RESUMEN
Objective:To investigate the role and mechanism of metformin in algesia of rats with type 2 diabetic neuropathic pain (DNP).Methods:Eighty sprague-dawley rats were randomly divided into normal control group ( n=15) and high-fat and high-glucose group ( n=65); normal diet and high-fat and high-sugar diet were given, respectively; before and 8 weeks after feeding, the body mass of rats and fasting blood glucose level were recorded, fasting insulin level was detected by ELISA, and insulin sensitivity index (ISI) was calculated. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) 8 weeks after feeding (baseline values) were measured in the high-fat and high-glucose group; after 12 h of fasting, intraperitoneal injection of streptozotocin (STZ, 35 mg/kg) was performed; 3 d after fasting, blood glucose was measured; 14 d after STZ injection, body mass was recorded and MWT and TWL were measured again: when MWT and TWL were ≤85% baseline values, it was defined that DNP model was successfully established ( n=45); and the left were into the diabetic painless group ( n=15). The rats with successful DNP were randomly divided into DNP group, DNP+vehicle group and DNP+metformin group ( n=15); 14 d after STZ injection, rats in the DNP+metformin group were given intraperitoneal injection of metformin (200 mg/kg) once daily for 14 consecutive d; DNP group did not accept any treatment, and rats in DNP+vehicle group were intraperitoneally injected with same amount of normal saline. MWT and TWL of all rats were measured 14 d after STZ injection, and 3, 7, 14 and 21 d after metformin injection. The expression levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α were detected by ELISA 7, 14 and 21 d after metformin injection. The fluorescence intensity of ionized calcium binding adaptor molecule-1 (Iba-1) in the spinal cord was detected by immunofluorescence staining, and the expression levels of Toll-like receptor 4 (TLR4), nuclear transcription factor (NF)-κB, phosphorylated (p)-NF-κB, adenylate activated protein kinase (AMPK), p-AMPK, and peroxisome proliferator activated receptor-γ coactivator (PGC)-1α in the spinal cord were detected by Western blotting 21 d after metformin injection. Results:(1) After 8 weeks of feeding, the body mass of rats in the high-fat and high-glucose group was significantly higher than that in the normal control group ( P<0.05); and the body mass of rats in the high-fat and high-glucose group was statistically lower than that in the normal control group 14 d after STZ injection ( P<0.05). Three d after STZ injection, the blood glucose level in high-fat and high-glucose group was significantly higher than that in normal control group ( P<0.05). After 8 weeks of feeding, the insulin level of high-fat and high-glucose group was statistically higher than that of normal control group, and the ISI in the high-fat and high-glucose group was significantly decreased as compared with that in the normal control group ( P<0.05). (2) As compared with those in the normal control group and diabetic painless group, MWT and TWL of DNP group and DNP+vehicle group were significantly decreased at each time point ( P<0.05). Three, 7, 14 and 21 d after metformin injection, MWT and TWL in DNP+metformin group were significantly increased as compared with those in DNP group and DNP+vehicle group ( P<0.05). (3) Seven, 14, and 21 d after metformin injection, the levels of IL-6, IL-1β and TNF-α in the spinal cord of rats in the DNP group and DNP+vehicle group were significantly increased as compared with those in the normal control group and diabetic painless group ( P<0.05); as compared with those in the DNP group and DNP+vehicle group, the levels of IL-6, IL-1β and TNF-α in the spinal cord of DNP+metformin group were significantly decreased ( P<0.05). (4) As compared with normal control group and diabetic painless group, the fluorescence intensity of Iba-1 and number of Iba-1 positive cells in the spinal cord tissues of DNP group and DNP+vehicle group were significantly increased ( P<0.05); while the fluorescence intensity of Iba-1 and number of Iba-1 positive cells in spinal cord tissues of DNP+metformin group were significantly decreased as compared with those in the DNP group and DNP+ vehicle group ( P<0.05). (5) As compared with those in the normal control group and diabetic painless group, the TLR4 and p-NF-κB protein expressions and p-NF-κB/NF-κB values in the spinal cord tissues of DNP group and DNP+vehicle group were significantly increased ( P<0.05); while those in the spinal cord tissues of DNP+metformin group were significantly decreased as compared with those in the DNP group and DNP+vehicle group ( P<0.05). As compared with those in the normal control group and diabetic painless group, the PGC-1α protein expression and p-AMPK/AMPK values in the spinal cord tissues of DNP group and DNP+vehicle group were significantly decreased ( P<0.05); while those in the spinal cord tissues of DNP+metformin group were significantly increased as compared with those in the DNP group and DNP+vehicle group ( P<0.05). Conclusion:Metformin, by activating AMPK/PGC-1α signaling pathway, may inhibit the TLR4/NF-κB expression, reduce the activation of microglia and the expressions of pro-inflammatory factors, and thus alleviate DNP.