Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 1. PUN alleviated liver injury in diet-induced NASH. (A) Serum ALT and AST levels and liver hydroxyproline concentrations. Data are presented as the mean ± SD (n = 10, from three independent experiments). (B, C) The H&E staining of liver tissues in the Control, vehicle and PUN (100, 300, 500 mg/kg) groups (original magnification, 200×). (D-E) Detection of ALT and AST concentrations in serum of PUN- treated mice after NASH. (F, G) H&E and Masson staining of liver tissues (original magnification, 200×). Similar results were obtained from three independent experiments. All data are presented as the mean ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Staining, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 2. PUN attenuated hepatic inflammatory response in mice with NASH. (A-C) Effects of PUN on NASH- induced serum TNF-α, IL-6, and IL-1β generation. Similar results were obtained from three independent experiments. (D-G) Effects of PUN on NLRP3, ASC, cleaved-caspase-1, and mature-IL-1β protein expression was measured by western blotting and quantification of protein expression was performed by densitometric analysis. Similar results were obtained from three independent experiments. All data are presented as the mean ± SEM (n = 6 in each group). *p < 0.05 and **P < 0.01 vs. Control group; ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Expressing, Western Blot, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 4. PUN upregulated the p62/Nrf-2 signaling in mice with NASH-induced liver injury. (A-C) Immunofluorescence stained analysis of Nrf2 and p-p62 in liver tissues (original magnification, 200×). (D-G) Liver tissue was analyzed by western blot for assessment of nuclear abundance of Nrf2, HO-1 and phosphorylation of p62 at ser349. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 5. PUN regulates up-regulated autophagy via the AMPK/mTOR/ULK1 signaling pathway. (A-E) Effects of PUN on protein abundance of Beclin-1, LC3, Atg5 and Atg7 measured by western blotting. (F-I) Effects of PUN on protein abundance of p-AMPK, p-mTOR and p-ULK1 measured by western blotting. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Quantitative Proteomics, Western Blot, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 6. PUN improved liver injury caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-B) Blood serum collected assessment of ALT and AST activities. (C-F) Liver tissues serum TNF-α, IL-6, and IL-1β and ROS generation. Similar results were obtained from three independent experiments. (G, H) Representative histological sections of liver were stained with H&E and Masson (magnification×400). Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Injection, Staining, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 7. PUN improved Txnip-NLRP3 signaling pathway caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-F) Effects of PUN on protein abundance of Txinp, NLRP3, ASC, Cleaved-Caspase1 and Mature-IL-1β measured by western blot. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Injection, Quantitative Proteomics, Western Blot, Control

Journal: Scientific reports

Article Title: Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis.

doi: 10.1038/s41598-025-98044-6

Figure Lengend Snippet: Fig. 8. PUN improved Autophagy caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-E) Effects of PUN on protein abundance of Beclin-1, Atg5, Atg7, LC3, p-AMPK, p-ULK1 and p-mTOR measured by western blot. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. NASH group.

Article Snippet: Antibodies against Nrf2, SQSTM1/p62 (phospho S349), p-ULK1, p-mTOR and p-AMPK were obtained from Abcam (Cambridge, UK); HO-1, Beclin1, LC3, Atg5, Atg7, NLRP3, ASC, Cleaved Caspase1, IL-1β, ULK1, mTOR and AMPK and β-actin were obtained from Proteintech; Test kits of malondialdehyde (MDA), reactive oxygen species (ROS), GSH, and superoxide dismutase (SOD), alanine transaminase (ALT), aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Techniques: Injection, Quantitative Proteomics, Western Blot, Control

Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: CMA is essential for maintaining the youthful state of the nucleus pulposus. a , b The protein and mRNA levels of L2A (LAMP2A) in human intervertebral discs with different degeneration grades. c The protein levels of L2A, P53, P21, and P16 in different senescent NPC models, N-Sen (Nutlin-3a-induced senescence), R-Sen (Replicative senescence, passage 9), and I-Sen (IL1B-induced senescence). d Immunofluorescence shows the levels of SA-β-gal and CMA activity in different senescent NPC models. CMA activity is represented by the average number of red spots per cell. e The protein levels of L2A, P53, P21, and P16 in NPC treated with ATRA or sg-L2A transfection. f Flow cytometry results of the cell cycle of each group. g The effect of CMA activation on IL1B-induced IDD. MRI imaging and histological staining were performed after sampling. h The grade of IDD in each group was evaluated by Pfirrmann scores, DHI, and histological scores, as described before. i Immunofluorescence showing levels of P53, P21, and P16 in the rat discs. j – l The effect of L2A-KD on rat IDD, evaluated by Pfirrmann grading and DHI. m The effect of L2A-KD on in vivo levels of P53, P21, and P16. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Immunofluorescence, Activity Assay, Transfection, Flow Cytometry, Activation Assay, Imaging, Staining, Sampling, In Vivo

Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: Elevated DYRK1A drives premature senescence in CMA-downregulated NPC. a TMT-MS analysis showing that DYRK1A levels were increased in NPC after L2A knockout. b Schematic diagram of the sequence of DYRK1A. c The protein level of DYRK1A in NPC treated with ATRA, si-L2A, or sg-L2A. d Immunofluorescence shows the level of DYRK1A in NPC after L2A knockout. e Immunoblotting shows the levels of L2A, DYRK1A, P53, P21, and P16 in NPC treated as indicated. NPC were treated with IL1B (10 ng/mL, 48 h) and Har (Harmine, 20 μg/mL, 48 h). f Flow cytometry showing the cell cycle of NPC treated as indicated. The proportion of NPC in the S phase was used for comparison. g Relative BrdU levels of NPC treated as indicated, measured at 450 nm. h SA-β-gal fluorescence levels detected by Fluorescein diβ-D-galactopyranoside (FDG) in NPC treated as indicated. i Western blot showing the protein levels of DYRK1A and senescence markers in NPC of each group. j Flow cytometry showing the cell cycle changes of NPC treated as indicated. k Relative BrdU levels of NPC treated as indicated. l Immunofluorescence showing SA-β-gal levels in NPC after DYRK1A and L2A overexpression. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Knock-Out, Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Comparison, Fluorescence, Over Expression

Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: CMA induces a fate switch of senescent NPC from cellular senescence to apoptosis. a , b Immunofluorescence showing the effect of L2A overexpression on P53 and senescence levels in I-Sen NPC. c The effect of L2A overexpression on the cell cycle of NPC in the control or I-Sen group. d Western blot showing the effect of L2A overexpression on the levels of senescence-related proteins (P53, P21, P16) in I-Sen NPC. e Western blot showing the effect of L2A overexpression on the levels of SASP inflammatory factors (IL6, IL-8, IL1B, TNF) and MMP3 in I-Sen NPC. f After treatment with CMA activator AR7 in I-Sen NPC, RNA-seq revealed that apoptosis-related pathways were significantly enriched. g , h TUNEL staining shows the apoptosis level in I-Sen NPC after L2A overexpression. i , j Immunoblotting showing the effect of L2A overexpression on the expression of apoptosis-related proteins in I-Sen NPC. k , l The effect of L2A overexpression on mitochondrial membrane potential in NPC of each group. m The apoptosis levels in normal and senescent NPC (R-Sen and I-Sen) treated with AI-10 or combined with L2A overexpression. n The effect of L2A overexpression or combined with BNIP3 knockdown on the apoptosis of senescent NPC. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, ns indicates not significant

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Immunofluorescence, Over Expression, Control, Western Blot, RNA Sequencing, TUNEL Assay, Staining, Expressing, Membrane, Knockdown