Alisol F and 25-anhydroalisol F were isolated from A. orientale by one of the authors (Q. Ma) using our previously-established method and provided with a purity of 98.0% determined by high-pressure liquid chromatography. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, and penicillin-streptomycin-amphotericin (PSA) were purchased from Gibco BRL Co. Ltd. (Gaithersbug, MD, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dexamethasone (DXM), d -galactosamine (d -gal) and Trizol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The Griess reagent, the protein extraction kit and BCA protein assay kit were obtained from Beyotime Institute of Biotechnology (Beijing, China). Go Tag® qPCR Master Mix and GoScriptTM Reverse Transcription System were purchased from Promega (Madison, WI, USA). Rabbit polyclonal antibodies against inducible nitric oxide synthase (iNOS), COX-2, p-p38 (Thr180/Tyr182), p38, p-ERK1/2 (Thr202/Try204), ERK1/2, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-p65 (Ser536), p65, GAPDH, p-IκB-α (Ser32), IκB-α, p-STAT3 (Tyr705), STAT3, goat-IgG HRP, and lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse TNF-α, IL-1β, and IL-6 ELISA kit were from R and D systems (Abingdon, UK).
D galactosamine d gal
Manufactured by Merck Group
Sourced in United States
D-galactosamine (D-Gal) is a monosaccharide derived from galactose. It is used as a laboratory reagent in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Myd88, by Cell Signaling Technology (2 mentions)
Dmem basic, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mouse tnf α, by R&D Systems (1 mentions)
Penicillin streptomycin amphotericin, by Thermo Fisher Scientific (1 mentions)
Il 6 elisa kit, by R&D Systems (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps from escherichia coli 055 b5, by Merck Group (1 mentions)
Gotag qpcr master mix, by Promega (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Griess reagent, by Beyotime (1 mentions)
Tnf α, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
8 protocols using d galactosamine d gal
1
Alisol F and 25-anhydroalisol F Isolation and Effects
2
Evaluating Intestinal Barrier Function
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Fetal bovine serum (FBS) and DMEM basic were purchased from Gibco (NY, USA). Betaine hydrochloride (purity of 99%) was purchased from Juhua Group Co. (Zhejiang, China). Lipopolysaccharide (LPS, purity of 99%) and D-galactosamine (D-Gal, purity of 98%) were purchased from Sigma (St. Louis, USA). Antibodies against TLR4, (ZO)-1 and GAPDH were purchased from Proteintech (Hubei, China). Rabbit anti-rat/mice occludin and MyD88 were purchased from Cell Signaling Technology (Boston, USA). The Goat anti-rabbit fluorescent secondary antibody (IRDye800) was purchased from LI-COR Biosciences, Inc. (Lincoln, USA).
3
ACLF Rat Model via CCl4 and D-Gal
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ACLF models were created in rats by CCl4 (Sigma-Aldrich, USA) intraperitoneal injection for ten weeks, followed by venous injection with D-galactosamine (D-gal) (Sigma-Aldrich, USA). The rats were injected with CCl4 dissolved in olive oil (10%) into the abdominal cavity twice every week. Doses of CCl4 were modified according to the index of liver function and the body weight. At ten weeks, two rats from each group were killed and the presence of cirrhosis was confirmed. Then the remaining rats were injected with D-gal at a dose of 0.70 g/kg BW to induce ACLF.
4
Modeling Acute Liver Failure In Vitro
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The human liver cell line L02 obtained from China Center for Type Culture Collection (CCTCC) were cultured in DMEM medium (HyClone, USA) containing 10% heat-inactivated FBS (GIBCO, USA) and 100 U penicillin/100 g streptomycin (Sigma, USA) at CO2 atmosphere with a temperature of 37 °C and a humidity of 5%. TNF-α (100 ng/mL, Sigma, USA) combined with D-Galactosamine (D-Gal, 44 μg/mL, Sigma, USA) were used to establish the ALF model in vitro. Constructed NAMPT overexpression plasmid and enveloped it with lentiviral vectors (LV, GeneCreate, China). Besides, the cells were treated with the fresh media containing 1 mM AMPK activator 5-aminoimidazole-4-carboxamide-1-b-D-riboside (AICAR). After 24 h treatment, the cells were harvested for further experiments. Fusobacterium nucleatum (ATCC10953, Beijing, China) were cultured on tryptic soy under anaerobic conditions. Escherichia coli (E.coli) strain (Beijing, China) were cultured in Luria-Bertani (LB) medium.
5
Evaluating Oxidative Stress and Inflammatory Pathways
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The Reactive Oxygen Species (ROS) Assay Kit (cat.no. S0033S), Fluo-4 AM (cat.no S1060), and Nitrite Assay Kit (cat.no S0023) were purchased from Beyotime Biotechnology (Shanghai, China). d -Mannose, BSA, and sodium cyanoborohydride were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). LPS and d -galactosamine (d-Gal) were purchased from Sigma-Aldrich (United States ). The antibodies of p-p65 (3033s), p65 (8242s), p-IκB-α (4812), IκB-α (2859), p-p38 (4511s), p38 (8690s), and β-actin (3700) were purchased from Cell Signaling Technology (United States ). The antibodies of p-JNK (76572) and JNK (179461) were purchased from Abcam Co., Ltd. (USK). All other reagents were obtained from Nanjing Jiancheng Bioengineering Institute (Shanghai, China) unless otherwise stated.
6
Therapeutic Potential of Modified MSCs in ALF
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Male C57BL/6J wild type mice (6~8 weeks old) were obtained from the Animal Core Facility of Nanjing Medical University. Animals were bred in a pathogen-free facility. Mice were randomly divided into five groups: (1) control group (Ctrl): mice were intraperitoneally (i.p.) injected with PBS only; (2) ALF group (ALF): mice simultaneously received 600 mg/kg D -galactosamine (D-Gal) (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/kg LPS (Sigma-Aldrich) dissolved in PBS by i.p. injection; (3–5) cell transplantation groups: mice received 600 mg/kg D-Gal and 100 μg/kg LPS via i.p. injection, and then 2 × 106 MSC, MSC-COX2(+), or MSC-COX2(−) were injected via tail vein after 6 h, namely the MSC group, MSC-COX2(+) group, or MSC-COX2(−) group, respectively. For TAK1 inhibition, we administrated the specific TAK1 inhibitor 5z-7-ox (5 mg/kg, Sigma) to mice 1 h before LPS/D-Gal administration. To inhibit the EP4 receptor of PGE2, we administered EP4-specific antagonist (GW627368X, 20 mg/kg, Cayman Chemical, Ann Arbor, MI, USA) 1 h before LPS/D-Gal treatment and every 24 h following MSC infusion. Blood and liver tissues were collected at the indicated time for further analysis.
7
D-Galactosamine-Induced Acute Liver Injury
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Male SD rats at 6-8 weeks of age were provided by the Center for Experimental Animals of Shanghai General Hospital. In this experiment, all animal protocols were carried out in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, which were approved by the regulations set by the Ethics Committee of Shanghai General Hospital as well. Eight rats were randomly divided into the model and control groups. The rats in the model group were injected intraperitoneally with 1.5 g/kg d -galactosamine (D-Gal) (Sigma, St. Louis, MO, USA) to induce severe ALI. In this study, the ALI animal model refers to severe liver damage. The injection dose was determined according to previous animal studies [14 (link)]. Changes in liver function were monitored for 3 days, and then, the animals were sacrificed for histological evaluation.
8
Intestinal Barrier Dysfunction Pathway
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Fetal bovine serum (FBS) and DMEM basic were purchased from Gibco (NY, USA). Betaine hydrochloride (purity of 99%) was purchased from Juhua Group Co. (Zhejiang, China). Lipopolysaccharide (LPS, purity of 99%) and D-galactosamine (D-Gal, purity of 98%) were purchased from Sigma (St. Louis, USA).
Antibodies against TLR4 and (ZO)-1 were purchased from Proteintech (Hubei, China). Rabbit antirat/mouse occludin, β-actin, MyD88, TRAF6 and TNF-α antibodies were purchased from Cell Signaling Technology (Boston, USA). A goat anti-rabbit uorescent secondary antibody (IRDye800) was purchased from LI-COR Biosciences, Inc. (Lincoln, USA).
Antibodies against TLR4 and (ZO)-1 were purchased from Proteintech (Hubei, China). Rabbit antirat/mouse occludin, β-actin, MyD88, TRAF6 and TNF-α antibodies were purchased from Cell Signaling Technology (Boston, USA). A goat anti-rabbit uorescent secondary antibody (IRDye800) was purchased from LI-COR Biosciences, Inc. (Lincoln, USA).
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