Fig 1: KORs suppress MI activation by regulating the NLRP3/caspase-1 pathway. (a) Western blotting was carried out to determine the protein expression levels of NLRP3 inflammasome and pro-caspase-1 in LPS-stimulated RM cells treated with caspase-1 inhibitor (AC-YVAD-cmk). LPS-stimulated RM cells were coadministered with the KOR agonist (U50488H) and excessively activated NLRP3 triggered by TLR4 agonist (RS09). The relative expression levels of NLRP3/caspase-1 pathway-related proteins (b) and MI-polarization-associated proteins (c) were quantified with western blotting. (d) Levels of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-? (pg/ml), in RM cell supernatant were determined using ELISA. All data in charts were represented as mean ± standard deviation; “ns” was labeled when the comparison between groups (p value) was greater than 0.05, while “*” was labeled when the p value was less than 0.05.
Fig 2: BSHM improves the immunity of whole body and kidney and promotes the immune balance and homeostasis. (a) Representative pictures of HE staining (× 200) of spleens (n = 3). (b) The levels of plasma IL-6, IL-10, and IL-17A (n = 10). (c) Flow cytometry was used to determine the levels of both Th17 and Treg cells in peripheral blood (n = 10). (d) Representative pictures of flow cytometry (n = 3). (e) Representative pictures of immunofluorescence staining. Red area for Foxp3 and green area for RORγt (n = 3). (f) The positive rates of immunofluorescence staining were analyzed by ImageJ (n = 3). Notes: ∗∗P < 0.01 vs. WKY group; △P < 0.05 vs. SHR group; △△P < 0.01 vs. SHR group.
Fig 3: Butyric acid ameliorated symptoms of MF in rats. (A) HE staining was applied to stain the heart tissues. (B). HE staining was performed to stain the gut tissues. (C) The levels of IL-6, TNF-α, and IL-12 in plasma were determined by ELISA. (D–F). ELISA was used to detect the levels of LPS, 8-oxo-dG and CS. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 4: Anti-bacterial ability of PPHNs in prophylactic infection scheme. (A) Anti-bacterial efficacy of PPHNs in ACF. Mean ± S.E.M., n = 3. ***p < 0.001. (B) Anti-bacterial efficacy of PPHNs in the liver. The number of viable bacteria per gram of liver tissue was determined with colony formation assay. Mean ± S.E.M., n = 3. ***p < 0.001. (C) Immunofluorescent image of FITC-labeled phages in the liver. Nucleus (blue) was stained with DAPI. Scale bar = 20 μm. (D) Evaluation of serum IFN-γ. Bac, bacteria. Mean ± S.E.M., n = 3. **p < 0.01, ***p < 0.001. (E) Evaluation of serum IL-6. Bac, bacteria. Mean ± S.E.M., n = 3. **p < 0.01, ***p < 0.001. (F) Endotoxin release assay. Endotoxin level in the culture supernatant was determined by ELISA at various time points following phage infection of E. coli. Mean ± S.E.M., n = 3. **p < 0.01. (G) Infiltration of immune cells into the liver, revealed by representative images of liver sections after HE staining. Bac, bacteria. Scale bar = 20 μm.
Fig 5: KORs regulate MI polarization state in CPB rats. (a) For detection of MI polarization state transitions, immunofluorescent counterstaining of iNOS/IBA-1 and arg-1/IBA-1 was carried out (scale bar = 50 µm). Accumulated IBA-1 expressions were detected when CPB occurred, and the MI polarization transition from M1 toward M2 phenotype was also observed when U50488H was applied to CPB rats. (b) Contents of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-? (pg/ml), in rats' brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “*” was labeled when the comparative significance between groups (p value) was less than 0.05.
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