1. 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 tumorigenic cells [
5]. Therefore, a balanced rate of apoptosis is important for preventing adverse cellular conditions. Upon oxidative stress conditions, autophagy removes damaged organelles and protein aggregates of cells, which is critical for maintaining normal cellular homeostasis [
6]. Moreover, failure or impairment of autophagy in cells may cause an accumulation of soluble or aggregated tau protein, a decreased level of Beclin–1, and finally the increased expression of amyloid precursor protein (APP) and β–amyloid, which are related to the pathogenesis of Alzheimer’s disease and oxidative stress conditions [
7]. In addition, research has revealed that the preservation of autophagic activity is vital for preventing detrimental intracellular accumulation of damaged molecules [
8].
Licochalcone is a major biological active phenolic components of the medicinal herb licorice
Glycyrrhiza inflate [
9]. The anti-oxidative effect of licochalcone A was reported for its suppressive effect on the production of ROS and neuronal apoptosis to exert neuroprotective effects [
10]. Licochalcone D protected mouse heart from oxidative damage attributed to the activation of autophagy [
11]. Licochalcone E activated antioxidant gene-dependent pathways related to mechanisms of defense against oxidative stress and inflammatory responses [
12]. LCB (
Figure 1) has been shown to possess anti-oxidative, anti-inflammatory, anti-cancer, cardioprotective, and hepatoprotective activities [
13]. The anti-apoptotic effect of LCB was also reported in an alcohol-induced hepatocyte injury model [
14]. In fact, many herbal medicines have been reported to exert protective autophagic effects against cellular stressful conditions such as apoptosis and oxidative stress [
15]. With the reported pharmacological role of several chemical components of
Glycyrrhiza inflate, however, the pharmacological role of LCB in autophagy induction remain un-investigated. With the reported protective role of autophagy and apoptosis in neuroprotection [
16], this study adopted H
2O
2-induced PC-12 cells as an in vitro oxidative stress induction system for the investigation of the regulation of both autophagy and apoptosis by LCB. PC-12 cells, originated from pheochromocytoma and isolated from rat renal medulla, are neuronal–like and share similar secretary properties and embryological origins with neurons [
17]. They can be induced by nerve growth factor (NGF) to differentiate into neuronal–like cells; therefore, it is widely adopted as the neuronal model for the study of neuropathic pain, neurotoxicity, and neuro–degenerative diseases [
18]. In order to provide a theoretical basis for the development of a novel natural autophagic agent for its possible protective effect in neuroprotection, the pharmacological effects of LCB in term of its anti-apoptotic and autophagic ability were studied in both PC-12 cells and
C. elegans models.
3. 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 compounds 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 anti-neuroinflammatory 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
2O
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
2O
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
2O
2 were decreased by approximately 50%, LCB pretreatment significantly inhibited damage and increased cell viability upon H
2O
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
2O
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
2O
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
2O
2 in ATG7 wild–type MEFs but not in ATG7-deficient MEFs, suggesting that LCB attenuated H
2O
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
2O
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
2O
2-induced apoptosis in PC-12 cells, suggested LCB protected PC-12 cells from H
2O
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
2O
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
2O
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
2O
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
2O
2-induced 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.
4. Materials and Methods
4.1. Reagents and Antibodies
3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT), paraformaldehyde, bovine serum albumin (BSA), hydrogen peroxide (H2O2), 2′,7′– dichlorofluorescin diacetate (DCFH–DA), rapamycin, and sodium bicarbonate were obtained from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO, USA). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA). Licochalcone B was purchased from Herbest Biological Technology Co., Ltd. (Baoji, China). Hoechst 33342, Alexa Fluor 488–labeled goat anti-Rabbit IgG (H + L), the Total Superoxide Dismutase Assay Kit with NBT, Lipid Peroxidation MDA Assay Kit, Calcein/PI Cell Viability/Cytotoxicity Assay Kit, Autophagy Staining Assay Kit with MDC, and LDH Cytotoxicity Assay Kit were purchased from Beyotime Biotechnology Inc. (Shanghai, China). Compound C (CC) was purchased from Topscience Co., Ltd. (Shanghai, China). The Caspase–3 Assay Kit (Colorimetric) was purchased from Abbkine Scientific Co., Ltd. (Wuhan, China). Primary antibodies against apoptosis-related proteins (cleavage–caspase-3, #9664; and caspase-3, #14220) and autophagy-related proteins (LC3, #3868; Beclin–1, #3495; SIRT1, #8469; AMPK, #4150; P–AMPK, #50081; p62, #39749) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti–GAPDH (sc–47724), β–actin (sc–8432), rabbit (sc–2357), and mouse (sc–2005) IgG–horseradish peroxidase secondary antibodies and an Annexin V Apoptosis Detection Kit (sc–4252 AK) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4.2. 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% CO2.
4.3. Establishment of Cellular Oxidative Stress Model
In brief, PC-12 cells were plated in 6–well (2 × 105 cells/well) plates for overnight cell adhesion. For the detection of cellular oxidative stress, cell death, or mechanisms, 4 different treatment groups were included: (1) Cells pre-treated with different concentrations (from 10 to 40 μM) of LCB for 16 h without H2O2 treatment (LCB group), (2) cells pre-treated with different concentrations (from 10 to 40 μM) of LCB for 16 h with the addition of 900 μM of H2O2 for a further 6 h (LCB + H2O2 treatment group), (3) cells treated with 900 μM of H2O2 for 6 h (H2O2 treatment group), and (4) cells treated with solvent or vehicle alone (control treatment group) were set as the experimental conditions.
4.4. Cell Viability Assay
PC-12 cells were plated in a 96–well (5 × 103 cells/well) for overnight cell adhesion. To set up the oxidative stress cellular model, PC-12 cells were treated with 900 μM of H2O2 for 6 h has described. Cells was then pre-treated with different concentrations of LCB for 16 h prior to the exposure of H2O2. Then, 10 μL of MTT solution (Sigma, St. Louis, MO, 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.
4.5. 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.
4.6. Caspase–3 Activity Assay
PC-12 cells were treated with 900 μM of H2O2 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 × 106 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.
4.7. 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).
4.8. 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).
4.9. 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
2O
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].
4.10. 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.
4.11. 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 H2O2 (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.
4.12. 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.
4.13. 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.
4.14. 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).
4.15. 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.
4.16. 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.
4.17. 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).