Journal: World Journal of Hepatology

Article Title: Qushi Huoxue ointment ameliorates metabolic associated steatotic liver disease through autophagy activation and ferroptosis inhibition

doi: 10.4254/wjh.v18.i2.115763

Figure Lengend Snippet: Experimental design summary diagram. Metabolic associated steatotic liver disease mouse model was first established using the methionine-choline-deficient diet. Following Qushi Huoxue ointment (QSHXO) treatment, serum and liver tissue samples were collected to assess pathological changes, liver function markers, and inflammatory cytokine levels. Subsequently, the bioactive components of QSHXO in serum were identified using liquid chromatography-mass spectrometry/mass spectrometry analysis. Next, network pharmacology was applied to predict the potential target pathways of QSHXO in the treatment of metabolic associated steatotic liver disease, specifically focusing on autophagy and ferroptosis. These predicted targets were further validated through western blot, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and transmission electron microscopy. MCD: Methionine-choline-deficient; MASLD: Metabolic associated steatotic liver disease; QSHXO: Qushi Huoxue ointment; TNF-α: Tumor necrosis factor-α; IL-β: Interleukin-β; ELISA: Enzyme-linked immunosorbent assay; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TC: Total cholesterol; TG: Triglyceride; Nrf2: Nuclear factor erythroid 2-related factor 2; Lc3: Light chain 3; GPX4: Glutathione peroxidase 4.

Article Snippet: The following reagents and antibodies were used in this study: Alanine aminotransferase (ALT) test kit (C009-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); aspartate aminotransferase (AST) test kit (C010-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); total cholesterol (TC) test kit (A111-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); triglyceride (TG) test kit (A110-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); tumor necrosis factor-α (TNF-α, EK201BHS, LianKe Bio, Hangzhou, Zhejiang Province, China); interleukin (IL)-1β (EK282/4, LianKe Bio, Hangzhou, Zhejiang Province, China); oltipraz (B5958, APExBIO, TX, United States); Beclin1 (AF5128, 1:1000, Affinity, Shanghai, China); light chain 3 (LC3) (AF5402, 1:1000, Affinity, Shanghai, China); P62 (AF5384, 1:1000, Affinity, Shanghai, China); Nrf2 (AF0639, 1:800, Affinity, Shanghai, China); glutathione peroxidase 4 (GPX4) (A11243, 1:1000, Abclonal, Wuhan, Hubei Province, China); SLC7A11 (A2413, 1:1000, Abclonal, Wuhan, Hubei Province, China); and lamin B1 (BF8009, 1:2000, Affinity, Shanghai, China).

Techniques: Ointment, Liquid Chromatography, Mass Spectrometry, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry, Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: World Journal of Hepatology

Article Title: Qushi Huoxue ointment ameliorates metabolic associated steatotic liver disease through autophagy activation and ferroptosis inhibition

doi: 10.4254/wjh.v18.i2.115763

Figure Lengend Snippet: Qushi Huoxue ointment ameliorates liver injury and inflammatory response in metabolic associated steatotic liver disease mice. A: Serum levels of aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-α, and interleukin-β; B: Schematic diagram illustrating the relationship between hepatic lipid accumulation and liver injury. Data are presented as means ± SD; a P < 0.05, c P < 0.001. MASLD: Metabolic associated steatotic liver disease; QSHXO: Qushi Huoxue ointment; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TNF-α: Tumor necrosis factor-α; IL-β: Interleukin-β; TC: Total cholesterol; TG: Triglyceride.

Article Snippet: The following reagents and antibodies were used in this study: Alanine aminotransferase (ALT) test kit (C009-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); aspartate aminotransferase (AST) test kit (C010-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); total cholesterol (TC) test kit (A111-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); triglyceride (TG) test kit (A110-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); tumor necrosis factor-α (TNF-α, EK201BHS, LianKe Bio, Hangzhou, Zhejiang Province, China); interleukin (IL)-1β (EK282/4, LianKe Bio, Hangzhou, Zhejiang Province, China); oltipraz (B5958, APExBIO, TX, United States); Beclin1 (AF5128, 1:1000, Affinity, Shanghai, China); light chain 3 (LC3) (AF5402, 1:1000, Affinity, Shanghai, China); P62 (AF5384, 1:1000, Affinity, Shanghai, China); Nrf2 (AF0639, 1:800, Affinity, Shanghai, China); glutathione peroxidase 4 (GPX4) (A11243, 1:1000, Abclonal, Wuhan, Hubei Province, China); SLC7A11 (A2413, 1:1000, Abclonal, Wuhan, Hubei Province, China); and lamin B1 (BF8009, 1:2000, Affinity, Shanghai, China).

Techniques: Ointment

Journal: World Journal of Hepatology

Article Title: Qushi Huoxue ointment ameliorates metabolic associated steatotic liver disease through autophagy activation and ferroptosis inhibition

doi: 10.4254/wjh.v18.i2.115763

Figure Lengend Snippet: Experimental design summary diagram. Metabolic associated steatotic liver disease mouse model was first established using the methionine-choline-deficient diet. Following Qushi Huoxue ointment (QSHXO) treatment, serum and liver tissue samples were collected to assess pathological changes, liver function markers, and inflammatory cytokine levels. Subsequently, the bioactive components of QSHXO in serum were identified using liquid chromatography-mass spectrometry/mass spectrometry analysis. Next, network pharmacology was applied to predict the potential target pathways of QSHXO in the treatment of metabolic associated steatotic liver disease, specifically focusing on autophagy and ferroptosis. These predicted targets were further validated through western blot, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and transmission electron microscopy. MCD: Methionine-choline-deficient; MASLD: Metabolic associated steatotic liver disease; QSHXO: Qushi Huoxue ointment; TNF-α: Tumor necrosis factor-α; IL-β: Interleukin-β; ELISA: Enzyme-linked immunosorbent assay; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TC: Total cholesterol; TG: Triglyceride; Nrf2: Nuclear factor erythroid 2-related factor 2; Lc3: Light chain 3; GPX4: Glutathione peroxidase 4.

Article Snippet: The following reagents and antibodies were used in this study: Alanine aminotransferase (ALT) test kit (C009-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); aspartate aminotransferase (AST) test kit (C010-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); total cholesterol (TC) test kit (A111-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); triglyceride (TG) test kit (A110-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); tumor necrosis factor-α (TNF-α, EK201BHS, LianKe Bio, Hangzhou, Zhejiang Province, China); interleukin (IL)-1β (EK282/4, LianKe Bio, Hangzhou, Zhejiang Province, China); oltipraz (B5958, APExBIO, TX, United States); Beclin1 (AF5128, 1:1000, Affinity, Shanghai, China); light chain 3 (LC3) (AF5402, 1:1000, Affinity, Shanghai, China); P62 (AF5384, 1:1000, Affinity, Shanghai, China); Nrf2 (AF0639, 1:800, Affinity, Shanghai, China); glutathione peroxidase 4 (GPX4) (A11243, 1:1000, Abclonal, Wuhan, Hubei Province, China); SLC7A11 (A2413, 1:1000, Abclonal, Wuhan, Hubei Province, China); and lamin B1 (BF8009, 1:2000, Affinity, Shanghai, China).

Techniques: Ointment, Liquid Chromatography, Mass Spectrometry, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry, Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: World Journal of Hepatology

Article Title: Qushi Huoxue ointment ameliorates metabolic associated steatotic liver disease through autophagy activation and ferroptosis inhibition

doi: 10.4254/wjh.v18.i2.115763

Figure Lengend Snippet: Qushi Huoxue ointment ameliorates liver injury and inflammatory response in metabolic associated steatotic liver disease mice. A: Serum levels of aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-α, and interleukin-β; B: Schematic diagram illustrating the relationship between hepatic lipid accumulation and liver injury. Data are presented as means ± SD; a P < 0.05, c P < 0.001. MASLD: Metabolic associated steatotic liver disease; QSHXO: Qushi Huoxue ointment; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TNF-α: Tumor necrosis factor-α; IL-β: Interleukin-β; TC: Total cholesterol; TG: Triglyceride.

Article Snippet: The following reagents and antibodies were used in this study: Alanine aminotransferase (ALT) test kit (C009-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); aspartate aminotransferase (AST) test kit (C010-2-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); total cholesterol (TC) test kit (A111-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); triglyceride (TG) test kit (A110-1-1, Nanjing Jiancheng, Nanjing, Jiangsu Province, China); tumor necrosis factor-α (TNF-α, EK201BHS, LianKe Bio, Hangzhou, Zhejiang Province, China); interleukin (IL)-1β (EK282/4, LianKe Bio, Hangzhou, Zhejiang Province, China); oltipraz (B5958, APExBIO, TX, United States); Beclin1 (AF5128, 1:1000, Affinity, Shanghai, China); light chain 3 (LC3) (AF5402, 1:1000, Affinity, Shanghai, China); P62 (AF5384, 1:1000, Affinity, Shanghai, China); Nrf2 (AF0639, 1:800, Affinity, Shanghai, China); glutathione peroxidase 4 (GPX4) (A11243, 1:1000, Abclonal, Wuhan, Hubei Province, China); SLC7A11 (A2413, 1:1000, Abclonal, Wuhan, Hubei Province, China); and lamin B1 (BF8009, 1:2000, Affinity, Shanghai, China).

Techniques: Ointment

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects C2C12 myotubes from TNFα/IFNγ-induced muscle cell atrophy. (A-C) C2C12 myotubes were treated with GT for 48 h at the indicated concentration and stained with anti-MHC Ab. (A) Representative images were shown. (B) Average myotube diameter was measured by ImageJ software. (C) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (D-F) C2C12 myotubes were treated with GT at the indicated concentrations together with TNFα (20 ng/mL) and IFNγ (100U/mL) for 24 h, and then stained with anti-MHC Ab. (D) Representative images were shown. (E) Average myotube diameter was measured by ImageJ software. (F) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (G-I) C2C12 myotubes were pre-treated with TNFα (20 ng/mL) and IFNγ (100U/mL) for 24 h, and then treated with GT (100 ng/mL) for 24 h. Cells were stained with anti-MHC Ab. (G) Representative images were shown. (H) Average myotube diameter was measured by ImageJ software. (I) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (J-K) C2C12 myotubes were treated with GT (100 ng/mL) together with TNFα (20 ng/mL) and IFNγ (100U/mL). Cells were isolated, and the protein levels of Atrogin-1 and MuRF-1 were evaluated by western blot. (J) Representative images were shown. (K) Images were measured by ImageJ software. The data were shown as mean ± SEM ( n =3; * P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Concentration Assay, Staining, Software, Isolation, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects against cellular atrophy through the lysophosphatidic acid receptor (LPAR) and Gαi2 activation. (A) C2C12 myotubes were treated with GT (100 ng/mL) for 24 h and then the expression of LPARs were quantified by RT-PCR. The data were shown as mean ± SEM of three independent experiments (* P ≤ 0.05; ns, not significant). (B–D) C2C12 myotubes were treated with GT (100 ng/mL), TNFα (20 ng/mL) and IFNγ (100U/mL), or Ki16425 (10 μM) for 24 h and then stained with anti-MHC Ab. (B) Representative images were shown. (C) Average myotube diameters and (D) the number of nuclei per myotube of more than 100 myotubes from 10 randomly chosen fields for each condition were measured by ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05; *** P ≤ 0.0001). (E-F) C2C12 cells were transfected with control (non-targeting) (siCtrl) or Gαi2 siRNA. (E) mRNA level of Gαi2 was evaluated by RT-PCR and (F, G) protein level of Gαi2 was evaluated by western blot. The data were shown as mean ± SEM of 2 independent experiments ((* P ≤ 0.05; *** P ≤ 0.0001). (H, I) The transfected cells were differentiated to myotubes for 4 days and treated with GT (100 ng/mL) for 48 h. (H) Representative images were shown. (I) Average myotube diameters of more than 100 myotubes from 10 randomly chosen fields of each condition were measured by ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05;*** P ≤ 0.001; ns, not significant).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Software, Transfection, Control, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects C2C12 myoblast from oxidative stress through the reduction of ROS and inflammation genes. (A, B) C2C12 myoblast was incubated for 4 h with TNFα (20 ng/mL), GT (100 ng/mL), or N-acetyl cysteine (NAC) as indicated, and then ROS levels were measured by flow cytometry. NAC was used as a negative control. (A) Representative FACS profiles were shown and (B) the data were presented as mean ± SEM of four independent experiments (* P ≤ 0.05; ***P ≤ 0.0001). (C, D) C2C12 myotube was incubated with TNFα (20 ng/mL) and GT (100 ng/mL) for 4 h as indicated, and then ROS levels were measured by fluorescence microscope. (C) Representative fluorescence images were shown and (D) fluorescence intensity were quantified using the ImageJ software. The data were presented as mean ± SEM of at least 10 randomly chosen fields of each condition (*** P ≤ 0.0001). (E, F) C2C12 myoblast was incubated for 24 h with TNFα (20 ng/mL) or GT (100ng/mL) as indicated, and mitochondria ROS were measured by flow cytometry. (E) Representative FACS profiles were shown and (F) the data were presented as mean ± SEM of four independent experiments (* P ≤ 0.05; *** P ≤ 0.0001). (G, H) C2C12 myoblast was incubated for 24 h with TNFα (20 ng/mL) or GT (100ng/mL) as indicated, and mitochondrial membrane potential (ΔѰm) were measured by flow cytometry. (G) Representative FACS profiles were shown and (H) the data were presented as mean ± SEM of five independent experiments (* P ≤ 0.05). (I) The effect of GT on scavenging of hydroxyl radical was analyzed using iron (II)-dependent TBA reactive substance. Ascorbic acid (AA) was used as a positive control. Data were shown as mean ± SEM of three independent experiments (* P ≤ 0.05; *** P ≤ 0.0001). (J, K) C2C12 myotubes were treated for 24 h with TI (TNFα at 20 ng/mL and IFNγ at 100U/mL) or GT (100 ng/mL) as indicated, and then the levels of IL-6 (J) and Nox-2 (K) were quantified by RT-PCR. The data were shown as mean ± SEM of three to four independent experiments (*, P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Incubation, Flow Cytometry, Negative Control, Fluorescence, Microscopy, Software, Membrane, Positive Control, Reverse Transcription Polymerase Chain Reaction

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects against the atrophy of primary normal Human Skeletal Myoblasts (HSkM). (A–C) HSkM myoblast were differentiated to myotube for 7 days in differentiation media. Differentiated cells were treated with different concentrations of GT in the presence or absence of TNFα (10 ng/mL) for 3 days and then stained with anti-MHC Ab. (A) Representative images of myotube cultures were captured with a phase-contrast microscope (100x magnification). (B) Average myotube diameters and (C) the number of nuclei per myotube were quantified from more than 100 myotubes in 10 randomly chosen fields of each condition using ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05; *** P ≤ 0.0001). (D, E) HSkM myotubes were treated with GT (100 ng/mL) together with TNFα (10 ng/mL) for 8 h. Cells then were isolated, and the protein levels of Atrogin-1 and MuRF-1 were evaluated by western blot. (D) Representative images were shown. (E) Images were measured by ImageJ software. The data were shown as mean ± SEM ( n =3; * P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Staining, Microscopy, Software, Isolation, Western Blot

Journal: Science Advances

Article Title: Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway

doi: 10.1126/sciadv.adc9273

Figure Lengend Snippet: ( A ) DOT1L -depleted NSCLCs were infected with lentivirus encoding doxycycline (Dox)–inducible WT or mutant DOT1L (Tet-on system). ( B ) The cells generated in (A) were subjected to clonogenic survival assay by crystal violet staining 7 to 14 days after seeding. ( C ) The cells generated in (A) were seeded for tumorsphere formation assay and cultured for 7 to 14 days. Scale bars, 1000 μm. ( D ) Effect of doxycycline induction on the relative tumor volume in BALB/c-nu mice xenografted with NCI-H460-DOT1L-KD-DOT1L-WT/R231Q cells ( n = 10 mice per group). ( E ) Survival analysis of mice in the DOT1L WT and R231Q groups ( n = 20 mice per group and tumor volume > 2000 mm 3 ). ( F ) Western blotting analysis of DOT1L-flag and H3K79me2 expression in xenograft tumors from the CDX model in (D) ( n = 3). ( G ) Ki67 staining and TUNEL staining of the CDX tumors. Scale bars, 50 μm. Data are shown as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. In (A), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. Data from (B) to (D), (F), and (G) were analyzed using Student’s t test (independent samples t test). The log-rank (Mantel-Cox) test was used for (E), ** P < 0.01.

Article Snippet: Apoptosis in tumor sections of xenograft models was detected using the TUNEL Assay Kit-HRP-DAB (Abcam) according to the manufacturer’s protocol.

Techniques: Infection, Mutagenesis, Generated, Clonogenic Cell Survival Assay, Staining, Tube Formation Assay, Cell Culture, Western Blot, Expressing, TUNEL Assay

Journal: Science Advances

Article Title: Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway

doi: 10.1126/sciadv.adc9273

Figure Lengend Snippet: ( A ) Crystal violet staining of NCI-H460-DOT1L-WT/R231Q cells treated with SGC0946 (2.5 or 7.5 μM), binimetinib (2.5 or 7.5 μM), or the combination at the same concentrations for 7 to 14 days (top). Comparison of relative colony formation between groups at 7.5 μM drug concentration (bottom). ( B ) Clonogenic survival assay to evaluate the effects of DOT1Lis SGC0946 (1 μM), binimetinib (1 μM), or the combination at the same concentrations on cells transfected with constructs expressing the other DOT1L gain-of-function mutants (Y216C, S225L, N241T, F243L, and A1003G). The plates were photographed 7 to 14 days after seeding. ( C ) Tumor growth curves and images for four xenograft models ( n = 6 mice per group) that were treated with vehicle (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline, five times per week), SGC0946 (10 mg/kg, five times per week), binimetinib (15 mg/kg, five times per week), or the combination at the same doses through intraperitoneal administration and oral gavage. The graphs show the TIR%. ( D ) IHC staining for Ki67 and TUNEL staining of the NCI-H1299-DOT1L-R231Q CDX tumor tissues. Scale bars, 50 μm. ( E ) Levels of RAF1, DOT1L-flag, p-MEK, MEK, and H3K79me2 in tumors from the NCI-H1299-DOT1L-R231Q CDX mice ( n = 2). ( F ) Summary diagram describing that regulation gain-of-function DOT1L mutations increase the malignant phenotypes of lung cancer by regulating the MAPK/ERK signaling pathway. Data are shown as means ± SEM. * P < 0.05, as compared to the WT group. # P < 0.05, ## P < 0.01, and ### P < 0.001, as compared to the single treatment group. In (A), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test and Student’s t test (independent samples t test). In (C) and (D), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Apoptosis in tumor sections of xenograft models was detected using the TUNEL Assay Kit-HRP-DAB (Abcam) according to the manufacturer’s protocol.

Techniques: Staining, Comparison, Concentration Assay, Clonogenic Cell Survival Assay, Transfection, Construct, Expressing, Saline, Immunohistochemistry, TUNEL Assay