Ab100768

The cell culture supernatant of transfected PC12 cells underwent 5 μg/mL LPS treatment for 12 h were collected by centrifugation at 12,000xg for 10 min at 4°C and the levels of tumor necrosis factor‐α (TNF‐α) (ab100785), interleukin‐6 (IL‐6) (ab234570), IL‐1β (ab100768) (Abcam) and IL‐8 (Cat#RAB1147, Sigma‐Aldrich) were detected by using the corresponding commercial ELISA kits according to the manufacturer's instructions.

Inflammation markers including TNF-α (ab46070), IL-1β (ab100768), IL-6 (ab100772), IL-10 (ab133112), and ICAM-1 (CD54) (ab100763) ELISA kits were obtained from Abcam Co., Eugene, OR, USA. As for the apoptotic signaling markers, cleaved caspase-3 (KHO1091) was purchased from Thermo Fisher Scientific Inc. Waltham, MA, USA, and caspase-9 (LS-F4141) was acquired from Biocompare, San Francisco, CA, USA. These markers were measured according to the manufacturer’s instructions using a SpectraMax i3X (Molecular devices San Jose, CA, USA) microplate reader.

Sixteen 18-week-old GK rats were used in this study. The rats were randomly divided into two groups of eight rats each. The rats in the BD3526 group received 2 ml of 50 mg/ml lyophilized BD3526 metabolites daily by gavage, whereas the rats in the control group received 2 ml of 50 mg/ml skim milk powder daily by gavage. The animals were individually caged with free access to a normal chewing bar and drinking water. The animals were kept alone in cages and allowed to eat freely. All animals were kept at 25°C with a 12-h light/dark cycle. The gavage experiments lasted for 4 weeks.
All rats were fasted for 8 h prior to administration of a glucose tolerance test. During the experiment, each rat was intraperitoneally injected with 5 g/kg of glucose solution. Blood glucose was monitored at 0, 15, 30, 60, and 120 min. Serum insulin was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, ab100578) after the rats were killed. IL-1β was measured using an ELISA kit (Abcam, ab100768). The specific experimental methods were conducted in strict accordance with the manufacturer's instructions.
The experiments were performed in strict accordance with the experimental protocol. After dissection of the rats, the intestinal samples were immediately frozen at −80°C for long-term preservation prior to RNA extraction and cytokine detection.