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Jun 25, 2019 - The mechanisms underlying neurodegenerative diseases are not fully understood yet. However, an increasing amount of evidence has ...
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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Lipoamide Ameliorates Oxidative Stress via Induction of Nrf2/ARE Signaling Pathway in PC12 Cells Yanan Hou, Xinming Li, Shoujiao Peng, Juan Yao, Feifei Bai, and Jianguo Fang*

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State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China ABSTRACT: The mechanisms underlying neurodegenerative diseases are not fully understood yet. However, an increasing amount of evidence has suggested that these disorders are related to oxidative stress. We reported herein that lipoamide (LM), a neutral amide derivative of lipoic acid (LA), could resist oxidative stress-mediated neuronal cell damage. LM is more potent than LA in alleviating hydrogen peroxide- or 6-hydroxydopamine-induced PC12 cell injury. Our results reveal that LM promotes the nuclear accumulation of NFE2-related factor 2 (Nrf2), following with the activation of expression of Nrf2governed antioxidant and detoxifying enzymes. Notably, silencing Nrf2 gene annuls the protection of LM, which demonstrates that Nrf2 is engaged in this cytoprotection. Our findings suggest that LM might be used as a potential therapeutic candidate for oxidative stress-related neurological disorders. KEYWORDS: lipoamide, oxidative stress, antioxidant, Nrf2, neuroprotective effect

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INTRODUCTION The common neurodegenerative disorders are affecting more and more people around the world.1 Although the underlying mechanism of neurodegeneration is not fully understood, accumulating evidence has supported that oxidative stress, caused by the excessive generation of reactive oxygen species (ROS) or/and the impaired antioxidant defense system, plays a critical role in these diseases.2,3 This is likely due to the vulnerability of the brain to oxidative stress as these tissues contain redox-active metal ions and plenty of polyunsaturated fatty acids and consume a lot of oxygen.2,4 ROS are produced endogenously from diverse metabolic processes.5,6 At low levels, ROS are important message molecules and involved in many signal transduction pathways.7,8 Since ROS are highly reactive, abnormal elevation of ROS level leads to oxidative stress and causes oxidative damage of various cellular components.9,10 So, mechanisms that exert a protective effect against oxidative injury may be of great importance in restoring the homeostasis of the ROS production and elimination. NFE2-related factor 2 (Nrf2) plays a vital role in antioxidant defense systems, and governs a battery of downstream antioxidant and detoxifying genes, such as NAD(P)H:quinone oxidoreductase 1(NQO1), thioredoxin reductase 1 (TrxR1), glutamate-cysteine ligase (GCL), thioredoxin 1 (Trx1), and heme oxygenase 1 (HO-1).11,12 Nrf2, the redox-sensitive nuclear factor, is ubiquitinated and proteasomal degraded by Kelch-like ECH-associated protein 1 (Keap1) because this inhibitory partner of Nrf2 anchors it tightly under basal conditions.13,14 Once cells are undergoing electrophilic or oxidative stress, the ability of Keap1 to target Nrf2 is impaired because certain cysteine residues may be changed. In this condition, the stabilized Nrf2 tends to escape from the control of Keap1 and then translocates and accumulates in the nucleus and makes a complex with the small Maf proteins to promote the expression of antioxidant response elements (ARE)mediated antioxidant and detoxifying genes.15,16 An effective © XXXX American Chemical Society

strategy is brought out that the inhibition of oxidative stress could be carried out by promoting the expression of Nrf2 and its downstream antioxidant enzymes. Lipoic acid (LA), a natural nutrient and antioxidant, has been regarded as a potent agent to prevent or treat some disorders, such as radiation injury,17 neurodegenerative diseases,18 and diabetes.19 Lipoamide (LM) is the neutral amide of LA, and these two compounds have similar structures and biological capacities.20 Lv et al. have reported that LA could promote the functional recovery through inducing the Nrf2/HO-1 pathway.21 Several other mechanism studies have also documented that LA displayed its protective effect via induction of Nrf2.22−24 Besides, studies have demonstrated that LM could act as an indirect antioxidant by stimulating phase II enzymes in ARPE-19 cells, a human epithelial cell line.25 Here we determined the differences between LA and LM in the cellular neuroprotection of PC12 cells. At the same concentrations, LM attenuated hydrogen peroxide (H2O2)- or 6-hydroxydopamine (6-OHDA)-induced oxidative injury to PC12 cells, while LA had little effect. In addition, LM enhanced Nrf2 expression level and its nuclear translocation. The expression levels of several antioxidant enzymes were also promoted. Furthermore, once the expression of Nrf2 was abolished, the protective effect of LM also vanished. Our results demonstrate that LM may have the potential to be an agent in the therapy of some neurological disorders.

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MATERIALS AND METHODS

Reagents and Chemicals. The purity of both LA and LM was more than 98%, and they were provided by Aladdin (Shanghai, China). Dimethyl sulfoxide (DMSO) was used to prepare 100 mM Received: Revised: Accepted: Published: A

April 29, 2019 June 25, 2019 June 25, 2019 June 25, 2019 DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry stock solutions of LA and LM, and the final concentration of DMSO is always no more than 0.4%. Tween 20, bovine serum albumin (BSA), phenylmethylsulfonyl fluoride (PMSF), Lipo6000 transfection reagent, and sodium orthovanadate (Na3VO4) were obtained from Beyotime (Nantong, China). Fetal bovine serum (FBS) and NADPH were provided by HyClone (South Logan, UT) and Roche (Mannheim,Germany), respectively. Primary antibodies for Nrf2, NQO1, lamin, actin, and Trx1 were purchased from Sangon (Shanghai, China). Primary antibody for HO-1 was obtained from Proteintech (Chicago, IL). Primary antibody for TrxR1, anti-mouse, and anti-rabbit antibodies were all provided by Santa Cruz Biotechnology (Santa Cruz, CA). Trx protein from recombinant E. coli and TrxR1 protein from recombinant rat were prepared as mentioned in our previous work.26,27 Chemicals employed in this study were all of analytical grade. Other reagents were products of Sigma-Aldrich (St. Louis, MO) unless otherwise mentioned. Cell Culture. PC12 cells, usually employed to study neurodegenerative disorders, were provided by Shanghai Institute of Cell Biology (Shanghai, China). Unless otherwise specified, PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (added with 100 units mL−1 penicillin and streptomycin, 10% FBS, and 2 mM glutamine). PC12 cells were maintained at 37 °C in 5% CO2. HEK293T (embryonic kidney-293T) cells were obtained from the same institute, and they were cultured in the same conditions as PC12 cells. As for PC12-shNrf 2 and PC12-shNT cells, they were cultured and treated according to our published work.27 MTT Assay. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in phosphate buffered saline (PBS) to prepare the 5 mg/mL stock solution and stored at −20 °C before it was used to measure the cell viability. PC12 cells (1 × 104 per well) were seeded in a 96-well plate and cultured for 1 day. Then various concentrations of compounds were used to treat cells for indicated time courses. After the culture medium was discarded, MTT (0.5 mg/ mL) in fresh medium was added to each well and incubated for 4 h at 37 °C. Subsequently, 100 μL of buffer (5% isobutanol, 0.1% HCl, and 10% SDS) per well was used to solubilize the formazan product at 37 °C. Then absorbance at 570 nm in each well was quantified by using a Multiskan GO (Thermo Scientific, Vantaa, Finland). Relative cell viability was calculated compared to the control (treated with an equal amount of DMSO). To measure the protective effect, LA- or LM-treated (no toxicity concentrations, 24 h) cells were exposed to 500 μM H2O2 or 200 μM 6-OHDA for 12 h. Additionally, the cell viability was measured by the MTT assay. This method is also used in measurement of shNrf 2 and shNT cell viability. LDH Leakage Assay. Once the integrity of the cell membrane is damaged, the cytosolic enzyme lactate dehydrogenase (LDH) is released into the medium. Here the LDH leakage assay was used to detect cell viability in this study. In brief, PC12 cells (2 × 105 cells per well) were seeded in a 12-well plate. After 1 day, cells were incubated with LM (50, 100 μM) for 24 h and then treated with 500 μM H2O2 or 200 μM 6-OHDA for 12 h. Culture medium was collected for LDH activity assay as mentioned in the published work.27 Hoechst 33342 Staining. The dye Hoechst 33342 was used to measure the cell apoptosis. In brief, PC12 cells (4 × 105 cells per well) were seeded in a 6-well plate and cultured for 24 h. Subsequently, cells were incubated with LM (50, 100 μM) for 24 h and then treated with 500 μM H2O2 or 200 μM 6-OHDA for 5 h. Then, the medium was replaced by FBS-free medium, and 5 μg/mL Hoechst 33342 was added. After staining PC12 cells for 15 min in the dark, cells were washed by PBS for at least three times. Then a fluorescence microscope was used to get photographs of each sample. Caspase-3 Activity Assay. This assay mentioned in our published work was also carried out to measure PC12 cell apoptosis.27 In brief, PC12 cells (1 × 106 cells per dish) were cultured in 60 mm dishes for 1 day and then incubated with LM (0, 50, 100 μM) for 24 h. Subsequently, cells were exposed to H2O2 (500 μM) or 6-OHDA (200 μM) for 12 h. Then cells were collected, resuspended, and lysed. The protein amount in the supernatant was quantified by Bradford method. Sample, buffer, and N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide

(Ac-DEVD-pNA) were mixed, and they were stored at 37 °C for 4 h. Cell apoptosis was measured by comparing the absorbance of each group at 405 nm. Intracellular ROS Measurement. 2′,7′-Dichlorfluorescein diacetate (DCFH-DA), a fluorescent probe, has often been employed to monitor the levels of oxidative stress for its sensitivity to ROS production. PC12 cells (2 × 105 cells per well) were cultured in a 12well plate for 24 h. Then cells were pretreated with LM (0, 50, 100 μM) for 24 h and exposed to 500 μM H2O2 or 200 μM 6-OHDA for 5 h. Subsequently, 10 μM DCFH-DA was added to each well and incubated for 30 min in the dark. Then intracellular ROS were measured by acquiring images by means of a Leica inverted fluorescence microscope. qRT-PCR. This method was carried out as described in our previous work.28 All primer sequences were provided by Invitrogen Life Technologies (Shanghai, China), and their sequences were as follows: GAPDH, forward 5′-CAGTGCCAGCCTCGTCTCAT-3′, reverse 5′-AGGGGCCATCCACAGTCTTC-3′; TrxR1, forward 5′ACTGCTCAATCCACAAACAGC-3′, reverse 5′-CCACGGTCTCTAAGCCAATAGT-3′; Trx1, forward 5′-CCTTCTTTCATTCCCTCTGTGAC-3′, reverse 5′-CCCAACCTTTTGACCCTTTTTAT-3′; NQO1, forward 5′-TCACCACTCTACTTTGCTCCAA-3′, reverse 5′-TTTTCTGCTCCTCTTGAACCTC-3′; GCLC, forward 5′-CAAGGACAAGAACACACCATCT-3′, reverse 5′-CAGCACTCAAAGCCATAACAAT-3′; GCLM, forward 5′-GGCACAGGTAAAACCCAATAGT-3′, reverse 5′-TTCAATGTCAGGGATGCTTTCT-3′; HO-1, forward 5′-GCCCTGGAAGAGGAGATAGAG-3′, reverse 5′-TAGTGCTGTGTGGCTGGTGT-3′. PC12 cells (1 × 106 cells per dish) were cultured in 60 mm dishes for 1 day and then incubated with LM (100 μM) for indicated time courses. Complementary DNA (cDNA) was generated from 50 ng RNA by using a Primescript RT reagent kit (TaKaRa, Dalian, China). The expression levels of mRNA were measured on a Mx3005PRT−PCR System (Applied Biosystems, Foster City, CA) by using the Power SYBR Green PCR Master Mix (TaKaRa, Dalian, China). Experiments were all independently performed three times. Western Blotting. PC12 cells were cultured in 100 mm dishes for 1 day. Then, several concentrations of LM were added and incubated for the indicated time courses. Then, total, cytoplasmic, and nuclear proteins were extracted from treated cells as mentioned in our previous work.27 The protein concentration of each sample was evaluated by Bradford method. Protein samples with equivalent amounts were loaded on SDS-PAGE for electrophoresis, and then PVDF membranes (Millipore, Bedford, MA) were used for transferring the separated proteins. These membranes were blocked with blocking solution (5% no-fat milk and 0.05% Tween-20 in PBS) for 1 h at room temperature and then stored in indicated primary antibodies (1:1000 dilution) overnight at 4 °C. Subsequently, membranes were kept in the corresponding secondary antibody (1:2000 dilution) for 1 h at room temperature. Imagequant LAS4000 (GE Healthcare, Munich, Germany) was used to record the bands, and ImageJ software was employed to measure the relative protein levels. Determination of NQO1, TrxR, Trx, HO-1 Activities and Total Thiol, Total Glutathione (GSH) Levels. For the assessment of NQO1, TrxR and Trx activities, total thiol level, and total GSH level, the detailed process was carried out as the protocols in our published work.27,29 Briefly, treated cells were washed, collected, and lysed, and the protein concentrations were measured by using the Bradford method. For measurement of total glutathione, collected cells were lysed and sonicated, and then the supernatant was used for total glutathione level assay according to the earlier work.30 For the measurement of NQO1, TrxR, Trx activities, and total thiol level, collected PC12 cells were lysed and vortexed, and then the protein in supernatant was used for assay according to the earlier published work.27 For the assessment of HO-1 activity, the method following a spectrophotometric procedure with some modifications was employed.31 In brief, treated PC12 cells were collected and lysed in buffer A (0.25 M sucrose in 20 mM Tris-HCl, pH 7.4, stored at 4 °C), B

DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry sonicated for 30 s, and the Bradford method was used to measure the protein amount in each sample. A 300 μL solution including 1 mg/ mL sample, 1 mM NADPH, 25 μM hemin, and excessive liver cytosolic fraction (lysed in buffer A, containing biliverdin reductase) was assembled. The mixture was vortexed several times and stored at 37 °C for 30 min in darkness. The negative control was prepared by substituting NADPH with an equal volume of buffer A. Then the reaction was stopped by adding 1 volume of chloroform. The mixture was vortexed for 30 s, followed by centrifuging at 4 °C for 10 min at 3000g. Chloroform phase was taken out, and its OD464 and OD530 were read. Considering the extinction coefficient of 40 mM−1/cm with OD464OD530, bilirubin concentration was calculated,and it represents the HO-1 activity. TrxR Activity Assay in Live Cells. To specifically and conveniently detect the intracellular TrxR activity, a fluorescent probe TRFS-Green was synthesized as described in the earlier work.32 When TRFS-Green reduction was catalyzed by TrxR, green fluorescence will occur. In brief, PC12 cells (2 × 105 cells per well) were cultured in a 12-well plate for 24 h and incubated with LM (50 μM, 100 μM) for 20 h, and new medium with 10 μM TRFS-Green was added. After 4 h, the TrxR activity was assessed by acquiring images using a Leica inverted fluorescence microscope. TrxR activity was indicated by the relative fluorescence intensity. Reporter Gene Assay. The pARE-luciferase plasmid (Beyotime, Nantong, China) was employed to transfect PC12 cells and HEK293T cells according to the manufacturer’s protocols. After cells were selected with G418 (0.5 mg/mL), they were incubated with LM (0, 50 μM, 100 μM) or tert-butylhydroquinone (tBHQ) (0, 20 μM, 40 μM) for 24 h. In this experiment, tBHQ was used as a positive control. Then cell lysates were prepared, and luciferase activity was measured with a Firefly Luciferase Reporter Gene Assay Kit (Beyotime, Nantong, China). Statistics. Unless otherwise mentioned, data in all experiments were performed at least three times and expressed as mean ± SD. One-way ANOVA was used to evaluate the differences between multiple groups, and P < 0.05 was considered as statistical significance.

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RESULTS LM Attenuated H2O2- or 6-OHDA-Induced Injury in PC12 Cells. In order to study the cytotoxicity of LA and LM, PC12 cells were incubated with various concentrations of LA or LM for 24 h, and then the relative cell viability was detected by using the MTT assay. Figure 1B illustrated that LA up to 1000 μM and LM up to 100 μM did not exert significant toxic effects. Two cell damage models, H2O2 model and 6-OHDA model were used in the evaluation of the cellular protection of LA and LM. H2O2 is a type of ROS, and it is widely used in the induction of oxidative stress. As for 6-OHDA, it causes death of dopaminergic neurons in substantia nigra pars compacta progressively mimicking Parkinson’s disease (PD). Studies have reported that 6-OHDA mediates neurodegeneration mainly through the induction of oxidative stress.33 At first, we selected serial concentrations and indicated treatment times of H2O2 or 6-OHDA on the injury of PC12 cells to establish the cytoprotective assay. As shown in Figure 1C, for the H2O2 model, the optimal time and concentration were 12 h and 500 μM, which leads to about 55% cell death. In the 6-OHDA model, the optimal time and concentration were 12 h and 200 μM, which leads to about 45% cell death as shown in Figure 1D. Therefore, these optimal conditions were used in the subsequent experiments. To assess the cytoprotective effect of LA and LM on PC12 cells, we chose 10, 50, 100 μM as the suitable nontoxic concentrations. Results in Figure 1E,F demonstrated that pretreatment with 50, 100 μM LM for 24 h could effectively and dose-dependently rescue PC12 cells

Figure 1. LM inhibited H2O2- or 6-OHDA-induced PC12 cell injury. (A) Chemical structures of LA and LM. (B) PC12 cell viability following LA or LM treatment at different concentrations. (C,D) Cytotoxicity evaluation of H2O2 (C) or 6-OHDA (D) for indicated times of treatment on PC12 cells. (E,F) Protection of LA or LM against PC12 cell injury induced by H2O2 (E) or 6-OHDA (F). (G,H) Inhibition of H2O2(G)- or 6-OHDA (H)-induced LDH release by LM. Experiments were all carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group. ^ P < 0.05 and ^^ P < 0.01, versus H2O2- or 6-OHDAtreated groups, respectively.

from cell injury caused by H2O2 or 6-OHDA, while LA had little protective effect. Similarly, Figure 1G,H showed that PC12 cells pretreated with 50, 100 μM LM significantly restored H2O2 or 6-OHDA-induced LDH leakage. The LDH assay could be considered as a verification experiment of the MTT assay, because LDH leakage indicated the integrity of the cell membrane. In summary, these experiments demonstrated that pretreatment of PC12 cells with LM could dosedependently improve H2O2- or 6-OHDA-induced cell viability decrease. LM Ameliorated Apoptosis in PC12 Cells. Hoechst 33342 staining and caspase-3 activity assay were both employed to assess the PC12 cells apoptosis because the condensed nucleus detected by Hoechst 33342 and the activation of caspase-3 are regarded as the marks of apoptotic cells. As shown in Figure 2A,B, almost no condensed nucleus C

DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. LM suppressed ROS generation caused by H2O2 (A) or 6OHDA (B). ROS accumulation was determined by DCFH-DA. (Scale bars represent 50 μm.)

Figure 2. LM rescued PC12 cells from apoptosis. (A,B) Suppression of LM on H2O2 (A)- or 6-OHDA (B)- induced cell apoptosis. Hoechst 33342 staining assay was employed to detect the apoptotic cells. (Scale bars represent 100 μm.) (C,D) The activity of caspase-3 was detected in H2O2 (C) and 6-OHDA (D) injury models. Experiments were all carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group. ^ P < 0.05 and ^^ P < 0.01, versus H2O2- or 6-OHDA-treated groups, respectively.

was detected in the control groups, while bright condensed nuclei (indicated by arrows) occurred clearly in the H2O2- or 6-OHDA-damaged groups. Pretreatment with 50, 100 μM LM significantly suppressed the cell apoptosis. In addition, the results of caspase-3 activity assay demonstrated that pretreatment of LM decreased H2O2- or 6-OHDA-induced caspase-3 promotion dose-dependently (Figure 2C,D), which was consistent with the Hoechst 33342 staining assay. LM Suppressed ROS Accumulation. ROS accumulation in living cells was detected by DCFH-DA because it is a sensitive and convenient fluorescent probe. As shown in Figure 3A,B, ROS accumulation rapidly increased when cells were cultured with H2O2 or 6-OHDA for 5 h. However, pretreatment with 50, 100 μM LM significantly attenuated ROS accumulation compared with the H2O2- or 6-OHDA-treated groups. These results revealed that LM could suppress the intracellular ROS accumulation. LM Promoted Expression of Detoxifying or Antioxidant Genes. Since LM could effectively rescue PC12 cells from oxidative injury, the redox-sensitive factor Nrf2 and the antioxidant system may be involved in this process. The expression levels or activities of several Nrf2-governed antioxidant and detoxifying enzymes (NQO1, TrxR1, Trx1, GSH, HO-1, and thiol) were determined. Figure 4A showed that exposure to LM resulted in a significant increase of genes of the antioxidant enzymes mentioned above. Among these

Figure 4. LM upregulated the expression of antioxidant genes and molecules. (A) The promotion of several phase II genes. (B,C) Effects of LM on protein expression of TrxR1, NQO1, Trx1, and HO-1 in PC12 cells. All experiments were carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group.

genes, the mRNA levels of HO-1 and TrxR were most inducible, for they were 4- to 5-fold elevated by LM. Other genes were induced by LM sustainably, and this action can last as long as 12 h. GCLC and GCLM expression was also assessed, as both of them act as important roles in the biosynthesis of GSH.34,35These data suggested that LM could efficiently induce the transcription of Nrf2-governed detoxifying or antioxidant genes. LM Upregulated and Restored the Antioxidant System. As Figure 4A had shown that Nrf2-governed phase II genes were upregulated by LM, we then examined these gene products. The NQO1, TrxR1, Trx1, and HO-1 protein expression assays were employed to evaluate the effect of LM. Results in Figure 4B,C showed that the corresponding proteins expression was promoted remarkably by LM in a dosedependent manner. Then Figure 5A−F indicated that the D

DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. LM rescued H2O2- or 6-OHDA-induced decline of several phase-II enzymes and antioxidants. Proteins extracted from treated PC12 cells were used to evaluate the activities of NQO1 (A), TrxR (B), Trx (C), HO-1 (F) and the levels of total GSH (D) and total thiol (E). All experiments were carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group. ^ P < 0.05 and ^^ P < 0.01, versus H2O2- or 6-OHDAtreated groups, respectively. Figure 5. LM promoted the antioxidant system in PC12 cells. Proteins were extracted from LM-treated PC12 cells. Then the activities of NQO1 (A), TrxR (B), Trx (C), HO-1 (F), the levels of total GSH (D), and total thiol (E) were measured. All experiments were carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group. (G) TRFS-Green probe was used to detect TrxR activity in LM-treated PC12 cells. (Scale bar represents 50 μm.)

Nrf2 Was Involved in the Protection of LM. To verify the role of Nrf2 in cytoprotection, we further examined the expression levels of nuclear, cytosolic, and total Nrf2 protein by using Western blotting analysis. LM treatment improved the expression of nuclear Nrf2 remarkably, and the peak occurred at 4 h (Figure 7A,C). The total Nrf2 also increased slightly, and the peak occurred at 8 h (Figure 7A,B). Although Nrf2 protein in cytoplasm decreased time-dependently (Figure 7A,D), it may be due to the Nrf2 nuclear translocation. These data demonstrated that LM promoted the Nrf2 nuclear translocation and may upregulate the expression of downstream antioxidant genes. To confirm this, PC12-ARE cells and HEK-293T-ARE cells, two stable cell lines transfected with pARE-luciferase plasmid, were treated with tBHQ or LM for 24 h. Results in Figure 8A,B showed that LM induced ARE-luciferase reporter activity dose-dependently in both PC12-ARE cells and HEK-293TARE cells, which is similar to that of tBHQ, a well-known Nrf2 agonist. These data suggested that LM has the ability to activate Nrf2-dependent transcription. To study whether the protective effect of LM was mediated by Nrf2, we employed PC12-shNrf 2 and PC12-shNT cells in this experiment. As shown in Figure 8A,B, the content of Nrf2 in PC12-shNrf 2 cells was about 40% of that in PC12-shNT cells. Then the MTT assay was employed to evaluate the LM protective effect in these two cell lines. Results in Figure 8C,D

activities of TrxR1, Trx1, HO-1, NQO1, and the levels of GSH and total thiol increased significantly after cells were incubated with LM. In our lab, the TRFS-Green probe was designed and synthesized to detect the intracellular TrxR activity. Figure 5G revealed that LM promoted the TrxR activity, which is a supplementary experiment of Figure 5B. These results demonstrated that LM could effectively promote the expression of a series of endogenous protective molecules. Figure 6 showed that a serious decrease of NQO1 (Figure 6A), TrxR1 (Figure 6B), Trx1 (Figure 6C), GSH (Figure 6D), HO-1 (Figure 6E), and total thiol (Figure 6F) levels occurred when cells were cultured with H2O2 or 6-OHDA alone. While, we also found that pretreatment with LM (100 μM) remarkably rescued the decline of these antioxidant molecules and restored the redox homeostasis. In summary, these data supported that the neuroprotection of LM is through the promotion of many antioxidant molecules. E

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might have the ability to activate Nrf2 factor and exert protective effect in PC12 cells. Here we employed PC12 cell as a model and demonstrated that LM has the abilities to rescue the cells from injuries caused by either H2O2- or 6-OHDA (Figures 1 and 2), suppress the intracellular ROS accumulation (Figure 3), upregulate a series of antioxidant enzymes (Figures 4 and 5), and restore the antioxidant system (Figure 6). Further mechanistic studies revealed that LM promotes the Nrf2 nuclear translocation and the downstream gene expression (Figured 7 and 8) and that knockdown of Nrf2 expression annuls the protective effect elicited by LM (Figure 9). In summary, these results suggested that LM protected PC12 cells from oxidative stress-induced damage by activating Nrf2 factors.

Figure 7. Expression level of Nrf2 was upregulated by LM in PC12 cells. LM-treated PC12 cells were collected, and their fractions were prepared. (A) Western blotting analysis was used to measure the total Nrf2, nuclear Nrf2, and cytosolic Nrf2. Quantification results of them were shown in (B−D), respectively. All experiments were carried out three times, and data were expressed as means ± SD. * P < 0.05 and ** P < 0.01, versus the control group.

Figure 9. Nrf2 was of great importance in cytoprotection. (A) Levels of Nrf2 expression in PC12-shNrf 2 and PC12-shNT cells. Quantification results of them were shown in (B). (C,D) Protection of LM against PC12-shNrf 2 and PC12-shNT cell injury caused by H2O2 (C) or 6-OHDA (D). All experiments were carried out three times, and data represent the means ± SD. ** P < 0.01, versus the control group. ^^ P < 0.01, versus H2O2- or 6-OHDA-treated groups.

Figure 8. ARE transcriptional activation was induced by LM in transfected PC12 cells (A) and HEK-293T cells (B). Transfected PC12 and HEK-293T cells were treated with tBHQ (20, 40 μM, positive control) or LM (50, 100 μM) for 24 h, and the cell lysates were used for luciferase activity measurement. All experiments were carried out three times, and data represent the means ± SD. * P < 0.05 and ** P < 0.01, versus the control group.

LM is better than LA in the protective effect, and it may be due to the presence of −CONH2. The resonance stabilization of the CO-N makes LM more stable than LA, and its lower acidity helps LM adapt to the physiological pH.20,43 In addition, Zhou et al. and Bilska et al. have documented that LM can easily penetrate the blood-brain barrier (BBB) because of its higher lipid solubility, and the ability of LM to adapt to the body environment exceeds that of LA.39,44 The little neuroprotection of LA may be partially due to its low bioavailability.45,46 Studies have also reported that LM had a greater antioxidant effect than LA, which may contribute to the protective effect of LM.25 Above all, we suggest that LM might be a potent central nervous system drug. Nrf2/ARE pathway is a pivotal defense mechanism against oxidative stress. One characterized mechanism controlling Nrf2 activation, termed as canonical activation, is through modifications of the Cys sulfhydryl groups in Keap1 by electrophiles or oxidizing species.47 The exact mechanism of activation of Nrf2 by LM is not clear. However, previous studies have suggested that LA may activate Nrf2 via oxidizing the sulfhydryl groups in the Keap1 protein.36,48 Thus, it is expected that LM works in the same way to activate Nrf2 and upregulate the expression levels of various antioxidant and

demonstrated that the protection of LM against cell injury caused by H2O2 or 6-OHDA almost vanished in the Nrf2silencing cells, highlighting the importance of Nrf2 in this cytoprotection.

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DISCUSSION LA widely distributes in many plants and animals, and it is often used as antioxidant and nutrient supplement.36 LA is also considered as a potent candidate in the prevention and treatment of a battery of disorders, such as radiation injury,17 diabetes,37 and some oxidative-stress-related diseases.18,38 Furthermore, many studies have disclosed that LA stimulates the expression of Nrf2-driven antioxidant species.23−25 In addition, Zhou et al. have reported that LM, the neutral amide of LA, may have a neuroprotective effect in an animal Parkinson’s disease model.39 However, many observations have shown that LM has better effective properties, such as in protection of macrophage-like cells from oxidative injury,40 in the prevention of thymocytes from etoposide-induced apoptosis,41 and in the protection of neuronal cells from glutamate-induced cell injury.42 Herein, we assumed that LM F

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Journal of Agricultural and Food Chemistry

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detoxifying enzymes, such as Trx1, NQO1, GSH, TrxR1, and HO-1. Figure 10 has illustrated the proposed mechanism for the protection of LM in PC12 cells.

Figure 10. Working model of the protective effect of LM on the induction of Nrf2 in PC12 cells.

In conclusion, these experiments illustrated that LM significantly protected PC12 cells from oxidative injury via activation of Nrf2 and its downstream detoxifying and antioxidant enzymes. We also found that LM is superior to LA in this protection, and the possible reasons are listed above. On the basis of our work, we can surmise that LM may be useful as a potential candidate against oxidative injury and a promising agent for preventing the neurodegenerative disorders.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Fax: +86 931 8915557. ORCID

Jianguo Fang: 0000-0002-2884-3363 Funding

We gratefully acknowledge the National Natural Science Foundation of China (21572093, 21778028) and the 111 project for the financial support. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.9b02680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX