Licochalcone B, a Natural Autophagic Agent for Alleviating Oxidative Stress-Induced Cell Death in Neuronal Cells and Caenorhabditis elegans Models

Autophagy has been implicated in the regulation of neuroinflammation and neurodegenerative disorders. Licochalcone B (LCB), a chalcone from Glycyrrhiza inflata, has been reported to have anti-cancer, anti-oxidation and anti-β–amyloid fibrillation effects; however, its effect in autophagy remain un-investigated. In the current study, the potential neuro-protective role of LCB in terms of its anti-oxidative, anti-apoptotic, and autophagic properties upon oxidative stress-induced damage in neuronal cells was investigated. With the production of reactive oxygen species (ROS) as a hallmark of neuroinflammation and neurodegeneration, hydrogen peroxide (H2O2) was adopted to stimulate ROS-induced cell apoptosis in PC-12 cells. Our findings revealed that LCB reduced cell cytotoxicity and apoptosis of PC-12 cells upon H2O2-stimulation. Furthermore, LCB increased the level of the apoptosis-associated proteins caspase-3 and cleaved caspase-3 in H2O2-induced cells. LCB effectively attenuated the level of oxidative stress markers such as MDA, SOD, and ROS in H2O2-induced cells. Most importantly, LCB was confirmed to possess its anti-apoptotic effects in H2O2-induced cells through the induction of ATG7-dependent autophagy and the SIRT1/AMPK signaling pathway. As a novel autophagic inducer, LCB increased the level of autophagy-related proteins LC3–II and decreased p62 in both neuronal cells and Caenorhabditis elegans (C. elegans) models. These results suggested that LCB has potential neuroprotective effects on oxidative damage models via multiple protective pharmacological mechanisms.


Introduction
A growing body of experimental and clinical evidence has confirmed that the high level of free radicals produced by oxidative stress is one of the pathogenic features in many neuronal disease models. Free radicals produced by oxidative stress affect the structure and function of neuronal cells [1], which leads to the progression of neurodegenerative diseases including Parkinson's and Alzheimer's disease [2]. Neurodegenerative diseases are characterized by the atrophy of specific nuclei and progressive loss of function of neurons, but their etiology and pathology remain unclear. Multiple risk factors including aging, external environmental, and internal genetic risk factors contribute cumulatively to the occurrence and progression of neurodegenerative diseases [3]. Generally, apoptosis is considered essential for the normal development and homeostasis of all multicellular organisms in vivo [4]. This form of programmed cell death is also important for the removal of damaged cells, resistance to bacterial infections, or the removal of potentially

LCB Attenuated H 2 O 2 -Induced Cell Death in PC-12 Cells
To investigate the effect of LCB on H 2 O 2 -induced cell death, the establishment of an H 2 O 2 -induced cell death model and the cytotoxicity of LCB were firstly examined in PC-12 cells. Based on the results in Figure 2A and Supplementary Figure S1, while no significant decrease in cell viability was observed in 5 to 40 µM of LCB treatment in PC-12 cells, 900 µM of H 2 O 2 treatment for 6 h was selected as the experimental conditions of the oxidative stressinduced cell death model in the subsequent experiments. In Figure 2B, the survival rate of PC-12 cells was rescued in a dose-dependent manner after 10 to 40 µM of LCB treatment in the H 2 O 2 -induced cells. With LDH and caspase-3 as the representative cytotoxicity markers and enzymes in the process of apoptosis, significant decreases in the levels of both markers in the H 2 O 2 -induced cells after LCB treatments are shown in Figures 2C and 2D. Furthermore, the protective effect of LCB was evaluated by morphological observation.
While obvious cellular shrinkage and reduced numbers of cells were observed in H 2 O 2treated PC-12 cells, a higher density of cells with a normal morphological phenotype was observed in the cells pre-treated with LCB before H 2 O 2 induction ( Figure 2E). Furthermore, a calcein/propidium iodide (PI) cytotoxicity assay using two different fluorescent stains was employed to distinguish live cells from dead cells. While green fluorescent calcein AM indicated live cells, propidium iodide (PI) stained dead cells in red. As shown in Figure 2F, although the number of live cells was severely decreased in the H 2 O 2 -induced group when compared to the control group, the number of dead cells was decreased after LCB treatments. These results confirmed that LCB attenuated H 2 O 2 -induced cell death in PC-12 cells.  (10, 20,

LCB Inhibited H 2 O 2 -Induced Oxidative Stress
Reactive oxygen species (ROS) is one of the crucial detrimental factors leading to neuronal cell damage and neurodegenerative diseases. Upon induction by H 2 O 2 , ROS production can be detected by DCFH-DA, which is oxidized by intracellular ROS to generate fluorescent DCF that can be detected by fluorescence microscopy and flow cytometry. PC-12 cells pre-incubated with increasing concentrations of LCB followed by the addition of H 2 O 2 were performed. As shown by fluorescence microscopic analysis in Supplementary Figure S2, the results showed that while H 2 O 2 increased the fluorescence intensity of DCF, LCB diminished the fluorescence intensity of DCF in a dose-dependent manner. The in-creased production of ROS and the anti-oxidative effect of LCB in H 2 O 2 -induced PC-12 cells were further confirmed by quantitating DCF fluorescence intensity using flow cytometry ( Figure 3A). Furthermore, important markers of oxidative stress induced by ROS production including malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. As seen in Figure 3B, the MDA level was increased 3.8-fold after H 2 O 2 challenge, while LCB (10 to 40 µM) attenuated the level of MDA to a level comparable to the control group. Consistently, SOD levels were significantly increased from 0.57 U/mL (H 2 O 2 group) to around 0.77 U/mL (H 2 O 2 + LCB 40 µM group) after LCB treatment ( Figure 3C). Thus, LCB was shown to suppress the level of ROS generation and lipid peroxidation and recover the level of superoxide dismutase for its protective anti-oxidation effect.

LCB Protected PC-12 Cells from H 2 O 2 -Induced Apoptotic Cell Death
Recent reports have confirmed that overproduction of ROS can result in the induction of oxidative stress and subsequently apoptosis. Anti-oxidative agents can be applied to inhibit or delay apoptosis and cell death [19]. As revealed by Hoechst 33342 fluorescent staining on DNA and nuclei in Figure 4A, while there was a decrease in the number of stained cells after H 2 O 2 treatment, the LCB treatment group showed an increase in the number of positive stained cells after H 2 O 2 induction, suggesting the possible effect of LCB on anti-apoptosis. To further investigate the effect of LCB on H 2 O 2 -induced apoptotic cell death, annexin V/PI was utilized to detect apoptotic cells. As shown in Figure 4B, the percentage of PI-positive (dead) cells was significantly increased after the exposure to H 2 O 2 , while pre-treatment of PC-12 cells with 10 to 40 µM of LCB before H 2 O 2 exposure showed a significant reduction in the percentage of PI-positive cells. Consistently, while the expression of cleaved apoptotic protein caspase-3 exhibited a significant increase in H 2 O 2 -induced PC-12 cells, the presence of LCB decreased the level of cleaved caspase-3 ( Figure 4C). These results indicated that LCB protects PC-12 cells from H 2 O 2 -induced damage through the anti-apoptotic signaling pathway.

LCB Induced Autophagy in PC-12 and C. elegans Models
As a key catabolic process for the lysosomal degradation of dysfunctional cytoplasmic components, autophagy is activated in response to oxidative stress for the protection of cells from apoptosis. In this study, the GFP-microtubule-associated protein light chain 3 (LC3) U87 stable cells were adopted to evaluate the autophagic effect of different concentrations (10-40 µM) and treatment times (0-24 h) of LCB. With the level of the autophagosomal LC3-II as the key marker for autophagic activity, LC3-II levels were detected by using both immunoblotting and immunofluorescence methods after LCB treatments. As shown in Figure 5A, the percentage of cells with GFP-LC3-II puncta formation was increased significantly in both time-and dose-dependent manners after LCB treatments. Figures 5B and 5C confirmed that LCB significantly increased the expression of LC3-II autophagic markers in PC-12 cells. To justify the novel autophagy role of LCB in the study, C. elegans was adopted as the in vivo model organism. Due to its small size and transparency for microscopy, together with the rapid growth and high fertility nature, C. elegans has emerged in the past decade as a well-adopted model to study various physiological and pathological contexts such as autophagy, apoptotic cell death, and RNA interference studies. Importantly, with its characteristic of simplicity, C. elegans was adopted for the study of autophagy, especially on tissue specificity and developmental processes [20]. To begin, two autophagic models of C. elegans expressing either GFP-LGG1/LC3 (LGG-1/LC3::GFP) or GFP-p62 (SQST-1/p62::GFP) were adopted. Consistent with the increased level of LC3-II in PC-12 cells and C. elegans ( Figure 5D), a decreased level of the autophagic substrate p62 was observed in the C. elegans model ( Figure 5D) after LCB treatments, confirming the induction of autophagic flux in both in vitro and in vivo models.

The Expression Profile of Autophagy-Related Genes in SH-SY5Y Human Cells after Treatment of LCB
To explore the signaling pathway of autophagy activated by LCB, SH-SY5Y cells were treated with LCB for 24 h. RNA extracted from SH-SY5Y cells with or without LCBtreatment was converted to cDNA for RT-PCR array analysis on 84 selected autophagyrelated genes with the expression profile shown in Figures 6A and 6B. As indicated in the LCB-treated group, 16 autophagic genes including ATG10, ATG4B, ATG4C, ATG5, BID, CTSD, DRAM2, GABARAPL1, HGS, IFNG, INS, MAP1LC3B, NPC1, PIK3CG, RB1, and B2M were found upregulated more than two fold when compared to the control group. As shown in Figure 6C, a heat map was adopted for differential genes expression showing that the mRNA level of the above 16 genes increased after LCB treatments. To further explore the mechanism of LCB-activated autophagy, the String database was adopted to search for its possible associated proteins and to predict that AMPK/SIRT1 signaling pathways may be linked to these 16 upregulated genes ( Figure 6D).

LCB Activated Autophagy via the AMPK/SIRT1 Signaling Pathway in PC-12 Cells
The cellular energy sensor, AMPK, and SIRT1, which plays an important role in activating autophagy were investigated. As shown in Figure 7A, the phosphorylation of AMPK at Thr272 and the SIRT1 protein level were increased by LCB in a dose-dependent manner. While Beclin-1 is one of the central regulators for the initiation stage of autophagosome membrane formation, p62 is one of the selective substrates for autophagy in the formation of cytoplasmic inclusion. By using rapamycin (rapa) as the positive control, Figure 7A shows that LCB was able to upregulate Beclin-1 and downregulate p62. Compound C (CC), a specific inhibitor of AMPK, abolished the effect of LCB on the protein expression of phosphorylation of AMPK and SIRT1 ( Figure 7B). Consistently, the autophagy markers including LC3II and p62 ( Figure 7B), which were modulated by LCB, were downregulated and upregulated, respectively, after CC treatments. Taken together, LCB may induce autophagy partially via the AMPK/SIRT1 signaling pathway in PC-12 cells.

LCB Reduced H 2 O 2 -Induced Apoptosis through AMPK/SIRT1-Mediated Autophagy
To further confirm the protective role of LCB-induced autophagy in H 2 O 2 -induced apoptotic cell death, the autophagic incidence of cells after LCB treatment was measured in an H 2 O 2 -induced cell model. As shown in Supplementary Figure S6, the MDC staining results demonstrated that autolysosomes were accumulated dose-dependently in the H 2 O 2induced PC-12 cells after LCB treatments. Therefore, the mechanistic pathway of LCBinduced autophagy and its effect on the apoptosis rate of H 2 O 2 -induced PC-12 cells were further investigated. While LCB significantly suppressed H 2 O 2 -induced apoptosis of PC-12 cells, CC abolished the anti-apoptotic effects of LCB as revealed by annexin V-PI staining flow analysis and the immunofluorescence assay of cleaved-caspase-3 in H 2 O 2induced PC-12 cells ( Figure 8A). While the cellular apoptosis of H 2 O 2 -induced PC-12 cells is closely associated with the cleaved-caspase-3 level, LCB was confirmed to suppress protein expression of cleaved-caspase-3 in the presence of H 2 O 2 , as shown in Figure 8B. Moreover, the role of autophagy gene ATG7 in LCB-induced autophagy was investigated in ATG7 wild-type and ATG7-deficient mouse embryonic fibroblasts (MEFs). As shown by the flow cytometry result in Figure 8C, LCB lowered significantly the apoptosis rate of H 2 O 2 -induced wild-type MEFs (ATG7+/+) but not ATG7-deficient MEFs (ATG7−/−). Taken together, these data suggested that LCB rescued cells from H 2 O 2 -induced apoptosis via ATG7 dependent autophagy and the AMPK/SIRT1-related pathway.

Discussion
With the critical role of oxidative stress in the pathogenesis of neuroinflammation, which is highly correlated with neurodegenerative diseases such as Alzheimer's and Parkinson's disease, the identification of natural anti-oxidative agents with protective effects in the inhibition of inflammation and apoptotic cell death of the brain is highly desirable [21]. With minimal side effects when compared to synthetic compounds, many natural com-pounds have been reported for their polypharmacological neuroprotective action via both anti-inflammatory and antioxidant activities. For example, the well-known polyphenol flavonoid, resveratrol, possesses neuroprotective effects via its potent antioxidant and antineuroinflammatory activity for the attenuation of neurotoxicity [22]. Licorice root is one of the most widely studied medicinal plants of the world. It is commonly applied as a herbal medicine. It has been traditionally used for the treatment of arthritis, heart diseases, lung diseases, gastric ulcer, and some microbial infections. Among them, a large number of biological active compounds was isolated from the species Glycyrrhiza [23]. In this study, our results have reported for the first time that LCB, a chalcone isolated from Glycyrrhiza inflate, enhanced autophagy and attenuated apoptotic cell death and oxidative stress, possibly via the modulation of the autophagic markers including SIRT1, AMPK, Beclin-1, LC3, and P62 in either the in vivo PC-12 cells or in vitro C. elegans models (Figure 9). H 2 O 2 is a reactive oxygen molecule that is involved in the pathogenesis of many neurological diseases and is commonly used as an inducer of oxidative damage and apoptotic cell death in cellular models. Excessive production of free radicals can lead to cellular damage and cause aging and dysfunction of the human body [24]. With the properties of high oxygen demand and metabolic rate, and a relatively low antioxidant defense system, brain tissue is more vulnerable to free radical attack than any other tissues or organs [25]. In the current study, the accumulation of reactive oxygen species in the cellular assay has demonstrated that H 2 O 2 treatment significantly induced apoptosis in PC-12 cells, and LCB protected PC-12 cells from apoptotic damages, as observed from morphology, apoptosis, and oxidative markers evaluation. Consistently, while the viability of PC-12 cells exposed to 900 µM of H 2 O 2 were decreased by approximately 50%, LCB pretreatment significantly inhibited damage and increased cell viability upon H 2 O 2 challenge. With malondialdehyde (MDA) as the final product of lipid peroxidation in cells, the elevation of free radicals can increase the level of MDA; therefore, MDA is commonly adopted as a biomarker of oxidative stress in disease models [26]. In the current H 2 O 2 -induced PC-12 cell model, the increase in both the ROS and MDA level were significantly alleviated after the treatment of LCB, confirming the anti-oxidative role of LCB in cells. With the close correlation of oxidative stress and apoptosis, LCB was confirmed to alleviate H 2 O 2 -induced cell apoptosis, as revealed by decreasing apoptosis-related proteins such as cleaved-caspase-3 in PC-12 cells. Importantly, LCB alleviated apoptosis induced by H 2 O 2 in ATG7 wild-type MEFs but not in ATG7-deficient MEFs, suggesting that LCB attenuated H 2 O 2 -stimulated cell apoptosis via the autophagy-gene-dependent mechanism. Furthermore, the cytoprotective effects of LCB are related to the regulation of the SIRT1/AMPK signaling pathway.
In fact, autophagy is a widespread biological phenomenon in eukaryotic cells for maintaining normal cellular homeostasis [27] and oxidative status [28]. In neurodegenerative diseases, autophagy removes toxic aggregated proteins and damaged organelles accumulated with ROS. In addition, autophagy also plays a regulatory role in apoptosis, and protects cells from external stresses such as nutrient deprivation or aggregation of pathogenic proteins [29]. Emerging evidence indicates that two programmed cell death modalities, autophagy and apoptosis, can antagonize or promote each other in different scenarios, and they are able to occur sequentially or co-exist in the same cell [30]. While the same inducing factors can induce autophagy or apoptosis in different cells, some molecules involved in autophagy and apoptosis may also intersect and play positive or negative roles in modulating autophagic and apoptotic programmed cell death [31].
In the current study, increased formation of autophagy marker (LC3) in both stable GFP-LC3-U87 cells and PC-12 cells were observed. Furthermore, the autophagic effect of LCB was confirmed in two autophagic in vivo model of C. elegans: LGG-1::GFP and SQST-1/p62::GFP. Therefore, to further explore the molecular mechanism of LCB-induced autophagy, a PCR array was adopted to assess the expression of 96 autophagy genes (including the house-keeping genes) after LCB treatment. Heap maps showed that the autophagic genes including MAP1LC3B, ATG10, ATG4B, ATG4C, ATG5, BID, CTSD, DRAM2, GABARAPL1, HGS, IFNG, INS, NPC1, PIK3CG, RB1, and B2M were found upregulated more than two fold when compared to the control group. Interesting, these autophagy genes are closely associated with SIRT1 and AMPK signaling pathways. SIRT1 plays a major role in the regulation of several transcription factors related to autophagy, and AMPK activates autophagy by acting as the cellular energy sensor [32]. Previous studies reported that the natural compound, resveratrol, activated AMPK-SIRT1 autophagy for the modulation of Parkinson's disease in a cell model [33]. Quercetin inhibited oxidative stress responses of high fat diet-induced atherosclerosis of rat by AMPK/SIRT1/NF-κB signaling [34]. With AMPK/SIRT1 working as the important signaling sensor of oxidative stress, AMPK-dependent GAPDH phosphorylation triggered Sirt1 activation and is required for autophagy induction upon glucose starvation [35]. In this study, LCB was confirmed to increase p-AMPK at Thr272 and SIRT1 protein expression. With the fact that Beclin-1 is also one of the central regulators for the initiation stage of autophagosome membrane formation, p62 can act as a receptor for vesicles that are going to be degraded by autophagy [36]. LCB increased protein expression of Beclin-1 but decreased p62. Consistently, CC could abolish the effect of LCB on the protein expression of phosphorylation of AMPK, SIRT1, LC3II, and p62, suggesting that LCB may induce autophagy via the AMPK/SIRT1 signaling pathway in PC-12 cells.
With the multiple protective effects of LCB in anti-oxidative stress-induced ROS production and cell death, the mechanistic action of LCB was further correlated with the induction of autophagy. While MDC can specifically label autophagosomes through ion trapping and specific binding to membrane lipids, the number of autophagic vacuoles on H 2 O 2 -induced PC-12 cells (with or without LCB treatment) revealed by MDC staining further confirmed the autophagic role of LCB in vitro. Again, the AMPK inhibitor, CC, could significantly abolish the protective effect of LCB in rescuing H 2 O 2 -induced apoptosis in PC-12 cells, suggested LCB protected PC-12 cells from H 2 O 2 -induced apoptosis via AMPK. To further validate this conclusion, the level of cleaved-caspase-3, a popular apoptosis protein marker, was restored in LCB-treated H 2 O 2 -induced cells with the presence of CC. While increased autophagy due to ROS production was reported to alleviate apoptosis [37], our findings confirmed the protective autophagic role of LCB in reducing H 2 O 2 -cellular damage in PC-12 cells in a pharmacological approach. While many studies have depicted the molecular role of ATG7 deficiency and impaired autophagy in diseases [38], our results demonstrated that LCB decreased H 2 O 2 -induced apoptosis in ATG7 wild-type cells but not in ATG7-deficient MEFs, suggesting the ATG7-dependent mechanism of LCB. Therefore, with the potential beneficial neuroprotective role LCB in protecting PC-12 cells from H 2 O 2induced apoptotic cell death via autophagy, our study has further clarified the traditional therapeutic role of LCB from a pharmacological point of view. However, the autophagic role of LCB in neuronal disease rodent models, and how it can regulate cell survival through the interaction of apoptosis and autophagy, still need to be further investigated.

Cell Culture
PC-12 obtained from the American Type Culture Collection (USA) was cultured in DMEM supplemented with 5% fetal bovine serum, 10% horse serum, 100 µg/mL streptomycin, and 100 U/mL penicillin purchased from Gibco (Grand Island, NY, USA). SH-SY5Y cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 U/mL penicillin. ATG7−/− and ATG7+/+ cell lines were kindly provided by Masaaki Komatsu (Juntendo University, Tokyo, Japan). All cell lines were placed at a 37 • C incubator supplied with 5% CO 2 . USA) was added into each well for a further incubation of 4 h at 37 • C. To dissolve the formazan, 150 µL of DMSO was added to each well and with its absorbance read by using the microplate spectrophotometer at 490 nm.

Lactate Dehydrogenase Release Assay
Cytotoxicity was measured as the level of lactate dehydrogenase (LDH) by using the LDH assay kit (Beyotime, Nantong, China) [39]. In brief, LDH reduced nicotinamide adenine dinucleotide (NAD) to NADH and formazan, which were quantified as the optical density at 490 nm by using the colorimetric method.

Caspase-3 Activity Assay
PC-12 cells were treated with 900 µM of H 2 O 2 for 6 h after pre-treated with different concentrations of LCB for 16 h. Cells was trypsinized, centrifuged at 600× g for 5 min at 4 • C, and the supernatant was carefully aspirated. Cells were washed twice with 1 mL of PBS and the supernatant removed. Then 5 × 10 6 cells were re-suspended in 50 µL cell lysis buffer and mixed with 5 µL Ac-DEVD-pNA. The plate was incubated for 60 min at 37 • C and with the optical density read at 405 nm by using the colorimetric method.

Cellular Autophagy Staining Kit
The induction of autophagy by LCB in PC-12 cells was quantitated by the autophagy staining kit which utilized monodansylcadaverine (MDC) as a fluorescent dye for the detection of autophagic vacuoles. In brief, 1 mL of MDC staining solution was added into each well and incubated with cells for 30 min at a 37 • C incubator and protected from light. The final FITC fluorescent signal was detected by flow cytometry (BD Pharmingen, San Diego, CA, USA).

Hoechst 33342 Staining
After LCB treatment, PC-12 cells seeded on the cover slides were washed with PBS buffer, fixed with 10% formaldehyde at room temperature for 10 min, and then stained with Hoechst 33342 solution in the dark for 5 min. After mounting, stained nuclei on the slides were observed under the fluorescence microscope (LEICA DM2500, Leica, Wetzlar, Germany).

Quantification of Superoxide Dismutase (SOD) and Malondialdehyde (MDA)
PC-12 cells were cultured in a 10 cm dish (5 × 10 5 cells) and pre-treated with different concentrations of LCB for 16 h. The cells were then exposed to H 2 O 2 (900 µM) for 6 h. SOD and MDA level in cells were evaluated by using spectrophotometry according to the manufacturer's assay protocol (Beyotime Institute of Biotechnology, Shanghai, China). The SOD activity was measured at the wavelength of 560 nm, and the level of MDA was detected by using the thiobarbituric acid method with the final absorbance measured at 532 nm [40].

Quantitation of Cellular Apoptosis
Apoptosis was detected by using an FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA). After LCB treatment for 16 h, PC-12 cells were washed with PBS and incubated with 100 µL of binding buffer containing annexin V-FITC and propidium iodide in the dark for 30 min at room temperature. The stained samples were analyzed by the FACS Calibur flow cytometer and with the results analyzed by the software Flow Jo 10.

Quantitation of ROS Production
ROS production was determined by using the 2,7-dichlorodihydrofluorescin diacetate (DCFH2-DA) staining assay (Abcam, Cambridge, UK). After pre-treatment of LCB for 16 h, PC-12 cells were incubated with 10 µM DCFH2-DA at 37 • C for 30 min before the induction of H 2 O 2 (900 µM) for 6 h. The PC-12 cells were re-suspended in PBS and analyzed by flow cytometry for the intensity of the FITC signal. The percentage of fluorescence-positive cells was recorded on a flow cytometer using excitation and emission filters of 488 and 525 nm, respectively.

Quantitative Real-Time Polymerase Chain Reaction (PCR)
After compound treatments, RNA was extracted from PC-12 cells, and the reverse transcription for cDNA was carried out by following the instructions of the cDNA synthesis kit (Transgene, Beijing, China). PCR was performed by using the FastTaq DNA Polymerase Kit (Transgene, Beijing, China). Each PCR reaction was prepared by adding TransScript SuperMix, gDNA Remover, RNase-free water, and 50 ng cDNA templates into a total volume of 25 µL. The real time PCR cycling protocol was performed at 94 • C for 5 min, followed by 40 cycles of 94 • C for 30 s, 60 • C annealing for 1 min, and with a final extension for 10 min at 72 • C by using the Vii7 ABI thermal cycler.

RT2 Profiler PCR Array
SH-SY5Y cells were treated with or without 20 µM LCB for 24 h. Then, 1 µg of RNA was extracted and reverse transcripted into cDNA. Then, 20 µL cDNA was added per well for the RT 2 Profiler PCR array analysis on human autophagy genes (Qiagen, Cat. no. PAHS-084Z). The Ct values were uploaded and analyzed by using the software available at the official website (http://www.qiagen.com/geneglobe (accessed on 8 July 2022)) with reference to the Ct values of the panel of housekeeping genes.

Western Blot Analysis
After treatments, cells were lysed in RIPA lysis buffer with the addition of protease and phosphatase inhibitors for protein extraction. The concentration of the total protein extract was determined by using the Bio-Rad DCTM Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Equal amounts (µg) of total protein lysate were loaded into each well of a 10% SDS-PAGE gel. The separated proteins were transferred to a nitrocellulose (NC) membrane. Membranes were blocked with 5% skim milk in TBST for 1 h at room temperature. Corresponding primary antibodies were added for overnight incubation of protein membrane at 4 • C. After the membrane was washed with TBST, secondary antibody was added to incubate with the membrane at room temperature for 1 h. Actin was used as the loading control for normalization of band intensity. The intensity of the protein signal was detected by using the GE Scanner (Belfast, ME, USA).

Gene Enrichment Analysis
The enrichment analysis was performed by STRING (https://string--db.org/) (accessed on 8 July 2022). Gene data collected were further listed (ATG10, ATG4B, ATG4C, ATG5, BID, CTSD, DRAM2, GABARAPL1, HGS, IFNG, INS, MAP1LC3B, NPC1, PIK3CG, RB1, B2M, SIRT1, and AMPK) and input into STRING. Gene correlation mapping was then generated according to enrichment scoring provided by STRING. The results of GO and KEGG pathway were considered for further analysis. Different colors of lines representing different gene interactions and source of data were presented.

Autophagy Assays in C. elegans
Autophagy assays in C. elegans were conducted as previously reported [41]. Briefly, the NGM medium of control and experimental groups was added with 100 µL of OP50 E. coli bacterial solution, blown dry, and set aside. The synchronized C. elegans DA2123 (LGG-1::GFP) and BC12921 (SQST-1/p62::GFP) were placed in an incubator at 20 • C for 16-20 h, centrifuged at 3000 rpm for 2 min, then with most of the supernatant removed until~1 mL of liquid was left. Then 10 µL of the sample was taken for microscopic observation and counting. According to the calculation of 80 C. elegans per NGM dish, the appropriate volume of liquid was added to the NGM dishes with or without 100 µM of LCB or rapamycin (20 µM) and then placed in the incubator at 20 • C after blowing dry on the ultra-clean bench for 48 h. The appropriate number of nematodes was picked and photographed under a fluorescence microscope (100× oil microscope) after anesthesia. Their fluorescence intensity was statistically analyzed.

Statistical Analysis
Data involved in the analysis of variance were obtained from at least 3 independent experiments. All data were expressed as mean and standard deviation (S.D.). Comparisons between the two groups in the experiment were statistically determined by using Student's t-tests. Comparisons between three and more were calculated by one-way analysis of variance (ANOVA) (GraphPad Prism 8.4, San Diego, CA, USA).

Conclusions
In conclusion, LCB has protective effects against H 2 O 2 -induced PC-12 cell death by reducing the apoptosis rate, LDH release, caspase-3 activity, ROS and MDA level, and protein expression of cleaved caspase-3/caspase-3 and p62. LCB also increased the level of SOD, MDC, LC3II/LCI ratio, Beclin-1, SIRT1, and P-AMPK in both PC-12 cells and C. elegans models. Our findings revealed that LCB protected cells from H 2 O 2 -induced apoptosis in an ATG7-dependent manner and via the SIRT1/AMPK signaling pathway, which has provided therapeutic implications for this herbal agent in reducing oxidative damage via neuroprotective autophagic mechanisms in neuronal cells.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ph15091052/s1, Figure S1: Establishment of cellular oxidative stress models; Figure S2: DCFH-DA assay for the measurement of ROS level; Figure S3: Western blot for protein expression of caspase-3; Figure S4: Western blot for protein expression of LC3 I/II; Figure S5: Mechanistic study on LCB by Western blot; Figure S6: MDC assay for autophagy detection.