Zerumbone

3T3-L1 fibroblasts (ATCC, Manassas, VA) or transduced cells were maintained and differentiated as described previously [57 (link)]. Briefly, 3T3-L1 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 25 mM glucose, 10% calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mM L-glutamine. The cytotoxic effects of zerumbone (> 98%, Z3902, Sigma Aldrich, St. Louis, MO) against 3T3-L1 cells were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay as previously described [58 (link)]. Following differentiation, cells were stained with Oil Red O (Sigma-Aldrich), dissolved in isopropanol, and quantified by measuring the optical absorbance at 500 nm. Images were collected using an Olympus microscope at a magnification of ×100 (Tokyo, Japan). To induce SIRT1 knockdown, 3T3-L1 cells were transduced with a lentiviral-based SIRT1 short hairpin RNA (shRNA; Genepharma, Shanghai, China). SIRT1 knockdown was confirmed by immunoblotting with an anti-SIRT1 antibody.

Zerumbone (ZER) was purchased from Sigma Aldrich (No.Z3902, St. Louis, MO, USA), dissolved in DMSO (Dimethyl sulfoxide). Bifendate (BIF) was purchased from Beijing Yuehe Pharmaceutical Factory (H11020980, Beijing, China). LPS was purchased from Beyotime Biotechnology (No.S1732, Shanghai, China). RPMI 1640 Medium was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Gaithersburg, MA, USA). The assay kits for ALT, AST, SOD, GSH-Px, GSH, and MDA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Bicinchoninic acid (BCA) assay kit and MTT were purchased from Solarbio (Beijing, China). Mouse IL-6 and TNF-α ELISA kits was purchased from ShangHai Haling Biological Technology Co., Ltd. (ShangHai, China). Rabbit anti-TLR4 antibody (#14358, 1:1000), rabbit anti-COX-2 antibody (#4842, 1:1000), rabbit anti-p-NF-κB p65 (Ser536) antibody (#3033, 1:1000), mouse anti-β-Actin antibody (#3700, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals and reagents were purchased from local firms.

Zerumbone (≥98% purity) and galantamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The target enzymes AChE, BChE, and their substrates, as well as elastase, trypsin, chymotrypsin, and their substrates were also obtained from Sigma-Aldrich (St. Louis, MO, USA). A BACE1 assay kit was obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

At two weeks after the injection of STZ, a group of eight rats were dosed by oral gavage once per day for three consecutive months with zerumbone (≥98%; Sigma-Aldrich, Inc.) doses of 20 or 40 mg/kg in a volume of 1.5 mL/kg distilled water. The dosage regime was selected based on a previous report demonstrating that zerumbone at these dosages is potentially effective in improving diabetic nephropathy in STZ-diabetic rats [15 (link)]. Another group of STZ-diabetic rats was treated orally for three months with 100 mg/kg/day fenofibric acid (purity ≥98.0%; Sigma-Aldrich, Inc.), which was based on studies which reported this can have beneficial effects on DR rats [20 (link)]. A vehicle-treated group of normal rats and STZ-diabetic rats were treated with 1.5 mL/kg distilled water only over the same treatment period. Animals had free access to standard rat diet (Harlan Teklad, Madison, WI, USA; Cat. No. 2018) and water throughout the entire period.
At the end of the three-month treatment, the rats were weighed, fasted overnight and anesthetized using an intraperitoneal injection of sodium pentobarbital (60 mg/kg). While under anesthesia, they were painlessly sacrificed and blood was collected from the abdominal aorta of each animal into heparin sample bottles. Rat eyes from each group were removed and the retinae were isolated.

Stock solutions containing ZER (zerumbone, Sigma-Aldrich, St. Louis, MO, USA) were prepared prior to each experiment. ZER crystals were dissolved in 1% dimethyl sulfoxide (DMSO—Sigma-Aldrich, St. Louis, MO, USA) to achieve a final concentration of between 4 and 1024 µg/mL [24 (link)].