The Cell Counting Kit-8 (CCK-8) (MA0218, Dalian, China) was provided by Meilun Biotechnology, with eltrombopag (EO) (SB-497115) and MHY1485 (HY-B0795) purchased from MedChemExpress. The triglyceride (TG) kit (A110-1-1), trace malondialdehyde (MDA) kit (A003-1), and trace reduced glutathione (GSH) kit (A006-2-1) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Improved Oil Red O staining kit (C0158S), 4% paraformaldehyde (P0099), bicinchonininc acid (BCA) protein concentration determination kit (P0012), ROS detection kit (S0033S), adenosine triphosphate (ATP) detection kit (S0026), mitochondrial membrane potential (MMP) detection kit (C2006), mitochondrial far-infrared fluorescence probe (C1032-50ug), Mito Tracker Green (C1048), Lyso Tracker Red (C1046), Hoechst 33342 (C1027), lipid droplet green fluorescence detection kit boron-dipyrromethene (BODIPY) 493/503 (C2053S), cell autophagy staining (monodansylcadaverine (MDC) method) detection kit (C3018S), along with adenovirus expression of monomeric Cherry (mCherry)-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 beta (LC3B) fusion protein (Ad-mCherry GFP-LC3B) (C3011-1mL) kit were purchased from Beyotime (Shanghai, China). The apoptosis detection kit (556547) was purchased from BD (Beijing, China).

L02 cells (Shanghai Institute of Life, Chinese Academy of Sciences) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (supplemented with double antibiotics and nonessential amino acids) at 37°C under 5% CO₂. Cells in exponential growth phase (3–10 passages) were seeded into various cell culture plates at moderate density for 24 h. The adherent cells were subjected to 9-trans-C18:1 (CAS: 112–79-8, E4637, Sigma-Aldrich, St. Louis, Missouri, United States) and/or sesamin (CAS: 607–80-7, SMB00705, Sigma-Aldrich, St. Louis, Missouri, United States) for 24 h. The study was divided into control, model, and intervention groups, respectively.

After L02 cells reach 80% coverage, they were digested and seeded into a 96 well plate. After adhesion, treatment with different fatty acids for 24 h was conducted. Then 10% CCK-8 medium were added into each well, followed by incubation at 37°C for 0.5–4 h. The absorbance at 450 nm was detected using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland).

The cultured L02 cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed twice with phosphate buffered solution (PBS), respectively. Then, the cells were subjected to an appropriate amount of staining washing solution for 20 s and Oil Red O staining working solution for 20 min successively, followed by washing with washing solution and PBS for 30 and 20 s, respectively. The cell nucleus was stained with hematoxylin for 1 min. Microscopic observations were conducted using a microscope (DMI4000B, Leica, Germany). After dissolving intracellular lipid droplets with 100% isopropanol, semiquantitative analyses were performed by measuring the absorbance at 510 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland).

The treated L02 cells were digested and centrifuged at 2000 × g for 15 min. Subsequently, cell precipitates were collected and fixed with 3% glutaraldehyde fixative, dehydrated, infiltrated, embedded, ultra-thin sectioned, stained, successively. Finally, the ultrastructure was characterized using a transmission electron microscopy (TEM, HT7700, Hitachi, Nakahikari, Ibaraki, Japan).

The cultured and treated L02 cells were collected and washed twice with PBS. After being mixed with 300 μL PBS, the cells were sonicated in an ice water bath. The intracellular levels of TG (A110-1-1, Nanjing Jiancheng), MDA (A003-1, Nanjing Jiancheng), and GSH (A006-2-1, Nanjing Jiancheng) were detected according to the supplier’s instructions. A microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland) was applied to measure absorbances at specific wavelengths.

Intracellular ATP was quantified using a commercial reagent kit. After removing the culture medium, 200 μL of lysis buffer was added into each well of the 6-well plate to fully lyse the L02 cells. The supernatant was obtained by centrifugation at 4°C and 1.2 × 10⁴ g for 5 min. After 100 μL ATP detection working solution was added into each detection well, 20 μL sample or standard was added and mixed quickly. The absorbance was measured using a multifunctional enzyme-linked immunosorbent assay reader (SpectraMax i3x, Molecular Devices, San Jose, United States). The concentrations of ATP in the samples were calculated based on the standard curve.

After the medium removal, the L02 cells were washed once with PBS and incubated with 5 μM 2′,7′-dichlordihydrofluorescein diacetate (DCF-DA),1 × 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), 200 mM Mito Tracker, and 1× MDC fluorescent probes prepared in serum-free 1640 medium at 37°C for 30 min. Then wash them once with PBS and medium, and replace with fresh medium. The intracellular ROS, MMP, mitochondria, and autophagy degrees were observed under a fluorescence microscope (DMi8-M, Leica, Germany). The semiquantitative analyses were performed using the Image J software.

After removing the culture medium, L02 cells were washed once with pre-cooled PBS and then subjected to digestion with ethylene diamine tetraacetic acid (EDTA) free trypsin, washing, centrifuge, and resuspension in 1× Binding buffer. Each sample was added with 5 uL Annexin-V and PI in the dark and incubated at room temperature for 15 min. Then the apoptosis was analyze using a flow cytometer (FACS AriaIII, BD, United States) within 1 h.

Mito Tracker Green and Lyso Tracker Red were employed for the co-localization of mitochondria and lysosomes. After culture medium removal and washing once with PBS, L02 cells were added with Mito Tracker Green or Lyso Tracker Red storage solution diluted with culture medium at a ratio of 1:10000 and 1:13333, respectively, followed by incubation at 37°C for 30 min. After removing the staining solution and adding fresh culture medium, microscopic observations were carried out by a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Semiquantitative analyses were performed using the Image J software.

After adhesion, L02 cells cultured in confocal dishes were transfected with Ad-mCherry-GFP-LC3B at a multiplicity of infection of 40 for 48 h and subsequently subjected to different treatment based on grouping. After removing old and adding fresh culture medium, they were observed using a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Semiquantitative analyses were performed based on yellow and red spots.

Total RNA was extracted from cells using TriZol reagent (15596026, Invitrogen, Carlsbad, CA, United States), followed by reverse transcription of mRNA into cDNA using a 1st strand cDNA synthesis kit (Code No. 6210A). qPCR was performed on QantStudio 1 (Applied Biosystems, Walktham, MA, United States) using a 20 μL amplification system consisted of 10 μL premix (SsoFast EvaGreen Supermix, BioRad, CA, USA), 1 μL primer for each, 1 μL cDNA, and 7 μL deionized H₂O. The qPCR reaction conditions were set as following: 95°C 30s for 1 cycle, along with 95°C 5s, 60°C 30 s, and 72°C 30 s for 40 cycles. Subsequently, melting curve analyses were performed to avoid amplification of non-specific products. β-actin was employed as an internal reference gene, and the mRNA relative level of the targets were calculated using the 2^-ΔΔCt algorithm. The primer sequences used in this work were presented in the Table 1.

After removing the culture medium, L02 cells cultured in confocal dishes were washed thrice with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min successively, followed by washing thrice with PBS and 0.1% Triton X-100 addition. After being kept at room temperature for 10 min and washed thrice with PBS subsequently, they were added with protein free rapid blocking solution (PS108P, Epizyme, Shanghai, China) and blocked at room temperature for 30 min. After removing the blocking solution, an incubation overnight at 4°C with primary antibodies against TFEB (1:100, TN27339, Abmart, Shanghai, China) and lysosome-associated membrane protein 1 (LAMP1) (1:20021997-1-AP, Protein, Wuhan, China) was performed. After washing thrice with PBS (5 min for each), the cells were incubated at room temperature for 1 h in the dark with Cy3 labeled sheep anti rabbit IgG (H + L) secondary antibody (1:200, A0516, Beyotime, Shanghai, China), followed by washing thrice with PBS (5 min for each). Hoechst 33342 (C1025, Beyotime, Shanghai, China) was used for nuclear staining. Finally, microscopic observations were carried out using a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany).

The treated L02 cells were subjected to radio immunoprecipitation assay (RIPA) lysis buffer to extract total protein and a BCA assay kit for protein concentration measurement. Thirty μg protein was uploaded into each well and separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane at a constant current of 300 mA, followed by block with 5% skim milk. Then the membrane was incubated with primary antibodies against microtubule-associated protein 1 light chain 3 (LC3) (1:1000, 14600-1-AP, Proteintech, Wuhan, China), PTEN-induced putative kinase 1(PINK1) (1:1000, DF7742, Affinity Bio sciences, Changzhou, China), Parkin (1:1000, PAL060Hu01, Cloudclone, Wuhan, China), Sequestosome 1(SQSTM1/p62) (1:1000, PAD198Hu01, Cloud-clone, Wuhan, China), TFEB (1:800, TN27339, Abmart, Shanghai, China), LAMP1 (1:1000, 21997-1-AP, Protein, Wuhan, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, 10494-1-AP, Proteintech, Wuhan, China), and Histone H3 (1:1000, AF0863, Affinity, Jiangsu, China) on a shaker overnight at 4°C. After being washed with tris-buffered saline with Tween 20 (TBST) (5 min for each), the membrane was incubated with HRP labeled sheep anti rabbit secondary antibody (1:2000, SA00001-2, Proteintech, Wuhan, China) at room temperature for 1 h, followed by washing with TBST thrice (5 min for each). Finally, the protein bands were visualized using an ECL chemiluminescence assay kit (P0018S, Beyotime, Shanghai, China) in a chemiluminescence imaging instrument (5200 Multi, Tanon, Shanghai, China). The ImageJ software (NIH, Bethesda, MD, United States) was employed to analyze the densities of protein bands, with GAPDH acting as the reference gene.

Lipid droplets were stained using BODIPY fluorescent probe. After removing the culture medium, L02 cells cultured in a 24 well plate were washed twice with PBS and then fixed with 4% paraformaldehyde at room temperature for 15 min, followed by rinse twice with PBS. After adding 250 μL staining solution into each well, an incubation at room temperature in the dark for 20 min was performed. Hoechst 33342 was utilized for nuclear staining. After being washed twice with PBS, the cells were observed under a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany) and semi-quantitative analyses were performed using the Image J software.

All data were presented as means ± standard deviation (SD) and analyzed using the SPSS software (version 22.0, SPSS Inc., Chicago, Illinois, United States). One-way ANOVA (analyzes of variance) was employed for group comparison. LSD was applied for homogeneous variance and Dunnett T3 for the non-homogeneous. When p < 0.05, it was considered statistically significant.

L02 cells were treated with 9-trans-C18:1 at various concentrations to detect its cytotoxicity. The results of CCK-8 (Figure 1A) showed that 9-trans-C18:1 at 500 μM exerted no significant effect on cell viability, and led to significantly increased intracellular TG (Figure 1B) (p < 0.01), indicating the successful construction of the in vitro high-lipid model. In the absence or presence of 9-trans-C18:1 at 500 μM, sesamin at higher than 32 μM caused a significant decrease in cell viability (Figures 1C,D). The results of Oil Red O staining (sesamin at 4, 8, 16, and 32 μM, respectively) showed a dose-dependent decrease in intracellular lipid accumulation (Figure 1E). Therefore, 32 μM was selected as the working concentration of sesamin for subsequent experiments. These data indicate that sesamin reduced lipid accumulation in L02 cells treated with 9-trans-C18:1.

The ultrastructure of L02 cells were analyzed through TEM. As shown in Figure 2, the mitochondria in control group were oval and elongated, with clear cristae structures. Compared with control, intracellular lipid droplets in model group increased significantly, with significantly changed morphology and structure of mitochondria: higher proportion of slender mitochondria and blurred cristae. Partial mitochondrial cristae structures disappeared completely, showing shrinkage necrosis and vacuolization (Green arrow). Few mitochondria were engulfed by lysosomes and exhibited characteristic autophagic lysosomes (Red arrow), indicating low levels of cellular autophagy. Compared with the model group, intervention with sesamin resulted in a decreased volume ratio of intracellular lipid droplets by 51.3% (Figure 2B), an increase in mitochondrial quantity, a restoration of oval and elongated structures, and a clear cristae structure, indicating an improvement on mitochondrial homeostasis (Figure 2C). The increased lysosomes and autophagic lysosomes (Figure 2D) suggest an increase in cellular autophagy levels, conducive to timely removal of damaged mitochondria. The lipid toxicity caused by lipid accumulation may be the main reason for low-level cellular autophagy and premature degradation of damaged mitochondria. Therefore, it could be speculated that sesamin restores mitochondrial function by increasing mitophagy, thereby promoting lipid metabolism and reducing lipid accumulation.

Subsequently, the impact of sesamin on cellular function was analyzed. ROS serves as an important indicator for cellular oxidative damage. Compared with control, the increased fluorescence intensity in the model group indicates an increase in intracellular oxidative stress level after 9-trans-C18:1 treatment, which could be alleviated by sesamin intervention, demonstrating its mitigating effect on intracellular oxidative stress (Figure 3A). MDA can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. GSH content is an important indicator for body’s antioxidant capacity. Sesamin significantly downregulated the intracellular MDA (Figure 3B), while significantly upregulating the intracellular GSH (Figure 3C), suggesting the role of sesamin in diminishing 9-trans-C18:1 induced oxidative stress level. The weakened mitochondrial MMP acts as the primary indicator for mitochondrial dysfunction. Compared with control, 9-trans-C18:1 treatment resulted in a significant decrease in intracellular MMP, which could be reversed by sesamin (Figure 3E). The mitochondrial far-infrared fluorescence probe can detect cell apoptosis by detecting changes in MMP. The intensity of red fluorescence represents the function of mitochondria. As shown in Figure 3F. sesamin intervention enhanced the function of mitochondria, consistent with the MMP results. Decreased ATP levels usually indicates impaired or decreased mitochondrial function. The significantly reduced ATP levels in the model group were reversed by sesamin, indicating sesamin’s effects on restoring partial mitochondrial function (Figure 3D). The above suggests that sesamin can improve the severe oxidative stress and disrupted mitochondrial homeostasis induced by 9-trans-C18:1 in L02 cells, thereby restoring partial mitochondrial function and diminishing lipid accumulation.

MDC is one of the most commonly used fluorescent probes for detecting cellular autophagy. Compared with control, autophagy in the model group was enhanced in some cells under 9-trans-C18:1 induction, with autophagy level relatively low, however. Compared with the model group, sesamin improved the cellular autophagy, thereby alleviating the 9-trans-C18:1 induced lipid accumulation to some extent and the degree of cell damage (Figures 4A,B). Next, changes in mitophagy were examined through co-staining with MitoTracker and LysoTracker (Figures 4C,D). Compared with control, the model group showed a significant decrease in mitochondrial fluorescence intensity, indicating that the accumulated lipid droplets significantly reduced mitochondrial homeostasis, in consistence with the TEM observation results (Figure 2). The fluorescence intensity of lysosomes and Mito-Lyso co-localization coefficient in the model group were significantly lower than those in the S32 group, indicating low mitophagy level in the model group. In the S32 group, significant enhanced Lyso fluorescence and Mito-Lyso co-localization coefficient were observed, suggesting that sesamin activated intracellular lysosomes and improved mitochondrial homeostasis by enhancing mitophagy levels, thereby reducing lipid droplet accumulation. As shown in Figures 4E,F, compared with the model group, the apoptosis rate of cells significantly decreased after intervention with sesamin, suggesting that sesamin may alleviate the degree of cell damage by enhancing autophagy levels to clear damaged mitochondria.

PINK1/Parkin is a common mitophagy pathway which can be activated by mitochondrial dysfunction (27). Firstly, the mRNA levels of LC3 and p62 were detected by qPCR. Compared with control, the mRNA levels of LC3 and p62 were upregulated in the model group, which could be further upregulated by sesamin (Figure 5A). Next, protein levels of LC3, p62, PINK1, and Parkin were analyzed using Western blot. As shown in Figures 5B,C, in comparison with control, the significantly increased protein levels of p62 suggest low-level autophagy under 9-trans-C18:1, consistent with the results of TEM (Figure 2), MDC (Figures 4A,B), and Mito Lyso co localization (Figures 4C,D). Compared with the model group, sesamin further significantly upregulated the protein levels of LC3, PINK1, and Parkin, while downregulated that of p62, indicating that the PINK1/Parkin pathway was activated, thereby promoting impaired mitophagy and improving mitochondrial homeostasis.

TFEB is activated during lysosomal impairment, which is part of the lysosomal injury response and necessary for subsequent lysosomal recovery, probably promoting lysosomal biosynthesis. The autophagy flux was analyzed through adenovirus transfection to effectively track the fusion process of autophagosomes and lysosomes. The results (Figures 6A,B) showed that the control cells exhibited diffuse yellow fluorescence of mCherry-GFP-LC3B with few autophagosomes and autolysosomes, indicating a non-autophagic state. Cells in the model group presented a large number of yellow spots and few red spots, suggesting that the cells attempted to initiate autophagy to alleviate the impairment caused by 9-trans-C18:1. Compared to the model group, a large number of red spots induced by sesamin suggest an increase in the fusion of autophagosomes and lysosomes and initiated process of autophagosome to autophagosomes to restore partial mitochondrial function, thereby alleviating the oxidative damage induced by 9-trans-C18:1, consistent with the results of TEM (Figure 2). Direct or indirect inhibitor of TFEB, EO or MHY1485, could reverse this trend. The mRNA levels of LAMP1 and TFEB were significantly upregulated before and after sesamin intervention, suggesting the up-regulating effects of both elaidic acid and sesamin on them (Figure 5A). Compared with control, insignificant changes in the overall fluorescence intensity of TFEB but significantly elevated nuclear translocation rate in the model group were observed (Figures 6C,D), indicating a role of 9-trans-C18:1 in dephosphorylating TFEB and inducing nuclear translocation partially. In comparison with the model group, the overall fluorescence intensity of TFEB was significantly enhanced and the translocation rate was significantly increased after intervention with sesamin, suggesting the activating effect of sesamin on TFEB translocation to initiate autophagy. EO or MHY1485 counteracted this effect. Compared with the model group, sesamin significantly upregulated the overall protein levels of TFEB (Figures 7A,B) and that in the nucleus (Figures 7C,D), with that in the cytoplasm declined. EO or MHY1485 reversed this trend, consistent with the immunofluorescence results (Figures 6C,D). Therefore, it could be speculated that sesamin promotes autophagy via regulating the translocation of TFEB.

The immunofluorescence staining of LAMP1 (Figures 7E,F) found that compared with control, the fluorescence intensity of LAMP1 in the model group was enhanced, indicating increased lysosomes to a certain extent after 9-trans-C18:1 treatment. The fluorescence intensity of LAMP1 was significantly enhanced by sesamin, which could be reversed by EO or MHY1485. This suggests that sesamin augments the count of lysosomes, thereby initiating autophagy and alleviating cellular impairment induced by 9-trans-C18:1. The Western blotting (Figures 7A,B) results exhibited upregulate the protein level of LAMP1 by sesamin, consistent with the immunofluorescence results.

BODIPY 493/503 probe can be used to characterize intracellular lipid droplets. The results (Figures 8A,B) showed that compared to control, the green fluorescence of the model group significantly increased. The fluorescence intensity was significantly diminished by sesamin. However, EO or MHY1485 could reverse the effect of sesamin. This suggests that sesamin may alleviate lipid accumulation induced by 9-trans-C18:1 through increasing mitophagy, thereby restoring partial mitochondrial function and mitigating the cellular impairment. As aforementioned, sesamin may activate the autophagy pathway regulated by TFEB via increasing the number of lysosomes, accelerating the process of autophagosome to autophagosomes to remove damaged mitochondria, thereby relieving the degree of cell damage induced by 9-trans-C18:1 and reducing lipid accumulation in L02 cells.