Sedanolide Activates KEAP1–NRF2 Pathway and Ameliorates Hydrogen Peroxide-Induced Apoptotic Cell Death

Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death probably due to preventing the decrease in the mitochondrial membrane potential and the increase in caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Kelch-like ECH-associated protein 1 (KEAP1)–NRF2 pathway.


Introduction
Oxidative stress is defined as an imbalance between oxidants and antioxidants and is associated with the development of various disorders and diseases [1].Studies have revealed that reactive oxygen species (ROS) generated endogenously or exogenously triggers oxidative stress [2].Under homeostatic conditions, ROS plays an important role in such physiological cellular processes as proliferation, differentiation, and growth through signaling [3].In contrast, excess cellular levels of ROS can cause damage to membranes and macromolecules such as proteins, nucleic acids, and lipids, which is closely related to numerous pathological conditions including cancer, inflammatory diseases, and neurodegenerative diseases [4][5][6].Thus, it is widely believed that the suppression of excessive ROS generation and the resulting oxidative stress is imperative to prevent various diseases.
One of the major antioxidant defense systems is the Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) signaling pathway [7].Under physiological conditions, NRF2 is largely bound to KEAP1 in the cytoplasm and constantly degraded through the ubiquitin-proteasome pathway [8].In the presence of electrophiles or oxidants, NRF2 is released from KEAP1 and translocated into the nucleus where it subsequently binds to antioxidant response element (ARE), thereby initiating the expression of phase II detoxifying and antioxidant enzymes such as NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and superoxide dismutase [7,9,10].These antioxidant enzymes reduce oxidative stress and consequently attenuate cellular damage.Therefore, the KEAP1-NRF2 pathway plays a pivotal role in the maintenance of cellular function and redox balance.Some botanically derived and synthetic compounds have been found to activate the KEAP1-NRF2 pathway including curcumin, sulforaphane, resveratrol, and so on [11][12][13], and they are in different phases of clinical trials for the treatment of chronic diseases such as cancer and type 2 diabetes mellitus [14].Thus, the KEAP1-NRF2 pathway is a potential therapeutic target for treating disorders and diseases driven by oxidative stress [15].
Phthalides are a class of bioactive natural products that are widely distributed in plants, fungi, lichens, and liverworts [16].Recently, these compounds have attracted much attention owing to their complicated chemical structures and various pharmacological activities including antimicrobial, anti-inflammatory, antitumor, and antidiabetic activities [17].Sedanolide (Figure 1) is a phthalide-like compound and one of the main constituents of the volatile oil of celery [18,19].It was found to have mosquitocidal, nematicidal, and antifungal activities [20].In addition, sedanolide exhibits moderate anti-inflammatory effects through the inhibition of cyclooxygenases 1 and 2, which play crucial roles in the inflammatory process [21].An in vivo study suggested that sedanolide exhibits anticarcinogenic activity in female mice [22].Most recently, Li et al. reported that sedanolide protects mice against acetaminophen-induced liver injury by activating the Nrf2 pathway [23].Thus, although this compound may have the potential to prevent disorders and diseases, the precise mechanism by which it modulates intracellular signaling pathways is unknown.
constantly degraded through the ubiquitin-proteasome pathway [8].In the presence of electrophiles or oxidants, NRF2 is released from KEAP1 and translocated into the nucleus where it subsequently binds to antioxidant response element (ARE), thereby initiating the expression of phase II detoxifying and antioxidant enzymes such as NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and superoxide dismutase [7,9,10].These antioxidant enzymes reduce oxidative stress and consequently attenuate cellular damage.Therefore, the KEAP1-NRF2 pathway plays a pivotal role in the maintenance of cellular function and redox balance.Some botanically derived and synthetic compounds have been found to activate the KEAP1-NRF2 pathway including curcumin, sulforaphane, resveratrol, and so on [11][12][13], and they are in different phases of clinical trials for the treatment of chronic diseases such as cancer and type 2 diabetes mellitus [14].Thus, the KEAP1-NRF2 pathway is a potential therapeutic target for treating disorders and diseases driven by oxidative stress [15].
Phthalides are a class of bioactive natural products that are widely distributed in plants, fungi, lichens, and liverworts [16].Recently, these compounds have attracted much attention owing to their complicated chemical structures and various pharmacological activities including antimicrobial, anti-inflammatory, antitumor, and antidiabetic activities [17].Sedanolide (Figure 1) is a phthalide-like compound and one of the main constituents of the volatile oil of celery [18,19].It was found to have mosquitocidal, nematicidal, and antifungal activities [20].In addition, sedanolide exhibits moderate anti-inflammatory effects through the inhibition of cyclooxygenases 1 and 2, which play crucial roles in the inflammatory process [21].An in vivo study suggested that sedanolide exhibits anticarcinogenic activity in female mice [22].Most recently, Li et al. reported that sedanolide protects mice against acetaminophen-induced liver injury by activating the Nrf2 pathway [23].Thus, although this compound may have the potential to prevent disorders and diseases, the precise mechanism by which it modulates intracellular signaling pathways is unknown.
In this study, we show that sedanolide is a KEAP1-NRF2 pathway activator that upregulates the expression of antioxidant genes, using a cell-based assay and RNA sequencing (RNA-seq) analysis.We also demonstrate that sedanolide confers cytoprotective effects against apoptotic cell death and prevents the decrease in the mitochondrial membrane potential (MMP) and the increase in caspase-3/7 activity induced by hydrogen peroxide (H2O2)-mediated oxidative stress.

Sedanolide Activates ARE-Dependent Transcription
To determine whether sedanolide modulates the KEAP1-NRF2 pathway, we used the human hepatoblastoma cell line HepG2 in which activation of the KEAP1-NRF2 pathway can be monitored by the ARE-dependent transactivation of the luciferase gene [24].Red-emitting beetle luciferase (SLR3) was used for monitoring ARE-dependent transcription, and green-emitting beetle luciferase (ELuc) was used as an internal control to normalize SLR3 luminescence and to monitor viability (Figure 2A).We performed In this study, we show that sedanolide is a KEAP1-NRF2 pathway activator that upregulates the expression of antioxidant genes, using a cell-based assay and RNA sequencing (RNA-seq) analysis.We also demonstrate that sedanolide confers cytoprotective effects against apoptotic cell death and prevents the decrease in the mitochondrial membrane potential (MMP) and the increase in caspase-3/7 activity induced by hydrogen peroxide (H 2 O 2 )-mediated oxidative stress.

Sedanolide Activates ARE-Dependent Transcription
To determine whether sedanolide modulates the KEAP1-NRF2 pathway, we used the human hepatoblastoma cell line HepG2 in which activation of the KEAP1-NRF2 pathway can be monitored by the ARE-dependent transactivation of the luciferase gene [24].Redemitting beetle luciferase (SLR3) was used for monitoring ARE-dependent transcription, and green-emitting beetle luciferase (ELuc) was used as an internal control to normalize SLR3 luminescence and to monitor viability (Figure 2A).We performed measurements in the sedanolide concentration range of 12.5 to 100 µM because sedanolide did not show significant cytotoxicity in luciferases-expressing HepG2 cells measured by the water-soluble tetrazolium salt (WST-1) assay (Figure 2B).Real-time bioluminescence measurement revealed that sedanolide dose-dependently activated ARE-dependent transcription (Figure 2C).The ARE-dependent transcription in 100 µM sedanolide-treated cells was approximately 8-fold higher than that in untreated cells.In addition, whereas SLR3 luminescence intensity was increased following treatment with sedanolide in a dose-dependent manner (Supplementary Figure S1A), ELuc luminescence intensity did not significantly change (Supplementary Figure S1B), indicating that sedanolide does not nonspecifically upregulate overall transcription but specifically activates ARE-dependent transcription without producing toxicity.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 3 of 1 measurements in the sedanolide concentration range of 12.5 to 100 µM because sedanolide did not show significant cytotoxicity in luciferases-expressing HepG2 cells measured by the water-soluble tetrazolium salt (WST-1) assay (Figure 2B).Real-time bioluminescence measurement revealed that sedanolide dose-dependently activated ARE-dependent tran scription (Figure 2C).The ARE-dependent transcription in 100 µM sedanolide-treated cells was approximately 8-fold higher than that in untreated cells.In addition, wherea SLR3 luminescence intensity was increased following treatment with sedanolide in a dose-dependent manner (Supplementary Figure S1A), ELuc luminescence intensity did not significantly change (Supplementary Figure S1B), indicating that sedanolide does no nonspecifically upregulate overall transcription but specifically activates ARE-dependen transcription without producing toxicity.

Sedanolide Induces Nuclear Translocation of NRF2
The transactivation of ARE-mediated genes requires the translocation of NRF2 into the nucleus [25].To determine whether the nuclear translocation of NRF2 is induced by the treatment with sedanolide, intracellular localization in sedanolide-treated cells was examined by Western blot analysis (Figure 3A,B) and immunofluorescence staining (Figure 3C).Luciferases-expressing HepG2 cells were exposed to various concentrations of sedanolide for 24 h, and nuclear and cytoplasmic NRF2 levels were determined by treatment with specific antibodies.As shown in Figure 3A, sedanolide dose-dependently increased the nuclear translocation of NRF2.To further verify this result, we examined the dynamics of NRF2 in a time-dependent manner following exposure to sedanolide.Sedanolide induced the nuclear translocation of NRF2 as early as 1 h after treatment, and the translocation reached its peak at 8 h (Figure 3B).In addition, we utilized fluorescence microscopy to further confirm the sedanolide-induced nuclear translocation of NRF2.Upon treatment with sedanolide, the fluorescence signals from NRF2 (red) and nuclei (blue) almost overlapped, demonstrating that NRF2 was translocated into the nucleus by the treatment with sedanolide (Figure 3C).Collectively, these results indicated that sedanolide triggers the nuclear translocation of NRF2.

Sedanolide Induces Nuclear Translocation of NRF2
The transactivation of ARE-mediated genes requires the translocation of NRF2 into the nucleus [25].To determine whether the nuclear translocation of NRF2 is induced by the treatment with sedanolide, intracellular localization in sedanolide-treated cells was examined by Western blot analysis (Figure 3A,B) and immunofluorescence staining (Figure 3C).Luciferases-expressing HepG2 cells were exposed to various concentrations of sedanolide for 24 h, and nuclear and cytoplasmic NRF2 levels were determined by treatment with specific antibodies.As shown in Figure 3A, sedanolide dose-dependently increased the nuclear translocation of NRF2.To further verify this result, we examined the dynamics of NRF2 in a time-dependent manner following exposure to sedanolide.Sedanolide induced the nuclear translocation of NRF2 as early as 1 h after treatment, and the translocation reached its peak at 8 h (Figure 3B).In addition, we utilized fluorescence microscopy to further confirm the sedanolide-induced nuclear translocation of NRF2.Upon treatment with sedanolide, the fluorescence signals from NRF2 (red) and nuclei (blue) almost overlapped, demonstrating that NRF2 was translocated into the nucleus by the treatment with sedanolide (Figure 3C).Collectively, these results indicated that sedanolide triggers the nuclear translocation of NRF2.(C) Immunofluorescence analysis of NRF2 proteins.The cells were treated with 100 µM sedanolide for 24 h.Then, the cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100.NRF2 was probed with a primary anti-NRF2 antibody and visualized with a goat anti-rabbit IgG Alexa Fluor 594 secondary antibody.Nuclei were stained with NucBlue.Fluorescence images were obtained by using a BZ-X710 fluorescence microscope.Insets show high-magnification images of the boxed areas.

Sedanolide Activates the NRF2 Pathway
To further investigate the biological effects of sedanolide, we analyzed the transcriptomic changes of luciferases-expressing HepG2 cells treated with sedanolide for 24 h and compared them with those of control cells.RNA-seq analysis indicated that 32 genes were upregulated and 28 genes were downregulated (fold change > 1.5) in the sedanolide-treated cells (Figure 4A).The volcano plot of differentially expressed genes (DEGs) revealed that several NRF2 downstream genes, including GCLC, GCLM, PTGR1, ABCC3, SLC5A11, GPX2, NQO1, TXNRD1, GSTA1, GSTA2, and SRXN1, were significantly upregulated by the treatment with sedanolide (Figure 4B).In addition, we performed pathway enrichment analysis of DEGs in the sedanolide-treated cells using Wikipathways database [26].As shown in Figure 4C, critical pathways involved in the antioxidant activity, such as the NRF2 pathway (WP2884) and glutathione (GSH) metabolism (WP100), were upregulated by the treatment with sedanolide, suggesting that sedanolide treatment induces antioxidant properties in the cells.
control.(C) Immunofluorescence analysis of NRF2 proteins.The cells were treated with 1 sedanolide for 24 h.Then, the cells were fixed with 4% PFA and permeabilized with 0.1% Tr 100.NRF2 was probed with a primary anti-NRF2 antibody and visualized with a goat anti IgG Alexa Fluor 594 secondary antibody.Nuclei were stained with NucBlue.Fluorescence were obtained by using a BZ-X710 fluorescence microscope.Insets show high-magnification of the boxed areas.

Sedanolide Activates the NRF2 Pathway
To further investigate the biological effects of sedanolide, we analyzed the scriptomic changes of luciferases-expressing HepG2 cells treated with sedanolide fo and compared them with those of control cells.RNA-seq analysis indicated that 32 were upregulated and 28 genes were downregulated (fold change > 1.5) in the sedan treated cells (Figure 4A).The volcano plot of differentially expressed genes (DEG vealed that several NRF2 downstream genes, including GCLC, GCLM, PTGR1, A SLC5A11, GPX2, NQO1, TXNRD1, GSTA1, GSTA2, and SRXN1, were significantly u ulated by the treatment with sedanolide (Figure 4B).In addition, we performed pa enrichment analysis of DEGs in the sedanolide-treated cells using Wikipathways da [26].As shown in Figure 4C, critical pathways involved in the antioxidant activity as the NRF2 pathway (WP2884) and glutathione (GSH) metabolism (WP100), were u ulated by the treatment with sedanolide, suggesting that sedanolide treatment in antioxidant properties in the cells.

Sedanolide Protects against H 2 O 2 -Induced Cell Death
As the above-mentioned results led us to speculate that sedanolide could confer tolerance to oxidative stress, we determined whether sedanolide exerts a cytoprotective effect against H 2 O 2 -induced cell death.Luciferases-expressing HepG2 cells were pretreated with various concentrations of sedanolide for 24 h and then exposed to H 2 O 2 for an additional 24 h.The H 2 O 2 -induced cell death was assessed in terms of cell viability and cell membrane damage by the WST-1 assay and the lactate dehydrogenase (LDH) release assay, respectively.As shown in Figure 5A, when the cells alone were exposed to H 2 O 2 , their viability significantly decreased in a dose-dependent manner.On the other hand, pretreatment of the cells with sedanolide conferred significant cytoprotective effects against cell death induced by the subsequent treatment with H 2 O 2 .The cytoprotective effects of sedanolide against H 2 O 2 -induced cell death were also assessed by the LDH release assay (Figure 5B).LDH release from cells treated with H 2 O 2 was significantly decreased in the presence of sedanolide.These results suggested that sedanolide confers significant cytoprotective effects against H 2 O 2 -induced oxidative stress.
with log2-normalized fold changes (FCs) in sedanolide-treated cells (n = 3) or control cells (n plotted against −log10 FDR.Red dots: upregulated significant DEGs in sedanolide-treated cells > 1.5, FDR < 0.05); blue dots: downregulated significant DEGs in sedanolide-treated cells (FC < FDR < 0.05).(C) Pathway enrichment analysis in sedanolide-treated cells compared with con cells, as determined by the WikiPathways analysis.Upregulated genes belonging to the corresp ing pathways are summarized in the right panel.

Sedanolide Protects against H2O2-Induced Cell Death
As the above-mentioned results led us to speculate that sedanolide could confer erance to oxidative stress, we determined whether sedanolide exerts a cytoprotectiv fect against H2O2-induced cell death.Luciferases-expressing HepG2 cells were pretrea with various concentrations of sedanolide for 24 h and then exposed to H2O2 for an a tional 24 h.The H2O2-induced cell death was assessed in terms of cell viability and membrane damage by the WST-1 assay and the lactate dehydrogenase (LDH) release say, respectively.As shown in Figure 5A, when the cells alone were exposed to H2O2, t viability significantly decreased in a dose-dependent manner.On the other hand, pretr ment of the cells with sedanolide conferred significant cytoprotective effects against death induced by the subsequent treatment with H2O2.The cytoprotective effects o danolide against H2O2-induced cell death were also assessed by the LDH release a (Figure 5B).LDH release from cells treated with H2O2 was significantly decreased in presence of sedanolide.These results suggested that sedanolide confers significant c protective effects against H2O2-induced oxidative stress.

Sedanolide Reduces Cytosolic and Mitochondrial ROS Levels Increased by H 2 O 2
Based on the above results (Figure 5A,B left panels), a concentration of 2 mM H 2 O 2 was selected for all subsequent experiments to induce H 2 O 2 -mediated oxidative stress.To investigate whether sedanolide reduces oxidative stress, luciferases-expressing HepG2 cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 to detect cytosolic or mitochondrial ROS levels.As shown in Figure 6A, whereas a bright signal of 2 7 -dichlorofluorescein (DCF), which shows enhanced fluorescence during ROS generation, was observed in the H 2 O 2 -exposed cells (left bottom panel), the signal intensity was decreased by the pretreatment with sedanolide (right bottom panel).To quantify the effect of sedanolide on the cytosolic ROS levels, the fluorescence intensities of DCF were measured by flow cytometry (Figure 6B).Consistent with the observation by fluorescence microscopy, H 2 O 2 -induced ROS production was statistically attenuated by the pretreatment with sedanolide.Furthermore, mitochondrial ROS levels were assessed by using MitoSOX Red as the fluorescence probe and analyzed by fluorescence microscopy and flow cytometry.Consistent with the results of the cytosolic ROS levels, pretreatment with sedanolide decreased the production of mitochondrial ROS, which is increased in H 2 O 2 -treated cells (Figure 6C,D).We noted that the 24 h treatment with sedanolide did not cause any appreciable variation in the basal levels of both cytosolic and mitochondrial ROS.These results suggested that the cellular antioxidant capacity is enhanced by the treatment with sedanolide, probably owing to the upregulation of the NRF2 pathway and glutathione metabolism, as shown in Figure 4.
was selected for all subsequent experiments to induce H2O2-mediated oxidative stress.To investigate whether sedanolide reduces oxidative stress, luciferases-expressing HepG2 cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 to detect cytosolic or mitochondrial ROS levels.As shown in Figure 6A, whereas a bright signal of 2′7′-dichlorofluorescein (DCF), which shows enhanced fluorescence during ROS generation, was observed in the H2O2-exposed cells (left bottom panel), the signal intensity was decreased by the pretreatment with sedanolide (right bottom panel).To quantify the effect of sedanolide on the cytosolic ROS levels, the fluorescence intensities of DCF were measured by flow cytometry (Figure 6B).Consistent with the observation by fluorescence microscopy, H2O2-induced ROS production was statistically attenuated by the pretreatment with sedanolide.Furthermore, mitochondrial ROS levels were assessed by using MitoSOX Red as the fluorescence probe and analyzed by fluorescence microscopy and flow cytometry.Consistent with the results of the cytosolic ROS levels, pretreatment with sedanolide decreased the production of mitochondrial ROS, which is increased in H2O2-treated cells (Figure 6C,D).We noted that the 24 h treatment with sedanolide did not cause any appreciable variation in the basal levels of both cytosolic and mitochondrial ROS.These results suggested that the cellular antioxidant capacity is enhanced by the treatment with sedanolide, probably owing to the upregulation of the NRF2 pathway and glutathione metabolism, as shown in Figure 4.

Sedanolide Alleviates H 2 O 2 -Induced Apoptosis
Elevated ROS levels cause extensive and irreparable damage to proteins, nucleic acids, lipids, and organelles, which can lead to activation of the cell death process including apoptosis [27].We next investigated the cytoprotective effects of sedanolide against H 2 O 2induced apoptosis.Luciferases-expressing HepG2 cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 24 h.Then, the cells were stained with fluorescein isothiocyanate (FITC) Annexin V and Ethidium Homodimer III (EthD-III) and visualized under a fluorescence microscope.As shown in Figure 7A, FITC Annexin V and EthD-III signals could barely be detected in control and sedanolide-pretreated cells.Whereas strong fluorescence signals were visible in response to H 2 O 2 treatment, these signals were decreased by the pretreatment with sedanolide.We next used flow cytometry to assess the degree of H 2 O 2 -induced apoptosis in the absence or presence of sedanolide pretreatment.When the cells alone were exposed to H 2 O 2 , the numbers of early and late apoptotic cells and necrotic cells were increased, which were significantly decreased by the pretreatment with sedanolide (Figure 7B).

Sedanolide Alleviates H2O2-Induced Apoptosis
Elevated ROS levels cause extensive and irreparable damage to proteins, nucleic acids, lipids, and organelles, which can lead to activation of the cell death process including apoptosis [27].We next investigated the cytoprotective effects of sedanolide against H2O2induced apoptosis.Luciferases-expressing HepG2 cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 24 h.Then, the cells were stained with fluorescein isothiocyanate (FITC) Annexin V and Ethidium Homodimer III (EthD-III) and visualized under a fluorescence microscope.As shown in Figure 7A, FITC Annexin V and EthD-III signals could barely be detected in control and sedanolide-pretreated cells.Whereas strong fluorescence signals were visible in response to H2O2 treatment, these signals were decreased by the pretreatment with sedanolide.We next used flow cytometry to assess the degree of H2O2-induced apoptosis in the absence or presence of sedanolide pretreatment.When the cells alone were exposed to H2O2, the numbers of early and late apoptotic cells and necrotic cells were increased, which were significantly decreased by the pretreatment with sedanolide (Figure 7B).The signaling pathways of apoptosis are complex but always rely on the enhanced permeability of mitochondrial membranes and the activation of caspases [28].To explore the precise mechanisms involved in the anti-apoptotic effect of sedanolide, we further investigated the integrity of MMP and the activity of caspase-3/7 in the H 2 O 2 -treated luciferases-expressing HepG2 cells.MMP status was evaluated using a specific fluorescence probe, tetramethylrhodamine methyl ester (TMRM), which tends to accumulate in healthy mitochondria and produces a red-orange fluorescence signal upon excitation.Fluorescence microscopy observations revealed strong fluorescence in control cells (Figure 8A, left upper panel).Whereas the fluorescence signals were decreased in the H 2 O 2 -treated cells (Figure 8A left bottom panel) owing to the depolarization of the mitochondrial membrane, these signals were retained by the pretreatment with sedanolide (Figure 8A, right bottom panel).Flow cytometric analysis revealed that the number of cells containing healthy mitochondria was decreased by the treatment with 2 mM H 2 O 2 (Figure 8B).On the other hand, the decrease in the number of healthy mitochondria-containing cells was repressed by the pretreatment with sedanolide.crotic cells were analyzed by flow cytometry.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 24 h.Then, the cells were stained with FITC Annexin V and EthD-III.Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01.n.s., not significant.
The signaling pathways of apoptosis are complex but always rely on the enhanced permeability of mitochondrial membranes and the activation of caspases [28].To explore the precise mechanisms involved in the anti-apoptotic effect of sedanolide, we further investigated the integrity of MMP and the activity of caspase-3/7 in the H2O2-treated luciferases-expressing HepG2 cells.MMP status was evaluated using a specific fluorescence probe, tetramethylrhodamine methyl ester (TMRM), which tends to accumulate in healthy mitochondria and produces a red-orange fluorescence signal upon excitation.Fluorescence microscopy observations revealed strong fluorescence in control cells (Figure 8A, left upper panel).Whereas the fluorescence signals were decreased in the H2O2-treated cells (Figure 8A left bottom panel) owing to the depolarization of the mitochondrial membrane, these signals were retained by the pretreatment with sedanolide (Figure 8A, right bottom panel).Flow cytometric analysis revealed that the number of cells containing healthy mitochondria was decreased by the treatment with 2 mM H2O2 (Figure 8B).On the other hand, the decrease in the number of healthy mitochondria-containing cells was repressed by the pretreatment with sedanolide.Caspase-3/7 activity was examined by the CellEvent Caspase-3/7 Green Detection Reagent, which is a nucleic acid-binding dye that harbors the caspase-3/7 cleavage sequence and is fluorescent after being cleaved and bound to DNA.Fluorescence microscopy images showed a significant increase in the intensity of activated caspase-3/7 signals in the H 2 O 2 -treated cells (Figure 8C, left bottom panel) but not in the sedanolide-pretreated cells even in the presence of H 2 O 2 (Figure 8C, right bottom panel).Flow cytometry was used to quantify the ratio of cells possessing activated caspase-3/7.As shown in Figure 8D, whereas treatment with H 2 O 2 significantly increased the ratio of cells containing activated caspase-3/7, pretreatment with sedanolide repressed the activation of these caspases.Taken together, these results suggested that sedanolide can protect against H 2 O 2 -induced apoptosis by improving ROS-mediated mitochondrial dysfunction and suppressing the ROS-activated caspase-3/7 signaling pathway.

Discussion
Phthalides are widely distributed in plants and fungi and exhibit a broad spectrum of biological activities, including antimicrobial, anti-inflammatory, antitumor and antidiabetic activities.However, there are few studies on the bioactivity of sedanolide.In this study, we investigated the cytoprotective effects of sedanolide against H 2 O 2 -induced oxidative stress and explored the mechanism underlying the cytoprotective effects of sedanolide.We found that sedanolide activates the KEAP1-NRF2 pathway, which is followed by the significant upregulation of antioxidant genes.Furthermore, we showed that although H 2 O 2 treatment decreased cell viability as a result of ROS generation, the pretreatment with sedanolide attenuated the H 2 O 2 -induced mitochondrial damage and apoptotic cell death by reducing cytosolic and mitochondrial ROS generation.
Initially, we revealed that the treatment with sedanolide triggered the nuclear translocation of NRF2 and then activated the ARE-dependent transcription.As sedanolide has an electrophilic α, β-unsaturated carbonyl moiety (Figure 1), it may bind covalently with the thiol group of KEAP1.In general, oxidative stress or electrophilic compounds rapidly lead to the nuclear translocation of NRF2 and the subsequent induction of cytoprotective antioxidant response [29].After redox homeostasis was achieved, NRF2 was exported from the nucleus to terminate the antioxidant response and then subjected to degradation.Consistent with these observations, we previously found that 5-hydroxy-4-phenyl-butenolide, an NRF2 activator, rapidly increased the ARE-dependent transcription and then gradually decreased it after prolonged incubation [30].In contrast, as shown in Figure 2C, real-time bioluminescence measurement revealed that the ARE-dependent transcriptional activity increased by the treatment with sedanolide lasted for at least 72 h.Although we do not know the mechanism behind this, we offer a plausible explanation in relation to the nuclear export system.It has been reported that the nuclear export and degradation of NRF2 is controlled by two independent mechanisms involving KEAP1-and Fyn-mediated systems.In the first mechanism, after the induction of NRF2-dependent genes, KEAP1 translocates into the nucleus to mediate the dissociation of NRF2 from ARE and the subsequent formation of the KEAP1-NRF2 complex.This process triggers the export of NRF2 from the nucleus, resulting in the degradation of NRF2 and the termination of NRF2 signaling [31].In the second mechanism, Fyn, a tyrosine kinase, phosphorylates NRF2 tyrosine 568, which results in the export of NRF2 into the cytosol where NRF2 is degraded by ubiquitination [32].Hence, it may be possible that sedanolide also inhibits the nuclear translocation of KEAP1 or the phosphorylation of NRF2, leading to the nuclear translocation of NRF2 and the maintenance of ARE-dependent transcriptional activity.
As shown in Figure 4, the mRNA expression levels of antioxidant enzymes belonging to the GSH system, such as GPX2, GCLC, GCLM, GSTA1, GSTA2, and MGST1, were significantly increased by the treatment with sedanolide.The GSH system, a major thioldependent antioxidant system, participates in the defense against oxidative stress through the efficient removal of various ROS by glutathione peroxidase (GPX) [33].Glutamate cysteine ligase (GCL) is the key rate-limiting enzyme in the biogenesis of GSH and is composed of glutamate cysteine ligase catalytic (encoded by GCLC) and glutamate cysteine ligase modifier (encoded by GCLM) subunits [34].Whereas Gclc-knockout mice were embryonic lethal, the conditional knockout resulted in critical GSH depletion and increased oxidative stress [35].Lim et al. have reported that the increased oxidative stress in Gclm-knockout mice is correlated with decreased capacity for GSH synthesis [36].On the other hand, GST plays an important role in oxidative stress tolerance including mediating the conjugation of GSH with a wide variety of electrophilic toxic compounds, and its overexpression confers tolerance to H 2 O 2 -induced cell death [37,38].In this regard, the upregulation of GSH system-related genes is considered to be associated with cytoprotection against oxidative stress and toxic compounds [39,40].Consistent with these observations, the pretreatment with sedanolide ameliorates H 2 O 2 -induced oxidative stress through the inhibition of cytosolic and mitochondrial ROS accumulation.Taken together, our findings suggest that sedanolide confers tolerance to both oxidative and xenobiotic stress via the upregulation of the GSH system in cells.
In addition to the upregulation of GSH system-related genes, sedanolide increased the transcript levels of NQO1, TXNRD1 (encoding thioredoxin reductase 1), SRXN1 (encoding sulfiredoxin 1), AKR1B10 (encoding aldo-keto reductase family 1 member B10), and AKR1C1 (encoding aldo-keto reductase family 1 member C1).NQO1 plays an important role in protecting cells against oxidative stress by scavenging superoxide and catalyzing the reductive metabolism of chemicals [41].TXNRD1 also plays a role in preventing oxidative stress by reducing the oxidation states of its substrates such as thioredoxin [42].SRXN1 detoxifies highly reactive peroxides, including H 2 O 2 , and assumes a crucial role in inhibiting cellular damage triggered by oxidative stress [43].Aldo-keto reductases, such as AKR1B10 and AKR1C1, are a family of enzymes that catalyze the conversion of aldehydes or ketones into their corresponding alcohols, leading to the inhibition of cytotoxic effects [44].Therefore, antioxidant enzymes regulated via the NRF2 pathway may be collectively involved in the cytoprotective effects of sedanolide.
The present study also showed that sedanolide suppressed H 2 O 2 -mediated cytosolic and mitochondrial ROS production and attenuated oxidative stress-induced apoptotic cell death.Apoptosis, a programmed cell death, is tightly regulated by a variety of factors and can be divided into extrinsic and intrinsic pathways.The former is initiated by death receptors, whereas the latter is associated with mitochondrial dysfunction [45].In the mitochondrion-mediated intrinsic apoptotic pathway, oxidative stress induces the depolarization of the mitochondrial membrane, resulting in the loss of MMP as the mitochondrial membrane pore opens.Thereafter, the mitochondria release cytochrome c, which activates the cytoplasmic caspase cascade and induces apoptotic cell death [46].Thus, the loss of MMP is indicative of mitochondrial dysfunction and is an early event in the onset of mitochondrion-mediated intrinsic apoptosis [47].To evaluate the preventive effect of sedanolide on mitochondrial dysfunction, MMP values were examined by fluorescence microscopy and flow cytometry (Figure 8A,B).We found that whereas the MMP values were significantly decreased in H 2 O 2 -treated cells, these values were restored through the pretreatment with sedanolide.
We also found that sedanolide repressed the increase in caspase-3/7 activity triggered by the treatment with H 2 O 2 .Caspases are intracellular cysteine protease enzymes that play a major role in the apoptotic mechanism, and caspase-3/7 is the final step in both extrinsic and intrinsic apoptotic pathways [48].Mouse embryonic fibroblasts lacking caspase-3/7 were resistant to both death receptor-initiated apoptosis and mitochondrion-mediated apoptosis [49].In addition, it has been reported that caspase-3/7 is required for the induction of efficient apoptosis by the activating feedback amplification of upstream apoptotic signals [50].Therefore, our findings suggest that the anti-apoptotic effects of sedanolide in H 2 O 2 -treated cells may be partially explained by the reduction in caspase-3/7 activity.However, although pretreatment with sedanolide completely abolished caspase-3/7 activation in H 2 O 2 -treated cells (Figure 8C,D), that only partially inhibited H 2 O 2 -induced apoptosis (Figure 7).These results suggest that sedanolide is ineffective in protecting cells from caspase-independent apoptosis such as apoptosis-inducing factor and endonuclease G-mediated pathways.
In conclusion, our results revealed that sedanolide exerts cytoprotective effects against H 2 O 2 -induced cell death.In addition, sedanolide inhibits excessive ROS generation and mitochondrial dysfunction probably owing to its ability to upregulate antioxidant enzymes belonging to the NRF2 pathway and glutathione metabolism.A recent study has suggested that sedanolide activates the NRF2 pathway [23].Our study describes for the first time the role of the cytoprotective effects of sedanolide in ameliorating H 2 O 2 -induced cell death.Taken together, the results suggest a potential mechanism underlying the cytoprotective effect of sedanolide.

Cell Culture and Treatment
Luciferases-expressing HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Fujifilm Wako Pure Chemical Co., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 1% Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA; Gibco, Grand Island, NY, USA), and 1 mM sodium pyruvate (Gibco) at 37 • C in a humidified atmosphere with 5% CO 2 .For experiments, cells were seeded in differently sized multi-well plates at a density of 1 × 10 5 cells/cm 2 and incubated for 24 h.Subsequently, the cells were pretreated with sedanolide (LKT laboratories, St. Paul, MN, USA) for 24 h and then exposed to H 2 O 2 (Fujifilm Wako Pure Chemical Co.) for an additional 24 h unless otherwise noted.Sedanolide was dissolved in dimethyl sulfoxide (DMSO) at 100 mM as a stock solution and stored at −30 • C until used.The final concentration of DMSO did not exceed 0.1% throughout the study.

Cytotoxicity Assay
Cell viability was determined using a Premix WST-1 Cell Proliferation Assay System (Takara Bio, Shiga, Japan) according to the manufacturer's instructions.Cell membrane damage was assessed using a Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's instructions.

Western Blot Analysis
Luciferases-expressing HepG2 cells were seeded in 6-well plates at 9 × 10 5 cells/well.After incubation for one day, the cells were treated with various concentrations of sedanolide.Cytoplasmic and nuclear fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.Protein levels were analyzed using the capillary electrophoresis western system Wes (ProteinSimple Co., San Jose, CA, USA) following the manufacturer's instructions.In brief, the protein samples were mixed with the 5× Fluorescent Master Mix (ProteinSimple Co.) and then heated at 95 • C for 5 min.Next, the heated samples, biotinylated protein ladder, primary antibodies, horseradish peroxide-conjugated anti-rabbit secondary antibody, and luminol-peroxide were loaded into the 12-230 kDa Wes Separation Module (ProteinSimple Co.).Primary antibodies Nrf2 (#NBP1-32822; Novus Biologicals, Centennial, CO, USA), lamin B1 (#12586; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (#2118; Cell Signaling Technology) were diluted to 1:50, 1:50, and 1:100, respectively.Protein levels were analyzed using Compass software (version 5.0.1) for Simple Western (ProteinSimple Co.).

Immunofluorescence Analysis
Luciferases-expressing HepG2 cells were seeded on coverslips placed in 24-well plates at 1.8 × 10 5 cells/well.After incubation for one day, the cells were treated with 100 µM sedanolide for 24 h.The cells were washed with PBS and fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Co.) for 15 min.Then, the cells were washed three times and permeabilized with 0.1% Triton X-100 for 10 min.After permeabilization, the cells were washed three times with PBS and blocked with 1% BSA for 1 h.After that, the cells were incubated with anti-Nrf2 antibody (1:200 dilution).The cells were washed three times with PBS and incubated for 1 h with goat anti-rabbit IgG Alexa 594 (1:1000 dilution; Thermo Fisher Scientific, Inc.).After washing with PBS, the cells were mounted with ProLong Glass Antifade Mountant with NucBlue Stain (Thermo Fisher Scientific, Inc.).Fluorescence images were obtained on a BZ-X710 fluorescence microscope and BZ-X Viewer (version 1.3.0.5) (Keyence, Osaka, Japan).

RNA Sequencing and Data Analysis
Luciferases-expressing HepG2 cells were seeded in 6-well plates at 9 × 10 5 cells/well.After incubation for one day, the cells were treated with 100 µM sedanolide for 24 h.Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.RNA-seq analysis was outsourced to Genome-Lead Co. Ltd. (Takamatsu, Japan).In brief, mRNA was purified from total RNA samples using a KAPA mRNA Capture Kit (KAPA Biosystems, Potters Bar, UK).Next, a cDNA library was prepared using an MGIEasy RNA Directional Library Prep Set (MGI Tech, Shenzhen, China).The libraries were sequenced on a DNBSEQ-T7RS platform (MGI Tech) using a DNBSeq-T7RS High-Throughput Sequencing Set (MGI Tech), and 150 bp paired-end reads were generated.The quality of the sequence data was evaluated using FastQC.Then, Trimmomatic was used to trim adaptor sequences and remove low-quality reads.The sequences were aligned to the GRCh38 human reference genome using the alignment tool STAR.Reads in transcripts per million (TPM) formats were computed using RNA-seq by Expectation Maximization (RSEM).To identify DEGs and altered pathways, read counts were uploaded and analyzed using the integrated Differential Expression and Pathway (iDEP.96)web-based applications [56].The DEGs were identified using the DESeq2 method with a false discovery rate (FDR) cutoff of 0.05 and a minimum fold change of 1.5.The analysis of enriched pathways in DEGs was conducted using the Wikipathways database [26] with the results being compiled in the form of histograms.Data are deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE245428.

Measurement of Cytosolic and Mitochondrial ROS Level
Luciferases-expressing HepG2 cells were seeded in 12-well plates at 3.6 × 10 5 cells/well.After incubation for one day, the cells were pretreated with sedanolide for 24 h and then exposed to H 2 O 2 for an additional 4 h. 2 ,7 -Dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich), which permeates cells and is oxidized by ROS into fluorescent DCF, was used to assess cytosolic ROS levels.After the treatment with H 2 O 2 , the cells were loaded with 20 µM DCFH-DA for 30 min at 37 • C in a humidified atmosphere with 5% CO 2 .Then, the cells were collected and analyzed using a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).For the visualization of DCF fluorescence, the cells were observed under a BZ-X710 all-in-one fluorescence microscope and BZ-X Viewer (version 1.3.0.5).
Luciferases-expressing HepG2 cells were seeded in 12-well plates at 3.6 × 10 5 cells/well.After incubation for one day, the cells were pretreated with sedanolide for 24 h and then exposed to H 2 O 2 for an additional 24 h.Fluorescent probe MitoSOX Red (Thermo Fisher Scientific, Inc.) was used to measure mitochondrial ROS production.After the treatment with H 2 O 2 , the cells were stained with 5 µM MitoSOX Red for 30 min at 37 • C in a humidified atmosphere with 5% CO 2 .Then, the cells were collected and analyzed using a FACSCalibur flow cytometer.For the visualization of MitoSOX Red, the cells were observed under a BZ-X710 all-in-one fluorescence microscope and BZ-X Viewer (version 1.3.0.5).

Apoptosis Assay
Luciferases-expressing HepG2 cells were seeded in 12-well plates at 3.6 × 10 5 cells/well.After incubation for one day, the cells were pretreated with sedanolide for 24 h and then exposed to H 2 O 2 for an additional 24 h.Cell death induced by H 2 O 2 was assessed using an Apoptotic/Necrotic Cells Detection Kit (Promokine, Heidelberg, Germany) according to the manufacturer's instructions.In brief, the cells were stained with FITC Annexin V and EthD-III for 15 min in the dark at room temperature.Cellular status was analyzed using a FACSCalibur flow cytometer.Data were collected from at least 10,000 gated events.Cellular status was determined as follows: early apoptosis (Annexin V positive/EthD-III negative), late apoptosis (Annexin V positive/EthD-III positive), necrosis (Annexin V negative/EthD-III positive), and healthy cells (Annexin V negative/EthD-III negative).For the visualization of FITC Annexin V and EthD-III fluorescence, the cells were observed under a BZ-X710 all-in-one fluorescence microscope and BZ-X Viewer (version 1.3.0.5).

Measurement of Mitochondrial Membrane Potential Changes
Luciferases-expressing HepG2 cells were seeded in 12-well plates at 3.6 × 10 5 cells/well.After incubation for one day, the cells were pretreated with sedanolide for 24 h and then exposed to H 2 O 2 for an additional 24 h.TMRM (Thermo Fisher Scientific, Inc.), a cationic fluorescent probe taken up by mitochondria in a membrane potential-dependent manner, was used to assess MMP.After the treatment with H 2 O 2 , the cells were loaded with 100 nM TMRM for 30 min at 37 • C in a humidified atmosphere with 5% CO 2 .Then, the cells were collected and analyzed using a FACSCalibur flow cytometer.For the visualization of TMRM, the cells were observed under a BZ-X710 all-in-one fluorescence microscope and BZ-X Viewer (version 1.3.0.5).

Quantification of Caspase-3/7 Activity
Luciferases-expressing HepG2 cells were seeded in 12-well plates at 3.6 × 10 5 cells/well.After incubation for one day, the cells were pretreated with sedanolide for 24 h and then exposed to H 2 O 2 for an additional 24 h.The percentage of cells containing active caspase-3/7 was determined using CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.After the treatment with H 2 O 2 , the cells were loaded with 5 µM reagent for 30 min at 37 • C in a humidified atmosphere with 5% CO 2 .Then, the cells were collected and analyzed using a FACSCalibur flow cytometer.For the visualization of fluorescence signals, the cells were observed under a BZ-X710 all-in-one fluorescence microscope and BZ-X Viewer (version 1.3.0.5).

Statistical Analysis
All assays were conducted in triplicate at least.Data are expressed as means ± standard deviations (SDs).Comparisons were performed by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey's multiple comparison test.GraphPad Prism 8.4.3 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis, and a p value less than 0.05 was considered statistically significant.

Figure 2 .
Figure 2. Effects of sedanolide on viability and ARE-driven bioluminescence of luciferases-express ing HepG2 cells.(A) Schematic drawing of reporter plasmid.HS4, HS4 insulator; TK, TK promoter ARE, antioxidant response element; pA, polyA signal.(B) Effects of sedanolide on cell viability measured by WST-1 assay.The results are expressed as percent of untreated controls (dotted hori zontal line).Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple compar ison test.* p < 0.05, ** p < 0.01, compared with untreated control.(C) Real-time bioluminescenc recordings of sedanolide-treated luciferases-expressing HepG2 cells.The cells were seeded in a 96 well white clear-bottom plate and treated with sedanolide at various concentrations.Biolumines cence was recorded in real time for 5 s at 30-minute intervals for 72 h.The time-dependent chang in ARE activation is expressed as a fold change where the relative bioluminescence intensity (SLR3/ELuc) of sedanolide-treated cells was normalized to that of untreated control cells at each time point.Dose dependencies are summarized in the area under the curves (AUCs) at the rightmos panel.Values are means ± SD, n = 3.

Figure 2 .
Figure 2. Effects of sedanolide on viability and ARE-driven bioluminescence of luciferases-expressing HepG2 cells.(A) Schematic drawing of reporter plasmid.HS4, HS4 insulator; TK, TK promoter; ARE, antioxidant response element; pA, polyA signal.(B) Effects of sedanolide on cell viability measured by WST-1 assay.The results are expressed as percent of untreated controls (dotted horizontal line).Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01, compared with untreated control.(C) Real-time bioluminescence recordings of sedanolidetreated luciferases-expressing HepG2 cells.The cells were seeded in a 96-well white clear-bottom plate and treated with sedanolide at various concentrations.Bioluminescence was recorded in real time for 5 s at 30-min intervals for 72 h.The time-dependent change in ARE activation is expressed as a fold change where the relative bioluminescence intensity (SLR3/ELuc) of sedanolide-treated cells was normalized to that of untreated control cells at each time point.Dose dependencies are summarized in the area under the curves (AUCs) at the rightmost panel.Values are means ± SD, n = 3.

Figure 3 .Figure 3 .
Figure 3. Effects of sedanolide on nuclear localization of NRF2 protein.(A) Western blot analysis of cytoplasmic and nuclear NRF2 protein in luciferases-expressing HepG2 cells treated with different concentrations of sedanolide for 24 h.(B) Western blot analysis of cytoplasmic and nuclear proteins in cells treated with 100 µM sedanolide for the indicated times.GAPDH and lamin B1 were used as loading control for cytoplasmic and nuclear proteins, respectively.Histogram plots (right panels) show the levels of NRF2 in the nuclei normalized to lamin B1.Values are means ± SD, n = 3, oneway ANOVA followed by Tukey's multiple comparison test.** p < 0.01, compared with untreated Figure 3. Effects of sedanolide on nuclear localization of NRF2 protein.(A) Western blot analysis of cytoplasmic and nuclear NRF2 protein in luciferases-expressing HepG2 cells treated with different concentrations of sedanolide for 24 h.(B) Western blot analysis of cytoplasmic and nuclear proteins in cells treated with 100 µM sedanolide for the indicated times.GAPDH and lamin B1 were used as loading control for cytoplasmic and nuclear proteins, respectively.Histogram plots (right panels) show the levels of NRF2 in the nuclei normalized to lamin B1.Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.** p < 0.01, compared with untreated control.(C)Immunofluorescence analysis of NRF2 proteins.The cells were treated with 100 µM sedanolide for 24 h.Then, the cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100.NRF2 was probed with a primary anti-NRF2 antibody and visualized with a goat anti-rabbit IgG Alexa Fluor 594 secondary antibody.Nuclei were stained with NucBlue.Fluorescence images were obtained by using a BZ-X710 fluorescence microscope.Insets show high-magnification images of the boxed areas.

Figure 4 .
Figure 4. Identification of significant DEGs induced by sedanolide in luciferases-expressing cells.(A) The number of significant DEGs.Red: upregulation, blue: downregulation.(B) V plot of RNA-seq results of sedanolide-treated cells versus control cells.Differential gene exp

Figure 5 .
Figure 5. Protective effect of sedanolide on H2O2-induced cell death in luciferases-expressing He cells.(A) WST-1 assay of H2O2-induced cytotoxicity in cells pretreated or not pretreated with danolide.(B) LDH release assay of H2O2-induced cell membrane damage in cells pretreated o pretreated with sedanolide.The cells were pretreated with sedanolide at various concentration 24 h and then exposed to H2O2 for an additional 24 h.Values are means ± SD, n = 3, two-way ANO followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01, compared with cells expose H2O2 at the corresponding concentrations without sedanolide pretreatment.

Figure 5 .
Figure 5. Protective effect of sedanolide on H 2 O 2 -induced cell death in luciferases-expressing HepG2 cells.(A) WST-1 assay of H 2 O 2 -induced cytotoxicity in cells pretreated or not pretreated with sedanolide.(B) LDH release assay of H 2 O 2 -induced cell membrane damage in cells pretreated or not pretreated with sedanolide.The cells were pretreated with sedanolide at various concentrations for 24 h and then exposed to H 2 O 2 for an additional 24 h.Values are means ± SD, n = 3, two-way ANOVA followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01, compared with cells exposed to H 2 O 2 at the corresponding concentrations without sedanolide pretreatment.

Figure 6 .
Figure 6.Effects of sedanolide on cytosolic and mitochondrial ROS production in luciferases-expressing HepG2 cells.(A,B) Effect of sedanolide on cytosolic ROS production in H2O2-treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 4 h.Then, the cells were stained with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and visualized under a fluorescence microscope (A) or analyzed by flow cytometry (B).(C,D) Effect of sedanolide on mitochondrial ROS production in H2O2-treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 24 h.Then, the cells were

Figure 6 .
Figure 6.Effects of sedanolide on cytosolic and mitochondrial ROS production in luciferasesexpressing HepG2 cells.(A,B) Effect of sedanolide on cytosolic ROS production in H 2 O 2 -treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 4 h.Then, the cells were stained with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and visualized under a fluorescence microscope (A) or analyzed by flow cytometry (B).(C,D) Effect of sedanolide on mitochondrial ROS production in H 2 O 2 -treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 24 h.Then, the cells were stained with MitoSOX Red and visualized under a fluorescence microscope (A) or analyzed by flow cytometry (B).Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01.n.s., not significant.

Figure 7 .
Figure 7. Inhibitory effect of sedanolide on H 2 O 2 -induced apoptosis in luciferases-expressing HepG2 cells.(A) Representative images of apoptotic and necrotic cell death by fluorescence microscopy using FITC Annexin V and EthD-III.(B) The percentages of early apoptotic, late apoptotic, and necrotic cells were analyzed by flow cytometry.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 24 h.Then, the cells were stained with FITC Annexin V and EthD-III.Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.* p < 0.05, ** p < 0.01.n.s., not significant.

Figure 8 .
Figure 8.Effect of sedanolide on MMP and caspase-3/7 activity in luciferases-expressing HepG2 cells.(A,B) Effect of sedanolide on MMP in H2O2-treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 24 h.Then, the cells were stained with TMRM and visualized under a fluorescence microscope (A) or analyzed by flow cytometry (B).(C,D)Effect of sedanolide on caspase-3/7 activity in H2O2-treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H2O2 for an additional 24 h.Then, the cells were

Figure 8 .
Figure 8.Effect of sedanolide on MMP and caspase-3/7 activity in luciferases-expressing HepG2 cells.(A,B) Effect of sedanolide on MMP in H 2 O 2 -treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 24 h.Then, the cells were stained with TMRM and visualized under a fluorescence microscope (A) or analyzed by flow cytometry (B).(C,D) Effect of sedanolide on caspase-3/7 activity in H 2 O 2 -treated cells.The cells were pretreated with sedanolide for 24 h and then exposed to 2 mM H 2 O 2 for an additional 24 h.Then, the cells were stained with CellEvent Caspase-3/7 Green Detection Reagent and visualized under a fluorescence microscope (C) or analyzed by flow cytometry (D).Values are means ± SD, n = 3, one-way ANOVA followed by Tukey's multiple comparison test.** p < 0.01.n.s., not significant.