2024 Volume 47 Issue 11 Pages 1953-1960
Cellular aging causes declining cell functionality, gradually disrupting cellular homeostasis. Mitochondria are crucial in numerous metabolic processes, including the electron transport chain and fatty acid β-oxidation. Mitochondrial dysfunction is closely linked to aging-related liver dysfunction because it impairs fatty acid metabolism, potentially leading to nonalcoholic fatty liver disease. We demonstrated that neferine-induced autophagy suppressed the aging phenotype in proliferative and replicative aging-induced cells and aging liver tissue by reactivating mitochondrial function. Pharmacological analyses revealed that neferine-induced autophagy via the death-associated protein kinase 1 (DAPK1) and c-Jun N-terminal kinase (JNK) signaling pathways despite the lack of AMP activated protein kinase (AMPK) signaling activation. Furthermore, neferine stimulated ATP production and β-oxidation activity in aging cells. Our in vivo experiments demonstrated that oral administration of neferine rejuvenated aging liver tissue, suppressed fatty acid accumulation in the liver, and reduced senescence-associated β-galactosidase activity. Thus, neferine rejuvenated aging cells and liver tissue by inducing autophagy to reactivate mitochondrial function.
Older adults are particularly vulnerable to acute liver injuries, hepatic viral infections, and nonalcoholic fatty liver disease (NAFLD), with the latter characterized by excessive fat accumulation in hepatocytes.1) Furthermore, diminished liver regeneration capability and immune system dysfunction predisposes older adults to hepatic fibrosis.2) Liver dysfunction is increasingly acknowledged as a leading cause of liver disease worldwide, particularly in Western countries.2,3) Its progression in the aging population is due to the synergistic impact of factors such as insulin resistance, lipid dysregulation, inflammation, and oxidative stress, and a recent study has highlighted the significant role of mitochondrial dysfunction in this context.4)
Although mitochondrial dysfunction is involved in the progression of liver dysfunction across various stages, the underlying mechanisms of aging-related excessive fat accumulation in hepatocytes remain unelucidated. Such dysfunction is associated with decreased mitochondrial fatty acid β-oxidation (FAO), leading to triglyceride accumulation within hepatocytes.5) Therefore, restoring aging-related mitochondrial homeostasis and protein expression patterns may potentially ameliorate liver dysfunction. However, comprehensive investigations on this topic are limited, and the effectiveness of mitochondrial activation in preventing aging-related accumulation of fat in liver tissue is unclear.
We previously reported that an important factor in aging-related mitochondrial dysfunction was the disruption of proteostasis caused by the accumulation of insoluble intracellular aggregates.6) Furthermore, we suggested that autophagic degradation of these insoluble aggregates reactivated mitochondria in aging cells, and that lotus germ extract activated mitochondria and exerted antiaging effects by inducing autophagy. Lotus germ extract contains neferine, which is crucial to these processes.6) Neferine is a bisbenzylisoquinoline alkaloid extracted from the seed embryos of Nelumbo nucifera, and it exhibits anti-inflammatory, antioxidant, and anti-cancer effects.7) Recent studies have indicated that neferine can induce autophagy, a cellular process that degrades and recycles cellular components, by modulating key signaling pathways. Specifically, neferine activates the c-Jun N-terminal kinase (JNK) pathway, leading to the initiation of autophagy.8)
Here, we used a pharmacological approach to investigate whether neferine, a compound known to activate mitochondria by inducing autophagy, mitigates aging, attenuates aging-related functional decline, and alleviates structural damage in liver tissue.
In this study, we used aging-induced proliferative and replicative NB1RGB cells (human diploid fibroblasts) to investigate the antiaging effects of neferine and its underlying mechanisms. Although the viability of aging NB1RGB cells slightly decreased following treatment with 5 and 10 µM neferine, treatment with 1 µM neferine was not cytotoxic (Fig. 1A). Therefore, subsequent experiments were performed using 1 µM neferine to further explore its potential antiaging mechanisms.
(A) Aging cells were treated with the indicated neferine concentration for 24 h. Then, cell viability was determined using an MTT assay. (B) Aging NB1RGB cells treated with 1 µM neferine for 3 d were positive for SA-β-gal staining (scale bar, 50 µm). (C) Aging NB1RGB cells treated with 1 µM neferine for the indicated times underwent immunoblotting using the indicated antibodies. Neferine phosphorylated JNK in the early stages of incubation. (D–F) Neferine-induced autophagy was crucial for rejuvenating aging cells. Aging NB1RGB cells were transfected with siControl or siATG7 for 24 h and treated with either DMSO or 1 µM neferine for 24 h. For immunoblotting, the treated cells were incubated with the indicated primary antibodies (D). The SA-β-gal activity of the treated cells was also measured (E, F). Data are presented as the mean ± standard deviation (S.D.) of three simultaneously performed experiments. p-Values were calculated using ANOVA followed by post hoc analysis with Tukey HSD test (A) or two-way ANOVA followed by post hoc analysis with Tukey HSD test (F). * p < 0.05 and ** p < 0.01.
We investigated whether neferine impacted senescence-associated β-galactosidase (SA-β-gal), a marker of cellular aging, in aging fibroblasts. The number of SA-β-gal-positive NB1RGB cells decreased following neferine treatment (Fig. 1B), suggesting that neferine rejuvenated aging cells. Because no definitive conclusion was reached regarding whether neferine-induced autophagy directly contributed to its antiaging effects, we continued our investigations in this direction. The AMP activated protein kinase (AMPK), JNK, and death-associated protein kinase 1 (DAPK1)–Beclin1 pathways are well-established pathways of autophagy induction.9) Thus, we used immunoblotting analysis to investigate the involvement of neferine in activating these pathways. We found that neferine treatment increased LC3-II expression (a marker of autophagy induction) and the levels of phosphorylated JNK and DAPK1 but did not induce AMPK phosphorylation (Fig. 1C). These results suggested that neferine-induced autophagy via JNK, and DAPK1 pathway activation.
Next, we investigated whether neferine-dependent autophagy induction was related to the suppression of the aging phenotype. Aging NB1RGB cells were treated with or without autophagy-related protein 7 (ATG7) small interfering RNA (siRNA) and/or neferine, and LC3-II levels were monitored. Neferine treatment increased LC3-II protein expression, and ATG7 knockdown by siRNA reduced the neferine-induced increase in LC3-II protein expression (Fig. 1D). Notably, ATG7 knockdown significantly suppressed the decrease in the number of SA-β-gal-positive cells induced by neferine treatment in aging fibroblasts (Figs. 1E, 1F). These results indicated that autophagy induction was key to the antiaging effects of neferine.
Neferine Activates Mitochondrial Function and Increases FAO Activity via Autophagy Induction in Aging CellsMitochondrial function plays a critical role in energy production and lipid metabolism and declines with age.5) Our experimental results suggested the restoration of mitochondrial function by neferine-induced autophagy. Mitochondrial transmembrane potential (ΔΨm) is an indicator of mitochondrial function. We analyzed the effect of aging on ΔΨm in NB1RGB cells using the fluorescent probe JC-1. Potential-dependent accumulation of JC-1 in healthy mitochondria forms aggregates that exhibit a red fluorescence. If the mitochondrial membrane is depolarized, the JC-1 monomers remain in the cytosol and exhibit a green fluorescence. Neferine treatment increased the ΔΨm in aging fibroblasts, as demonstrated by fluorescence microscopy and microplate reader analysis (Figs. 2A, 2B). Because ATP production is related to mitochondrial function, we used a luciferase assay to evaluate the effect of neferine on ATP production, revealing increased ATP levels in neferine-treated aging fibroblasts (Fig. 2C).
(A, B) Neferine increased ΔΨm. The cells were incubated with JC-1 for 30 min and assessed using (A) fluorescence microscopy (red, JC-1 aggregates; green, JC-1 monomers) or (B) a microplate reader. ΔΨm was determined as the ratio between the red fluorescence intensity (activated mitochondria) and the green fluorescence intensity (depolarized mitochondria). (C) ATP levels were determined using a luminescence-based assay. (D) FAOBlue assay showed that neferine stimulated FAO activity. (E) Aging NB1RGB cells were transfected with siControl or siATG7 for 24 h and then treated with DMSO or 1 µM neferine for 24 h before measuring FAO activity. (F) Aging NB1RGB cells were treated with DMSO (n = 3) or 1 µM neferine (n = 3) for 24 h and NCoR1 expression was detected by immunoblotting. (G) Aging NB1RGB cells were treated with 1 µM of neferine for 24 h; their RNA was then subjected to qPCR. Data are presented as the mean ± S.D. of three simultaneously performed experiments. p-Values were calculated using Student t-test (B–D, G) or two-way ANOVA followed by post hoc analysis using Tukey HSD test (E). * p < 0.05 and ** p < 0.01.
Mitochondrial FAO is a critical metabolic process in which fatty acids are degraded to produce energy. Aging and mitochondrial FAO are strongly associated, and aging-related impaired FAO function leads to incomplete FAO, thereby exacerbating mitochondrial damage.10) Thus, FAO abnormalities are considered to accelerate cellular aging and increase the risk of tissue aging and associated diseases.11) We used FAOBlue dye to investigate the FAO capacity in aging fibroblasts, which was reflected by the fluorescence intensity of the FAOBlue dye and measured using a fluorescence plate reader. Neferine treatment upregulated FAO activity in aging fibroblasts (Fig. 2D). To investigate whether the neferine-induced increase in FAO activity was associated with autophagy induction, we suppressed ATG7 expression using ATG7 siRNA. Neferine treatment of aging fibroblasts with ATG7 siRNA under the same conditions resulted in increased FAO activity, but the inhibition of autophagy induction by suppressing ATG7 expression attenuated the increase in neferine-induced FAO activity (Fig. 2E). These results suggested that neferine activated mitochondrial function and increased FAO in aging cells.
It has been shown previously that autophagy causes degradation of NCoR1, the nuclear receptor corepressor of peroxisome proliferator-activated receptor α (PPARα), thereby activating PPARα and enhancing fatty acid β-oxidation.12) Therefore, we investigated whether neferine treatment alters NCoR1 expression in aging fibroblasts. As shown in Fig. 2F, neferin treatment decreased the expression level of NcoR1, which was expected to activate PPARα-related signaling pathways. mRNA expression levels of factors related to β-oxidation were examined by real-time quantitative PCR (qRT-PCR), and the expression levels of CPT1B and CPT2 increased (Fig. 2G). The increased expression of CPT1B and CPT2 suggested that these factors are involved in the stimulation of neferin-induced β-oxidation. These results indicated that neferine stimulates β-oxidation activity via autophagy induced degradation of NCoR1.
Neferine Suppresses Liver Injury and Fatty Acid Accumulation Caused by Aging-Related Metabolic AbnormalitiesWe investigated age-related changes in the liver and kidneys of mice. Previous studies have reported that fatty liver results from lipid accumulation in the liver and that age-related damage occurs in glomeruli and other renal structures.13) Histological analysis of liver and kidney tissues using hematoxylin–eosin (H&E) staining revealed that aged mice exhibited intracellular structures resembling lipid droplets in the liver, compared to young mice. Additionally, damage to renal structures, including glomeruli, was observed in the kidneys of aged mice (Fig. 3A). We compared hepatic NCoR1 expression levels between young and aged mice using immunohistochemistry. Our results indicate that NCoR1 expression is elevated in the livers of aged mice compared with young mice (Fig. 3B). Furthermore, serum triglyceride concentrations were measured and found to be significantly increased in the aged mice (Fig. 3C). These results suggest that fat accumulation in the liver and renal tissue injury are induced in aged mice.
(A–C) Mice aged 7 weeks (young) and 92 weeks (aged) were subjected to H&E staining (A), immunohistochemical detection of NCoR1 expression (B), or measurement of serum triglyceride levels (C) (scale bar, 100 µm). (D–H) Mice aged 50 weeks were administered DMSO (n = 4) or 1 mg/kg of neferine (n = 5) orally once every 2 d for 92 d. Body weight (D), liver weight (E), serum triglyceride levels (F), and serum ALT activity (G) were measured. (H) Liver tissue sections of neferine- or DMSO-treated aging mice underwent H&E staining (scale bar, 100 µm). Histological examination revealed that neferine inhibited lipid accumulation in liver tissue. Data are presented as the mean ± S.D. (C–G). p-Values were calculated using Welch’s t-test (C–G). * p < 0.05 and ** p < 0.01.
Using our cellular aging model, we found that neferine improved the aging phenotype by inducing autophagy and restoring mitochondrial function, particularly FAO. On the basis of these findings, we hypothesized that neferine attenuated aging-related liver damage. To test our hypothesis, aging mice (50 weeks old) were orally administered 1 mg/kg of neferine every other day for 92 d to determine whether this treatment ameliorated aging-dependent liver damage. Although there were no significant changes in body weight (Fig. 3D), liver weight decreased during the treatment period (Fig. 3E). Histological analysis of liver and kidney tissues using H&E staining revealed the presence of liver intracellular structures resembling lipid droplets and damage to renal structures, including glomeruli in the dimethyl sulfoxide (DMSO)-treated (control) mice. In contrast, the neferine-treated mice showed an almost complete lack of these structures, similar to young mice. (Fig. 3F). Moreover, neferine treatment significantly reduced serum triglyceride levels and ALT activity (Figs. 3G, 3H), suggesting that neferine inhibited liver injury and the accumulation of neutral fats caused by aging-related metabolic abnormalities.
Neferine Ameliorates the Aging Liver StateWe used immunoblotting analysis to investigate autophagy induction in liver tissue following neferine administration. The results showed that neferine treatment induced DAPK1 expression, which we had already confirmed at the cellular level, and increased the LC3-II/LC3-I ratio based on band intensity. These findings suggested that neferine-induced autophagy in liver tissue (Figs. 4A, 4B). To evaluate the effects of neferine on liver tissue aging, sections were stained for SA-β-gal activity. The liver sections of mice that were administered neferine showed reduced SA-β-gal activity compared with mice that were administered DMSO, indicating suppression of the liver aging phenotype by neferine (Figs. 4C, 4D). Finally, we examined the expression level of NCoR1 in liver tissues, and as shown in Fig. 4E, the nuclear expression of NCoR1 was observed in the liver of aged mice, and the nuclear expression of NCoR1 was decreased in the aged mice treated with neferine. Thus, we could confirm not only the serum TG concentration and HE-stained liver image, but also the decreased expression of NCoR1, which is important for regulation of fatty acid oxidative activity. These findings suggested that neferine ameliorated the aging state and suppressed liver injury in naturally aging mice by autophagy induction. Therefore, neferine is a promising candidate for the improvement of aging-related deterioration of liver function.
(A–E) Mice aged 50 weeks were orally administered DMSO (n = 4) or 1 mg/kg of neferine (n = 5) once every 2 d for 92 d. (A) Neferine-induced autophagy in liver tissue. Sections from each liver underwent immunoblotting. The intensity of the LC3-II/LC3-I bands was determined (the levels of LC3-II are reported relative to those of LC3-I) (B). (C) Neferine suppressed SA-β-gal expression in liver tissue. The indicated liver fragments were stained using an SA-β-gal assay kit. The blue intensity was measured using ImageJ (D). (E) Liver tissue sections from neferin- or DMSO-treated aging mice were subjected to immunohistochemistry to detect NCoR1 expression levels (scale bar, 100 µm). Data are presented as the mean ± S.D. (B, D). p-Values were calculated using Welch’s t-test (B, D). * p < 0.05.
We found that neferine suppressed fatty acid accumulation in liver tissue and recovered liver damage in an aging-related liver dysfunction mouse model. Furthermore, neferine administration reduced serum triglyceride levels and serum ALT activity and decreased liver weight, demonstrating therapeutic benefits against aging-induced liver dysfunction. Lastly, neferine treatment reactivated FAO via autophagy induced NCoR1 degradation in aging cells and did not inhibit cell growth.
Neferine has numerous pharmacological properties, including antitumor, antidepressant-like, and antiarrhythmic effects.14) This study is the first to highlight the potential of neferine for treating aging-related liver damage. Although there are several reports, including our study, of neferine-induced autophagy, we demonstrated that autophagy induction was a crucial factor in the antiaging effects of neferine and the restoration of mitochondrial FAO. Abnormalities in autophagy lead to mitochondrial dysfunction and a consequent decrease in FAO capacity.15) Because we found that mitochondrial dysfunction was related to aging, it is plausible that neferine improves mitochondrial function and increases FAO capacity by inducing autophagy. In particular, neferine decreases the expression of NCoR1, which is important for increasing FAO capacity, suggesting that neferine promotes β-oxidation by activating the NCoR1-PPARα pathway.
Aging induces several physiological changes in liver tissue, such as the declining regenerative capacity of hepatocytes due to cellular senescence, chronic low-level inflammation, and lipid accumulation. The characteristic lipid accumulation in NAFLD can progress to hepatitis, cirrhosis, and even hepatocellular carcinoma. Thus, inhibition of lipid accumulation in hepatocytes is crucial for maintaining hepatic homeostasis.16,17) Because the mitochondrial β-oxidation pathway is primarily involved in lipid metabolism in cells, aging-related mitochondrial dysfunction contributes to lipid accumulation. The relationship between autophagy and aging-related liver function decline has been the topic of several studies.18) Autophagy dysfunction was observed in NAFLD hepatocytes, and the administration of compounds that induce autophagy, such as carbamazepine and rapamycin, has been reported to improve NAFLD symptoms in high-fat diet mouse models.19) Our research demonstrated that neferine administration ameliorated liver damage induced by natural aging (NAFLD-like phenotype) in mice. Until now, the relationship between autophagy induction and improved liver function in naturally aged mice was unclear. However, our results suggest that mitochondrial activation via autophagy induction ameliorates aging-associated decline in liver function. It is known that aging causes deterioration of kidney function due to damage to the glomeruli and other structures.20) In this study, we showed that neferine treatment suppressed kidney tissue damage, suggesting that neferine may restore function in tissues including the kidney as well as the liver. Moreover, it is speculated that long-term administration of neferine may affect the lifespan of mice. However, further studies on its effects in other organs and lifespan are still needed.
In conclusion, neferine rejuvenates aging cells by reactivating mitochondria via autophagy induction and is a potential therapeutic agent for aging-related liver dysfunction.
NB1RGB (human fibroblast) (Riken BRC, Ibaraki, Japan) cells were cultured in MEMα (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell cultures were split at a ratio of 1 : 4 every three days and maintained at 37 °C with 5% CO2. We categorized each cell line into two types based on the number of days in culture: NB1RGB: young cells: from 8 to 20 d and aging cells: from 60 to 70 d.
Detection of SA-β-Gal ActivityCellular SA-β-gal activity was measured using a senescence detection kit (#ab65351, Abcam, Cambridge, England) following the manufacturer’s instructions. All images were captured with a microscope (IX73, Olympus, Tokyo, Japan) and processed using Adobe Photoshop software.
JC-1 StainingMitochondrial membrane potential (ΔΨm) was measured using JC-1 staining (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions and a previous report.21) The results are presented as the ratio of fluorescence at 535/590 nm to that at 485/535 nm (aggregate fluorescence to monomer fluorescence) using a fluorescence microplate reader and i-control 1.11 software (Infinite M200, TECAN, Kanagawa, Japan). All images were captured with a fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) and processed using Adobe Photoshop software.
ATP AssayIntracellular ATP levels were measured using the CellTiter-Glo 2.0 assay kit #G9241 (Promega, Madison, WI, U.S.A.) following the manufacturer’s protocol. ATP production was normalized to the total protein content.
Cell Viability AssayCell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were treated with the indicated conditions and incubated with MTT solution (1 mg/mL) for 2 h. Isopropanol/HCl was then added to a final concentration of 50%/20 mM, and absorbance at 570 nm was measured using a spectrophotometer.
Immunoblotting AnalysisDAPK1, P-AMPK, total-AMPK, P-JNK, LC3, NCoR1 and ATG7 (Cell Signaling, Danvers, MA, U.S.A.), and β-actin (Sigma-Aldrich, St. Louis, MO, U.S.A.). The antibodies were diluted at a ratio of 1 : 1000, except for anti-β-actin (1 : 10000). Secondary antibodies were purchased from Promega (anti-rabbit and anti-mouse at 1 : 5000).
FAOBlue AnalysisNB1RGB cells were seeded in 96-well plates, cultured for 24 h, and treated with neferine (1 µM) in MEMα medium for 24 h. After washing the cells with HBSS, FAOBlue (Funakoshi Co., Ltd.) diluted in HBSS (final concentration: 5 µM) was added and allowed to react for 30 min at 37 °C. Finally, the fluorescence intensity (excitation, 420 ± 10 nm; emission, 460 ± 10 nm) was measured using a fluorescence plate reader (transmission-type measurement). All results were corrected according to the fluorescence intensity of cell-free FAOBlue solution (5 µM).
qRT-PCRqRT-PCR was performed as previously described.22) The total RNA in each reaction was normalized using β-actin cDNA as an internal control. The following forward and reverse primers for human were used: CPT1A, 5′-CAATCGGACTCTGGAAATG-3′ & 5′-CCGCTGACCACGTTCTTC-3′; CPT1B, 5′-GGTCCAGTTTACGGCGATAC-3′ & 5′-CCTCTCATGGTGAACAGCAA-3′; CPT2, 5′-TGACCAAAGAAGCAGCAATG-3′ & 5′-GAGCTCAGGCAAGATGATCC-3′; CACT, 5′-GTGTCCAAGTGGATTGAGCA-3′ & 5′-TACACCCTGGGCTTTCTCAC-3′; LCAD, 5′-TGCAATAGCAATGACAGAGCC-3′ & 5′-CGCAACTACAATCACAACATCAC-3′.
siRNA-Mediated Gene TargetingNB1RGB cells were transfected with siRNA for siATG7 (the siRNA SMARTpool for human ATG7 from Dharmacon, Lafayette, CO, U.S.A.) and controls (Santa Cruz, Santa Cruz, CA, U.S.A.) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol.
Aging-Dependent Liver Dysfunction Mouse ModelAll experimental protocols and surgical procedures were approved by the Animal Research Committee of Kochi University (Permit Number: P-00040). Animal experiments were conducted according to the guidelines for animal experimentation of Kochi University and the recommendations of the ARRIVE guidelines.
The mice were housed and bred in an animal room and exposed to a 12-h light/dark cycle with access to food and water ad libitum. C57BL/6 mice (aged 50 weeks) were orally administered 1 mg/kg of neferine once every 2 d for 92 d, and body weight was measured daily. C57BL/6 mice (7 weeks and 92 weeks of age) were evaluated for liver and kidney status. Liver tissue was collected and stored in 4% paraformaldehyde. Following embedding in paraffin wax blocks, sections of liver and kidney tissue were cut, stained with H&E, and mounted (PathoMount; FUJIFILM Wako Pure Chemical Corporation) prior to microscopic examination using a microscope (BZ-X800, KEYENCE). For immunohistochemical analysis, the sections underwent microwave treatment in 0.01 mol/L citric acid buffer for antigen retrieval, followed by incubation with 0.3% hydrogen peroxide to eliminate endogenous peroxidase. The sections were then blocked with 2.5% goat serum for 10 min, incubated for 12 h with the anti-NCoR1 antibody in 3% bovine serum albumin, and subsequently treated for 1 h with peroxidase-labeled polymer-conjugated goat anti-rabbit immunoglobulins (FUJIFILM Wako Pure Chemical Corporation). Afterward, the sections were exposed to 3,3′-diaminobenzidine and finally stained with Mayer’s hematoxylin. The samples were mounted using PathoMount (FUJIFILM Wako Pure Chemical Corporation) and examined under a microscope (BZ-X800, KEYENCE,). Other liver sections underwent immunoblotting analysis using primary antibodies against DAPK1, LC3, and HSP60 (loading control).
Maturement of Serum Triglyceride Levels and Serum ALT ActivityWe used commercially available enzymatic kits to measure the levels of serum triglyceride (#632-50991; FUJIFILM Wako Pure Chemical Corporation) and serum ALT activity (#700260; Cayman Chemical, Ann Arbor, MI, U.S.A.) according to the manufacturer’s protocol.
Detection of SA-β-Gal Activity in Liver TissueWe determined SA-β-gal activity in liver tissue using senescence detection kit #ab65351 (Abcam). Briefly, liver tissue was placed in 1.5 mL tubes and fixed with 0.5 mL Fixative Solution for 15 min at room temperature. Then 50 µL Staining Solution (prepared according to the manufacturer’s protocol) was added to each tube, incubated for 18 h at 37 °C, and washed with 1 × phosphate-buffered saline (PBS). Photographs were taken to assess SA-β-gal activity in each liver tissue sample in terms of area and the intensity of the blue staining.
Statistical AnalysesDifferences in mean values were evaluated using Student’s t-test or Welch’s t-test (depending on whether the sample sizes or variances between groups differed) for unpaired data to assess differences between two groups. For comparisons involving more than two compounds on one cell type, ANOVA was used, and for comparisons involving two cell types treated with more than one compound, two-way ANOVA was used, followed by the Tukey HSD test. A p-value of <0.05 was considered statistically significant. Statistical analyses were performed using Mac statistical analysis software (Esumi Co., Tokyo, Japan).
This work was partially supported by JSPS and Takeda Science Foundation. We thank the Division of Biological Research, Science Research Center, Kochi University for the use of research instruments.
S. K. conducted most of the experimental work. Y. Maejima, Y. Morioka, Z. A. K. B. E., and Y. S. made experimental contributions. S. K. and T. N. designed the experimental plans and analyzed and interpreted the data. T. N. designed and directed the project. S. K. and T. N. wrote the manuscript.
The authors declare no conflict of interest.
All data that support the findings of this study are available from the corresponding author upon reasonable request.
