Fig 1: Effect of the TSPO ligands on inflammatory chemokine CCL8. BV-2 cells were exposed to 100 ng/mL of LPS for 24 h with or without our novel TSPO ligands compared to PK 11,195 (25 µM each). CCL8 levels (pg/mL) were calculated using a standard calibration curve and are presented as mean ± SD; four replicates in each. ANOVA followed by Bonferroni’s post-hoc test was performed. *** p < 0.001 compared to all other groups.
Fig 2: Validation of epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines. A Western diet (WD) significantly increased the levels of eWAT-associated pro-inflammatory cytokines in both eWAT and plasma. (A–E) Western blot analysis validated a marked increase in the expression of eWAT-associated pro-inflammatory cytokines in the WD group compared with that in the controls. Specifically, the levels of CCL8, CCL9, CXCL13, and IL-18 were significantly upregulated in the eWAT of WD group, indicating heightened inflammatory activity in this tissue. (F–I) Corresponding ELISA analysis of plasma samples showed increased concentrations of these cytokines in the WD group, with significant increases observed for CCL8, CCL9, CXCL13, and IL-18. Importantly, in the eWAT-removal group, plasma levels of these cytokines were reduced to levels comparable to the sham, confirming that eWAT is a primary source of the increased circulating cytokines observed in the WD group. Both the control (normal diet-fed) and the WD group underwent the same surgical procedure without eWAT removal. The results are presented as mean ± SD with a sample size of n = 9 for western blot quantification and n = 5 for plasma ELISA assay. Asterisks denote statistically significant differences compared with the control group, with *p < 0.05 and ***p < 0.001, ns = no significant difference, One-way analysis of variance followed by Dunnett’s post hoc test.
Fig 3: Identification of epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines using AB array and QuantSeq 3′-mRNA sequencing. (A) Heatmap of cytokine expression in eWAT using the AB array. A total of 115 cytokines were significantly upregulated in the WD group compared with those in the control group, as indicated by the color scale representing relative expression levels (blue: downregulation, red: upregulation). (B) Heatmap of cytokine expression in eWAT determined using QuantSeq 3′-mRNA sequencing. In this analysis, 132 cytokines showed significant upregulation in the WD group compared with those in the control group, with similar color coding for relative expression changes. (C) A subset of five cytokines, including CCL8, CCL9, CXCL13, and IL-18, were consistently increased across both datasets (AB array for eWAT and QuantSeq for plasma) and were identified as eWAT-associated pro-inflammatory cytokines contributing to systemic and neuroinflammation in the WD group. (D) Protein–protein interaction (PPI) network analysis of differentially expressed genes (DEGs) was constructed using known interaction data. The network highlights significant interactions among key pro-inflammatory cytokines, illustrating the interconnected roles of these factors in inflammatory signaling. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs, with significant pathways related to inflammation, such as cytokine–cytokine receptor interaction and the chemokine signaling pathway. The x-axis denotes the number of genes associated with each pathway, while the size and color of the dots represent gene count and statistical significance, respectively.
Fig 4: Epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines induce significant neurotoxicity in HT22 cells. (A) The viability of HT22 hippocampal neuronal cells was not affected after 24 h of direct culture with eWAT-associated cytokines (CCL8, CCL9, CXCL13, and IL-18) at varying concentrations (25, 50, and 100 ng/mL). The results indicate no direct cytotoxic effects of these cytokines on hippocampal neurons under these conditions. (B) To evaluate the cytotoxicity of eWAT-associated cytokine in BV2 microglia, each cytokine was treated at a concentration of 50, 100, and 200 ng/ml. eWAT-associated cytokines did not induce cytotoxicity in BV2 microglial cells after 24 h of exposure, but they significantly increased microglial proliferation. (C) Treatment with each cytokine at a concentration of 100 ng/mL and analysis by ELISA showed that eWAT-associated pro-inflammatory cytokines significantly increased IL-6 secretion in BV2 microglial cells compared to those of untreated controls. (D) Intracellular reactive oxygen species (ROS) levels were measured in BV2 cells using a DCFDA fluorescence assay. Treatment with eWAT-associated cytokines led to a significant increase in ROS production, indicating oxidative stress–mediated microglial activation. (E) Treatment of HT22 hippocampal neurons with conditioned medium (CM) containing 100 ng/mL of each eWAT-associated cytokine to BV2 microglia significantly decreased cell viability, demonstrating the neurotoxic effect of cytokine-activated microglia on neurons. The data are presented as mean ± SD, with sample sizes ranging from n = 3 to n = 6 per group. Statistical significance is indicated by asterisks (*, compared with the control at 24 h): *p < 0.05, **p < 0.01, and ****p < 0.0001, ns = no significant difference, One-way analysis of variance followed by Dunnett’s post hoc test.
Supplier Page from Abcam for Mouse MCP2 ELISA Kit (CCL8)