| Popis: |
Objective Investigating the effect of miR-135a-5p in regulating of CXCL12 on the formation of morphine tolerance in C57BL/6J mice. Methods Totally 64 mice were randomly divided into the following groups: control (NS) group, morphine tolerance (MT) group, CXCL12 inhibitor (CXCL12-siRNA) group, inhibitor negative control (NC-siRNA) group, miR-135a-5p agonist (miR-agomir) group, agonist negative control (miR-NC) group, miR-135a-5p agonist+CXCL12 overexpression (miR-agomir+LV-CXCL12) group, and miR-135a-5p agonist+CXCL12 over-expression negative control (miR-agomir+LV-control) group. Morphine tolerance models were established by subcutaneous injection. The tail-flick test was used to measure the thermal tail-flick latency (TL) before and after drug administration in each group. After modeling, the mice were euthanized under anesthesia, and L4~L5 spinal cord tissues were collected. RT-qPCR method was used to detect the expression of miR-135a-5p and CXCL12 mRNA, Western blot was used to detect the expression of CXCL12 proteins, and a dual luciferase reporter gene assay was used to detect the targeting relationship between miR-135a-5p and CXCL12. Results 1)The morphine-tolerant mouse model demonstrated a significant increase in CXCL12 mRNA and protein expression(P＜0.05) and a significant downregulation of miR-135a-5p (P＜0.05) compared to the NS group. 2)Silencing Cxcl12 and over-expression of miR-135a-5p both increased the thermal pain threshold in morphine-tolerant mice, significantly slowed down the occurrence and progression of morphine tolerance. 3)Compared to the miR-NC group, the miR-agomir group showed a decrease in both CXCL12 mRNA and protein expression(P＜0.05). 4)Dual-luciferase reporter gene experiments demonstrated that miR-135a-5p was targeted at Cxcl12. 5)Over-expression of CXCL12 reversed the thermal pain-protective effect of miR-135a-5p in morphine-tolerant mice. Conclusions It is demonstrated by the results above that miR-135a-5p alleviates the formation of morphine tolerance by suppressing the expression of CXCL12. |
