Effects of targeted silencing ANX A3 on morphine analgesic tolerance and inflammation in rats / 解放军医学杂志

ABSTRACT

Objective: To investigate the effects and possible mechanisms of targeted silencing ANXA3 on morphine analgesia and inflammation in rats. Methods: Male SD rats were injected intrathecally with 15 μg morphine for 7 days to establish a chronic morphine analgesic tolerance model. Thirty rats were randomly divided into morphine group, negative control group and ANXA3 siRNA group. The maximum possible percentage of analgesic effect (%MPE) was calculated by measuring the paw withdrawal thermal latency (PWL) of rats in each group at the 1st, 3rd, 5th and 7th day of the experiment. On the 7th day, rats were anesthetized and sacrificed after behavioral test, and the spinal cord enlargement of rats in each group were collected. The mRNA and protein levels of ANXA3 were detected by RT-PCR and Western blotting, and of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined by RT-PCR and ELISA kits. In addition, Western blotting was used to evaluate the phosphorylation levels of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). Results: After injection of morphine, no significant difference existed in %MPE between the negative control group and the morphine group, while the %MPE was obviously higher in ANXA3 siRNA group than in the negative control group (P<0.05) on the 3rd day (78.27% ± 8.50% vs. 71.75% ± 5.98%), 5th day (64.37% ± 25.94% vs. 43.56% ± 2.69%) and 7th day (39.96% ± 3.27% vs. 13.81% ± 1.25%) after morphine injection. The relative expression levels of ANXA3 mRNA and protein showed no significant difference between morphine group and the negative control group, but was lower in the relative expression levels of ANXA3 mRNA and protein in ANXA3 siRNA group than in the negative control group (0.47 ± 0.09 vs. 1.14 ± 0.08, 0.38 ± 0.05 vs. 1.15 ± 0.21, P<0.05). Compared with morphine group, no significant difference existed in TNF-α, IL-1β and IL-6 protein concentrations in the negative control group. The protein concentrations of TNF-ct, IL-1β and IL-6 were lower in ANXA3 siRNA group than in the negative control group [(210.15 ± 19.86) pg/g vs. (394.93 ± 54.18) pg/g], [(313.42 ± 42.19) pg/g vs. (534.26 ± 49.78) pg/g] and [(434.68 ± 49.15) pg/g vs. (794.92 ± 98.32) pg/g, P<0.05]. Compared with morphine group, no significant difference existed in the relative expressive levels of p-ERK and p-JNK in the negative control group. The relative expressive levels of p-ERK and p-JNK proteins were obviously lower in ANXA3 siRNA group than in the negative control group (0.55 ± 0.04 vs. 1.07 ± 0.12 and 0.68 ± 0.07 vs. 1.15 ± 0.04, respectively. P<0.05). Conclusion Targeted silencing ANXA3 may enhance the morphine analgesic effect and reduce inflammatory response by regulating the phosphorylation level of ERK and JNK signaling pathway.