Journal: Journal of Cellular and Molecular Medicine

Article Title: Casticin From Sanse Powder‐ In Vitro Release Medium Inhibits FAK / PI3K / AKT Phosphorylation Reducing Synovial Senescence and Alleviates Inflammation in Knee Osteoarthritis

doi: 10.1111/jcmm.71391

Figure Lengend Snippet: SSP‐IVRM alleviates synovial inflammation and pathological progression in KOA rats. (A) ELISA was used to detect the levels of IL‐1β, IL‐6, CXCL1 and CXCL3 in the serum of rats in each group; (B) Representative images of HE, Masson and Sirius Red staining of knee synovial tissues from each group, along with Krenn's scoring; (C) Representative images of IL‐1β immunofluorescence in knee synovial tissues from each group; (D) Western blot analysis of the protein expression levels of MMP3, MMP9, COL I and TGF‐β in the synovial tissues of rats in each group. (E) qRT‐PCR analysis of MMP3, MMP9, COL1A1 and TGF‐β mRNA expression in synovial tissues. Statistical graphs are presented as Mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus KOA group.

Article Snippet: DMEM high‐glucose medium (Cat# C11995500CP) and fetal bovine serum (Cat# 10099‐141) were purchased from GIBCO (USA); LPS (Cat# L2880) was obtained from Sigma‐Aldrich (USA); Casticin (Cat# B20835 , ≥ 98%) was purchased from Yuanye Biotechnology (China); MIR002 (Cat# HY‐143412), Y15 (Cat# HY‐12444) and VSLRGDTRG acetate (Cat# HY‐P10941A) were obtained from MCE (China); Senescence‐Tracker Senescent Cell Fluorescent Probe (Cat# C0603), Enhanced Mitochondrial Membrane Potential Assay Kit (Cat# C2003S), Reactive Oxygen Species Assay Kit (Cat# S0035S) and Cell Counting Kit‐8 (Cat# C0038) were purchased from Beyotime Biotechnology (China); Rat CXCL1 ELISA Kit (Cat# E‐EL‐R0003), Rat CXCL3 ELISA Kit (Cat# E‐EL‐R0697), Rat IL‐6 ELISA Kit (Cat# E‐EL‐R0015) and Rat IL‐1β ELISA Kit (Cat# E‐EL‐R0012) were obtained from Elabscience (China); TGF‐β Antibody (Cat# 3711), CDC2 Antibody (Cat# 77055), FAK Antibody (Cat# 3285), PI3 Kinase p85 Antibody (Cat# 4292), AKT Antibody (Cat# 9272) and Phospho‐FAK (Tyr397) Antibody (Cat# 3283) were purchased from CST (USA); Phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) Antibody (Cat# AF3242) was obtained from Affinity (China); Phospho‐AKT (Ser473) Antibody (Cat# 80455‐1‐RR), IL‐1β Antibody (Cat# 26048‐1‐AP), IL‐6 Antibody (Cat# 21865‐1‐AP), MMP3 Antibody (Cat# 17873‐1‐AP), MMP9 Antibody (Cat# 10375‐2‐AP), p53 Antibody (Cat# 10442‐1‐AP), p21 Antibody (Cat# 10355‐1‐AP) and GAPDH Antibody (Cat# 60004‐1‐Ig) were purchased from Proteintech (China).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Western Blot, Analysis, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Casticin From Sanse Powder‐ In Vitro Release Medium Inhibits FAK / PI3K / AKT Phosphorylation Reducing Synovial Senescence and Alleviates Inflammation in Knee Osteoarthritis

doi: 10.1111/jcmm.71391

Figure Lengend Snippet: CAS alleviates synovial inflammation and cellular senescence in KOA rats. (A) Representative images of HE, Masson and Sirius Red staining of synovial tissue from rat knee joints in each group, along with Krenn's scores; (B) Serum levels of IL‐1β, IL‐6, CXCL1 and CXCL3 in each group of rats as measured by ELISA; (C) Representative immunofluorescence images of IL‐1β in synovial tissue from rat knee joints in each group; (D) Protein expression levels of MMP3, MMP9, COL I, TGF‐β, p53, p21, CDC2 and CCNA1 in synovial tissue from each rat group as determined by Western Blot. (E) qRT‐PCR analysis of the mRNA expression levels of MMP3, MMP9, COL1A1, TGF‐β, p53, p21/CDKN1A, CDC2 and CCNA1 in synovial tissues from each rat group. Statistical graphs are presented as Mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus KOA group.

Article Snippet: DMEM high‐glucose medium (Cat# C11995500CP) and fetal bovine serum (Cat# 10099‐141) were purchased from GIBCO (USA); LPS (Cat# L2880) was obtained from Sigma‐Aldrich (USA); Casticin (Cat# B20835 , ≥ 98%) was purchased from Yuanye Biotechnology (China); MIR002 (Cat# HY‐143412), Y15 (Cat# HY‐12444) and VSLRGDTRG acetate (Cat# HY‐P10941A) were obtained from MCE (China); Senescence‐Tracker Senescent Cell Fluorescent Probe (Cat# C0603), Enhanced Mitochondrial Membrane Potential Assay Kit (Cat# C2003S), Reactive Oxygen Species Assay Kit (Cat# S0035S) and Cell Counting Kit‐8 (Cat# C0038) were purchased from Beyotime Biotechnology (China); Rat CXCL1 ELISA Kit (Cat# E‐EL‐R0003), Rat CXCL3 ELISA Kit (Cat# E‐EL‐R0697), Rat IL‐6 ELISA Kit (Cat# E‐EL‐R0015) and Rat IL‐1β ELISA Kit (Cat# E‐EL‐R0012) were obtained from Elabscience (China); TGF‐β Antibody (Cat# 3711), CDC2 Antibody (Cat# 77055), FAK Antibody (Cat# 3285), PI3 Kinase p85 Antibody (Cat# 4292), AKT Antibody (Cat# 9272) and Phospho‐FAK (Tyr397) Antibody (Cat# 3283) were purchased from CST (USA); Phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) Antibody (Cat# AF3242) was obtained from Affinity (China); Phospho‐AKT (Ser473) Antibody (Cat# 80455‐1‐RR), IL‐1β Antibody (Cat# 26048‐1‐AP), IL‐6 Antibody (Cat# 21865‐1‐AP), MMP3 Antibody (Cat# 17873‐1‐AP), MMP9 Antibody (Cat# 10375‐2‐AP), p53 Antibody (Cat# 10442‐1‐AP), p21 Antibody (Cat# 10355‐1‐AP) and GAPDH Antibody (Cat# 60004‐1‐Ig) were purchased from Proteintech (China).

Techniques: Staining, Tissue, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR, Analysis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Casticin From Sanse Powder‐ In Vitro Release Medium Inhibits FAK / PI3K / AKT Phosphorylation Reducing Synovial Senescence and Alleviates Inflammation in Knee Osteoarthritis

doi: 10.1111/jcmm.71391

Figure Lengend Snippet: CAS improves inflammation and cellular senescence in LPS‐induced rat synovial fibroblast‐like cells. (A) CCK‐8 assay to evaluate the cytotoxic effects of CAS on FLS; (B) ELISA to measure the levels of IL‐1β, IL‐6, CXCL1 and CXCL3 in the culture media of rat FLS from each group; (C) Representative immunofluorescence images of IL‐1β and IL‐6 in FLS from each group; (D) Western blot analysis of protein expression levels of MMP3, MMP9, COL I, TGF‐β, p53, p21, CDC2 and CCNA1 in FLS from each group; (E) CM‐H2DCFDA probe to assess ROS levels in FLS from each group; (F) JC‐1 probe to detect mitochondrial membrane potential levels in FLS from each group; (G) Representative images of senescent cells labelled by Senescence‐Tracker probe in FLS from each group. Statistical graphs are presented as Mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LPS group.

Article Snippet: DMEM high‐glucose medium (Cat# C11995500CP) and fetal bovine serum (Cat# 10099‐141) were purchased from GIBCO (USA); LPS (Cat# L2880) was obtained from Sigma‐Aldrich (USA); Casticin (Cat# B20835 , ≥ 98%) was purchased from Yuanye Biotechnology (China); MIR002 (Cat# HY‐143412), Y15 (Cat# HY‐12444) and VSLRGDTRG acetate (Cat# HY‐P10941A) were obtained from MCE (China); Senescence‐Tracker Senescent Cell Fluorescent Probe (Cat# C0603), Enhanced Mitochondrial Membrane Potential Assay Kit (Cat# C2003S), Reactive Oxygen Species Assay Kit (Cat# S0035S) and Cell Counting Kit‐8 (Cat# C0038) were purchased from Beyotime Biotechnology (China); Rat CXCL1 ELISA Kit (Cat# E‐EL‐R0003), Rat CXCL3 ELISA Kit (Cat# E‐EL‐R0697), Rat IL‐6 ELISA Kit (Cat# E‐EL‐R0015) and Rat IL‐1β ELISA Kit (Cat# E‐EL‐R0012) were obtained from Elabscience (China); TGF‐β Antibody (Cat# 3711), CDC2 Antibody (Cat# 77055), FAK Antibody (Cat# 3285), PI3 Kinase p85 Antibody (Cat# 4292), AKT Antibody (Cat# 9272) and Phospho‐FAK (Tyr397) Antibody (Cat# 3283) were purchased from CST (USA); Phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) Antibody (Cat# AF3242) was obtained from Affinity (China); Phospho‐AKT (Ser473) Antibody (Cat# 80455‐1‐RR), IL‐1β Antibody (Cat# 26048‐1‐AP), IL‐6 Antibody (Cat# 21865‐1‐AP), MMP3 Antibody (Cat# 17873‐1‐AP), MMP9 Antibody (Cat# 10375‐2‐AP), p53 Antibody (Cat# 10442‐1‐AP), p21 Antibody (Cat# 10355‐1‐AP) and GAPDH Antibody (Cat# 60004‐1‐Ig) were purchased from Proteintech (China).

Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot, Analysis, Expressing, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Casticin From Sanse Powder‐ In Vitro Release Medium Inhibits FAK / PI3K / AKT Phosphorylation Reducing Synovial Senescence and Alleviates Inflammation in Knee Osteoarthritis

doi: 10.1111/jcmm.71391

Figure Lengend Snippet: CAS reduces the inflammatory state of rat synovial fibroblast‐like cells by lowering cellular senescence levels. (A) Representative images of senescent positive cells in rat FLSs from each group detected using the Senescence‐Tracker probe; (B) Mitochondrial membrane potential levels in rat FLSs from each group measured by the JC‐1 probe; (C) Intracellular ROS levels in rat FLSs from each group assessed with the CM‐H2DCFDA probe; (D) Protein expression levels of MMP3, MMP9, COL I, TGF‐β, p53, p21, CDC2 and CCNA1 in rat FLSs from each group detected by Western blotting (WB); (E) Representative immunofluorescence images of IL‐1β and IL‐6 in rat FLSs from each group; (F) Levels of IL‐1β, IL‐6, CXCL1 and CXCL3 in the culture supernatant of rat FLSs from each group measured by ELISA. Statistical graphs are presented as Mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LPS group; △ p < 0.05, △△ p < 0.01, △△△ p < 0.001 versus MIR002 group.

Article Snippet: DMEM high‐glucose medium (Cat# C11995500CP) and fetal bovine serum (Cat# 10099‐141) were purchased from GIBCO (USA); LPS (Cat# L2880) was obtained from Sigma‐Aldrich (USA); Casticin (Cat# B20835 , ≥ 98%) was purchased from Yuanye Biotechnology (China); MIR002 (Cat# HY‐143412), Y15 (Cat# HY‐12444) and VSLRGDTRG acetate (Cat# HY‐P10941A) were obtained from MCE (China); Senescence‐Tracker Senescent Cell Fluorescent Probe (Cat# C0603), Enhanced Mitochondrial Membrane Potential Assay Kit (Cat# C2003S), Reactive Oxygen Species Assay Kit (Cat# S0035S) and Cell Counting Kit‐8 (Cat# C0038) were purchased from Beyotime Biotechnology (China); Rat CXCL1 ELISA Kit (Cat# E‐EL‐R0003), Rat CXCL3 ELISA Kit (Cat# E‐EL‐R0697), Rat IL‐6 ELISA Kit (Cat# E‐EL‐R0015) and Rat IL‐1β ELISA Kit (Cat# E‐EL‐R0012) were obtained from Elabscience (China); TGF‐β Antibody (Cat# 3711), CDC2 Antibody (Cat# 77055), FAK Antibody (Cat# 3285), PI3 Kinase p85 Antibody (Cat# 4292), AKT Antibody (Cat# 9272) and Phospho‐FAK (Tyr397) Antibody (Cat# 3283) were purchased from CST (USA); Phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) Antibody (Cat# AF3242) was obtained from Affinity (China); Phospho‐AKT (Ser473) Antibody (Cat# 80455‐1‐RR), IL‐1β Antibody (Cat# 26048‐1‐AP), IL‐6 Antibody (Cat# 21865‐1‐AP), MMP3 Antibody (Cat# 17873‐1‐AP), MMP9 Antibody (Cat# 10375‐2‐AP), p53 Antibody (Cat# 10442‐1‐AP), p21 Antibody (Cat# 10355‐1‐AP) and GAPDH Antibody (Cat# 60004‐1‐Ig) were purchased from Proteintech (China).

Techniques: Membrane, Expressing, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Casticin From Sanse Powder‐ In Vitro Release Medium Inhibits FAK / PI3K / AKT Phosphorylation Reducing Synovial Senescence and Alleviates Inflammation in Knee Osteoarthritis

doi: 10.1111/jcmm.71391

Figure Lengend Snippet: CAS inhibits FAK/PI3K/AKT phosphorylation process to reduce synovial cell senescence and improve synovial inflammation. (A) Representative immunofluorescence images of p‐FAK and p‐PI3K in FLS from each rat group; (B) Western blot analysis of protein expression levels of p‐FAK, FAK, p‐PI3K, PI3K, p‐AKT and AKT in FLS from each group; (C) Western blot analysis of protein expression levels of MMP9, COL I, TGF‐β, p53, p21, CDC2 and CCNA1 in FLS from each group; (D) Assessment of mitochondrial membrane potential in FLS from each group using the JC‐1 probe; (E) Detection of ROS levels in FLS from each group using the CM‐H2DCFDA probe; (F) Representative images of senescent FLS detected with the Senescence‐Tracker probe in each group; (G) ELISA analysis of IL‐1β, IL‐6, CXCL1 and CXCL3 levels in the culture medium of FLS from each group. Statistical graphs are presented as Mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LPS group; △ p < 0.05, △△ p < 0.01, △△△ p < 0.001 versus CAS group.

Article Snippet: DMEM high‐glucose medium (Cat# C11995500CP) and fetal bovine serum (Cat# 10099‐141) were purchased from GIBCO (USA); LPS (Cat# L2880) was obtained from Sigma‐Aldrich (USA); Casticin (Cat# B20835 , ≥ 98%) was purchased from Yuanye Biotechnology (China); MIR002 (Cat# HY‐143412), Y15 (Cat# HY‐12444) and VSLRGDTRG acetate (Cat# HY‐P10941A) were obtained from MCE (China); Senescence‐Tracker Senescent Cell Fluorescent Probe (Cat# C0603), Enhanced Mitochondrial Membrane Potential Assay Kit (Cat# C2003S), Reactive Oxygen Species Assay Kit (Cat# S0035S) and Cell Counting Kit‐8 (Cat# C0038) were purchased from Beyotime Biotechnology (China); Rat CXCL1 ELISA Kit (Cat# E‐EL‐R0003), Rat CXCL3 ELISA Kit (Cat# E‐EL‐R0697), Rat IL‐6 ELISA Kit (Cat# E‐EL‐R0015) and Rat IL‐1β ELISA Kit (Cat# E‐EL‐R0012) were obtained from Elabscience (China); TGF‐β Antibody (Cat# 3711), CDC2 Antibody (Cat# 77055), FAK Antibody (Cat# 3285), PI3 Kinase p85 Antibody (Cat# 4292), AKT Antibody (Cat# 9272) and Phospho‐FAK (Tyr397) Antibody (Cat# 3283) were purchased from CST (USA); Phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) Antibody (Cat# AF3242) was obtained from Affinity (China); Phospho‐AKT (Ser473) Antibody (Cat# 80455‐1‐RR), IL‐1β Antibody (Cat# 26048‐1‐AP), IL‐6 Antibody (Cat# 21865‐1‐AP), MMP3 Antibody (Cat# 17873‐1‐AP), MMP9 Antibody (Cat# 10375‐2‐AP), p53 Antibody (Cat# 10442‐1‐AP), p21 Antibody (Cat# 10355‐1‐AP) and GAPDH Antibody (Cat# 60004‐1‐Ig) were purchased from Proteintech (China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Analysis, Expressing, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: AI‐Designed TREM1‐Targeted LYTAC Nanoparticles Reprogram the Neuroimmune Microenvironment in Traumatic Brain Injury

doi: 10.1002/advs.77972

Figure Lengend Snippet: Systemic TREM1 ablation attenuates neuroinflammation and neurological deficits after TBI. (a) Schematic timeline of the experimental design. WT and Trem1 −/− mice were subjected to CCI or sham surgery, followed by longitudinal behavioral and histological assessments. (b, c) Analysis of the neuroinflammatory profile in the perilesional cortex at 3 days post‐TBI. (b) Representative images of inflammatory cytokines in TBI lesions detected by the Proteome Profiler Array Mouse Cytokine Array Panel A in WT and Trem1 −/− mice at 3 days post‐TBI. (c) Quantification of inflammatory cytokines in TBI lesions of WT and Trem1 −/− mice at 3 days post‐TBI or sham treatment by ELISA ( n = 5 mice per group, two‐way ANOVA followed by Tukey's multiple‐comparison test). (d) Representative immunofluorescence images of macrophages (IBA1 + , green) and the reactivation marker CD86 (red) in the TBI cortex. Trem1 ablation significantly reduced reactive polarization (scale bar, 50 µm). (e) Evaluation of neuronal apoptosis. Representative co‐staining of TUNEL (green) and NeuN (red) (left) ( n = 3 mice per group, scale bar, 100 µm). Yellow arrows indicate apoptotic neurons. (f) Assessment of white matter integrity. Representative images of Myelin Basic Protein (MBP, green) and NeuN (red) (left) and quantification of MBP fluorescence intensity (right) (scale bar, 100 µm). (g) Representative immunofluorescence images of hippocampal neurons (NeuN, red) showing preserved cellular density in Trem1 −/− mice ( n = 3 mice per group; scale bar, 100 µm). (h) Brain water content analysis at 1day and 3 d post‐TBI, indicating alleviated cerebral edema in Trem1 −/− mice ( n = 6 mice per group; two‐way ANOVA followed by Tukey's multiple‐comparison test). (i, j) Assessment of blood‐brain barrier (BBB) permeability. (i) Representative images of Evans Blue extravasation. (j) Quantification of dye leakage ( n = 6 mice per group; two‐way ANOVA followed by Tukey's multiple‐comparison test). (k, l) Molecular validation of tissue preservation. (k) Representative Western blots of the neuronal marker beta3‐tubulin, synaptic marker Synaptophysin (Syn), and tight junction protein Occludin. l, Densitometric quantification normalized to GAPDH ( n = 3 independent experimental replicates; two‐way ANOVA followed by Tukey's multiple‐comparison test). (m–o) Assessment of sensorimotor function. m, Latency to fall in the accelerating Rotarod test ( n = 8 mice per group; two‐way ANOVA followed by Tukey's multiple‐comparison test). (n) Performance in the fixed‐speed (30 rpm) Rotarod test ( n = 8 mice per group, one‐way ANOVA followed by Tukey's multiple‐comparison test). o, Modified Neurological Severity Score (mNSS) trajectory ( n = 8 mice per group; two‐way ANOVA followed by Tukey's multiple‐comparison test). (p, q) Evaluation of anxiety‐like behavior and exploratory activity in the Open Field Test (OFT). (p) Representative locomotor tracking plots. (q) Quantification of the percentage of time spent in the center zone ( n = 8 per group; two‐way ANOVA followed by Tukey's multiple‐comparison test). Data are presented as mean ± SD. Each dot represents an individual mouse, except in l, where each dot represents an independent experimental replicate. Statistical tests are specified for each panel above. Exact p values are provided in the Source Data. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: IL‐1β ELISA kit , ABclonal , # RK00006.

Techniques: Analysis, Enzyme-linked Immunosorbent Assay, Comparison, Immunofluorescence, Marker, Staining, TUNEL Assay, Fluorescence, Permeability, Biomarker Discovery, Tissue, Preserving, Western Blot, Modification, Activity Assay

Journal: Advanced Science

Article Title: AI‐Designed TREM1‐Targeted LYTAC Nanoparticles Reprogram the Neuroimmune Microenvironment in Traumatic Brain Injury

doi: 10.1002/advs.77972

Figure Lengend Snippet: Generative AI‐driven design and mechanistic validation of a TREM1‐targeting LYTAC degrader. (a) Schematic of the RFdiffusion‐guided pipeline for de novo TREM1‐binding peptide design. Peptide backbones were generated around the Ig‐like domain of TREM1 and subsequently screened by AlphaFold3, ZDOCK, HDOCK, and Rosetta‐based energetic analysis to identify high‐ranking candidates. (b) Predicted structural models of Pep15, Pep17, and Pep457 in complex with TREM1, showing the predicted binding interfaces. (c) Temperature‐related intensity change (TRIC) assays showing concentration‐dependent target engagement of Pep15, Pep17, and Pep457 with TREM1. Left, schematic illustration of the TRIC assay principle; right, dose‐response curves of the indicated peptides ( n = 3 independent experiments). (d) ELISA‐based binding analysis of Pep15, Pep17, and Pep457 to immobilized TREM1. Top, schematic of the ELISA workflow; bottom, quantitative binding curves ( n = 3 independent experiments). (e) Surface plasmon resonance (SPR) analysis of Pep457 binding to TREM1. Top, schematic of the SPR assay; bottom, representative sensorgrams obtained at the indicated Pep457 concentrations. (f) Structural model of the bifunctional TREM1‐LYTAC, in which Pep457 engages TREM1 and the mannose‐6‐phosphate (M6P) moiety recruits the cation‐independent mannose‐6‐phosphate receptor (CI‐M6PR). (g) Representative confocal images showing partial colocalization of TREM1‐LYTAC with lysosomes following cellular uptake. TREM1‐LYTAC, green; lysosomes, red; nuclei, blue. Arrows indicate representative colocalized puncta. Scale bars as indicated. (h) ELISA quantification of cellular TREM1 protein following treatment with vehicle, scrambled‐LYTAC, TREM1‐LYTAC, or TREM1‐LYTAC in the presence of bafilomycin A1 (BafA1) or MG132 ( n = 5 independent experiments). (i) Representative flow‐cytometric histograms showing cell‐surface TREM1 expression after the indicated treatments. (j) Trem1 mRNA expression measured by qPCR after the indicated treatments ( n = 3 independent experiments; one‐way ANOVA followed by Tukey's multiple‐comparison test). (k) Requirement of both functional modules for TREM1 reduction. Cellular TREM1 protein levels were quantified after treatment with Pep457 plus M6P, Pep457 alone, M6P alone, TREM1‐LYTAC in the presence of CI‐M6PR blockade, or conjugated TREM1‐LYTAC ( n = 5 independent experiments; one‐way ANOVA followed by Tukey's multiple‐comparison test). (l) Plasma pharmacokinetic profile of free TREM1‐LYTAC after intravenous administration. Fluorescence intensity was fitted to a monoexponential decay model, yielding an apparent half‐life of 1.13 h ( n = 3 mice). (m) Proposed mechanism of TREM1‐LYTAC‐mediated degradation. TREM1‐LYTAC bridges cell‐surface TREM1 to CI‐M6PR, thereby promoting receptor internalization and lysosomal degradation. Data are mean ± SD. Each dot represents an independent experimental replicate or an individual animal. Statistical tests are specified for each quantitative panel above. Exact p values are provided in the Source Data. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: IL‐1β ELISA kit , ABclonal , # RK00006.

Techniques: Biomarker Discovery, Binding Assay, Generated, Analysis, Concentration Assay, Drug discovery, Enzyme-linked Immunosorbent Assay, SPR Assay, Confocal, Expressing, Comparison, Functional Assay, Clinical Proteomics, Fluorescence

Journal: Advanced Science

Article Title: AI‐Designed TREM1‐Targeted LYTAC Nanoparticles Reprogram the Neuroimmune Microenvironment in Traumatic Brain Injury

doi: 10.1002/advs.77972

Figure Lengend Snippet: Engineering and validation of a BBB‐targeted, pH‐responsive nanodevice for spatiotemporal delivery of TREM1‐LYTAC. (a) Schematic illustration of APTL‐NP fabrication. TREM1‐LYTAC was conjugated to a generation‐4 PAMAM dendrimer through a terephthalaldehyde‐derived acid‐labile linker, followed by surface functionalization with Angiopep‐2 and PS‐PEG‐NHS to enable LRP1‐directed BBB targeting and improve systemic stability. (b) Representative transmission electron microscopy images of PAMAM‐based precursor particles and APTL‐NP (Scale bars, 1 µm). (c) Dynamic light scattering profiles showing the apparent hydrodynamic size distributions of G4 PAMAM and APTL‐NP under the indicated measurement conditions. (d) Time‐dependent changes in hydrodynamic diameter and polydispersity index of APTL‐NP incubated in PBS at pH 7.4 and 37°C. (e) pH‐dependent zeta‐potential profile of APTL‐NP. (f) Nanoparticle tracking analysis showing pH‐dependent particle size and concentration distributions after incubation at pH 7.5, 7.0, or 6.5. (g) pH‐responsive release of TREM1‐binding‐competent TREM1‐LYTAC from APTL‐NP. Released free TREM1‐LYTAC was separated by ultrafiltration and quantified using a TREM1‐binding ELISA with free TREM1‐LYTAC standards ( n = 3 independent experiments). (h) Representative EdU staining images of BMECs, HT22 cells, and NHAs treated with vehicle or APTL‐NP. (i) Quantification of EdU‐positive cells in the indicated cell types( n = 6 independent experiments, two‐way ANOVA followed by Tukey's multiple‐comparison test). (j) Representative flow‐cytometric plots for apoptosis analysis in BMECs, HT22 cells, and NHAs after treatment with vehicle or APTL‐NP. (k) Quantification of apoptotic cells in the indicated cell types( n = 6 independent experiments, two‐way ANOVA followed by Tukey's multiple‐comparison test). (l) Cell viability after 24 h or 48 h exposure to the indicated concentrations of APTL‐NP ( n = 3 independent experiments, two‐way ANOVA followed by Tukey's multiple‐comparison test). (m) Plasma pharmacokinetic profile of APTL‐NP after intravenous administration. The fluorescence signal was fitted to a monoexponential decay model, yielding an apparent half‐life of 8.38 h ( n = 3 mice). (n) Representative photographs and quantification of hemolysis induced by the indicated concentrations of APTL‐NP ( n = 3 independent experiments, one‐way ANOVA followed by Tukey's multiple‐comparison test). (o, p) Plasma C3a and C5a concentrations at the indicated time points after administration of vehicle or APTL‐NP ( n = 6 mice per group, Two‐way ANOVA followed by Tukey's multiple‐comparison test). (q) Representative H&E‐stained sections of the spleen, heart, lung, liver, and kidney from vehicle‐ and APTL‐NP‐treated mice (Scale bar, 100 µm). Data are presented as mean ± SD where applicable. For in vitro assays, n denotes independent experimental replicates; for in vivo analyses, n denotes individual mice. Statistical tests are specified for each quantitative panel above. Exact p values are provided in the Source Data. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: IL‐1β ELISA kit , ABclonal , # RK00006.

Techniques: Biomarker Discovery, Derivative Assay, Stability, Transmission Assay, Electron Microscopy, Dynamic Light Scattering Assay, Incubation, Zeta Potential Analyzer, Analysis, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Comparison, Clinical Proteomics, Fluorescence, In Vitro, In Vivo

Journal: Advanced Science

Article Title: AI‐Designed TREM1‐Targeted LYTAC Nanoparticles Reprogram the Neuroimmune Microenvironment in Traumatic Brain Injury

doi: 10.1002/advs.77972

Figure Lengend Snippet: Key resources table.

Article Snippet: IL‐1β ELISA kit , ABclonal , # RK00006.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software