apoptosis pathways—such as nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein (Keap1), anti-heme oxygenase 1 (HO-1), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), B-cell lympho-ma-2 (Bcl-2) protein, and Bcl-2-associated X protein (Bax). These findings suggest that pectolinarin has the potential to be used as a plant material for functional foods to be applied in the treatment of neurodegenerative diseases, such as Alzheimer’s disease, by mitigating oxidative stress-induced damage to neuronal cells.


Introduction
The oxidative and antioxidant systems in human bodies are in dynamic equilibrium [1]. The imbalance between pro-oxidation and anti-oxidation overproduces reactive oxygen species (ROS), which induce oxidative stress [2]. ROS is a general term for substances that are more unstable than oxygen and mainly includes superoxide (O 2− ), hydroxyl (·OH), and hydrogen peroxide (H 2 O 2 ) [3]. ROS combine with purines and pyrimidines in deoxyribonucleic acid (DNA), causing damage to the DNA double helix structure and causing oxidative stress, aging, and cancer [4,5]. Oxidative stress can also cause neuronal cell death and neuroinflammation in the brain, resulting in cognitive impairment and memory problems as well as neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease [6,7].
With increases in the oxidative-redox system. it eventually loses its equilibrium-with age and the influence of irregular diets resulting in an excess of ROS production and oxidative stress in our bodies [8,9]. Besides this, neurodegenerative diseases such as AD have been shown to be oxidative stress-related diseases [10,11]. H 2 O 2 stimulates astrocytes in the cortex and hippocampus, which significantly increases iNOS, and iNOS catalyzes nitric oxide (NO) production in the brain [12]. In addition, oxidative stress is also associated with the expression of Nrf2. Nrf2 is an antioxidant response regulator that is released from Keap1, translocates to the nucleus, activates protective gene transcription, and promotes their expression [13]. Studies have shown that inadequate Nrf2 expression has been found in the brains of human AD patients [14,15]. Therefore, the interaction of Keap1 and Nrf2 appears to be related to their antioxidant capacity, which protects neurons from oxidative stress [16].
In human cells, apoptosis is divided into internal pathways and external pathways, with the internal pathway undergoing molecular changes such as the activation of Bcl-2 and the inactivation of the pro-apoptotic effector Bax [17,18]. Apoptosis can help in the removal of damaged or dysfunctional brain cells, such as neurons that have accumulated toxic proteins [19]. This process is important for maintaining the overall health and function of the brain. However, excessive or abnormal apoptosis may contribute to the progression of neurodegenerative diseases [20]. Therefore, studying the apoptosis mechanisms of SH-SY5Y neuronal cells after treatment with the neurotoxic fragment Aβ [25][26][27][28][29][30][31][32][33][34][35] is important.
SH-SY5Y cells, which are human-origin neuronal cells, are commonly used in the study of neurodegenerative diseases [21][22][23]. Many studies have reported the use of SH-SY5Y cells for studying oxidative stress-induced inflammation and apoptosis [24][25][26]. H 2 O 2induced oxidative stress in the SH-SY5Y cell model has been used to confirm the protective effects of active compounds [27,28]. Meanwhile, pectolinarin is a flavonoid that has been identified as an active compound in a number of plants, including Cirsium japonicum var. ussuriense, Lippia rubella, and Cirsium nipponicum (Maxim.) [29][30][31]. Anti-inflammatory, antiapoptosis, anti-bacterial, and anti-tumor properties have been reported in pectolinarin [32][33][34]. Moreover, some previous studies have demonstrated the pharmacokinetic profile and intestinal absorption of pectolinarin to verify its physiochemical properties. According to Mehta and Dhapte [35], pectolinarin in rat plasma-following the oral administration of Cirsium japonicum DC. extract-reached the maximum possible concentration quickly. Chen et al. [36] reported that the absorption of pectolinarin in the jejunum was the highest among the parts of the small intestine, using the everted rat intestinal sac method. In this study, we investigated the ·OH and NO radical scavenging ability of pectolinarin in vitro and the mechanisms of pectolinarin against H 2 O 2 -induced oxidative stress in SH-SY5Y neuronal cells.

NO Radical Scavenging Activity of Pectolinarin
The results for the NO radical scavenging activity of pectolinarin are shown in Table 2. Pectolinarin (1, 2.5, 5, and 10 µg/mL) showed dose-dependent increases in NO radical scavenging activity-at 23.21%, 24.50%, 30.40%, and 35.19%, respectively. These results suggest that pectolinarin possesses antioxidant properties that make it capable of scavenging NO radicals.
suggest that pectolinarin possesses antioxidant properties that make it capable o enging NO radicals.

Effects of Pectolinarin against H2O2-Induced Lactate Dehydrogenase Release in SH-S Cells
The effects of pectolinarin on H2O2-induced LDH release in SH-SY5Y cells are in Figure 1b. In comparison with the normal group-in which 0% of the LDH w leased-in the control group, this significantly increased to 52.30%. Moreover, tre with different concentrations (1, 2.5, 5, and 10 µg/mL) of pectolinarin in H2O2-induc Values are mean ± SD. The different letters (a-f) between groups represent significant differences as determined by the Tukey HSD multiple range test (p < 0.05).

Effects of Pectolinarin against H 2 O 2 -Induced Lactate Dehydrogenase Release in SH-SY5Y Cells
The effects of pectolinarin on H 2 O 2 -induced LDH release in SH-SY5Y cells are shown in Figure 1b. In comparison with the normal group-in which 0% of the LDH was released-in the control group, this significantly increased to 52.30%. Moreover, treatment with different concentrations (1, 2.5, 5, and 10 µg/mL) of pectolinarin in H 2 O 2 -induced SH-SY5Y cells dose-dependently decreased the level of LDH release to 32.59%, 20.83%, 9.87%, and 5.10%, respectively. These results show that pectolinarin inhibits H 2 O 2 -induced LDH release in SH-SY5Y cells.

Effects of Pectolinarin against H 2 O 2 -Induced ROS Formation in SH-SY5Y Cells
The dichlorofluorescein diacetate (DCF-DA) assay was used to confirm the inhibitory effects of pectolinarin against H 2 O 2 -induced ROS formation, and the results are presented in Figure 2. ROS production was significantly higher in the H 2 O 2 -induced control group (Figure 1c) when compared with the normal group, confirming that H 2 O 2 caused oxidative stress in SH-SY5Y cells. As shown in Figure 2b, when the ROS production in the control group was 100%, the ROS production in the normal group was 78.28%-indicating that when the SH-SY5Y cells were exposed to H 2 O 2 (500 µM) for 24 h and then reacted with DCF-DA, the ROS production in the control group increased by 21.72% compared with the normal group. The intensity of ROS production at 60 min ( Figure 1d) showed that treatment with different concentrations (1, 2.5, 5, and 10 µg/mL) of pectolinarin dosedependently decreased the levels of ROS to 85.65%, 80.92%, 79.32%, and 78.37% when the ROS production of the control group was set as 100%. These results show that pectolinarin inhibits H 2 O 2 -induced ROS formation in SH-SY5Y cells.

Effects of Pectolinarin against H2O2-Induced ROS Formation in SH-SY5Y Cells
The dichlorofluorescein diacetate (DCF-DA) assay was used to confirm the inhib effects of pectolinarin against H2O2-induced ROS formation, and the results are pres in Figure 2. ROS production was significantly higher in the H2O2-induced control g ( Figure 1c) when compared with the normal group, confirming that H2O2 caused oxid stress in SH-SY5Y cells. As shown in Figure 2b, when the ROS production in the co group was 100%, the ROS production in the normal group was 78.28%-indicatin when the SH-SY5Y cells were exposed to H2O2 (500 µM) for 24 h and then reacted DCF-DA, the ROS production in the control group increased by 21.72% compared the normal group. The intensity of ROS production at 60 min ( Figure 1d) showed treatment with different concentrations (1, 2.5, 5, and 10 µg/mL) of pectolinarin dos pendently decreased the levels of ROS to 85.65%, 80.92%, 79.32%, and 78.37% whe ROS production of the control group was set as 100%. These results show that pectol inhibits H2O2-induced ROS formation in SH-SY5Y cells.

Effects of Pectolinarin on Oxidative Stress-Related Protein Expression in H2O2-Induced SY5Y Cells
The action mechanisms of oxidative stress-related proteins such as HO-1, Nrf2 Keap1 were measured using a Western blot to investigate the protective effects of pe narin against H2O2-induced oxidative stress in neural cells. The protein expression o 1 and Nrf2 was significantly lower in the H2O2-induced control group compared wi normal group, as shown in Figure 2. However, treatment with pectolinarin (1, 2.5, 5 10 µg/mL) in H2O2-induced SH-SY5Y cells increased Nrf2 and HO-1 protein expre In addition, Keap1 protein expression was significantly higher in the H2O2-induced trol group than in the normal group. On the other hand, treatment with pectolinar 2.5, 5, and 10 µg/mL) dramatically reduced Keap1 protein expression. These r

Effects of Pectolinarin on Oxidative Stress-Related Protein Expression in H 2 O 2 -Induced SH-SY5Y Cells
The action mechanisms of oxidative stress-related proteins such as HO-1, Nrf2, and Keap1 were measured using a Western blot to investigate the protective effects of pectolinarin against H 2 O 2 -induced oxidative stress in neural cells. The protein expression of HO-1 and Nrf2 was significantly lower in the H 2 O 2 -induced control group compared with the normal group, as shown in Figure 2. However, treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) in H 2 O 2 -induced SH-SY5Y cells increased Nrf2 and HO-1 protein expression. In addition, Keap1 protein expression was significantly higher in the H 2 O 2 -induced control group than in the normal group. On the other hand, treatment with pectolinarin (1, 2.5, 5, Molecules 2023, 28, 5826 5 of 13 and 10 µg/mL) dramatically reduced Keap1 protein expression. These results suggested that pectolinarin has antioxidant properties via the regulation of oxidative stress pathway proteins in H 2 O 2 -induced SH-SY5Y cells.

Effects of Pectolinarin on Inflammation-Related Protein Expression in H 2 O 2 -Induced SH-SY5Y Cells
Inflammation-related protein expression in H 2 O 2 -induced SH-SY5Y cells was assessed to confirm the mechanisms of pectolinarin. Figure 3 showed that H 2 O 2 -induced cells had significantly increased expression of IL-1β, iNOS, and COX-2 proteins compared with the untreated normal SH-SY5Y cells. Furthermore, pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly reduced the expression of the iNOS, COX-2, and IL-1β proteins. These results show that pectolinarin inhibits inflammation in H 2 O 2 -induced SH-SY5Y cells by downregulating the inflammation pathway-related proteins iNOS, COX-2, and IL-1β. suggested that pectolinarin has antioxidant properties via the regulation of oxidative stress pathway proteins in H2O2-induced SH-SY5Y cells.

Effects of Pectolinarin on Inflammation-Related Protein Expression in H2O2-Induced SH-SY5Y Cells
Inflammation-related protein expression in H2O2-induced SH-SY5Y cells wa assessed to confirm the mechanisms of pectolinarin. Figure 3 showed that H2O2-induced cells had significantly increased expression of IL-1β, iNOS, and COX-2 proteins compared with the untreated normal SH-SY5Y cells. Furthermore, pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly reduced the expression of the iNOS, COX-2, and IL-1β proteins These results show that pectolinarin inhibits inflammation in H2O2-induced SH-SY5Y cell by downregulating the inflammation pathway-related proteins iNOS, COX-2, and IL-1β.

Effects of Pectolinarin on Apoptosis-Related Protein Expression in H2O2-Induced SH-SY5Y Cells
The mechanisms of apoptosis-related proteins were studied using Bax and Bcl-2 pro tein expression. As shown in Figure 4, H2O2-induced SH-SY5Y cells had significantly higher Bax protein expression than normal SH-SY5Y cells, but treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly decreased the protein expression of Bax. On the othe hand, treatment with H2O2 decreased Bcl-2 protein expression in SH-SY5Y cells compared with normal cells, and treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly increased the protein expression of Bcl-2. Furthermore, the Bax/Bcl-2 protein expression was significantly higher in the H2O2-induced control group. These findings show tha treatment with pectolinarin protects SH-SY5Y cells from H2O2-induced damage by mod ulating the expression of Bax and Bcl-2, which are proteins associated with apoptosis.

Effects of Pectolinarin on Apoptosis-Related Protein Expression in H 2 O 2 -Induced SH-SY5Y Cells
The mechanisms of apoptosis-related proteins were studied using Bax and Bcl-2 protein expression. As shown in Figure 4, H 2 O 2 -induced SH-SY5Y cells had significantly higher Bax protein expression than normal SH-SY5Y cells, but treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly decreased the protein expression of Bax. On the other hand, treatment with H 2 O 2 decreased Bcl-2 protein expression in SH-SY5Y cells compared with normal cells, and treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly increased the protein expression of Bcl-2. Furthermore, the Bax/Bcl-2 protein expression was significantly higher in the H 2 O 2 -induced control group. These findings show that treatment with pectolinarin protects SH-SY5Y cells from H 2 O 2 -induced damage by modulating the expression of Bax and Bcl-2, which are proteins associated with apoptosis. Molecules 2023, 28, x FOR PEER REVIEW 6 of 13

Discussion
Under physiological conditions, the production and clearance of ROS in organisms are normal physiological functions [37]. Excessive ROS production in the body can readily lead to oxidative stress [38]. ROS consists of free radicals such as ·OH, O2− and other nonradicals such as H2O2 [39]. Although H2O2 is a toxic by-product, it is involved in many physiological processes such as cell proliferation, differentiation, and immune regulation [40]. However, high concentrations of H2O2 can promote cell apoptosis and oxidative stress in the brain, leading to neurodegenerative diseases such as AD [41,42].
The OH radical, as a by-product of ROS, has high reactivity and a strong oxidation capacity, which can damage cells or tissues in a short period of time [43]. Excess NO production in the brain can cause neuroinflammation and neurodegenerative diseases [44]. The present results showed that the ·OH and NO radical scavenging activity of pectolinarin increased dependent on its concentration, with its ·OH scavenging activity exceeding 80% at concentrations of 2.5, 5, and 10 µg/mL. These results suggest that pectolinarin has antioxidant properties for the scavenging of ·OH and NO radicals.
Several studies have found that throughout the natural aging process, various food intakes or physiological activities cause an increase in the excessive production of ROS, which promotes the deposition of Aβ and accelerates the formation of senile plaques [45,46]. The MTT assay was used in our present study to confirm the protective effects of pectolinarin against H2O2-induced oxidative stress in SH-SY5Y cells. The MTT assay is a standard approach for evaluating mitochondrial damage by measuring cell viability, and importantly, is well-known for assessing cell metabolism-in contrast with the sulforhodamine B assay, which assesses the protein contents [47]. The DCF-DA assay was used to confirm the inhibitory effects of pectolinarin against H2O2-induced ROS formation in SH-SY5Y cells. Dichlorofluorescein (DCF) is a highly fluorescent chemical formed by the oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) by ROS [48]. Meanwhile, we used the LDH cytotoxicity kit to confirm any inhibitory effects on LDH release. After being treated

Discussion
Under physiological conditions, the production and clearance of ROS in organisms are normal physiological functions [37]. Excessive ROS production in the body can readily lead to oxidative stress [38]. ROS consists of free radicals such as ·OH, O2− and other non-radicals such as H 2 O 2 [39]. Although H 2 O 2 is a toxic by-product, it is involved in many physiological processes such as cell proliferation, differentiation, and immune regulation [40]. However, high concentrations of H 2 O 2 can promote cell apoptosis and oxidative stress in the brain, leading to neurodegenerative diseases such as AD [41,42].
The OH radical, as a by-product of ROS, has high reactivity and a strong oxidation capacity, which can damage cells or tissues in a short period of time [43]. Excess NO production in the brain can cause neuroinflammation and neurodegenerative diseases [44]. The present results showed that the ·OH and NO radical scavenging activity of pectolinarin increased dependent on its concentration, with its ·OH scavenging activity exceeding 80% at concentrations of 2.5, 5, and 10 µg/mL. These results suggest that pectolinarin has antioxidant properties for the scavenging of ·OH and NO radicals.
Several studies have found that throughout the natural aging process, various food intakes or physiological activities cause an increase in the excessive production of ROS, which promotes the deposition of Aβ and accelerates the formation of senile plaques [45,46]. The MTT assay was used in our present study to confirm the protective effects of pectolinarin against H 2 O 2 -induced oxidative stress in SH-SY5Y cells. The MTT assay is a standard approach for evaluating mitochondrial damage by measuring cell viability, and importantly, is well-known for assessing cell metabolism-in contrast with the sulforhodamine B assay, which assesses the protein contents [47]. The DCF-DA assay was used to confirm the inhibitory effects of pectolinarin against H 2 O 2 -induced ROS formation in SH-SY5Y cells. Dichlorofluorescein (DCF) is a highly fluorescent chemical formed by the oxidation of 2 ,7 -dichlorodihydrofluorescein (DCFH) by ROS [48]. Meanwhile, we used the LDH cytotoxicity kit to confirm any inhibitory effects on LDH release. After being treated with pectolinarin, the SH-SY5Y cells showed an antioxidant effect by increasing cell viability and reducing ROS production. Besides this, treatment with pectolinarin lowered LDH secretion. These results suggest that pectolinarin can protect against H 2 O 2 -induced cell death, ROS production, and LDH increase.
Nrf2 is a transcription factor that regulates gene expression in antioxidant defense, detoxification, and anti-inflammatory pathways [49]. HO-1 is an enzyme that catalyzes heme breakdown to produce biliverdin, carbon monoxide, and iron. Biliverdin is then metabolized further to bilirubin, which has powerful antioxidant and anti-inflammatory properties [50]. Under normal conditions, Keap1 binds to Nrf2 and regulates Nrf2 activity by promoting ubiquitin-mediated degradation [51]. Studies have shown that activation of the Nrf2/HO-1 system can protect neurons against oxidative stress and prevent the development of neurodegenerative diseases [52,53]. In our present results, treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) in H 2 O 2 -induced SH-SY5Y cells showed a significantly increase in HO-1 and Nrf2 protein expression. In addition, treatment with pectolinarin (1, 2.5, 5, and 10 µg/mL) significantly decreased the expression of Keap1 protein. AD is closely related to the overproduction of ROS and oxidative damage in the brain, and research has shown that regulating the Nrf2/HO-1 pathway can also reduce ROS production, which is consistent with the results of this experiment [54]. These results suggest that pectolinarin has neuroprotective effects against oxidative stress induced by H 2 O 2 through the regulation of antioxidant-related proteins in SH-SY5Y cells.
Inflammation is an immunological response to oxidative stress caused by an excess of ROS. NO is produced by the deamination of arginine under the action of iNOS and enters the adjacent cell through the cell membrane to cause inflammation [55]. COX-2 is an enzyme involved in the production of prostaglandins, as well as the promotion of inflammation and oxidative stress. Inhibiting COX-2 expression may have anti-aging effects by reducing inflammation and oxidative stress [56]. IL-1β is a biologically active small molecule protein that engages in cell signal transmission during the inflammatory response and produces additional pro-inflammatory cytokines [57]. This study found that H 2 O 2 -induced SH-SY5Y cells exhibited considerably higher levels of iNOS, COX-2, and IL-1β expression than H 2 O 2 -untreated SH-SY5Y cells. Furthermore, treatment with pectolinarin significantly decreased the expression of iNOS, COX-2, and IL-1β proteins. During the pathogenesis of AD, the inflammatory response causes neuronal damage and cell death. These proteins are involved in cell signaling in this pathogenesis process, which promotes the inflammatory response [58]; therefore, iNOS, COX-2, and IL-1β may be key players in the etiology of AD. These results suggest that pectolinarin protects H 2 O 2 -induced SH-SY5Y cells through regulating inflammation-related proteins.
Apoptosis is an active process that is regulated by a variety of genes [59]. H 2 O 2 is the source of ·OH that can cross the cell membrane of the brain and cause DNA injury, lipid peroxidation, and oxidative stress-ultimately accelerating apoptosis in the brain [60]. Previous research has investigated apoptosis induction using various methods, such as by TUNEL assay, flow cytometry, and Western blots. Zhao et al. [61] confirmed that H 2 O 2 -treatment significantly induced apoptosis in SH-SY5Y cells using the TUNEL assay and flow cytometry with an Annexin V-FITC/PI kit. Moreover, Tian et al. [62] reported that H 2 O 2 -induced SH-SY5Y cells increased the expression ratio of Bax/Bcl-2, which is a valuable predictor of apoptosis. In the present study, the effect of pectolinarin on H 2 O 2 -induced apoptosis was studied using Western blots for the Bax/Bcl-2 ratio. The Bcl-2 protein family is classified into two categories: the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. The main mechanism of these two proteins is that Bax improves the permeability of the mitochondrial membrane and promotes cytochrome C protein release, ultimately leading to neuronal cell death [63,64]. On the other hand, Bcl-2 protects neuronal cells against death, apoptosis, and oxidative stress stimulation [65]. Our study showed that Bax/Bcl-2 protein expression was significantly increased in the H 2 O 2induced control group, but treatment with pectolinarin significantly reduced the Bax/Bcl-2 protein expression. These results suggest that pectolinarin protects H 2 O 2 -induced SH- SY5Y cells by regulating apoptotic pathway proteins. These results show that pectolinarin could regulate the expression of Bax and Bcl-2 proteins, reducing H 2 O 2 -induced oxidative stress and apoptosis in SH-SY5Y cells. This suggests that pectolinarin, as a potential natural compound in functional foods for use against AD, exhibits anti-oxidative stress and cytoprotective effects.

OH Scavenging Assay
The method of Gutteridge was used to test pectol [66]. After mixing 10 mM FeSO4·ּ H2O-EDTA, 10 mM 2-d tolinarin for each concentration, 10 mM H2O2 was adde 37 °C for 4 h before adding 2.8% TCA and 1.0% TBA solu ice cooling for 10 min, and measuring absorbance at 490

NO Radical Scavenging Assay
The method of Marcocci [67] was used to assess pectolinarin's NO radical scavenging ability. Each pectolinarin concentration was dissolved in methanol, and a 5 mM sodium nitroprusside solution was added and incubated at room temperature for 150 min. After the reaction, the mixed solution was placed in a 96-well plate at a ratio of 1:1 with Griess reagent and reacted at room temperature with light protection for 30 min, and the absorbance was measured at 540 nm. The formula was as follows: NO radical scavenging activity (%) = (Absc − Abss)/Absc × 100 Absc: Absorbance of control, Abss: Absorbance of sample.

Cell Culture
SH-SY5Y human neuronal cells were cultured in DMEM medium containing 10% FBS and 100 units/mL penicillin-streptomycin in an incubator at 37 • C and 5% CO 2 humidity. Cells were cultured in a T75 flask, rinsed with phosphate-buffered saline (PBS, PH = 7.4) until they reached a growth state of more than 80% differentiation, and then 2 mL of trypsin-EDTA mixture was added to separate the attached cells before centrifugaton at 1000 rpm for 3 min.

Cell Viability
The MTT technique was used to assess cell viability [68]. When the cells were more than 80% differentiated, 2.5 × 10 5 cells/well were seeded in a 96-well plate and incubated at 37 • C for 24 h. After 24 h, pectolinarin in different concentrations was added to the plate for each well and cultured for 4 h before being treated with H 2 O 2 (500 µM) and cultured for another 24 h. After 24 h incubation, the medium was removed from each well, 200 µL of 5 mg/mL MTT solution added, and the plate incubated for 4 h at 37 • C. After that, the MTT solution was removed and DMSO added for 30 min. The absorbance was measured at 540 nm (Thermo Fisher Scientific, Waltham, MA, USA).

Measurement of ROS Production
The DCF-DA assay was used to measure ROS production according to Wang and Zhu's method [69]. When the cells had been differentiated by more than 80%, they were seeded at 2.5 × 10 5 cells/well in a black 96-well plate and cultured at 37 • C for 24 h. After 24 h, pectolinarin at different concentrations was added to the plate for each well and cultured for 4 h before being treated with H 2 O 2 (500 µM) and cultured for 24 h. After 24 h, cells were treated with 80 µM of DCF-DA solution after removing the medium from each well, incubating for 30 min at 37 • C, and then measuring the fluorescence at an excitation of 480 nm and emission of 535 nm using a FLUO starOPTIMA (BMG labtech, Ortenberg, Germany).

Measurement of LDH Release Activity
The LDH release was assessed using an LDH cytotoxicity detection kit, following the manufacturer's protocol (Takara Bio Inc., Kusatsu, Japan). The cell supernatant was combined with the LDH reaction mixture (catalyst: dye solution = 1:45) and incubated at room temperature for 30 min. The absorbance was measured at 490 nm (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis
All results are expressed as mean ± standard deviation. Statistical significance was determined using one-way analysis of variance followed by Duncan's multiple test on the SPSS statistics program (version 25, SPSS Inc., Chicago, IL, USA). Statistical significance was considered as p < 0.05.

Conclusions
In conclusion, pectolinarin had in vitro antioxidant capabilities for ·OH and NO radicals scavenging, increased cell viability, and decreased ROS formation in H 2 O 2 -induced SH-SY5Y cells. In addition, pectolinarin inhibited LDH release in H 2 O 2 -induced SH-SY5Y cells. Furthermore, pectolinarin regulated oxidative stress, inflammation, and apoptosisrelated protein expression and provided neuroprotection against H 2 O 2 -induced oxidative stress in SH-SY5Y cells. Therefore, pectolinarin has the potential to be used as a plant material for functional foods, to be applied for the treatment of oxidative stress-related neurodegenerative diseases. In the future, we will investigate the protective mechanisms of pectolinarin against inflammation and oxidative damage in aging mouse brain models, in order to provide a theoretical basis for future research and the development of AD functional foods.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.