Paeonol alleviates neuropathic pain by modulating microglial M1 and M2 polarization via the RhoA/p38MAPK signaling pathway

Abstract

Background: This study aimed to investigate the potential mechanism of paeonol in the treatment of neuropathic pain.

Methods: Relevant mechanisms were explored through microglial pseudotime analysis and the use of specific inhibitors in cell experiments. In animal experiments, 32 SD rats were randomly divided into the sham operation group, the chronic constrictive injury (CCI) group, the ibuprofen group, and the paeonol group. We performed behavioral testing, ELISA, PCR, Western blotting, immunohistochemistry, and immunofluorescence analysis.

Results: The pseudotime analysis of microglia found that RhoA, Rock1, and p38MAPK were highly expressed in activated microglia, and the expression patterns of these genes were consistent with the expression trends of the M1 markers CD32 and CD86. Paeonol decreased the levels of M1 markers (IL1β, iNOS, CD32, IL6) and increased the levels of M2 markers (IL10, CD206, ARG-1) in LPS-induced microglia. The expression of iNOS, IL1β, RhoA, and Rock1 was significantly increased in LPS-treated microglia, while paeonol decreased the expression of these proteins. Thermal hyperalgesia occurred after CCI surgery, and paeonol provided relief. In addition, paeonol decreased the levels of IL1β and IL8 and increased the levels of IL4 and TGF-β in the serum of CCI rats. Paeonol decreased expression levels of M1 markers and increased expression levels of M2 markers in the spinal cord. Paeonol decreased IBA-1, IL1β, RhoA, RhoA-GTP, COX2, Rock1, and p-p38MAPK levels in the spinal dorsal horn.

Conclusion: Paeonol relieves neuropathic pain by modulating microglial M1 and M2 phenotypes through the RhoA/p38 MAPK pathway.

Keywords: CCI; Paeonol; Pseudotime; RhoA; p38MAPK; single-cell sequencing.

FIGURE 1

Expression levels of microglia M1, M2 markers and RhoA/p38MAPK expression levels in spinal cord after peripheral nerve injury. (A) Cell types from single‐cell sequencing data. Red dots represent microglia. (B) UAMP plot showing microglia grouped into 9 clusters. (C) UAMP plot of expression levels of microglial activation marker Aif1 (IBA‐1). (D, E) UAMP plot of expression levels of M1 markers Fcgr2b and Cd86 in microglial cells. (F, G) UAMP plot of the expression levels of M2 markers Mrc1 and Clec10a in microglial cells. Purple represents high expression, white represents low expression. (H–K) Violin plot of expression of Rhoa, Rock1, Rock2, and p38MAPK (MAPK14) in microglia.

FIGURE 2

Pseudotime analysis of microglia. (A) Differentiation trajectories of microglia. Display by cell type. (B) Differentiation trajectories of microglia. The abscissa is the pseudotime series. From left to right are microglia from the initial state to the final state. There are 3 branch nodes, divided into 7 states. (C) Microglia initial state to final state is shown. The brighter the blue, the “older” the cell state is in pseudotime. (D) Changes in the expression levels of RhoA, Rock1, Rock2, and MAPK14 in the pseudotime differentiation trajectories of microglia. (E) Clustering and expression trends of RhoA, Rock1, Rock2, and MAPK14 with the M1 markers Fcr2b and CD86 and the M2 markers Mrc1 and Clec10a.

FIGURE 3

Flow cytometry and immunohistochemistry for M1 and M2 markers. (A–D) Three‐dimensional map of RhoA, Rock1, Rock2, and p38MAPK interactions with paeonol. (E) Determination of the effect of paeonol on the viability of GMI‐R1 cells by the CCK‐8 method. (F) Determination of the proportion of M1 microglia (CD32) by flow cytometry. (G) Determination of the proportion of M2 microglia (CD206) by flow cytometry. (H–J) Immunofluorescence results of CD32 and CD206. #Compared with the control group. *Compared with the LPS group. n = 3.

FIGURE 4

qRT–PCR and Western blot analysis of GMI‐R1 cells. (A) qRT–PCR results of the M1 phenotype proinflammatory factors TNF‐α, IL1β, IL6, iNOS, and CD32. (B) qRT–PCR results of the M2 phenotype anti‐inflammatory factors IL10 and ARG‐1. (C and D) Western blot results of Rhoa/p38MAPK pathway. #Compared with the control group. *Compared with the LPS group. n = 3.

FIGURE 5

H&E staining, behavioral tests, serum inflammatory factor levels, and sciatic nerve immunohistochemical results. (A) Results of hot plate experiments (n = 8). (B–F) Serum inflammatory factor levels (n = 3). #Compared with the Sham group. *Compared with the CCI group. n = 3. (G) H&E staining of sciatic nerves. Red arrows represent myelin structural destruction, and blue arrows represent inflammatory cell infiltration. (H) Liver H&E staining. (I) Kidney H&E staining. (J) Immunohistochemistry for RhoA, COX2, nNOS, and IL1β. #Compared with the Sham group. *Compared with the CCI group. n = 3.

FIGURE 6

Expression levels of M1 and M2 markers and the RhoA/p38MAPK pathway in the spinal cord. (A) Spinal cord immunofluorescence results for CD32 and CD206. (B) Western blot and qPCR results of expression levels of M1 markers and M2 markers in the spinal cord. (C) Western blot results of RhoA, Rock1, Rock2, p38MAPK, p‐p38MAPK, COX2, IL1β, and IBA‐1 in the dorsal horn of the spinal cord. #Compared to the Sham group. *Compared to CCI Group. n = 3.