*2.2. In Vivo Analysis*

#### 2.2.1. Experimental Animals and Grouping

The protocol was approved by the Ethical Committee and the Laboratory Animal Center of China medical university, Shenyang, Liaoning, China. Eighteen 10-week-old specific pathogen-free (SPF) male mice from the Jackson Laboratory (Bar Harbor, ME, USA) were maintained at a constant temperature (22–25 ◦C) in a 12 h light/dark cycle and fed with a standard laboratory diet and water. Six non-diabetic BLKS/jdb/m male mice were selected as the control group (db/m, *n* = 6, weight: 18.4–10.7 g). Twelve spontaneous diabetic BLKS/jdb/db male mice were randomly separated into the diabetic group (db/db, *n* = 6, weight: 35.9–40.9 g) and the curcumin-treated diabetic group (db/db + C, *n* = 6, weight: 35.7–40.3 g). Each group was kept in the same cage. The curcumin-treated group was intragastrically given a dose of 200 mg/kg/d curcumin for 10 weeks, and the control group and diabetic group were given equivalent volumes of solution (normal saline + 0.1% DMSO) for 10 weeks. Curcumin was dissolved in DMSO, then diluted with normal saline to make the content of DMSO 0.1% and administered daily by gavage. All groups were sacrificed at 20 weeks, and mandibles (3 mm × 3 mm) were harvested and fixed in 4% paraformaldehyde solution for 7 days at 4 ◦C in the dark. The Block sections were sequentially dehydrated in ascending serial concentrations of ethanol, cleared with xylene twice, and embedded in poly(methyl methacrylate). Serial sections with a thickness of 70–80 μm were cut parallel to the coronal plane and prepared for fluorescence microscopy and histological staining. All analyses of in vivo experiments were made by an experienced pathologist (blind to the treatments of the mice) in order to characterize any changes.
