Matrine

Matrine (purity >98%) and fluoxetine were bought from Sigma and dissolved in 1% DMSO in 0.9% saline. LY294002 was purchased from Tocris, rapamycin was purchased from Target Mol, and they were dissolved in 1% DMSO in ACSF. The doses of Matrine (15, 30, 60, and 120 mg/kg), fluoxetine (20 mg/kg), LY294002 (10 nmol/site), and rapamycin (0.2 nmol/site) were chosen based on previous reports (Jiang et al., 2015 (link); Gong et al., 2016 (link); Ludka et al., 2016 (link); Jiang et al., 2017 (link)). Matrine and fluoxetine were i.p. injected in a volume of 10 mL/kg. LY294002 and rapamycin were intracerebroventricularly (i.c.v.) infused.

Matrine (purity >98%), MTT, Triton X-100 and dimethyl sulfoxide (DMSO) were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-phosphorylated (p)-Akt (cat. no. 66444-1-1g), anti-Akt (cat. no. 55230-1-AP), anti-PTEN (cat. no. 22034-1-AP), anti-E-cadherin (cat. no. 20874-1-AP) and anti-vimentin (cat. no. 10366-1-AP) antibodies were all purchased from Wuhan Sanying Biotechnology (Wuhan, China). Anti-MMP2 (cat. no. 40994), anti-MMP9 (cat. no. 3852), anti-Snail (cat. no. 3879), anti-Slug (cat. no. 9585) antibodies and DyLight™ 594 Phalloidin (cat. no. 12877) were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-β-actin (cat. no. TA-09) antibody was purchased from OriGene Technologies, Inc. (Beijing, China). Radio immunoprecipitation assay (RIPA) lysis buffer and DAPI staining solution were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).

Anti-Bcl-2, anti-p-JAK1, anti-JAK1, anti-p-STAT3, anti-STAT3, anti-β-actin, anti-cleaved caspase-3, anti-caspase-3 and HRP-conjugated anti-rabbit antibodies and the IL-6 kit were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Matrine (≥99% purity by HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Afatinib was purchased from SYNkinase (San Diego, CA, USA).

An experimental animal model of DbCM was induced as described previously in 7-week oldmale and female (1:1) Sprague-Dawley rats (weight, 180–220 g) provided by Animal Experimental Center of Xi'an Jiaotong University (10 (link)). The rats were raised in independent cages in a 12-h light/dark cycle, 25±2°C and 50% humidity. Animals had access to standard chow and clean water ad libitum. Intraperitoneal injection of streptozotocin (STZ; 65 mg/kg body weight) was employed to induce diabetes in rats. Prior to the induction of diabetes, rats were administered with matrine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) orally at a dose of 300 mg/body weight per day for 10 days. Further details of the treatments, and the treatment groups, are provided in Table I. In the present study, all animal experimental procedures were approved by the Experimental Animal Use Ethics Committee of Xi'an Jiaotong University, and were performed in accordance with guidelines for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research.

Morphine sulfate and methadone hydrochloride were purchased from the Darou Pakhsh Pharmaceutical Company (Tehran, Iran). Naloxone hydrochloride and matrine were obtained from the Sigma-Aldrich and Ningxia Zijinghua companies respectively. For dilution, all drugs and extracts were dissolved in normal saline. The drugs and extracts were prepared immediately before use and injected subcutaneously in a volume of 5 ml/kg. The doses of total extract, alkaloid fraction, matrine and methadone were as follows. Total extract: 100, 200 and 300 mg/kg; alkaloid fraction: 5, 10 and 20 mg/kg; matrine: 5, 15 and 30 mg/kg; methadone: 10 mg/kg.