2.1 Animals
Male Sprague–Dawley rats (weighing 180-200 g) were obtained from the Laboratory Animal Center of Fujian Medical University (license no. SCXK (Min) 2016-0002, Fujian, China). All animals were housed in a temperature- and humidity-controlled environment on a 12-hour light/dark cycle with free access to food and water. Before the experimental procedures, animals were randomly allocated into different groups. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fujian Medical University (Fujian, China) in accordance with the animal care guidelines of NIH (Bethesda, MD, USA). All animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020).
2.2 Drugs
KM (molecular formula: C20H22N2O; molecular weight: 306.1804; PubChem CID: 91895267; purity >99%, HPLC) was isolated from Gelsemium elegans Benth . via pH-zone-refining countercurrent chromatography, as described previously (Su et al., 2011); gabapentin (1201301) and streptozotocin (STZ; S0130) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
2.3 Induction ofdiabetic neuropathic pain and assessment
DNP can be readily induced by the systemic administration of STZ (Schnedl et al., 1994). In brief, rats were fasted overnight and then administered an intraperitoneal (i.p.) injection of 70 mg/kg fresh STZ dissolved in 0.1 M citrate buffer solution (pH 4.5) (Courteix et al., 1993). Animals in the control group received an equal volume of citrate buffer. The blood glucose levels from the tail vein were assessed using a One Touch Ultra Easy glucometer (Life Scan Inc., Milpitas, CA) 72 hours after STZ injection. Rats with blood glucose levels ≥16.7 mmol/L were considered diabetic and subsequently included in the study (Jiang et al., 2019). Diabetic rats with neuropathic pain were defined according to the ratio of the 21st-day mechanical withdrawal threshold (MWT) to the baseline MWT < 0.8 as determined by a von Frey test (Fox et al., 1999).