Introduction
Autophagy is a cellular process in which the cell degrades and recycles its components to maintain homeostasis. As a fundamental catabolic process, autophagy involves the lysosome-mediated degradation of damaged, unnecessary, or dysfunctional cellular components [1,2]. Autophagy also plays a critical role in cellular metabolism and in regulating cell death and survival [3,4]. Under normal physiological conditions, most eukaryotic cells exhibit basal autophagy. In contrast, autophagy-deficient cells display extensive abnormal protein accumulation and mitochondrial disorganization, indicating that autophagy plays a critical role in the clearance of damaged organelles and protein aggregates that are resistant to degradation by the ubiquitin-proteasome system, thereby contributing to the maintenance of cellular homeostasis [5,6]. Genetic and mechanistic studies have highlighted autophagy’s involvement in various human health conditions, including cancer [7,8], neurodegenerative diseases [9,10], age-related disorders, and inflammatory and infectious diseases [11,12].
Hypericum hookerianum Wight & Arnott (family Hypericaceae), widely allocated across Asian countries such as China, Bhutan, India, Myanmar, Nepal, Thailand, and Vietnam [13], is well known in folklore medicine for its diverse therapeutic properties, including antioxidant [14,15], anticancer [16,17], antidepressants [18,19], neuroprotection [20], and antifungal activities [21-23]. In this study, we investigated the impact of sesamin on the activation of autophagy.
Materials and Methods
1. Cell culture
The HeLa and cells were obtained from the Korea Cell Line Bank and cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. Cells were maintained at 37℃ in a humidified incubator with 5% CO2.
2. Autophagy screening method
The plasmid pMRX-IP-GFP-LC3-RFP-LC3ΔG, containing an autophagic flux probe with an internal control, was obtained from Addgene (Cat. No. 84572). The HeLa cell line expressing this construct was designated as HeLa-LC3BΔG. Cells were seeded at a density of 30,000 HeLa-LC3BΔG cells per well in a black-walled, clear-bottom 96-well plate (Greiner). After 24 hours of incubation, cells were treated with Sesamin and a positive control. Following a 24-hour treatment period, cells were fixed with 4% paraformaldehyde for 10 minutes and washed with Dulbecco’s phosphate-buffered saline (DPBS). Fluorescence intensities for green fluorescent protein (GFP) (488 nm ± 9, 514 nm ± 15) and red fluorescent protein (RFP) (584 nm ± 9, 612 nm ± 15) were measured using a Tecan Infinite 1000 Series plate reader (Tecan).
The HEK293-LC3-Hibit cell was provided by Promega (Cat. No. GA1040). Cells were seeded at a density of 20,000 HEK293-LC3-Hibit cells per well in a white-walled, flat-bottom 96-well plate (Greiner). After 18 hours of incubation, cells were treated with sesamin and a positive control. Following a 24-hour treatment period, the nano buffer was added for each well, and the luminescent intensity was measured by the Glomax machine (Promega).
3. Acridine orange staining
HeLa cells were seeded in a black-walled, clear-bottom 96- well plate at a density of 5,000 cells per well. After 12 hours of incubation, cells were treated with sesamin for 24 hours. Following treatment, the culture medium was removed, and the wells were washed with DPBS. Cells were then stained with 2 µM acridine orange for 15 minutes, followed by another DPBS wash before being photographed using the Operetta high-content imaging system (Perkin Elmer). The fluorescence intensity was estimated using the Harmony 3.5 program (Perkin Elmer).
4. Lysosome pH measurement
HeLa cells were seeded at a density of 8,000 cells per well in a black-walled, clear-bottom 96-well plate. After overnight incubation, the culture medium was replaced, and cells were treated with sesamin 50 µM, rapamycin 25 µM, bafilomycin A1 20 nM, or a combination of sesamin 50 µM and bafilomycin A1 20 nM. After 24 hours of incubation, half of the culture medium was removed, and 50 µL of 2 µM DND-160 (L7545; Thermo Fisher Scientific) prepared in fresh medium was added to each well. Cells were incubated for 15 minutes and subsequently washed with DPBS. Fluorescence was measured at excitation/ emission wavelengths of 329/540 nm (yellow) and 329/440 nm (blue), with a bandwidth of 10 nm, using the Tecan Infinite 1000 Series plate reader. Imaging was performed using the Operetta high-content imaging system. The fluorescence intensity was estimated using the Harmony 3.5 program.
5. Immunoblot analysis
HeLa cells (2 × 105) were seeded in a 6-well plate (Corning) and incubated overnight. After overnight growth, the cells were treated with compounds, and after 24 hours, the cells were harvested. First, cells were lysed using cell extraction buffer (Invitrogen) along with a complete protease inhibitor cocktail (Sigma-Aldrich) and phenylmethanesulfonyl fluoride (1 mM). Cells were lysed for 30 minutes and spun down at 14,000 rpm for 20 minutes. The supernatant containing total protein was quantified using the BCA protein assay kit (Invitrogen). Furthermore, a total of 10 µg of protein was loaded onto an SDS-PAGE gel and electrophoresed at 100 V for 120 minutes. Proteins were then transferred onto a polyvinylidene fluoride membrane (Millipore) at 100 V for 90 minutes. Membranes were blocked with either 5% skimmed milk or 3% bovine serum albumin (for phosphorylated proteins) for 1 hour at room temperature. Primary antibodies (1:1,000 dilution) were incubated overnight at 4℃, followed by incubation with secondary antibodies (1:1,000 dilution) at room temperature for 90 minutes. Chemiluminescent signals were detected using the SuperSignal West Pico Plus reagent (Thermo Fisher Scientific), and images were captured.
6. Data analysis
All graphs were generated using GraphPad Prism 10 software (GraphPad). Three independent experiments present data as mean ± standard deviation. Statistical significance was determined using one-way nonparametric analysis of variance.
Results
1. Identification of sesamin- an autophagy activator from H. hookerianum
To characterize the autophagy-promoting activity of individual constituents isolated from H. hookerianum, we subjected all 28 purified compounds to the puncta-formation assay (Fig. 1A and 1B, Supplementary Table 1, Supplementary Fig. 1). Autophagy activation is traditionally assessed by monitoring the turnover of lipidated LC3B (LC3B-II) via immunoblotting. While effective, this method is labor-intensive, time-consuming, and resource-demanding. As a result, cell-based reporter assays utilizing fluorescently tagged LC3B have emerged as a more practical alternative for high-throughput screening. Initial systems employed GFP-LC3B constructs to visualize autophagosome formation, as GFP-LC3B is incorporated into autophagosomal membranes upon induction of autophagy. However, the GFP signal is quenched in the acidic environment of autolysosomes, limiting the ability to monitor autophagic flux. To address this limitation, dual-tagged RFP-GFP-LC3B constructs were developed. While this system allows differentiation between autophagosomes (GFP+/RFP+) and autolysosomes (GFP–/RFP+), it does not quantitatively assess autophagic flux due to the persistence of non-lipidated LC3 forms. To overcome these constraints, the GFP-LC3B-RFP-LC3ΔG reporter assay was established [1,2]. In the GFP-LC3B-RFP-LC3ΔG reporter assay, GFP is fused to full-length LC3B, whereas RFP is fused to an LC3B variant lacking the N-terminal glycine residue (LC3ΔG). Upon induction of autophagy, the cysteine protease ATG4 cleaves full-length LC3B at its N-terminus, releasing GFP-LC3B and RFP-LC3ΔG [24,25]. The liberated GFP-LC3B conjugates to phosphatidylcholine and is incorporated into autophagosomal membranes, ultimately undergoing lysosomal degradation. In contrast, RFP-LC3ΔG lacks the essential glycine required for lipidation and remains cytosolic. Consequently, the ratio of GFP to RFP fluorescence provides a quantitative measure of autophagic flux, with a decrease in the GFP/RFP signal ratio reflecting increased autophagic degradation. This system enables more accurate and dynamic measurement of autophagic flux by distinguishing between functional autophagy and static autophagosome accumulation. HeLa-GFP-LC3B-RFP-LC3ΔG cells were incubated with either vehicle (dimethyl sulfoxide [DMSO]) or 50 µM of each compound for 24 hours. Following treatment, cells were fixed, and automated high-content imaging was performed on the Operetta platform.
Quantitative image analysis revealed that sesamin (HH28), which was isolated from dichloromethane extract, has been previously reported for its neuroprotective effects. This result identifies sesamin as a potent activator of autophagy among the 28 H. hookerianum’s single compounds.
2. Sesamin activates autophagy and autophagic flux
Autophagy activation leads to the formation of autophagosome vesicles, which sequester unwanted cellular components for degradation. LC3B is predominantly recruited to the inner membrane of autophagosomes. Therefore, HeLa-GFP-LC3B-RFP-LC3ΔG cells were utilized to examine autophagic vesicle formation. Sesamin treatment induces a significant decline in the GFP/RFP ratio and a marked increase in LC3B puncta, indicating the formation of autolysosomes (Fig. 2B–2D, Supplementary Fig. 2). Torin-1 and bafilomycin A1 were used as an autophagy activator and inhibitor, respectively. Furthermore, exposure to sesamin led to a decreased luminescent signal in the Autophagy-LC3B-HiBiT assay (Fig. 2E). Upon autophagy activation, LC3BI undergoes processing and conversion to LC3BII, which is subsequently recruited to the inner membrane of the autophagosome. Therefore, the LC3BII/LC- 3BI ratio serves as a marker of autophagy. As illustrated in the figure, sesamin increased the ratio of LC3BII/LC3BI levels as determined by immunoblotting (Fig. 3A and 3B). On the other hand, p62 facilitates the delivery of ubiquitinated cargo for autophagic degradation through its C-terminal ubiquitin-associated or LC3-interacting region domain [26]. Consequently, p62 levels increase during the early stages of autophagy, but it is degraded along with its cargo inside the autolysosome. Therefore, p62 is widely used as a marker of autophagic flux. The p62 expression increases after 6 hours treatment with sesamin, decreases after 12 hours, and disappears completely after 24 hours, indicating sesamin-induced autophagic flux (Fig. 3C and 3D).
3. Sesamin activates lysosome biogenesis
Autophagic flux during starvation is facilitated by increased lysosomal activity. Perinuclear lysosomes exhibit a more acidic environment, enhancing enzymatic function and promoting degradation [27]. For this reason, the acridine orange was used to determine the lysosome generation. The red color of acridine orange increases at an acidic pH and is used to describe acidic lysosomal formation. Sesamin treatment for 24 hours significantly increased acid-red fluorescent in a dosedependent manner, indicating that sesamin induces lysosome formation (Fig. 4A and 4B). Meanwhile, lysosomal pH was analyzed in HeLa cells following incubation with DMSO, 20 nM bafilomycin A1, 25 μM rapamycin, and 50 μM sesamin for 24 hours, after that the cell was stained by LysoSensor DND-160 for 15 minutes. A reduced yellow-to-blue ratio is indicative of increased pH. The treatment with sesamin induces an increase in the ratio of yellow-to-blue, indicating the pH reduction. However, treatment with bafilomycin A1, an autophagy inhibitor, decreases the yellow-to-blue ratio, indicating increased pH (Fig. 5A and 5B).
4. Sesamin shows autophagic neuroprotection against oxidative stress
In this study, the neuroprotective effect of sesamin against oxidative stress-induced neuronal cell death via autophagy activation was investigated. HT22 cells were treated with sesamin in the presence or absence of the autophagy inhibitor (chloroquine [CQ]), followed by glutamate-induced oxidative stress. Sesamin treatment significantly protected neuronal cells, whereas co-treatment with CQ abolished this effect. These results indicate that sesamin exerts its neuroprotective activity through autophagy activation under oxidative stress conditions (Fig. 6A and 6B).
Discussion
This study identified sesamin, a bioactive compound isolated from H. hookerianum , as a potent autophagy activator. Our findings demonstrate that sesamin promotes autophagy by increasing the LC3B-II/LC3B-I ratio and enhancing autophagic flux, which are hallmarks of effective autophagy induction.
Activation of lysosomal biogenesis by sesamin further strengthens its role as an autophagy enhancer. Lysosomes play a critical role in the final degradation of autophagic cargo, and their impairment is commonly observed in neurodegenerative diseases [27]. Our study demonstrates that sesamin increases lysosomal acidification, as evidenced by enhanced acridine orange staining and LysoSensor assays, indicating improved lysosomal function. This aligns with previous research suggesting that stimulating lysosomal activity can enhance the clearance of toxic protein aggregates, further underscoring sesamin’s potential in neuroprotection.
While our study provides strong evidence for the role of sesamin in autophagy activation further investigations are needed to elucidate its precise molecular mechanisms. Specifically, identifying the direct molecular targets of sesamin in the autophagy-lysosomal pathway will provide deeper insights into its mode of action. Additionally, in vivo studies using animal models will be crucial to validate our in vitro findings and assess the potential therapeutic application of sesamin.
Sesamin, isolated from H. hookerianum, is a potential autophagy activator. Sesamin significantly increased the autophagosome and reduced pH levels in HeLa cells. Moreover, sesamin promoted autophagic flux, as evidenced by increased LC3B-II and changed p62 expression time-dependent, along with enhanced lysosomal activity. These findings highlight sesamin as a promising natural compound for therapeutic strategies aimed at autophagy-related diseases. In vivo studies and further mechanistic investigations are needed to confirm the potential of sesamin for clinical application in autophagyrelated diseases.
