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).