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:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Clec7a Worsens Long-Term Outcomes after Ischemic Stroke by Aggravating Microglia-Mediated Synapse Elimination.

doi: 10.1002/advs.202403064

Figure Lengend Snippet: Figure 5. Clec7a promotes microglia-mediated synaptic phagocytosis after ischemic stroke. A) Schematic of the experimental design. B) PCR genotyping showed that Clec7ai∆MG (lanes 1, 3, 4, and 6) and WT littermate control (lane 7) mice were successfully generated by crossing Cx3cr1CreERT2 transgenic mice with Clec7afl/flmice. Heterozygotes (Ht) that contained both Clec7a WT and Clec7aflox bands are shown. C) Representative photographs of TTC staining (left) after tMCAO and quantification of the relative infarct volume (right). n = 7 mice per group. D) T2 images at 24 h after the onset of ischemia in both groups. n = 8 mice per group. E) DW images of both groups at 24 h after ischemic stroke. n = 8 mice per group. F) CD68+ lysosome content

Article Snippet: The purified human Clec7a protein (Cat. 1859-DC-050) and human MD2 protein (Cat. 1787-MD) were purchased from R&D Systems (USA).

Techniques: Control, Generated, Transgenic Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Clec7a Worsens Long-Term Outcomes after Ischemic Stroke by Aggravating Microglia-Mediated Synapse Elimination.

doi: 10.1002/advs.202403064

Figure Lengend Snippet: Figure 6. Inducible knockdown of microglial Clec7a rescues impaired neurological function after ischemic stroke. A–D) Long-term neurological dysfunc- tion after tMCAO was assessed with the mNSS (A), rotarod test (B), adhesive contact test (C), and adhesive removal test (D). E) Schematic diagram and representative navigation trajectories for learning and memory. F) The swimming speed is presented. G) The average escape latencies. H) The number of entries onto the platform. I) The duration in each zone. J) The number of entries into each zone. n = 10-13 mice per group. Mice aged 8–10 weeks were used for the experiments shown in this figure. Significance was calculated using either one-way ANOVA (F and H) or two-way ANOVA (A-D, G, and I,J), followed by Tukey’s multiple comparisons test. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 versus Sham; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus tMCAO; ns indicates no significant difference.

Article Snippet: The purified human Clec7a protein (Cat. 1859-DC-050) and human MD2 protein (Cat. 1787-MD) were purchased from R&D Systems (USA).

Techniques: Knockdown, Adhesive

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Clec7a Worsens Long-Term Outcomes after Ischemic Stroke by Aggravating Microglia-Mediated Synapse Elimination.

doi: 10.1002/advs.202403064

Figure Lengend Snippet: Figure 7. Microglial Clec7a knockout restores neuronal activity and synaptic transmission after ischemic stroke. A) Representative traces of the action potentials recorded from ischemic penumbra neurons in +180 pA current injection of Clec7afl/fland Clec7ai∆MG mice subjected to tMCAO and sham operation. Scale bar, 20 mV, 50 ms. B) Statistics showed that as the injection current increased (from 0 to +360 pA), the number of spikes was significantly increased in tMCAO Clec7afl/flmice compared to tMCAO Clec7ai∆MG and sham mice (Clec7afl/flgroup, n = 16 neurons in 3 mice, Clec7ai∆MG group, n = 16 neurons in 3 mice; Clec7afl/fl+ tMCAO group, n = 22 neurons in 5 mice, Clec7ai∆MG + tMCAO group, n = 24 neurons in 6 mice). C) Representative PPR traces of Clec7afl/fland Clec7ai∆MG mice subjected to tMCAO and sham operation after two consecutive stimuli. Scale bar, 100pA, 10 ms. D) Statistical analysis showed that the ratios of tMCAO Clec7afl/flmice was increased compared to tMCAO Clec7ai∆MG and sham mice (Clec7afl/flgroup, n = 10 neurons in 3 mice, Clec7ai∆MG group, n = 12 neurons in 3 mice; Clec7afl/fl+ tMCAO group, n = 12 neurons in 5 mice, Clec7ai∆MG + tMCAO group, n = 16 neurons in 6 mice). Statistical significance was determined by two-way ANOVA (B) and one-way ANOVA (D) followed by Tukey’s multiple comparisons test. Data are presented as mean ± SD. *p < 0.05 and ***p < 0.001 versus Sham; ###p < 0.001 versus tMCAO.

Article Snippet: The purified human Clec7a protein (Cat. 1859-DC-050) and human MD2 protein (Cat. 1787-MD) were purchased from R&D Systems (USA).

Techniques: Knock-Out, Activity Assay, Transmission Assay, Injection