Research Paper Volume 12, Issue 11 pp 11004—11024

Figure 5. cJun-induced Mlxipl upregulation protectively inhibited inflammation in the SDH and improved mechanical allodynia. (A) The schematic illustrates the timing of the main experimental process. (B and D–F) QPCR (B) and western blot (D) were performed before and after surgery. CJun or p-cJun in the ipsilateral SDH was upregulated after SNI surgery. Quantification of the western blot (E-F). N = 5. ***P < 0.001, **P < 0.01, *P < 0.05 vs. SNI BL Ipsi; ^$$$P < 0.001, ^$$P < 0.01, ^$P < 0.05 vs. SNI BL Contra. (C and G–J) QPCR(C), western blot (G) and immunofluorescence (I) were performed at day 7 after Sham or SNI surgery. CJun or p-cJun was significantly upregulated in the ipsilateral SDH. cJun was co-localized with Mlxipl (white arrows). Quantification of the western blot (H) and immunofluorescence (J). N = 5. ^$$$P < 0.001, ^$$P < 0.01 vs. SNI Contra; ^###P < 0.001, ^##P < 0.01 vs. Sham Ipsi. (K–O) Intrathecal injections of cJun small interfering RNA (sicJun) or lentivirus encoding cJun (LvcJun) were performed at day 7 after SNI surgery. The injections were carried out once a day for 7 consecutive days and then cJun expression was detected by qPCR (K) and western blot (L). Knockdown of cJun inhibited the expression of Mlxipl and p-cJun. Overexpression of cJun promoted the expression of Mlxipl and p-cJun (L-O). Quantification of the western blots (M–O). N = 5. ^$$$P < 0.001, ^$$P < 0.01 vs. vehicle; ^###P < 0.001, ^##P < 0.01 vs. sicJun+OeMlxipl; ***P < 0.001, ^**P < 0.01 vs. LvcJun+shMlxipl. (P–S) The effect of cJun on neuroinflammation (P–R) and mechanical allodynia (S), with or without the pre-microinjection of OeMlxipl or shMlxipl. N = 5. ^$$$P < 0.001, ^$$P < 0.01 vs. vehicle; ^###P < 0.001, ^##P < 0.01 vs. sicJun+OeMlxipl; ***P < 0.001, ^**P < 0.01 vs. LvcJun+shMlxipl. BL, baseline (before surgery); ipsi, ipsilateral; Contra, contralateral; SNI, spare nerve injury; SDH, spinal dorsal horn.