Research Article pubs.acs.org/chemneuro
Cite This: ACS Chem. Neurosci. 2019, 10, 2956−2966
Xanthohumol Analogues as Potent Nrf2 Activators against Oxidative Stress Mediated Damages of PC12 Cells Feifei Bai,† Baoxin Zhang,*,† Yanan Hou,† Juan Yao,† Qianhe Xu,† Jianqiang Xu,‡ and Jianguo Fang*,† †
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State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, 222 South Tianshui Road, Lanzhou 730000, China ‡ School of Life Science and Medicine & Panjin Industrial Technology Institute, Dalian University of Technology, Panjin Campus, Panjin 124221, China ABSTRACT: The nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor controlling a series of cytoprotective genes, is closely associated with scavenging the reactive oxygen species and maintaining the intracellular redox balance. Accumulating evidence has indicated that activation of Nrf2 is efficient to block or retard oxidative stress mediated neurodegenerative disorders. Small molecules that contribute directly or indirectly to the Nrf2 activation thus are promising therapeutic agents. Herein, we screened xanthohumol and its analogues, and two analogues (11 and 12) were disclosed to possess low cytotoxicity and rescue PC12 cells from the hydrogen peroxide or 6-hydroxydopamine induced injuries. Molecular mechanism studies demonstrated that compounds 11 and 12 are potent Nrf2 activators by promoting the nuclear accumulation of Nrf2 and enhancing the cellular antioxidant defense system. More importantly, genetically silencing the Nrf2 expression shuts down the observed cytoprotection conferred by both compounds, supporting the critical involvement of Nrf2 for the cellular actions of compounds 11 and 12. KEYWORDS: Nrf2, reactive oxygen species, neurodegenerative disorders, xanthohumol analogues
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INTRODUCTION Reactive oxygen species (ROS) are produced from various physiological processes and have been proven to be essential messenger molecules involved in diverse signaling pathways.1−3 However, massive accumulation of ROS resulting from the imbalance between ROS formation and elimination leads to oxidative stress, which has been entangled in neurodegenerative disorders.4−6 A vast amount of research shows that additional increment of exogenous antioxidants or upregulation of cellular antioxidant defense systems could effectively alleviate neurodegenerative diseases.7,8 The Kelchlike ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and antioxidant response elements (ARE) signaling pathway is one of the main pathways to regulate intracellular redox balance.9,10 Under normal conditions, Keap1, existing as a homodimer, interacts with one molecule of Nrf2 to form a Keap1-Nrf2 complex with two different affinity binding sites (ETGE motif and DLG motif) and represses the activation of Nrf2. Meanwhile, Keap1 also associates with a functional E3 ubiquitin ligase complex (Rbx1) by the scaffold protein (Cullin3), which facilitates Rbx1mediated polyubiquitination of Nrf2. The ubiquitinated Nrf2 is subsequently degraded via 26S proteasome.11 Under stress conditions, Keap1 rich in active sulfhydryl groups of cysteine residues is modified, which affects the low-affinity DLG motif interaction of Nrf2, resulting in disturbance of the Keap1-Nrf2© 2019 American Chemical Society
Cul3 assembly and Nrf2 ubiquitination. Nrf2 releases from the Keap1-Nrf2 complex and accumulates in the cytosol. Then it transfers into the nucleus and subsequently binds to ARE by heterodimerizing with small Maf proteins (musculoaponeurotic fibrosarcoma) to modulate the transcription of a vast array of antioxidant and phase II detoxifying genes.10,12 In addition, AMP-activated kinase that participates in cellular redox and energy homeostasis, protein kinase C that phosphorylates Nrf2, and glycogen synthase kinase 3β that could be involved in the regulation of cell differentiation, proliferation, and apoptosis have been implicated in modulation of Nrf2.12−14 A variety of biomolecules such as glutathione (GSH), NAD(P)H quinone oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1) could prevent the cells from oxidative injury and elevate cell survival. Therefore, activating Nrf2 is a promising therapeutic strategy for neurodegenerative diseases.15,16 Due to the vital function of Nrf2 in antioxidant defense, Nrf2-targeting agents have been explored extensively for the past years.17,18 It has been reported that several classes of Nrf2 activators, either extracted natural products or synthetic small molecules such as 6-dehydrogingerdione,19 honokiol,20 resveratrol (Res),21 quercetin,22 sulforaphane,23 xanthohumol Received: March 22, 2019 Accepted: May 22, 2019 Published: May 22, 2019 2956
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
Research Article
ACS Chemical Neuroscience
Figure 1. Structures of compounds Xn and 1−23.
(Xn),24 and dithiolethiones,25 exert impact on the expression of cytoprotective enzymes. Xn, a naturally occurring ingredient isolated from hops (Humulus lupulus L.) was found to exhibit abundant pharmacological activities including antimicrobial,26 anti-inflammatory,27 antioxidant,28,29 anticancer,30 neuroprotective,31 cardiovascular protection,32 antimenopausal syndrome,33 antiobesity,34 and cancer chemoprevention.35 In previous work, we revealed that Xn was an efficient Nrf2 activator and displayed neuroprotection against oxidative insults of PC12 cells.19 In view of this, we modified Xn and synthesized a series of analogues. With further study of their neuroprotective effects, we surprisingly found that compounds 11 and 12 showed lower toxicity and more excellent protective effects against injuries of PC12 cells caused by hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA) compared with Xn. Further studies revealed that both compounds raised nuclear accumulation of Nrf2 and promoted the expression of downstream antioxidant molecules of Nrf2 to reduce intracellular ROS level and relieve H2O2- or 6-OHDA-caused apoptosis in PC12 cells. In addition, the Nrf2 knockdown experiment in PC12 cells eliminated the protective response of 11 and 12, further indicating that Nrf2 was essential for the protective response of compounds 11 and 12 toward the cells.
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group (compounds 1−10). Meanwhile, some compounds with the removing prenyl group (compounds 11−23) were synthesized to analyze the function of the prenyl group on activity. The structures of compounds are shown in Figure 1, and the detailed synthetic procedure and characterization of compounds including 1H NMR, 13C NMR spectra and mass spectra were reported in our previous publications.36 The purity of final compounds was assessed by HPLC and was found to be higher than 95%. Initial Screening. Initially, we adopted PC12 cells (a neuronlike rat pheochromocytoma cell line) to assess the cytotoxicity of Xn analogues by the MTT assay. As shown in Figure 2, compounds 1−5, 7−10, 15, 18, and 21 exhibited strong toxicity to PC12 cells at the concentrations of 5, 10, and 20 μM; Xn and compounds 6, 22, and 23 presented apparent cytotoxicity at concentration of 20 μM, while compounds 11− 14, 16, 17, 19, and 20 displayed negligible toxicity toward the cells at the maximum concentration (20 μM). Additionally, compound 13 was nontoxic and appeared to contribute to the cell growth, as the cell viability of 13 increased to 118% at 20 μM. Next, we applied the H2O2 injury model and 6-OHDA injury model, two established models of neurodegenerative disorders in cells, to probe whether Xn analogues alleviated oxidative damage to PC12 cells. 6-OHDA, a neurotoxic synthetic organic compound, selectively destroying dopaminergic and noradrenergic neurons in the brain, is used to induce Parkinson’s model in laboratory.37 Figure 3 indicated that all compounds at different concentrations (0.5 μM and 1 μM) showed little protection on PC12 cells against oxidative lesions. As is well-known, the hydroxyl group could improve
RESULTS
Chemistry. Based on our previous work that Xn protected PC12 cells from H2O2- or 6-OHDA-caused oxidative damage, we synthesized diverse compounds based on the structure of Xn that contained a Michael acceptor structure and prenyl 2957
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
Research Article
ACS Chemical Neuroscience
Figure 2. Effects of Xn and its analogues on PC12 cells. The cells were subjected to different compounds at the concentrations of 5, 10, and 20 μM for 24 h, and the cytotoxicity of the compounds to the cells was measured by the MTT assay. All data from three independent experiments are represented as means ± SD; *P < 0.05, and **P < 0.01 vs the control group.
Figure 4. Cytoprotective effects of Xn, 11, 12, 13, 20, and 23 against H2O2- or 6-OHDA-caused injury. PC12 cells were stimulated with different compounds for 24 h, and the original medium was replaced with the fresh medium including H2O2 (A) or 6-OHDA (B), and then the cell survival was measured via the MTT assay. Data from three independent experiments are represented as means ± SD; **P < 0.01 vs the control group; ∧P < 0.05 and ∧∧P < 0.01 vs the group only treated with H2O2 or 6-OHDA. Figure 3. Cytoprotection of Xn analogues against H2O2- or 6-OHDAcaused damage. The cells were subjected to different compounds at different concentrations for 24 h, and the original medium was removed. The fresh medium including H2O2 (A) or 6-OHDA (B) was added for 12 h, and then the cell viability was measured by the MTT assay. Data from three independent experiments are represented as means ± SD; **P < 0.01 vs the control group; ∧P < 0.05 and ∧∧P < 0.01 vs the group only treated with H2O2 or 6-OHDA.
Defense of PC12 Cells against H2O2- and 6-OHDACaused Injury by 11 and 12. Inspired by the above results, 11 and 12 were chosen to further investigate whether they exert protective response on PC12 cells against the H2O2- and 6-OHDA-caused insults. As shown in Figure 4A, compared with the H2O2-treated group, pretreatment with 11 or 12 for 24 h significantly protected PC12 cells from oxidative damage in a dose-dependent manner. For instance, the PC12 cells subjected to H2O2 led to about 46% cell death; however, in the group treated with 11 at 10 μM (12 at 10 μM) before H2O2 exposure, the cell survival was increased to ∼77% (∼83%). As shown in Figure 4B, 6-OHDA decreased the cell viability to 58% of the control, while, if the cells were preprotected with 11 or 12, the cell viability was obviously increased in a concentration-dependent manner. Compounds 11 and 12 (10 μM) increased the cell survival to ∼76% and ∼81%, respectively. To further confirm the effects of 11 and 12 on the cell growth, we checked the activity of lactate dehydrogenase (LDH) in culture medium. LDH is a cytoplasmic enzyme that catalyzes pyruvate to lactic acid and NADH to NAD+ during glycolysis. Once the integrity of the cell membrane is disrupted, LDH will be released from the cytoplasm into the culture medium. Thus, detecting the level of LDH in the culture medium could indirectly determine the toxicity of the compounds toward the cells.38,39 As shown in Figure 5, the LDH activities of the H2O2- and 6-OHDA- treated group were
the antioxidant activity of the molecule. Among the tested compounds, 11 and 12 contained phenol groups on the phenyl ring B, which might increase the antioxidant abilities of them. Meanwhile, compounds 11, 12, 20, and 23 displayed similar protective activities on PC12 cells. On the basis of these reasons, we selected compounds 11, 12, 13, 20, and 23 for the follow-up studies. The results (Figure 4) revealed that the PC12 cells stimulated with H2O2 led to about 46% cell death and compound 13 did not increase the cell viability to PC12 cells. However, treatment of the cells with compounds 11, 12, 20, and 23 (5 μM) for 24 h before H2O2 exposure significantly decreased the cell death. In addition, an obvious increase in the cell survival was not observed after pretreating with 20 and 23 (10 μM), while an evident drop in the cell death was shown by pretreatment with 11 and 12 (10 μM). In the 6-OHDA injury model, the cytoprotective activities of 11, 12, 13, 20, and 23 were similar to the results obtained from the H2O2 damage model. 2958
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
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connection between apoptosis and protective activity of 11 and 12, we detected nuclear morphology and caspase-3 activity. Hoechst 33342 is a member of the Hoechst stains, blue fluorescent dyes employed to stain DNA, which couples to the minor groove of double-stranded DNA with a preference for sequences rich in adenine and thymine.37 As shown in Figure 6A, B, no obvious apoptotic nuclei were detected in the control cells, while the PC12 cells treated with H2O2 or 6-OHDA showed the bright blue fluorescence. The result indicated that both H2O2 and 6-OHDA triggered apoptosis. However, a distinct decrease of fluorescence intensity was observed in PC12 cells incubated with H2O2 or 6-OHDA after 11 or 12 exposures. We also tested the activity of intracellular caspase-3. H2O2 and 6-OHDA led to the improvement of activity of caspase-3. Nevertheless, upon pretreatment of PC12 cells with 11 or 12, a visible drop of the caspase-3 activity was exhibited (Figure 6C, D) in a dosedependent manner. Altogether, compounds 11 and 12 markedly prevented the apoptosis arising from H2O2 or 6OHDA. Preclusion of Intracellular ROS Accumulation. Oxidative stress occurs once the growth of ROS produced by 6OHDA or exogenously supplemented with H2O2 exceeds the ability of the cell itself to eliminate oxidants, resulting in apoptosis.1 In order to identify whether two compounds mitigate oxidative stress in cells, the level of ROS was assessed. 2′,7′-Dichlorfluorescein diacetate (DCFH-DA), a nonfluorescence dye that can permeate freely through the cell
Figure 5. Cytoprotection of compounds 11 and 12 against oxidative injury. The cells were stimulated with different concentrations of compounds 11 and 12 for 24 h, and then exposed to H2O2 or 6OHDA for 12 h. The LDH activity in the medium was measured. All data from three independent experiments are represented as means ± SD **; P < 0.01 vs the control group; ∧P < 0.05 and ∧∧P < 0.01 vs the group only treated with H2O2 or 6-OHDA.
nearly 2.6-fold and 2.3-fold that of the control, respectively. However, if the cells were subjected to 11 or 12 before H2O2 or 6-OHDA incubation, the LDH activity was remarkably decreased. The results were in tune with the data obtained by MTT assay. Collectively, compounds 11 and 12 could effectively defend against cellular damage caused by H2O2 or 6-OHDA. Reduction of Cell Apoptosis Induced by H2O2 or 6OHDA. Apoptosis, programmed cell death, has the characteristics of nuclear chromatin condensation in morphology and activation of cytosolic caspase proteins. To explore the
Figure 6. Inhibition of H2O2- or 6-OHDA-induced apoptosis by 11 and 12. The cells were stimulated with different concentrations of 11 and 12 for 24 h, and then H2O2 (A) or 6-OHDA (B) was added for another 12 h. Then the cells were stained by Hoechst 33342. Scale bars: 100 μm. (C, D) PC12 cells were subjected to various concentrations of 11 and 12 for 24 h, and subsequently treated with H2O2 (C) or 6-OHDA (D) for 12 h. The caspase-3 activity was measured by a colorimetric assay. All data from three independent experiments are represented as means ± SD **P < 0.01 vs the control group; ∧∧P < 0.01 vs the group only treated with the H2O2 or 6-OHDA. 2959
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
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ACS Chemical Neuroscience
Figure 7. Alleviation of the accumulation of intracellular ROS by 11 and 12. Images show the level of ROS by DCFH-DA staining. Scale bars: 50 μm.
explanation that 11 and 12 efficiently reduced the accumulation of intracellular ROS and the cytoprotective activity of 12 was superior to 11. Analysis of the chemical structures of 11 and 12 revealed that they contained the α,β-unsaturated ketone structure, which was the reaction center of many potent Nrf2 activators. Thus, we assumed that compounds 11 and 12 could also effectively induce Nrf2 to alleviate oxidative stress. To test this hypothesis, we analyzed the expression of downstream antioxidant/detoxifying genes of Nrf2 at the mRNA level after incubating the PC12 cells with 11 or 12 (10 μM) for 0, 3, 6, and 12 h. Figure 9 shows that stimulation of
membrane, has been widely used in the detection of intracellular ROS. The probe diffuses into the cell and then DCFH-DA is hydrolyzed to nonfluorescent 2′,7′-dichlorodihydrofluorescin (DCFH) by the esterase. DCFH reacts with the oxidants to produce a visible green fluorescence.40 The intensity of green fluorescence in cells reflects the production of ROS. As shown in Figure 7, the cells exposed to H2O2 or 6OHDA showed bright green fluorescence, suggesting a burst of ROS, while a negligible fluorescence was observed in the control cells. However, both 11 (12) preprotection and H2O2 (6-OHDA) stimulation apparently weakened green fluorescence. The results disclosed here that compounds 11 and 12 could efficiently prevent the accumulation of intracellular ROS. Promotion of Antioxidant Gene Expression. For the sake of exploring the function that compounds 11 and 12 afforded protection against cellular damage induced by H2O2 or 6-OHDA, we evaluated the antioxidative ability of compounds to scavenge free radicals in vitro. Due to significant antioxidant activity, Res was used as a positive control.41 The ABTS•+ radicals could be eliminated by 11, 12 and Res in a dose-dependent manner, while Res displayed better capacity than compounds 11 and 12 (Figure 8A). Figure 8B demonstrates that the capacity for scavenging DPPH free radicals enhanced as the concentration of molecules increased (11, 12, and Res). Meanwhile, the scavenging ability of 12 was most prominent (10 μM) among three compounds. This could be closely related to its structure with two phenolic hydroxyl groups on the phenyl ring B. The results supported an
Figure 9. Promotion of phase II gene transcription by 11 (A) and 12 (B) in PC12 cells. PC12 cells bred in 60 mm dishes for 1 day were subjected to compounds (10 μM) for 0, 3, 6, and 12 h. Phase II gene transcription was evaluated and standardized using the expression of GAPDH as an internal control by RT-PCR as described in the Methods. Data from three independent experiments are represented as means ± SD; *P < 0.05 and **P < 0.01 vs the vehicle group.
the cells with 11 or 12 could dramatically increase the expression of phase II genes, with the greatest upregulation detected at 12 h. Among all the tested genes, the elevation of HO-1 was most obvious (compound 11), as about 14-fold increase was shown. The improvement of glutamate cysteine ligase catalytic subunit (GCLC) was the most remarkable, as more than 15-fold promotion was observed. Meanwhile, HO-1, glutamate cysteine ligase modifier subunit (GCLM), thioredoxin reductase 1 (TrxR1), and NQO1 were significantly increased (compound 12). The results disclosed that both compounds 11 and 12 promoted the transcription of antioxidant genes mRNA in PC12 cells. Upregulation of the Intracellular Antioxidant System. Based on the above results that a series of antioxidant/
Figure 8. Scavenging activities of ABTS (A) and DPPH (B) free radicals in vitro by 11 and 12. Data from three independent experiments are represented as means ± SD; *P < 0.05 and **P < 0.01 vs the vehicle group. 2960
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
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ACS Chemical Neuroscience
Figure 10. Upregulation of antioxidant system by 11 and 12 in PC12 cells. Induction of total GSH (A) and activities of HO-1 (B), NQO1 (C), Trx (D), and TrxR (E). The cells were exposed to various concentrations of 11 and 12 for 24 h and then the level of total GSH and the activities of antioxidant enzymes were measured. Prevention of total GSH consumption (F, K) and rescue of HO-1 (G, L), NQO1 (H, M), Trx (I, N), and TrxR (J, O) activities by 11 and 12. PC12 cells were treated with 11 or 12 for 24 h, and subsequently exposed to 500 μM H2O2 or 200 μM 6OHDA for 12 h. The cells were collected, and the activities of antioxidative products were measured. (P) Determination of the intracellular TrxR activity by TRFS-green. Scale bar: 50 μm. All data from three independent experiments are represented as means ± SD; **P < 0.01 vs the control group; ∧∧P < 0.01 vs group only treated with the H2O2 or 6-OHDA.
11 or 12. Our findings explained that compounds 11 and 12 could upregulate intracellular antioxidant defense system. Elevation of Nrf2 Nuclear Translocation. Translocation of Nrf2 transferring from the cytoplasm to the nucleus is a vital fundamental for the transcription of Nrf2-driven antioxidant genes.20 Hence, we investigated whether two compounds promoted nuclear accumulation of Nrf2. Figure 11B indicates that, upon incubation of the cells with compounds 11 or 12 (10 μM) for 0, 2, 4, and 8 h, the total Nrf2 was notably increased. Meanwhile, Nrf2 in cytoplasmic was decreased, and after 2 h treatment, both 11 and 12 increased nuclear Nrf2 by ∼5- and ∼4.8-fold, respectively. The results showed that compounds 11 and 12 expedited the process of the Nrf2
detoxifying genes were increased, we subsequently focused our attentions on a train of corresponding products exerting antioxidant action. As shown in Figure 10A−E, after stimulation the PC12 cells with 11 and 12, total GSH and the activities of HO-1, NQO1, TrxR, and thioredoxin (Trx) were increased in a dose-dependent way. In addition, we also applied the probe, TRFS-green, developed by our laboratory, to further determine the activity of intracellular TrxR (Figure 10P).42 The increment of fluorescence indicates an increase in TrxR activity. Treatment of the cells with H2O2 or 6-OHDA led to a decrease in activities of biomolecules (Figure 10), while a visible increase in the level of GSH and activities of antioxidant enzymes was displayed in cells preprotected with 2961
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
Research Article
ACS Chemical Neuroscience
control nontargeting shRNA (PC12-shNT). The results (Figure 12) show a clear promotion of the cell viability in PC12-shNT cells after 11 (12) pretreatment, while the deficiency of the protective effect was detected in PC12shNrf2 cells. In short, Nrf2 played an indispensable role in the cytoprotective action of compounds 11 or 12. Medicinal Chemical Properties of Compounds 11 and 12. Some medicinal chemical properties, such as cLogP (noctanol/water partition coefficient), could affect the ability of compounds to pass the blood−brain barrier (BBB).43 Next, we detected cLogP of compounds 11 and 12. The cLogP values of 11 and 12 were 1.87 and 1.76, respectively. The general criterion of cLogP for a molecule to penetrate the BBB is between 0 and 5. Thus, it was likely that compounds 11 and 12 could cross the BBB.
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Figure 11. Impulse of nuclear translocation of Nrf2 by 11 and 12. PC12 cells were exposed to 11 or 12 (10 μM) for 0, 2, 4, and 8 h and then collected. The samples of corresponding proteins were prepared as described in the Methods. Total Nrf2, cytosolic Nrf2, and nuclear Nrf2 (A) were analyzed by Western blot. Quantifications of band intensity shown in (B)−(D) were done using ImageJ. All data from three independent experiments are represented as means ± SD; **P < 0.01 vs the control group.
DISCUSSION AND CONCLUSION It is reported that the low levels of ROS could act as signaling molecules involved in the regulation of specific pathways, while excessive ROS would cause lipid peroxidation, protein modification, and DNA damage. The damage to lipid, proteins, and DNA is closely related with various diseases.44 Nrf2, as far as we know, governs the expression of target genes concerned with cellular defense against damage by oxidants and electrophiles and the modulation of cellular redox homeostasis and proteostasis.45−47 Activators of the Nrf2/ARE pathway upregulate a series of antioxidant molecules, which is beneficial to oxidative stress related diseases involving inflammation, diabetes, and neurodegenerative disorders.48−50 The past decades have witnessed some successes in the treatment of neurodegenerative diseases with Nrf2 activators, such as dimethyl fumarate approved by the U.S. Food and Drug Administration for treating patients with multiple sclerosis.51,52 Xn, an active ingredient derived from hops, has attracted growing attention from medicinal scientists. Previous studies have indicated that Xn could promote neuronal differentiation and neurite outgrowth and exert neuroprotective effects in ischemic stroke of rats.31,53 However, there are limited studies about Xn analogues on neuroprotective functions. In our previous work, we found that Xn showed neuroprotection against oxidative stress mediated injury in PC12 cells via activation of Nrf2. Inspired by the result, we developed two different classes of Xn analogues and discovered that two compounds (11 and 12) with low cytotoxicity effectively rescued PC12 cells from oxidative stress induced damage. Some of the compounds contained the prenyl group (Xn and compounds 1−10), and other compounds removed the prenyl group (compounds 11−23). The initial cytotoxicity and neuroprotective effects of these compounds are shown in Figures 2−4. On the basis of these results, we analyzed the structure−activity relationship (SAR). The prenyl group played a significant role in the cytotoxicity of compounds, which could be due to the presence of the prenyl group improving the lipophilicity of compounds and making it easy for the compounds to penetrate the cell membranes, as Xn showed stronger toxicity than 11 at the concentration of 20 μM (2 > 15, 1 > 14, 6 > 19, 7 > 20, 4 > 17, 9 > 22, 3 > 16). Introducing electron-withdrawing groups on the phenyl ring led to the double bond being more electron-deficient, increasing the toxicity of molecules, as 21−23 were highly toxic. Owing to an increase in lipophilicity, the methoxy group also could affect the toxicity of the compounds (15 and 18). According to Figures 3 and 4, compounds that removed the
nuclear translocation to elevate downstream target genes transcription of Nrf2. Prerequisite of Nrf2 for the Cytoprotection of 11 and 12. Is Nrf2 a necessary condition for compounds to exert antioxidant effects? We determined the effects of compounds on the PC12-shNT cells and PC12-shNrf2 cells by the MTT assay after pretreatment of the cells with compounds 11 or 12 for 24 h in the H2O2 or 6-OHDA damage model. Silencing of the Nrf2 expression was evaluated in Figure 12A, B via Western blot. The level of Nrf2 protein in PC12 cells transfected with shNrf2 (PC12-shNrf2) was significantly decreased to ∼30% of that in those transfected with the
Figure 12. Prerequisite of Nrf2 for the neuroprotection of 11 and 12. (A, B) Evaluation of Nrf2-knockdown efficiency by different shRNA via the Western blot. The cells were subjected to various concentrations of 11 or 12 for 24 h, the original medium was replaced with fresh medium including H2O2 (C) or 6-OHDA (D), and then the cell survival was measured by the MTT assay. All data from three independent experiments are represented as means ± SD; **P < 0.01 vs the control group; ∧∧P < 0.01 vs the group only treated with H2O2 or 6-OHDA. 2962
DOI: 10.1021/acschemneuro.9b00171 ACS Chem. Neurosci. 2019, 10, 2956−2966
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nuclear translocation of Nrf2 and subsequently improved the expression of Nrf2-driven defensive genes as well as corresponding antioxidant products. Importantly, our studies on SAR and action mechanism would pave the way for the exploitation of Xn analogues as potential neuroprotective agents.
prenyl group could be beneficial to defend PC12 cells against oxidative damage, as compound 20 had a better cytoprotective effect than 7 did. The phenol group on the phenyl ring B is important for the cytoprotection, as 11 and 12 displayed protective potency against oxidative damage, while 13 appeared to provide no protection. The relationship between structure and activity revealed here will provide guidance for further modification of Xn with improved bioactvity. In this work, we found that two compounds 11 and 12, at nontoxic concentrations as feasible neuroprotectants, drastically reduce intracellular ROS and effectively rescue oxidative stress-mediated apoptosis, resulting in an increase in the cell viability (Figures 3−7). It has been reported that several natural products containing electrophilic unsaturated ketones could alkylate the sulfhydryl groups of Keap1, as Xn alkylated the 151 cysteine residue of Keap1.54 In analogy to the activation of Nrf2 by these compounds, we reasoned that 11 and 12 with the Michael acceptor moiety also could alkylate the sulfhydryl groups of the Keap1 protein to form a covalent adduct, resulting in the configuration change of Keap1. Then, Rbx1-mediated ubiquitinatied degradation of Nrf2 was disturbed.55−57 By dissociation from the Keap1-Nrf2 complex, Nrf2 translocated into the nucleus and subsequently bound to the ARE in the promoter of a battery of its target genes, which aided in upregulation of the antioxidant defense system against oxidative injury.58 Our results showed (Figure 11) that the expression of Nrf2 in the nucleus was promoted after 2 h treatment, suggesting that the two compounds might decrease Nrf2 degradation via the 26S proteasome. The thioredoxin system and the glutathione system are fundamental guards in defense against oxidative stress.11,59,60 According to the results, the expression of phase II genes as well as homologous gene products including HO-1, NQO1, TrxR, Trx, and GSH were significantly upregulated (Figures 9 and 10). This demonstrated that induction of Nrf2 by 11 (12) could modulate the thioredoxin system and the glutathione system. Nrf2 is a master regulator of cellular antioxidant response and plays a pivotal part in the protection against oxidants and electrophiles. The cytoprotection of 11 and 12 was completely restrained in PC12-shNrf2 cells, highlighting the important role of Nrf2 under physiological conditions (Figure 12). This is likely the main underlying mechanism for cytoprotection of compounds 11 and 12 against oxidative damage. There are many Nrf2 activators, such as curcumin, quercetin, and celastrol, that have a Michael acceptor or phenolic groups.61 It has been reported that curcumin had low solubility in aqueous solutions and showed instability under physiological conditions.62 Quercetin (25−100 μM) increased the cell survival and reduced either the amounts of reactive oxygen species (ROS) or DNA damage by activating Nrf2.63 Celastrol showed great cytotoxicity toward the cells at low concentration (
