Fig 1: Effects of arbutin and Ag490 on LPS-stimulated IEC6 and RAW 264.7 cells (A) IEC-6 cells were given arbutin (100 and 500 µM) for 12 h after treatment with LPS (1 µg/ml) for 12 h and the level of p-STAT3 was determined by western blot analysis (B) IEC-6 cells were pretreated with AG490 (50 µM) for 1 h, treated with LPS (1 µg/ml) for 12 h, and given arbutin (100 and 500 µM) for 12 h. Then the level of p-STAT3 was detected by western blot analysis (C, D) IEC-6 cells were pretreated with AG490 (50 µM) for 1 h, LPS (1 µg/ml) for 12 h, and then treated with arbutin (500 µM) for 12 h, respectively. The TNF-a and IL-6 levels were detected by ELISA (E) RAW 264.7 cells were pretreated with AG490 (50 µM) for 1 h, treated with LPS (1 µg/ml) for 12 h, and given arbutin (500 µM) for 12 h, and the p-STAT3 expression was detected by immunofluorescence as described in the Materials and Methods. Data were expressed as mean ± SD. *p < 0.05 compared between the indicated groups; **p < 0.01 Compared with sham group; #p < 0.05 compared with LPS control group; ##p < 0.01 compared with LPS control group; NS p > 0.05 compared between the indicated groups.
Fig 2: Early MC exposure caused pathological outcomes in the small intestine and elevated levels of systemic IL-6 in both WT mice and NSG™ mice. Normalized mRNA expression of (A) TLR2, TLR4, REG3G, and (B) CD28, CD57, IL-7, and PD1 against 18S in the small intestine of CONTROL and MC groups of mice and showed as the fold change of the CONTROL group (***p < 0.001). (C) IL-6 (pg/mL) levels were measured in the serum of both CONTROL and MC mice groups and represented as bar graphs (****p < 0.001). Normalized mRNA expression of (D) CD28, CD57 against 18S in the small intestine of Hu-CONTROL (NSG™ mice treated with vehicle only) and Hu-MC (NSG™ mice orally administered with MC for 2 weeks) groups of mice and showed as the fold change of the Hu-CONTROL group (***p < 0.001). (E) IL-6 (pg/mL) levels were measured in the serum of both Hu-CONTROL and Hu-MC mice groups and represented as bar graphs (****p < 0.001). All data were represented as mean ± SEM, statistical significance was tested using unpaired t-test between the two groups, followed by Bonferroni Dunn Post hoc corrections.
Fig 3: Malfunctioning of macrophages in TP-induced indirect hepatotoxicity. (A) Schematic presentation of the experimental procedure to investigate the time-dependent release of inflammatory factors in mice treated with TP (500 µg/kg) and LPS (0.1 mg/kg). (B) Serum endotoxin content in mice treated with TP and LPS (n = 8) 6 h after LPS injection. (C–E) Changes in serum TNF-a, IL-6, and IL-1ß in mice after the treatment of TP and LPS (n = 8). Results were expressed as means ± SEM. Statistical analysis was performed using One-way ANOVA and Two-way ANOVA followed by Tukey’s Multiple Comparison Test. p < 0.05 was considered to be statistically different.
Fig 4: The cGAS pathway promotes the maturation and activation of BMDCs. (A) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. (B) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. (C) Culture supernatants were collected after 24 h; the expressions of TNF-a, IL-6, IL-10, and IL-12p70 were assayed by ELISA. All data are expressed as mean ± SD, (* p < 0.05; ** p < 0.01; n.s.: no statistical significance).
Fig 5: Inflammation evaluation. (A) Clinical photographs of hind legs of the control group and CAIA group mice. Swelling of the ankle was observed in the CAIA group. (Score 3) (B) Clinical severity of arthritis in control group and CAIA group after collagen antibody cocktail injection. Severity of arthritis in each foot was scored from 0 (no swelling) to 4 (erythema and severe swelling of the entire tarsal joint), and the total of 4 feet (0–16). In the CAIA group, the score was about 12 on day 6 of antibody administration. *P < 0.05. (C) Histological analysis of the hind paw of each group on day 14. The knee joint was stained with hematoxylin and eosin (a) and Safranin O (b) (1: tibia, 2: femur). Arrows indicate increased inflammatory cells. (scale bar: 100 µm) (D) Blood IL-1ß levels (pg/ml) in the control and CAIA groups. In the CAIA group, the IL-ß concentration was 24.5 (pg/ml) *P < 0.05. (E) Blood IL-6 levels (pg/ml) in the control and CAIA groups. There was no significant (NS) difference between the control group and the CAIA group. NS, not significant.
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