Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 2. Clec7a-MAPK/NF-κB-NLRP3 Inflammasome Axis might be an effective target of L-THP against NP. (A) Heat maps of DEGs in the CCI group compared with the Sham group (Left) and CCI+l-THP group (Right) by RNA-seq technology (n = 4 rats/group). In clustering analysis, upregulated and downregulated genes are colored red and blue, respectively. (B) Volcano plots of DEGs in the CCI group compared with the Sham group (Left) and CCI+l-THP group (Right). Blue represents downregulated genes; red represents upregulated genes (|Fold change|>1.2, p < 0.05). (C) Venn plots show the total number of identified DEGs in the spinal cord of the Sham, CCI, and CCI+l-THP groups. The overlapped area represented 156 of 636 altered genes in CCI, which were increased by L-THP treatment (Left). The overlapped area represented 24 of 613 altered genes in CCI, which were attenuated by L-THP treatment (Right). (D) qRT-PCR validation profiles of six randomly selected genes. The fold change values of relative expression patterns of different genes are shown on the left Y axis for qRT-PCR, and the FPKM of DEGs were shown on the right Y axis for RNA-seq. (E) The top 20 biological process(BP) terms in the enrichment analysis. (F) 23 enrichment KEGG signaling pathways. (G) Venn diagrams were severally generated by intersecting the DEGs in 3 biological process terms or 4 KEGG pathways related to inflammatory responses.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques: RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Protein-Protein interactions, Generated

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 3. Intragastric administration of L-THP significantly attenuated CCI-induced neuropathic pain by regulating Clec7a-MAPK/NF-κB-NLRP3 inflammasome-induced pyroptosis axis. Pregabalin was used as a positive control drug. (A) Timeline schematic of the experimental paradigm. (B) Repetitive administration of L-THP at 32 and 64 mg/kg at postoperative 6–10 days significantly ameliorated the mechanical withdrawal threshold in the CCI rat model (n = 12 rats/group). (C) qRT-PCR analysis shows the mRNA levels of Clec7a and NLRP3 in the ipsilateral spinal cord of CCI rats on postoperative 10 days(n = 3 rats/group with 3 technical replicates). (D) Protein expressions of Clec7a, p-Syk/Syk, p-NF-κB-p65/NF-κB-p65, p-ERK/ERK, p-JNK/JNK, p-p38/p38 MAPK, NLRP3, GSDMD-N and Caspase-1 in the ipsilateral spinal cord of CCI rats on postoperative 10 days were measured using western blot analysis (n = 3 rats/group with 3 technical replicates). Data are presented as mean ± SEM. Two-way repeated measures ANOVA and Tukey’s post hoc test were used to analyze data at different time points (B). One-way ANOVA was used to analyze data among multiple groups, followed by Tukey’s post hoc test for equal variances or Dunnett’s T3 post hoc test for unequal variances (C-D). **p < 0.01, ***p < 0.001 compared with the Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the CCI group; △p < 0.05, △△p < 0.01, △△△p < 0.001 compared with the CCI+l-THP+l group.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques: Positive Control, Quantitative RT-PCR, Western Blot

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 5. The overexpression of Clec7a partially inhibits the analgesic effect of L-THP. (A) Timeline schematic of the experimental paradigm. (B) Repetitive administration of L-THP at 64 mg/kg significantly ameliorated the mechanical withdrawal threshold (MWT) in the CCI rat model, while overexpression Clec7a reversed the MWT of CCI rats treated with L-THP (n = 4–10 rats/group). (C) Protein expressions of Clec7a, p-Syk/Syk, p-NF-κB-p65/NF-κB-p65, p-ERK/ERK, p-JNK/ JNK, p-p38/p38 MAPK, NLRP3, GSDMD-N and Caspase-1 in the ipsilateral spinal cord of CCI rats on postoperative 10 days were measured using western blot analysis (n = 3 rats/group with 3 technical replicates). (D) The concentrations of IL-1β and IL-18 in the serum of indicated groups were assessed by ELISA. Data are presented as mean ± SEM. Two-way repeated measures ANOVA and Tukey’s post hoc test were used to analyze data at different time points. One-way ANOVA was used to analyze data among multiple groups, followed by Tukey’s post hoc test for equal variances or Dunnett’s T3 post hoc test for unequal variances (C-D). **p < 0.01, ***p < 0.001 compared with the Sham group; △p < 0.05, △△p < 0.01, △△△p < 0.001 compared with the CCI group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the CCI+pLVSO5–NC group; §p < 0.05, §§p < 0.01, §§§p < 0.001 compared with the CCI+pLVSO5–Clec7a group.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques: Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 6. L-THP effectively inhibits the upregulation of Clec7a in spinal microglia. Representative images and quantitative analysis of co-localization of Clec7a with Iba-1 (A), GFAP (B) or NeuN (C) in the spinal cord. The tissues were collected from each group of rats on postoperative day 10. White arrows point to Clec7a- positive cells. Scale bars are indicated. Data are presented as mean ± SEM. One-way ANOVA was used to analyze data among multiple groups, followed by Tukey’s post hoc test for equal variances or Dunnett’s T3 post hoc test for unequal variances. *p<0.05, ***p<0.001 compared with the Sham group; ###p < 0.001 compared with the CCI group.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques:

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 8. L-THP was directly combined with the Clec7a protein. (A) The schematic diagram of the molecular docking of L-THP with Clec7a, shown as the 3D diagram. The ribbon and stick structure displays the predicted bonds between L-THP and Clec7a. (B) The interaction between L-THP and Clec7a was determined by SPR assay. The equilibrium dissociation constant was evaluated using BIA evaluation software.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques: SPR Assay, Software

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

doi: 10.1016/j.phymed.2023.155075

Figure Lengend Snippet: Fig. 9. The graphical abstract illus trates the proposed mechanism un derlying the effect of L-THP in CCI rats. Several pathways, including Clec7a-MAPK/NF-κB-NLRP3 axis, were activated and promoted the release of proinflammatory cytokines that further promoted neuroinflammation and neuropathic pain. L-THP attenuated CCI- induced neuropathic pain through inhibiting the Clec7a-MAPK/NF-κB- NLRP3 signaling pathway, inhibiting the excessive release of proin flammatory cytokines, and decreasing neuroinflammation in the spinal cord.

Article Snippet: First, the Clec7a protein (1859-DC-050, R&D System, USA) was immobilized on the flow cell after being diluted to 30 g/ml in the immobilization buffer (1 PBS-EP, pH 7.4).

Techniques: