Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Identification of obacunone as a potent renal cyst inhibitor. ( A ) Cytotoxicity of 12.5 μM candidate compounds on MDCK cells. n = 5. ( B ) Representative images of MDCK cysts treated without (Ctrl) or with 12.5 μM candidate compounds. Bar = 100 μm. ( C ) Diameters of MDCK cysts at day 12. n = 30. ( D ) Representative images of embryonic kidneys. Bar = 500 μm. ( E ) Cyst indices of embryonic kidneys at day 6. n = 6. ( F ) Volumes of embryonic kidneys at day 6. n = 6. ( G ) Representative images of kidneys from wild-type (WT) or kidney specific Pkd1 knockout mice (PKD) treated without or with 100 mg/(kg·d) candidate compounds. Bar = 0.5 cm. ( H ) Chemical structure of Y10. Data are presented as means ± SEM. ** p < 0.01, *** p < 0.001, versus Ctrl.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques: Knock-Out

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Inhibitory effect of obacunone on MDCK cyst formation and expansion. ( A ) Viability of MDCK cells treated without (Ctrl) or with obacunone. n = 5. ( B ) Number of total colonies in MDCK cyst model. n = 3. ( C ) Cyst formation rate. n = 3. ( D ) Representative images of MDCK cysts. Narrow black lines indicate obacunone treatment. Bar = 100 μm. ( E ) Diameters of MDCK cysts showing dose response of obacunone. n = 30. ( F ) Diameter of MDCK cysts showing reversible effect of obacunone. Arrow indicates the time of washout obacunone. n = 30. Data are presented as means ± SEM. * p < 0.05, *** p < 0.001 versus Ctrl. ### p < 0.001 versus 50 μM obacunone treatment without washout.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques:

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Inhibitory effect of obacunone on the enlargement of embryonic kidney cyst. ( A ) Representative images of embryonic kidneys treated without (Ctrl) or with obacunone. Narrow black lines indicate obacunone treatment. Bar = 500 μm. ( B ) Cyst indices (cyst area to embryonic kidney area). ( C ) Relative kidney volumes of embryonic kidneys on day 6. Data are presented as means ± SEM. n = 6. * p < 0.05, *** p < 0.001 versus Ctrl. ## p < 0.01 versus 50 μM obacunone treatment.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques:

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Inhibitory effect of obacunone on cyst development in PKD mice. ( A ) Weight curves of mice. ( B ) Representative images of the kidneys. Bar = 1 cm. ( C ) Kidney indices. ( D ) Hematoxylin- and eosin-stained pictures of kidneys. Bar = 400 μm. ( E ) Cyst indices. ( F ) LTL (green), DBA (red) and DAPI (blue) fluorescence staining of PKD kidneys. Arrows indicate the collecting ducts, asterisks represent the cyst area. Bar = 50 μm. Data are presented as means ± SEM. n = 8. ** p < 0.01 versus WT-Ctrl. ## p < 0.01, ### p < 0.001 versus PKD-Ctrl.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques: Staining, Fluorescence

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Effect of obacunone on NRF2 activity. ( A ) Immunofluorescence of NRF2 (red) and DAPI (blue) staining in mIMCD-3 cells treated without (Ctrl) or with forskolin (FSK) or obacunone (Oba). Bar = 10 μm. ( B ) Quantification of fluorescence intensities of NRF2 as in ( A ). n = 12 cells. ( C ) Representative Western blots of nuclear NRF2 in the kidneys of wild-type (WT) and PKD mice (PKD) treated without or with obacunone. ( D ) Quantification of nuclear NRF2 expression levels as in ( C ). Data are presented as means ± SEM. n = 6. * p < 0.05, ** p < 0.01 versus Ctrl. #### p < 0.0001 versus FSK. *** p < 0.001 versus WT-Ctrl. # p < 0.05 versus PKD-Ctrl.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Inhibition effect of obacunone on lipid peroxidation. ( A ) Representative Western blots of GPX4 and 4-HNE in the kidneys of wild-type (WT) and PKD (PKD) mice treated without (Ctrl) or with obacunone (Oba). ( B ) Quantification of GPX4 expression levels. ( C ) GSH-Px activity. ( D ) GSH content. ( E ) Quantification of 4-HNE expression levels. ( F ) MDA content. Data are presented as means ± SEM. n = 6. * p < 0.05, ** p < 0.01 versus WT-Ctrl. # p < 0.05, ## p < 0.01 versus PKD-Ctrl.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques: Inhibition, Western Blot, Expressing, Activity Assay

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: Inhibitory effect of obacunone on abnormal cell proliferation. ( A ) Representative Western blots of mTOR signaling pathway proteins in the kidneys of wild-type (WT) and PKD mice (PKD) treated without (Ctrl) or with obacunone (Oba). ( B ) Relative phosphorylation levels of mTOR protein. ( C ) Relative phosphorylation levels of S6 protein. ( D ) Representative Western blots of MAPK signaling pathway proteins. ( E ) Quantification of H-Ras protein expression levels. ( F ) Quantification of B-Raf protein expression levels. ( G ) Relative phosphorylation levels of ERK protein. ( H ) Representative Western blots of PCNA. ( I ) Quantification of PCNA protein expression levels. ( J ) Immunofluorescence staining of Ki-67 (red) and DAPI (blue) in the PKD kidneys. Arrows represent the Ki-67 positive cells, asterisks represent the cyst area. Bar = 50 μm. ( K ) Quantification of Ki-67 positive cells. Data are presented as means ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 versus WT-Ctrl. # p < 0.05, ## p < 0.01, ### p < 0.001 versus PKD-Ctrl.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

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

Journal: Antioxidants

Article Title: Obacunone Retards Renal Cyst Development in Autosomal Dominant Polycystic Kidney Disease by Activating NRF2

doi: 10.3390/antiox11010038

Figure Lengend Snippet: The suggested pharmacological mechanism of obacunone in ADPKD. See text for details.

Article Snippet: DMEM/F-12 medium containing 10 μM forskolin (Sigma-Aldrich, Saint Louis, MO, USA, Cat# F6886), without or with candidate compounds, was added to each well and replaced every 12 h. To detect the influence of obacunone (Selleck, Houston, TX, USA, Cat# S3784) on the formation of MDCK cysts, different concentrations of obacunone were supplied into the medium at day 0, and the numbers of cell colonies and cysts (larger than 50 μm) in different groups were counted using a Nikon microscope (Nikon, Tokyo, Japan, ECLIPSE Ti2-U) at day 6.

Techniques:

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone alleviated AOM/DSS-induced colitis in CRC mice. Experimental schematic overview of the procedures used for generating the AOM/DSS mouse model (A). After intraperitoneal injection with AOM (10 mg/kg), 2.5% DSS was added to drinking water for 7 days, followed by administration of tap water. The cycle was repeated another twice. All mice were sacrificed after 12 weeks of AOM injection. Changes in body weights of individual mice (B) and scores of bloody diarrhoea (C) were measured three times a week during the experiment. After sacrifice, the colon and spleen weights were measured (D, E). Colon lengths of mice were determined (F, G). Values are expressed as means ± SD ( n ≥ 6). vs Vehicle group, ### p < 0.001; vs A/D group, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: Injection

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone inhibited the viability of human colon cancer cells. Human colon cancer cells were treated with different concentrations of obacunone for 48 h, and subjected to the MTT assay for detection of cell viability. (A) Caco2 cells, (B) HT-29 cells, (C) SW480 cells, (D) HCT-116 cells. Values are expressed as means ± SD ( n = 4). vs control group, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: MTT Assay, Control

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone prevented tumour growth in a CRC model. Representative views of mouse colons (A). Statistical data on tumour number and tumour size in mice (B–D). Values are expressed as means ± SD ( n ≥ 6). vs Vehicle group, ### p < 0.001; vs A/D group, * p < 0.05; ** p < 0.01.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques:

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone reduced histologic pathological changes in CRC. H&E staining of the colon from different groups (A). Macroscopic inflammation scores of H&E staining (B). The arrows indicate the neoplastic areas or inflammatory areas. Values are presented as means ± SD ( n ≥ 6). vs Vehicle group, ### p < 0.001; vs A/D group, ** p < 0.01.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: Staining

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone inhibited the expression of inflammatory cytokines in CRC. ELISA evaluation of inflammatory cytokine (IL-6, TNF-α, NO) levels in different groups (A). IHC staining for assesment of the COX-2 level (B). qPCR analysis of relative mRNA expression of inflammatory cytokines (C). Values are presented as mean ± SD ( n ≥ 6). vs Vehicle group, ## p < 0.01, ### p < 0.001; vs A/D group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone suppressed the protein levels of pro-inflammatory cytokines and phosphorylation of the NF-κB complex. Protein levels of TNF-α, iNOS, COX-2 (A) and NF-κB complex (C) in colon tissue were measured via WB. Relative densities of these proteins were calculated (B, D). Values are presented as means ± SD ( n = 3). vs Vehicle group, # p < 0.05; ### p < 0.001; vs A/D group, ** p < 0.01; *** p < 0.001.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: Phospho-proteomics

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone inhibited the cell proliferation in vivo . PCNA (A) and BrdU (B) levels in different groups were detected via IHC staining. Relative mRNA expression of the cell cycle-associated genes in colonic tissue (C). Values are presented as means ± SD ( n = 4). vs Vehicle group, # p < 0.05; ## p < 0.01; vs A/D group, * p < 0.05.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: In Vivo, Immunohistochemistry, Expressing

Journal: Pharmaceutical Biology

Article Title: Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer

doi: 10.1080/13880209.2020.1812673

Figure Lengend Snippet: Obacunone suppressed mRNA and protein levels of cell cycle-associated genes in vitro . Flow cytometry was used to analyze the cell cycle of Caco2 cells treated with or without obacunone (A). Relative cell numbers in the G1 and G2 phases (B). Levels of proteins related to the cell cycle in Caco2 cells were determined via WB and their relative densities calculated (C). Relative levels of mRNAs associated with the cell cycle in Caco2 cells treated with/without obacunone were assessed via qPCR (D). Values are presented as means ± SD ( n = 3). vs control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 5-Bromo-2-deoxyuridine (BrdU, ab142567) was obtained from Abcam (Cambridge, MA) and obacunone (purity ≥ 98%) from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Techniques: In Vitro, Flow Cytometry, Control