2.1.1 Animals
The animal experiment was approved by the Guiding Prin-ciples for the Care and Use of Laboratory Animals published by the National Science and Technology Commission of China. Forty-five male Sprague-Dawley rats (7–8 weeks old, 200–240 g) were purchased from Liaoning Changsheng Biotechnology Co. LTD. (Beijing, China) (SCXK (Liaoning) 2020-0001) and housed in 15 cages in a facility with a 12-h/12-h artificial light–dark cycle, a temperature of 22–24°C, and a humidity level of 40%. The cage bedding was changed daily.
2.1.2 Induction of diabetes mellitus and drug treatment
Fifteen rats were randomly assigned to the control group and fed a standard diet. The remaining rats were fed a high-carbohydrate/high-fat diet for 6 weeks and were then intraperitoneally injected with STZ (Sigma-Aldrich, St Louis, MO, USA) (30 mg/kg). After 1 week, rats with a fasting glucose level ≥ 16.7 mmol/L were considered to be diabetic and continued to be fed a high-carbohydrate/high-fat diet. The diabetic rats were randomly divided into a DPN group and an ALA group, with 15 rats in each group. The rats in the ALA group were intragastrically administered an ALA suspension (60 mg/kg/day) for 12 weeks. The rats in the control group and DPN group were intragastrically administered an equal volume of distilled water every day for 12 weeks. After the treatment, the motor nerve conduction velocity and mechanical pain threshold of the diabetic rats in the ALA group and the DPN group were observed to be reduced to varying degrees: the rats in both the ALA group and the DPN group were therefore considered to have DPN15.
2.1.3 Blood glucose level and weight analysis
The diabetic rats having been identified, fasting glucose levels and body weight were measured and recorded every 4 weeks.
2.1.4 Mechanical pain threshold measurement
The mechanical pain threshold was measured by using an electronic von Frey (type 2390, 90 g probe, 0.8 mm diameter; IITC Life Science Inc. Woodland Hills, CA, USA) after the 12-week treatment period. The rats were placed on wire mesh, covered with a glass box, and allowed to acclimate to their surroundings for 15 min. A stainless-steel filament was applied vertically to the plantar surface of the hind paw. Rapid paw withdrawal by the rats was considered to be a positive response, and the force that elicited the response was recorded (grams). Paw withdrawal caused by the physical activity of the rats was considered to be a negative response. Measurements were taken at intervals of 3–5 min. The response of each paw was assessed three times.
2.1.5 Motor Nerve conduction velocity
After the 12-week treatment period, the MNCV of the rats was measured. The rats were anesthetized with isoflurane (3% volume) before being fixed in the prone position. The sciatic nerve was exposed through an incision, and the skin between the biceps femoris and semitendinous muscles was separated from the muscles on the experimental side. Electrodes were implanted in two sites of the sciatic nerve notch approximately 10 mm apart. Platinum wire electrodes were placed directly under the sciatic nerve trunk in the right leg for stimulation, after which the data were recorded. The sciatic nerve was stimulated with a single square-wave pulse (intensity: 1.2 V, width: 1 ms) using an experimental system (BL-420s, Taimeng, Sichuan, China). The MNCV was measured and calculated as follows: MNCV = D/L (m/s).
2.1.6 Immunofluorescence assay
For fluorescence staining, sciatic nerve and dorsal root ganglions sections underwent routine deparaffinization and rehydration and were then subjected to antigen retrieval by incubation with citric acid (0.01 mol/L, pH 6.0) in a microwave oven at approximately 90°C for 20 min. Next, the sections were blocked with non-immunoreactive serum and then incubated with antibodies against KIF5A (Abcam,USA,1:500), DYNC1I2 (Proteintech,USA,1:100), p-AMPK(Immunoway,USA,1:100), p-CREB(Abcam,USA,1:100) at 4°C overnight and subsequently incubated with secondary antibodies at 37°C for 1 h. After being stained, sections were incubated with DAPI for nuclear staining and sealed for photography using an inverted fluorescence microscope (CTS SP8, Leica, Germany).
2.1.7 Western blotting for the detection of related proteins in tissues
Sciatic nerve and dorsal root ganglion tissues were homogenized in RIPA buffer, and protein samples were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4°C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands.
2.2.1 Cell culture assays in vitro
NSC34 cells were maintained in culture media with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in an incubator with a humidified atmosphere and 5% CO2. NSC34 cells were divided into the Control group, DPN group, ALA group. Except the Control group, the other groups were added 50mmol·L− 1 glucose and 250µmol·L− 1 palmitic acid. ALA group were given 250µM ALA16. Relevant indexes were detected 24h after administration.
2.2.2 Immunofluorescence analysis
The dried and sterilized glass slides were placed on a 24-well petri dish, the cells were spread evenly on the glass slides in the wells, and 4% paraformaldehyde was added and incubated for 20 min at room temperature. Then 0.25% Triton X-100 was added and incubated for 15 min for permeabilization, and 10% normal goat serum was added and incubated for 30 min for blocking. Subsequently, the cells were incubated with the primary antibody KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) overnight at 4°C, followed by the relevant secondary antibody at 37°C in the dark for 1 h for fluorescence staining. DAPI was used for nuclear staining. β-tubulin Ⅲ was used to measure and characterize the length of neurons and axons. After immunofluorescence staining, 15 different neurons were randomly selected from each group of cells, and the axonal length of the neurons was detected using Image J.
2.2.3 Western blotting for the detection of related proteins in cells
NSC34C cells were homogenized in RIPA buffer, and protein samples were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4°C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands.