Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction

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: JCI Insight

Article Title: Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.183483

Figure Lengend Snippet: ( A ) Schematic of lineage labeling AT2 cells in WT, HPS1, HPS2, and HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice. ( B ) From images in , quantification of EYFP + proSP-C – cells as a percentage of EYFP + lineage cells in WT, HPS1, HPS2, and HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice at 8 and 24 weeks of age. ( C ) IF staining of whole-mount lung tissue for EYFP, AGER, and LAMP3 in WT, HPS1, HPS2, and HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice at 8 weeks of age. Dashed line boxes represent squamous EYFP + AGER + LAMP3 – AT2-derived AT1 cells with AGER and EYFP and merged images shown below. ( D ) IF staining of HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice at 8 and 24 weeks of age, with arrows indicating cuboidal EYFP + AGER – LAMP3 – cells with EYFP and LAMP3 images shown below. HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ 8-week images ( C ) and demonstrated again ( D ) capture simultaneous presence of squamous EYFP + AGER + LAMP3 – and cuboidal EYFP + AGER – LAMP3 – cells. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: *** P < 0.001; **** P < 0.0001. n = 3–4 per group per time point. Scale bars in C and D , 20 μm (inset boxes equal scale to main image). Schematic created with BioRender.com.

Article Snippet: IF staining was performed with antibodies for proSP-C (Invitrogen, PA5-71680), NKX2.1 (Invitrogen, MA5-13961), GFP (Abcam, ab6673), LAMP3 (Synaptic Systems, 391 005), AGER (R&D Systems, Bio-Techne, MAB1179), KRT8 (DSHB), CLDN4 (Invitrogen, PA5-32354), LGALS3 (Invitrogen, 14-5301-82), KRT19 (Invitrogen, SA30-06), and HTII-280 (Terrace Biotech).

Techniques: Labeling, Staining, Derivative Assay

Journal: JCI Insight

Article Title: Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.183483

Figure Lengend Snippet: ( A ) IF staining of paraffin-embedded lung tissue for EYFP, KRT8 or CLDN4, and LAMP3 in WT and HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice at 8 weeks of age. Dashed line boxes represent EYFP + cells with inset boxes displaying EYFP, KRT8 or CLDN4, and LAMP3 staining. ( B ) IF staining of whole-mount lung tissue for EYFP, KRT19, and LAMP3 in WT, HPS1, HPS2, and HPS1/2 Sftpc CreERT2/+ R26R EYFP/+ mice at 8 weeks of age. White arrows indicate squamous EYFP + KRT19 – LAMP3 – AT2-derived AT1 cells, and yellow solid arrows indicate cuboidal EYFP + KRT19 + cells; dashed line boxes and insets display EYFP and KRT19 and merged images. ( C ) Schematic for alveolosphere culture system with feeder-free alveolar maintenance media (AMM) and alveolar differentiation media (ADM) and treatment with pifithrin-α (PFTα). ( D ) Bright-field imaging and ( E ) IF staining of alveolospheres for AGER and LAMP3 generated with AT2 cells from WT or HPS1/2 mice with and without treatment with PFTα at day 14 (D14) of culture. ( F ) Relative gene expression of selected reprogrammed transitional cell ( Krt8 , Cldn4 ) and AT1 cell ( Hopx ) genes in alveolospheres at day 0 (D0), day 7 (D7), and day 14 (D14) of culture with and without treatment with PFTα. Relative gene expression compared with WT at D0. Each point represents 4 replicate wells per time point for a total of n = 3–4 mice. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: * P < 0.05; ** P < 0.01. Scale bars in A , B , and E 20 μm (inset boxes equals to main image); D 1 mm. Schematic created with BioRender.com.

Article Snippet: IF staining was performed with antibodies for proSP-C (Invitrogen, PA5-71680), NKX2.1 (Invitrogen, MA5-13961), GFP (Abcam, ab6673), LAMP3 (Synaptic Systems, 391 005), AGER (R&D Systems, Bio-Techne, MAB1179), KRT8 (DSHB), CLDN4 (Invitrogen, PA5-32354), LGALS3 (Invitrogen, 14-5301-82), KRT19 (Invitrogen, SA30-06), and HTII-280 (Terrace Biotech).

Techniques: Staining, Derivative Assay, Imaging, Generated, Gene Expression