Trilobatin Protects Against Oxidative Injury in Neuronal PC12 Cells Through Regulating Mitochondrial ROS Homeostasis Mediated by AMPK/Nrf2/Sirt3 Signaling Pathway

Oxidative stress-induced neuronal cell damage is a crucial factor in the pathogenesis of mitochondria-associated neurological diseases. Therefore, elimination of overproduction of mitochondrial reactive oxygen species (mtROS) may be a potential strategy for prevention and treatment of neurological diseases. In the present study, the neuroprotective effects of trilobatin (TLB), a novel small molecule monomer derived from Lithocarpus polystachyus Rehd, and its underlying mechanisms were investigated in vitro using hydrogen peroxide (H2O2)-induced oxidative stress model in a neuron-like PC12 cell. The findings revealed that pre-treatment with TLB dramatically concentration-dependently suppressed H2O2-induced PC12 cells damage by enhancing cell viability, repressed reduction of mitochondrial membrane potential (MMP) and decreased mtROS overgeneration, thereby deferring cell apoptosis. Further study demonstrated that TLB not only increased the enzymatic activities of glutathione peroxidase (GPx), isocitrate dehydrogenase 2 (IDH2),superoxide dismutase 2 (SOD2) and deacetylation of SOD2, but also activated silent mating-type information regulation 2 homolog 3 (Sirt3) within the mitochondria and thereby upregulating forkheadboxO3a (FoxO3a), which regulated mitochondrial DNA genes, then led to improving complex I activity and adenosine triphosphate (ATP) synthesis. What’s more, TLB up-regulated p-adenosine monophosphate-activated protein kinase (AMPK) level, the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), and ERRα. Intriguingly, TLB failed to mitigate H2O2-induced PC12 injury in the presence of the AMPK inhibitor (Compound C), indicating that the beneficial effects of TLB on the regulation of mtROS homeostasis were reliance on AMPK -Sirt3 signaling pathway. Moreover, TLB also facilitated nuclear factor erythroid 2-related factor 2 (Nrf2) and promoted antioxidant gene expression in turn, and knockdown of Nrf2 by siRNA dramatically reduced the neuroprotective effects of TLB. Notably, AMPK inhibitor abolished the activation of Nrf2 and Sirt3, whereas, knockdown of Nrf2 blocked the upregulation of Sirt3, but it did not affect p-AMPK level. In conclusion, our findings demonstrate that TLB protects against oxidative injury in neuronal PC12 cells through regulating mtROS homeostasis in the first time, which is, at least partly, mediated through the AMPK/Nrf2/Sirt3 signaling pathway.

Oxidative stress-induced neuronal cell damage is a crucial factor in the pathogenesis of mitochondria-associated neurological diseases. Therefore, elimination of overproduction of mitochondrial reactive oxygen species (mtROS) may be a potential strategy for prevention and treatment of neurological diseases. In the present study, the neuroprotective effects of trilobatin (TLB), a novel small molecule monomer derived from Lithocarpus polystachyus Rehd, and its underlying mechanisms were investigated in vitro using hydrogen peroxide (H 2 O 2 )-induced oxidative stress model in a neuron-like PC12 cell. The findings revealed that pre-treatment with TLB dramatically concentrationdependently suppressed H 2 O 2 -induced PC12 cells damage by enhancing cell viability, repressed reduction of mitochondrial membrane potential (MMP) and decreased mtROS overgeneration, thereby deferring cell apoptosis. Further study demonstrated that TLB not only increased the enzymatic activities of glutathione peroxidase (GPx), isocitrate dehydrogenase 2 (IDH2), superoxide dismutase 2 (SOD2) and deacetylation of SOD2, but also activated silent mating-type information regulation 2 homolog 3 (Sirt3) within the mitochondria and thereby upregulating forkheadboxO3a (FoxO3a), which regulated mitochondrial DNA genes, then led to improving complex I activity and adenosine triphosphate (ATP) synthesis. What's more, TLB up-regulated p-adenosine monophosphate-activated protein kinase (AMPK) level, the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), and ERRα. Intriguingly, TLB failed to mitigate H 2 O 2 -induced PC12 injury in the presence of the AMPK inhibitor (Compound C), indicating that the beneficial effects of TLB on the regulation of mtROS homeostasis were reliance on AMPK -Sirt3 signaling pathway. Moreover, TLB also facilitated nuclear factor erythroid 2-related factor 2 (Nrf2) and promoted antioxidant gene expression in turn, and knockdown of Nrf2 by siRNA dramatically reduced the neuroprotective effects of TLB. Notably, AMPK inhibitor abolished the activation of Nrf2 and Sirt3, whereas, knockdown of Nrf2 blocked the upregulation of Sirt3, but it did not affect p-AMPK level.

INTRODUCTION
There is general recognition that considerable neurological diseases are characterized by a progressive death of neurons, accompanied with a decrease in antioxidant ability and/or an augment in oxidative stress, which is termed as a disequilibrium between the antioxidant enzymes system and the production of reactive oxygen species (ROS; . ROS, by definition, is generally taken to encompass the initial species generated by oxygen reduction including superoxide (O •− 2 ) or hydrogen peroxide (H 2 O 2 ) as well as their secondary reactive products (Forman et al., 2015), and accumulating evidence supports a dual role of ROS as a ''double-edged sword'' that may be either beneficial or deleterious in living systems (Poprac et al., 2017). ROS at a low level appears to exert the beneficial effect in normal physiological conditions and multiple cellular signaling pathways. Nonetheless, it is widespread presumed that excessive ROS is a major contributor to the pathogenetic mechanisms underlying neurological diseases, such as Alzheimer's disease, cerebral stroke, Parkinson's disease (Kalogeris et al., 2014;Liu et al., 2015). Therefore, application of antioxidant which is termed as ''a substance that, when present at a low concentration compared with that of an oxidizable substrate, inhibits oxidation of the substrate'' to deter oxidative stress-induced neuron demise may be a potential strategy to fight neurological diseases (Halliwell and Gutteridge, 1995). Nevertheless, the exploitation of natural or synthetic antioxidant was considered promising therapeutic candidates for neurological diseases has been challenged in spite of promising preclinical data and a great deal of public interests.
Mitochondria are the sites of the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS), through which numerous adenosine triphosphate (ATP) are generated by the electron transport chain (ETC). Likewise, ROS are produced as byproducts of ETC, and incomplete decrease of molecular oxygen in the ETC leads to mitochondrial ROS (mtROS) generation (Shadel and Horvath, 2015). Hence, mitochondria are not only major sites of ROS production, but also tend to oxidative injury (Hung et al., 2018). Therefore, it is unsurprising that mtROS plays a critical role, and targeting to mtROS may be an important approach to affect cellular redox signaling pathways in the neurological diseases. A growing body of evidence indicates that silent mating-type information regulation 2 homolog 3 (Sirt3), localized to the mitochondrial matrix, is a mitochondrial nicotinamide adenine dinucleotide (NAD) + -dependent protein (Brown et al., 2013). Importantly, Sirt3 can not only orchestrate mitochondrial oxidative metabolism, but also modulate mtROS homeostasis by regulating the mitochondrial complexes such as complex I and complex III in the ETC, which are considered as the principal sites of mtROS production, as well as the main antioxidant enzymes of mitochondria including manganese superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and isocitrate dehydrogenase 2 (IDH2; . In addition, recent studies report that Sirt3 is activated by its upstream target of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which is directly regulated by adenosine monophosphate-activated protein kinase (AMPK), sequentially regulates mitochondrial antioxidant enzymes and forkheadboxO3a (FoxO3a)-regulated mitochondrial DNA transcription to limit the production of mtROS (Liu S.-G. et al., 2017). Notably, nuclear factor erythroid 2-related factor 2 (Nrf2), a principal key transcription factor, is recognized as a main stress responder that transcriptionally activates antioxidant and cytoprotective genes through binding to antioxidant response element (ARE) motifs in mtROS homeostasis. Intriguingly, Nrf2 is not only a regulator of Sirt3 expression, but also is activated by AMPK (Satterstrom et al., 2015;Lv et al., 2017). Thus, it reasonable to assume that AMPK/Nrf2/Sirt3 signaling pathway plays an imperative role in regulating the mtROS homeostasis.
Currently, natural antioxidants have gained extensive focus and exhibit beneficial effects on neurological diseases. Chinese herbs are plentiful resources to develop novel natural antioxidants. Lithocarpus polystachyus Rehd, a traditional Chinese folk medicine, has been used for a long time to prevent or treat various diseases (Hou et al., 2011;Zhou et al., 2013). Trilobatin (TLB; Figure 1A), a small molecule monomer, is considered as a novel bioactive compound of Lithocarpus polystachyus Rehd. Previous studies have demonstrated that TLB exhibited anti-oxidant and anti-inflammatory activities (Fan et al., 2015;, but until now, the protective effect of TLB on oxidative stress-induced cellular mitochondrial dysfunction and apoptosis in neuron still remains a mystery. Under this scenario, the aim of this study was designed to assess whether TLB exerts salutary effects on H 2 O 2 -induced PC12 cell injury and its underlying mechanisms. In the current study, we found, for the first time, that TLB protected against H 2 O 2 -induced PC12 cell injury was through reducing mtROS production and apoptosis, which, at least in part, was via activation of AMPK/Nrf2/Sirt3 signaling pathway. These findings not only offer novel evidence concerning the neuroprotective effects of TLB, but also provide a new possible candidate for treating neurological diseases.

Cell Culture
The neuron-like highly differentiated rat pheochromocytoma line PC12, which is a clonal cell line derived from a rat adrenal medulla, and has been broadly used as a cellular model of neuronal development and neurological diseases due to its neuronal properties, such as neurotransmitter release, neurite growth, response to nerve growth factor . PC12 cells were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml), and incubated in a humidified atmosphere of 5% CO 2 at 37 • C.

MTT Assay
Cell viability was determined using MTT assay as described in prior study. Briefly, 400 µM H 2 O 2 treated PC12 cells (at a density of 2 × 10 4 cells/well in 96-well plates) for 48 h was employed as oxidative stress model in vitro as described in our previous report (Gao et al., 2017). The PC12 cells were pre-treated with indicated concentrations of TLB for 1 h prior to expose to 400 µM H 2 O 2 for another 48 h. Thereafter, the cells were incubated with MTT (5 mg/ml) for an additional 4 h. The medium was replaced with DMSO (150 µl) was added, and absorbance at 490 nm was evaluated by a microplate reader. Cell viability was expressed as the percentage of MTT reduction relative to the absorbance of the control cells. In parallel experiments, NAC, a thiol antioxidant, was used as a positive control for antioxidant capacity that suppresses cell demise, for compare with the effect exerted by TLB. The AMPK inhibitor 10 µM Compound C was added to the cell cultures 1 h or knockdown of Nrf2 prior to TLB treatment. Cell viability was expressed as a percentage compared with the control.

LDH Assay
Release of cytosolic LDH into the extracellular medium was considered as an indicator of cytotoxicity, thus cytotoxicity was measured by an LDH assay kit according to the product manual. Briefly, PC12 cells were treated as mentioned above and then collected supernatant of each well and centrifuged at 2000× g for 20 min. Absorbance was read at 490 nm wavelength and all values of % LDH released were normalized to the control. In addition, cellular morphologic changes were observed using phase contrast microscopy.

Hoechst 33342 Staining
In brief, PC12 cells were treated as mentioned above, these cells were washed with PBS, fixed with 4% formaldehyde and then stained with 10 µg/ml Hoechst 33342 for 20 min at room temperature in the dark. After washing with PBS, the nuclei were visualized using fluorescence microscopy (Olympus IX73; Olympus). The mean fluorescence intensity was quantized by the Image Pro Plus software.

Annexin V-FITC/PI Staining
The cells were treated as described above, then washed with PBS and resuspended in the binding buffer containing Annexin V-FITC and PI for 15 min in the dark. Thereafter, the cells were detected by Navios flow cytometry (Beckman Coulter, Brea, CA, USA). The percentage of apoptotic cells was counted using Navios software (Beckman Coulter, Brea, CA, USA).

Caspase 3/7 Activity Assay
PC12 cells were treated as previously mentioned, the caspase 3/7 activity was determined using the Caspase-Glo 3/7 Assay according to the manufacturer's instructions (Zeng et al., 2017). PC12 cells were lysed in lysis buffer and centrifuged at 12,500× g for 5 min, thereafter 50 ml of 2× substrate working solution added into 15 ml of cell lysate were co-cultured in 96-well plates at room temperature for 30 min. Then the fluorescence intensity was detected using multimode reader with excitation at 490 nm and emission at 520 nm. All values of caspase 3/7 activities were presented as a percentage compared with the control.

Measurement of ROS Level and Mitochondrial O 2
•-PC12 cells were treated as mentioned above. Intracellular ROS generation was determined using DCFH-DA (Wedgwood et al., 2013). In brief, the cells were incubated with 20 µM DCFH-DA in DMEM for 30 min in the dark, rinsed twice with PBS solution and the fluorescence was observed and recorded using a fluorescent microscope at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Quantification of ROS level was evaluated using the Image Pro Plus software. All values of ROS level were expressed as a percentage compared with the control. Furthermore, the generation of mitochondrial O •− 2 was determined using MitoSOX Red, a selective fluorescent indicator for detecting O •− 2 generation within mitochondria. Briefly, PC12 cells were treated as mentioned above, cells were washed with balanced salt solution (HBSS) and stained with MitoSOX Red (5 µM) for 20 min in the dark at 37 • C. Thereafter, the cells loaded with Mito Tracker green dye (200 nM) for 20 min under darkness at 37 • C. Whereafter, the cells were washed with PBS and visualized by fluorescence microscopy (Olympus IX73; Olympus, Tokyo, Japan) with excitation/emission (510/580 nm) filters.

Measurement of Mitochondrial Membrane Potential (MMP)
Rhodamine 123 dye was applied to monitor mitochondrial integrity as previously mentioned (Gao et al., 2017). After pre-treated with or without TLB, PC12 cells were stained with 2 µg/ml rhodamine 123 for 20 min in the dark, and then were washed with PBS. The density of green fluorescence (excitation 485, emission 595 nm) was visualized using fluorescence microscopy (Olympus IX73; Olympus), and then the mean fluorescence intensity was measured using the Image Pro Plus software. All values of green fluorescence density were expressed as a percentage compared with the control.

Determination of Cellular NADH and NAD + Concentrations
PC12 cells were treated as mentioned above. NAD + and NADH concentrations were measured using the NAD/NADH Assay Kit according to the manufacturer's instructions.

Measurement of Mitochondrial Enzyme Activities
PC12 cells were treated as mentioned above. Briefly, mitochondria were isolated using a mitochondria isolation kit for cultured cells according to the manufacturer's instructions. The sample protein concentrations were determined using BCA protein quantification kit and adjusted the final sample protein concentration is 5.5 mg/ml protein. Then, centrifuged the sample for 20 min at 12,000× g in a cold centrifuge. The mitochondrial enzyme activities of complex I, SOD2, GPx, IDH2 and the content of MDA were measured according to manufacture's instruction of the assay kits, respectively.

ATP Quantification
In brief, PC12 cells were treated and mitochondria were isolated as described above. The homogenized samples were centrifuged at 16,000× g for 20 min, the supernatant was used to detect ATP levels by luminescent ATP detection assay kit according to the manufacturer's protocols. The activity of the ATP synthase enzyme was measured in absorbance at 340 nm wavelength. The activity rate is expressed as the change in absorbance at 340 nm/min/amount of sample loaded into the well.

Extraction of Nuclear and Cytosolic Protein
Nuclear and cytosolic proteins were performed following a process reported previously with a nuclear extraction Kit (Jin et al., 2014). Nuclear and cytosolic proteins were fractionated according to the instructions of the manufacturer. Briefly, following treatment, cells were harvested and centrifuged at 300× g for 5 min at 4 • C. The cell pellets were mixed with hypotonic buffer containing phosphatase and protease inhibitors. After 10 min incubation on ice, 10% Nonidet P-40 Assay Reagent was added to the cells. Nuclei were recovered by centrifugation (14,000× g, 30 s), and the supernatant was kept as cytoplasmic extract at −80 • C until use. The nuclei were extracted with Nuclear Extraction Buffer for 30 min on ice. Insoluble material was removed by centrifugation at 14,000× g for 10 min. Finally, the supernatant was used as a nuclear extract (Yoo et al., 2017).

Western Blot
Western blot was performed as described previously (Gao et al., 2017). Briefly, the protein concentration was measured with BCA Protein Assay Kit. Samples with equal amounts of proteins were separated on 10% polyacrylamide gels, then the separated proteins were blotted onto PVDF membrane, and probed with selective antibody, respectively. The appropriate primary antibody

siRNA Transfection
Nrf2-siRNA was used to inhibit endogenous Nrf2 expression and a scrambled siRNA was used as a control. PC12 cells were seeded at 60% confluency into 6-well plates. Six microliter of Nrf2-siRNA and 18 µL lipofectamine TM RNAiMAX transfection reagent were diluted into 150 µL Opti-MEM I, and then gently mixed and incubated at room temperature for 15 min, then lipofectamine mixture was added to the Nrf2-siRNA mixture at a final concentration of 60 pM siRNA. Transfection of PC12 cells with scrambled siRNA served as a negative control. After incubation for 24 h at 37 • C, 2 ml of complete medium with 10% FBS was added to the transfected cells to replace transfection solution and the cells were exposed to H 2 O 2 at the same time. The knockdown of endogenous Nrf2 by siRNA was confirmed by Western blot. The transfected cells were cultured for 48 h, and then harvested for further analysis.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD). All experiments were performed in triplicate. The one-way ANOVA of variance followed by least significant difference post hoc test was applied to determine individual differences. Differences with P < 0.05 were considered statistically significant.

TLB Concentration-Dependently Protected Against H 2 O 2 -Induced Injury in PC12 Cells
Previous study confirmed that the treatment of 400 µM H 2 O 2 for 48 h resulted in significantly PC12 cell death (nearly 50%), thus, such concentration and incubation time were used for the following experiments. The results indicated that pre-treatment with 15, 30 and 60 µM of TLB markedly reduced H 2 O 2 -induced cell death in a concentration-dependent manner (66%, 76% and 93%, respectively; F (6,14) = 65.682, P < 0.001; Figure 1B). Moreover, in parallel, pre-treated PC12 cells with 15, 30 and 60 µM of TLB significantly attenuated H 2 O 2 -induced LDH leakage (from 34% to 26%, 20%, 14%, respectively; F (6,14) = 25.742, P < 0.001; Figure 1C). Furthermore, the protective effect of TLB was confirmed by morphologic observations, which showed that H 2 O 2 -treated cells resulted in dominant cellular shrinkage and floatation than that of control, whereas pre-treatment with TLB or NAC, the cells became normal morphology ( Figure 1D). Moreover, nuclei condensation was observed in PC12 cells after exposure to 400 µM H 2 O 2 by Hoechst 33342 staining, and pre-treatment with 15, 30 and 60 µM of TLB reduced these changes in nuclei ( Figure 1E). In addition, apoptosis cell was detected in PC12 cells after exposure to H 2 O 2 by annexin V/PI double staining and flow cytometry analysis. After H 2 O 2 treatment, the rate of early apoptosis increased to 17.5%. However, pre-treatment with 15, 30 and 60 µM of TLB decreased the rate of early apoptosis to 8%, 5%, 2.5%, respectively (F (6,14) = 160.148, P < 0.001; Figures 1F,G). It's worth noting that, Bax expression was increased dramatically in the cytosolic fraction, whereas, decreased notably in the mitochondrial fraction in PC12 cells after treated with H 2 O 2 . However, H 2 O 2 significantly decreased Bcl-2 expression in the cytosolic fraction, but there was slightly change of Bcl-2 expression in the mitochondrial fractions. Subsequently, the Bax/Bcl-2 ratio was significantly decreased in the mitochondrial fraction but increased in the cytosolic fraction in PC12 cells as a result of H 2 O 2 treatment. However, pre-treatment with TLB suppressed the increase in H 2 O 2 -induced Bax/Bcl-2 ratio both in cytosolic and mitochondrial fractions (F (4,10) = 79.661, P < 0.001; F (4,10) = 94.141, P < 0.001). Additionally, TLB also decreased the cyto c in the cytosolic fraction (F (4,10) = 47.137, P < 0.001; Figures 1H-K). Furthermore, caspase 3/7 activation is a main biomarker in the apoptosis progress of neuronal cells . The results revealed that treatment with H 2 O 2 for 48 h increased caspase 3/7 activity to 193% than that of control. In contrast, pre-treatment with 15, 30 and 60 µM of TLB markedly decreased caspase 3/7 activation to 171%, 137%, 113%, respectively (F (6,14) = 72.733, P < 0.001; Figure 1L). What's more, TLB (60 µM) alone did not exhibit cytotoxicity and lead to apoptosis of PC12 cells, and the protective effect of TLB was same or higher than those exerted by an equivalent concentration of positive agent, a classical antioxidant, NAC. These results indicated that TLB protected against oxidative stress-induced apoptosis in H 2 O 2 -stimulated PC12 cells.

TLB Restored H 2 O 2 -Induced Change of MMP
The mitochondrial membrane potential (MMP) was assessed by analyzing the green fluorescence intensity using rhodamine 123 staining as previous study (Gao et al., 2017). The results showed that pre-treatment with 15, 30 and 60 µM of TLB significantly inhibited the decrease of MMP induced by H 2 O 2 (from 27% to 42%, 68%, 89%, respectively; F (6,14) = 1312.749, P < 0.001; Figures 2A,B). These findings further confirmed that TLB protected against H 2 O 2 -induced PC12 cell death, at least partly, via restoring MMP function.

TLB Suppressed H 2 O 2 -Induced Accumulation of Mitochondrial O 2 •and Improved Mitochondrial Enzyme Activities
Accumulating excessive ROS is considered to be one of the major causes of cell injury induced by H 2 O 2 . The level of intracellular ROS was determined using staining with DCFH-DA fluorescent probe in PC12 cells. The results showed that 400 µM H 2 O 2 led to a dramatical augment in fluorescence intensity (279%) in the PC12 cells than that of control. However, the increase in ROS was dramatically inhibited by pre-treatment with TLB at different concentrations (219%, 152%, 119%, respectively; F (6,14) = 523.086, P < 0.001; Figures 3A,B). Since mitochondria are the main source of ROS, determining the fate of cells through regulating multiple signal transduction pathways and mitochondria are also vulnerable targets. The protective effect of TLB on regulation of mitochondrial homeostasis was detected by MitoSOX Red staining, which is a specific fluorescent probe for detection of the mtROS generation, owing to MitoSOX Red is easily oxidized by O •− 2 and displays red fluorescence within mitochondria. H 2 O 2 markedly promoted mitochondrial O •− 2 production than that of control. This effect was dramatically mitigated by TLB pre-treatment ( Figure 3C). Furthermore, MDA, a marker of lipid peroxidation was measured. The data showed that H 2 O 2 remarkably increased MDA content, while the increase in MDA was dramatically attenuated by TLB pre-treatment (F (6,14) = 97.404, P < 0.001; Figure 3D). In addition, the results revealed that H 2 O 2 significantly decreased enzymatic activities of SOD2 (F (6,14) = 201.632, P < 0.001), GPx (F (6,14) = 151.201, P < 0.001) and IDH2 (F (6,14) = 72.049, P < 0.001), whereas TLB pre-treatment sharply increased these enzymatic activities (Figures 3E-G). These findings indicated that TLB protected the neuronal cells against oxidative stress through attenuating excessive intracellular ROS and mitochondrial O •− 2 and its antioxidant role.

AMPK/Nrf2/Sirt3 Axis Was Involved in the Neuroprotective Effects of TLB on H 2 O 2 -Induced PC12 Cells Injury
To further investigate the relationship of AMPK, Nrf2 and Sirt3, Nrf2 siRNA or p-AMPK inhibitor were applied. The results  (Figures 7C,D). These findings indicated that AMPK/Nrf2/Sirt3 axis plays an essential role for the effect of TLB on regulation of mtROS homeostasis in PC12 cells.

DISCUSSION
The current study, for the first time, revealed that: (1) TLB, a novel small molecule monomer, derived from Lithocarpus polystachyus Rehd exerted neuroprotective effects against oxidative stress-induced neuronal cell apoptosis; (2) the beneficial effects of TLB was owed to attenuation of mitochondrial ETC injury and inhibition of O •− 2 generation within the mitochondria; (3) TLB not only activated AMPK/Sirt3 signaling pathway, but also facilitated nuclear translocation of transcription factor Nrf2 and in turn promoted antioxidant gene expression; and (4) Most importantly, the findings of current study, revealed that Nrf2 is the downstream of AMPK and upstream of Sirt3, respectively. AMPK/Nrf2/Sirt3 axis plays an imperative role in the neuroprotective effect of TLB on the oxidative injury in neuronal cells (Figure 8).
Oxidative stress is considered as a valid therapeutic strategy for neurological diseases since the particular vulnerability of the brain to oxidative injury and high levels of ROS may be involved in neuropathology. Oxidative stress occurs under the condition of imbalance of cellular redox homeostasis, and it is further related to a lot of pernicious outcome for the neuronal cells such as lipid peroxidation or even apoptosis. In the present study, H 2 O 2 , the most stable ROS, was used to induce oxidative stress in PC12 cells due to its characteristic with an extracellular messenger and an intercellular one, which alter several proteins, thus leading to mitochondrial injury and even cell apoptosis. Here, the results demonstrated that H 2 O 2 triggered cell injury, accompanied by mitochondrial O •− 2 generation and cell apoptosis via activating caspases and releasing proapoptotic factors including Bax and Cyto c from mitochondria, consisting with our previous study (Gao et al., 2017). Whereas, these effects were reversed by TLB pre-treatment. These findings highlighted that TLB-regulated pharmacological preconditioning effect in neuronal cells, at least in partly, through Caspase 3-dependent apoptosis pathway. Excessive mtROS production can lead to oxidative damage to cellular ingredients such as lipids, proteins and mitochondrial DNA and induce cellular senescence. Reduced ETC/OXPHOS ability can lead to cellular energy deprivation such as reduced ATP charge, altered mtROS production, or loss of MMP, with the precise consequence dictating the specific mitochondrial stresssignaling response . Thus, it appears possible that the beneficial effect of TLB might be related to regulation of mtROS homeostasis. To this end, mitochondrial O •− 2 level and ETC were determined in the present study. As expected, the results suggested that TLB attenuated mitochondrial O •− 2 generation, thereby restored MMP as evidenced by MitoSOX Red dying and rhodamine 123 staining. Furthermore, evidence indicates that Complex I, the major source of spontaneous O •− 2 , is the point of entrance anion production in the mitochondrial ETC for NADH reducing equivalents, and it not only engenders ROS but also maintains the proton gradient across the mitochondrial inner membrane and is needed for ATP synthesis. Moreover, since brain neurons are sensitive to deficiencies in OXPHOS, and complex I is particularly larger than other cell types (Pinto et al., 2012). Thus, defects in complex I are linked with multiple human neurological diseases (Papa and De Rasmo, 2013). Our results demonstrate that oxidative stress induced excessive ROS production by disrupting complex I, thereby reducing ATP synthesis and NAD + /NADH ratio, which is in consistent with the theory that complex I is the first step in energy metabolism and redox pathways (Martinvalet et al., 2008). However, TLB reversed these effects, further confirmed that TLB protected against oxidative stress-induced neuronal cell injury through inhibition of mitochondrial dysfunction. Additionally, the deleterious effects of oxidative stress can be deterred by antioxidant enzymes. Our findings manifested that TLB also enhanced the mitochondrial antioxidant enzyme activities of SOD2, GPx-1 and IDH2 to eliminate overproduction of ROS. Intriguingly, SOD2 is not only the forefront antioxidant enzyme that protects cells against excessive ROS generated in the mitochondria, but also a direct substrate of Sirt3, which is a NAD + -dependent deacetylase localized in the mitochondrial matrix, and can mediate ROS levels also by deacetylating ETC complexes to lower mtROS production and maximize ATP synthase (Karnewar et al., 2018). As expected, TLB activated Sirt3, thereby facilitated deacetylation of SOD2 in mitochondria and triggered its downstream gene IDH2 to scavenge mitochondrial O •− 2 . Furthermore, a body of evidence suggests that Sirt3 binds mtDNA through regulating FoxO3a, thereby elevated ETC capacity and ATP synthesis. Consistent with that theory, our findings revealed that TLB not only enriched Sirt3 in mitochondria, but also triggered mtDNA transcription, and then regulated redox status in neuronal cells. Moreover, it is noteworthy that UCP2, a new member of the mitochondrial uncoupling protein family, which negatively controls MMP and ATP production to attenuate mtROS when neuronal cells were challenged by oxidative stress (Suzuki et al., 2015). In keeping with the theory, the results in this study revealed that H 2 O 2 increased UCP2 expression, while, TLB blocked the increase in UCP2 expression, further confirmed TLB maybe targeted mitochondria to mediate proper balance between the productions of mitochondrial energy and mtROS. However, the underlying molecular mechanisms need to be further investigated.
To elucidate the mechanism of TLB-triggered Sirt3 activation, we investigated the activation of AMPK, PGC-1α, and ERRα. AMPK, a ubiquitous serine/threonine protein kinase, is known to regulate cellular metabolic balance, and it is activated in response to disruptions in cellular energy such as decreases in cellular ATP. Recently, AMPK has been reported to influence mtROS homeostasis by initiating PGC-1α, a downstream effector of AMPK. Hence, AMPK plays a crucial role in intracellular metabolism and may be a potential therapeutic target for mtROSrelated neurological diseases. Furthermore, previous evidence demonstrates that PGC-1α acts as an endogenous regulator of Sirt3 and activates Sirt3 by triggering ERRα to cause ERRα coupling with ERRE in the Sirt3 promoter region (Zhou et al., 2014). We therefore made an assumption that TLB controls mtROS homeostasis via activating the AMPK-PGC-1α-Sirt3 axis. As expected, the results indicated that H 2 O 2 decreased p-AMPK, in keeping with the theory that oxidative stress acts as a trigger for AMPK . Whereas, TLB promoted AMPK phosphorylation, thereby triggered activation of PGC-1α and ERRα, suggesting that AMPK-PGC-1α-Sirt3 axis was necessary for the protective effect of TLB on regulation of mtROS homeostasis in neuronal cells. Intriguingly, interaction between AMPK and Nrf2 also appears to play a positive role of oxidative stress-induced neuron demise, and Nrf2 interacts with PGC-1α to mediate crucial signaling pathway within mitochondria and triggers the activation of Sirt3 to exert neuroprotective effect under oxidative stress condition (Shah et al., 2016;Wang et al., 2017). Nrf2 is known as a transcription factor and its inactive form is kept in the cytoplasm bound to Keap-1. When the cell is in an oxidative stress condition, active sites of cysteine residues of Keap-1 were oxidized, thereby prevented Keap-1 from bonding with Nrf2. Then, Nrf2 translocates into the nucleus and bonds to ARE including HO-1 and NQO-1, which are the crucial genes in the brain and is activated by Nrf2 to protect against neurons cell injury (Shetty et al., 2017). We thus focused on the role of Nrf2 indepth. Our findings demonstrated that TLB decreased Keap-1, which is considered as cytoplasmic inhibitor of Nr2, and then promoted Nrf2 translocation from cytoplasm into nucleus, and improved its antioxidant genes. Most importantly, knockdown of Nrf2 partially abolished the protection of TLB, further confirmed that the protective mechanism of TLB is, at least in partly, through activating Nrf2. Based on the above findings, it is reasonable assumed that AMPK/Nrf2/Sirt3 axis was involved in the salutary effects of TLB on H 2 O 2 -induced PC12 cell injury. Intriguingly, we uncovered that Nrf2 and Sirt3 were the downstream genes of AMPK, and Nrf2 was the upstream of Sirt3, evidenced by AMPK inhibitor or knockdown of Nrf2. Importantly, as expected, TLB exerted neuroprotective effects on H 2 O 2 -induced neuronal cell injury, at least in partly, through mediating AMPK/Nrf2/Sirt3 axis. Together, these findings suggested that there exists crosstalk between oxidative stress, mitochondria, and apoptosis, however, the precise underlying mechanisms of reciprocity between AMPK, Nrf2 and Sirt3 were needed to further study.
Additionally, since TLB is a novel compound, we did primary research to confirm its antioxidative effects in PC12 cells, which was accepted as a common model for studying of neuronal properties (Liu et al., 2018). Although we have confirmed the neuroprotective effects of TLB in PC12 cells after oxidative stress insults, further studies are necessary to determine the neuroprotective role of TLB in other types of cells such as human neuron-like cells SH-SY5Y or mouse/rat primary neurons. Moreover, considering TLB is a small molecule monomer, we hypothesize that it could overcome blood brain barrier (BBB) in vivo although there is no direct evidence. Thus, whether TLB could cross the BBB and confer neurological diseases such as ischemic cerebral stroke in vivo is imperative to explore in our is further studies.

CONCLUSION
In summary, the results of this study demonstrate that TLB exerts its salutary effects on oxidative stress-induced neuronal apoptosis via mediating mtROS homeostasis and the AMPK/Nrf2/Sirt3 signaling pathway. To our knowledge, this is the first indication that TLB exhibits antioxidant properties to eliminate overproduction of mtROS and plays an imperative role in oxidative stress-induced neuronal demise, suggesting that TLB may be a promising natural radical scavenger for the prophylaxis and treatment of neurological diseases.

AUTHOR CONTRIBUTIONS
QG and JS designed the experimental approaches. JG performed all the other studies described herein, except the Western blot experiments conducted by SL and FX, Annexin V-FITC/PI staining by YL, and Quantitative real-time PCR analysis conducted by CL and YD. JG wrote the manuscript with the help from QG.