Targeting Forkhead box O1-aquaporin 5 axis mitigates neuropathic pain in a CCI rat model through inhibiting astrocytic and microglial activation

Figures & data

Figure 1. LPS triggered the FoxO1-AQP5 pathway in microglia and astrocytes. (a) and (b) WB examined the expression of FoxO1 and the downstream AQP5 and ERK/p38 MAPK signaling pathways in microglia and astrocytes induced with and without LPS. Data are presented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 (vs. Con group).

Figure 2. Inhibition of FoxO1 or AQP5 abated LPS-induced production of inflammatory factors in cells.

si-FoxO1, si-AQP5 and si-NC were transfected into LPS-induced microglia and astrocytes and cultured for 24 hours. A: WB evaluated the profiles of FoxO1, AQP5 and the ERK/p38 MAPK pathway in microglia and astrocytes. B-C: The contents of IL-6, IL-1β and TNF-α in microglia and astrocytes were monitored by ELISA. D-E: WB checked the protein expression of iNOS, COX2, and pNF-κB in microglia and astrocytes. *P < 0.05, **P < 0.01, ***P < 0.001 (vs. LPS+si-NC group).

Figure 3. CCI rats experienced NP, accompanied by increased inflammatory factors in spinal cord tissues. (a) and (b) PWT and PWL in CCI rats 1, 3, 7, 14 and 21 days after CCI. (c-f) IHC was employed to analyze iBA1-, GFAP-, and Caspase3-positive cells in microglia and astrocytes. (g) The expression of neutron IL-6, IL-1β and TNF-α in spinal cord tissues of CCI rats at 1, 7, and 21 days after operation was gauged by ELISA. (h) The levels of iNOS, COX2 and p-NF-κB in the spinal cord of CCI rats were measured by WB at 1, 7, and 21 days after the operation. Data are expressed as mean ±SEM (n = 5). **P < 0.01, ***P < 0.001 (vs Sham group).

Figure 4. FoxO1-AQP5 was up-regulated in spinal cord tissues of CCI rats. (a) WB assayed the expression of FoxO1, AQP5, p-ERK and p-p38 in the spinal cord tissue of CCI rats at 1, 7, and 21 days postoperatively. Data are expressed as mean ± SEM (n = 5). ***P < 0.001 (vs. Sham group).

Figure 5. Inhibition of FoxO1 attenuated nerve pain and inflammatory cytokine production in the spinal cord in CCI rats.

Resveratrol (10 μL), a Sirt1 agonist, was delivered intrathecally via microinjector into the brains of CCI rats on the first day after CCI surgery. (a) The protein profiles of FoxO1, AQP5, p-ERK and p-p38 in spinal cord tissues of CCI rats were measured with WB on the third day after CCI surgery. (b) and (c) PWT and PWL of CCI rats at 1, 3, 7, 14 and 21 days after CCI surgery. (d)-(h) IHC or immunofluorescence were performed to determine apoptosis of IBA1, GFAP in the spinal cord. Caspase3-positive cells and neurons in CCI rats on the third day after CCI. Scale bar = 50 μm. (i) The levels of IL-6, IL-1β and TNF-α in spinal cord tissues of CCI rats were analyzed by ELISA on the third day after surgery. (j) The profiles of iNOS, COX2, and p-NF-κB in spinal cord tissues of CCI rats were determined by WB. *P < 0.05, ***P < 0.001 (vs. Sham group), &&&&P < 0.001 (vs. CCI group).

Figure 6. The mechanism’s diagram.

FoxO1-AQP5 axis becomes upregulated in LPS-activated microglia and astrocyte, and enhances inflammatory cytokines expression.