Objective To investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in spinal dorsal horn astrocytes in morphine-tolerant mice.
Methods Twenty-four SPF male C57b1/6 mice, 6-8 weeks old, weighing 22-25 g, were divided into four groups by random number table method: normal saline group (group C), morphine group (group M), dichloroacetic acid (DCA) group (group D) and morphine + DCA group (group MD), 6 mice in each group. Group C was intrathecally injected with normal saline 10 μl, twice a day for 5 consecutive days; groups M, D and MD were intrathecally injected with normal saline containing morphine 10 μg, DCA 200 μg and morphine 10 μg + DCA 200 μg, twice a day for 5 consecutive days. The tail-flick latency (TFL) of mice was detected before the first administration and 1 hour after the first daily administration. The mice were sacrificed after the last administration, and the protein contents of PDK4, phosphorylated pyruvate dehydrogenase (p-PDH), p-S293-PDH, and p-S300-PDH in the dorsal horn of the spinal cord were detected by Western blot, and immunofluorescence histochemistry was used to detect the co-expression of PDK4 and astrocyte marker glial fibrillary acidic protein (GFAP) in the dorsal horn of spinal cord.
Results Compared with before the first administration, TFL was significantly prolonged in groups M and MD 1 hour after the first daily administration on day 1-5 (P < 0.05). Compared with group C, TFL was significantly prolonged 1 hour after the first daily administration on day 1-5 (P<0.05), and the protein contents of PDK4, p-S300-PDH, and p-S293-PDH in the dorsal horn of spinal cord were significantly increased in group M (P < 0.05); the TFL was significantly prolonged 1 hour after the first daily administration on day 1-5 (P < 0.05), and the protein content of p-S293-PDH in the spinal dorsal horn was significantly increased in group MD (P < 0.05). Compared with group M, the TFL was significantly shortened 1 hour after the first daily administration on day 1-5 in group D (P < 0.05); the TFL was significantly prolonged 1 hour after the first daily administration on day 3-5 in group MD (P < 0.05); the protein contents of PDK4, p-S293-PDH, and p-S300-PDH were significantly decreased in groups D and MD (P < 0.05). Compared with group D, the TFL was significantly prolonged 1 hour after the first daily administration on day 1-5 in group MD (P < 0.05), and the protein content of p-S293-PDH in the spinal dorsal horn was significantly increased (P < 0.05). PDK4 protein was expressed in spinal dorsal horn cells and co-expressed with the astrocyte marker GFAP.
Conclusion The content of PDK4 protein is increased in spinal dorsal horn astrocytes of morphine-tolerant mice, and the PDK4 inhibitor DCA inhibites the expression of downstream p-S293-PDH and p-S300-PDH proteins. |
