Polygonum Cuspidatum Alcohol Extract Exerts Analgesic Effects via the MAPK/ERK Signaling Pathway

Abstract

Objective: Traditional Chinese medicine Polygonum cuspidatum (PC) has significant effects on reducing pain. In this study, we investigated the analgesic effects of the alcohol extract of PC on three types of inflammatory pain and explored its mechanism.

Methods: Potential targets for the analgesic effects of the main active components of PC alcohol extract were screened by network pharmacology and molecular docking. Three different inflammatory pain mouse models (acetic acid twisting, formalin foot swelling, and xylene ear swelling) were used to study the analgesic effects of PC. The expression of latent signaling pathways in L_4-6 spinal cord tissues in formalin foot swelling mice was evaluated using real-time qPCR (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC) analyses.

Results: Network pharmacology analysis shows that PC analgesic mechanism is related to the MAPK/ERK signaling pathway. The five main active components of PC have good docking ability with JNK and p38. PC alcohol extract significantly reduced the pain behavior and alleviated inflammatory reactions in three mouse models, inhibited the mRNA and protein phosphorylation levels of JNK, ERK, p38, and CREB in spinal cord tissues.

Conclusion: PC alcohol extract can inhibit inflammation and alleviate pain, which is related to its inhibition of the MAPK/ERK signaling pathway in spinal cord. Thus, PC alcohol extract is a promising candidate for pain treatment.

Keywords: MAPK/ERK; Polygonum cuspidatum; analgesic; network pharmacology.

Figure 1

Image contrast diagram of PC alcohol extract and mixed standard. (A) HPLC chromatogram of alcohol extract of PC. (B) HPLC chromatogram of mixed standard. 1 polydatin, 2 resveratrol, 3 emodin-8-o-β-D-glucoside, 4 emodin, 5 physcion.

Figure 2

Five main active components of PC - analgesic network pharmacological study. (A) Venn diagram showing the analgesia common targets of the five main active components of PC. (B) Analysis of the PPI network of the five main active components of PC and analgesia common targets. (C) PC-component-target network diagram. (D) KEGG enrichment analysis of the five main active components of PC and analgesia common targets. (E) GO enrichment analysis of the five main active components of PC and analgesia common targets.

Figure 3

3D schematic diagram of the molecular docking simulation of the five main active components of PC with JNK/p38 (Human sources) molecules. Molecular docking mode diagram of polydatin with JNK (A) and p38 (B). Molecular docking mode diagram of resveratrol with JNK (C) and p38 (D). Molecular docking mode diagram of emodin-8-o-β-D-glucoside with JNK (E) and p38 (F). Molecular docking mode diagram of emodin with JNK (G) and p38 (H). Molecular docking mode diagram of physcion with JNK (I) and p38 (J).The dashed line represents the region where small molecules bind to proteins. Letters represent the names of amino acid residues. Numbers represent the length of hydrogen bonds.

Figure 4

Analgesic pharmacodynamics study of PC alcohol extract. (A) Twisting latency time of mice in the control (Ctrl), model (Mod), low-dose PC (HD), high-dose PC (HG), and positive control aspirin (AS) groups in the acetic acid twisting model. (B) Bar graphs of the number of twists in each group in the acetic acid twisting model. (C) Bar graphs of the twisting inhibition rate in each group in the acetic acid twisting model. (D) Bar graphs of the phase I licking time in each group in the formalin foot swelling model. (E) Bar graphs of the phase II licking time in each group in the formalin foot swelling model. (F) Differences in ear weight in each group in the xylene ear swelling model. (G) Images of the right foot mice in the Ctrl/Mod/HD/HG/AS groups in the formalin foot swelling model. Data represent the mean ± SD (n ≥ 5). ns: not significant vs Ctrl group, ^##P < 0.01 vs Ctrl group, **P < 0.01 vs Mod group.

Figure 5

Effects of PC alcohol extract on expression of genes associated with the inflammation and the MAPK/ERK signaling pathway. (A) ELISA analysis of the gene expression of the inflammatory factors 1L-6 in serum. (B) ELISA analysis of the gene expression of the inflammatory factors 1L-1β in serum. RT-qPCR analysis of the gene expression of the inflammatory factors TNF-α (C), NLRP3 (D), 1L-6 (E), and 1L-β (F) in the L_4-6 spinal cord of mice in the Ctrl/Mod/HD/HG/AS groups in the formalin foot swelling model. RT-qPCR analysis of the gene expression of JNK (G), ERK (H), p38 (I), and CREB (J) in the L_4-6 spinal cord of mice in the Ctrl/Mod/HD/HG/AS groups in the formalin foot swelling model. Data represent the mean ± SD of three independent experiments. ns: not significant vs Ctrl group, ^#P < 0.05 and ^##P < 0.01 vs Ctrl group, *P < 0.05 and **P < 0.01 vs Mod group.

Figure 6

Effects of PC alcohol extract on proteins associated with the MAPK/ERK signaling pathway. (A) Western blot analysis of the expression of JNK, p-JNK, ERK, p-ERK, p38/p-P38, CREB and p-CREB protein in the L_4-6 spinal cord tissues of mice in the Ctrl/Mod/HD/HG/AS groups in the formalin foot swelling model. (B–E) Quantification of the data shown in (A). IHC assay for the expression of p-JNK (F) and p-p38 (G) in the mice L_4-6 spinal cord tissues mice in the Ctrl/Mod/HD/HG/AS groups in the formalin foot swelling model. (H) Quantification of the data shown in (F). (I) Quantification of the data shown in (G). Data represent the mean ± SD of three independent experiments. ns: not significant vs Ctrl group, ^##P < 0.01 vs Ctrl group, *P < 0.05 and **P < 0.01 vs Mod group.

Figure 7

Mechanism by which PC alcohol extract exerts its analgesic effects through inflammation and via the MAPK/ERK signaling pathway.