Salvianolic Acid B Alleviates LPS-Induced Spleen Injury by Remodeling Redox Status and Suppressing NLRP3 Inflammasome

Figures

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  Figure 1. Schematic representation of the experimental procedures.

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  Figure 2. Effects of LPS and SAB on average body weight and immune organ indices of piglets. (A) Average body weight; (B) Spleen index; (C) Thymus index; (D) Mandibular lymph node index. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS, and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 3. Effects of LPS and SAB on blood routine indicators in piglets. (A) White blood cells; (B) Neutrophils; (C) Lymphocytes; (D) Monocytes; (E) Eosinophils; (F) Basophils. Results are expressed as the mean ± SEM (n = 4). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS, and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 4. Effects of LPS and SAB on spleen histomorphology and apoptosis in piglets. (A) The representative images of spleen stained with H&E with a magnification of 15× and 40×, a scale bar of 100 μm, red pulp (red boxes), white pulp (white boxes). (B) The representative images of spleen stained with TUNEL and DAPI with a magnification of 40× and a scale bar of 50 μm. (C) Quantitative analysis of the TUNEL-positive cell percentage of spleen. (D) Splenic caspase 3 activity. (E) Splenic caspase 8 activity. (F) Splenic caspase 9 activity. (G) Splenic mRNA expression of BCL2. (H) Splenic mRNA expression of BAX. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS, and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 5. Effects of LPS and SAB on splenic immune status in piglets. (A) Splenic MPO expression was detected by immunofluorescence analysis and the representative images were showed with a magnification of 60× and a scale bar of 50 μm; (B) Splenic MPO activity; (C) Splenic TNF-α level; (D) Splenic INF-γ level; (E) Splenic IL-1β level; (F) Splenic IL-18 level; (G) The mRNA expression of inflammation-related genes in the spleen. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 6. Effects of LPS and SAB on splenic redox status in piglets. (A) Splenic MDA content. (B) Splenic 8-OHdG content. (C) Splenic CAT activity. (D) Splenic GPX activity. (E) Splenic SOD activity. (F) Representative Western blot bands showing cytosolic and nuclear Nrf2 protein in the spleen. (G) Splenic relative expression of cytosolic Nrf2 level. (H) Splenic relative expression of nuclear Nrf2 level. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 7. Effects of LPS and SAB on splenic NLRP3 inflammasome activation in piglets. (A) Representative Western blot bands showing NLRP3 inflammasome-associated pathways. (B) Splenic relative expression of NLRP3. (C) Splenic relative expression of GSDMD. (D) Splenic relative expression of cleaved caspase 1. (E) Splenic relative expression of mature IL-β. (F) Splenic NF-κB p65 phosphorylation. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.

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  Figure 8. Effects of LPS and SAB on splenic mitochondrial structure and function in piglets. (A) Representative TEM images of spleen illustrating splenic ultrastructural features. (B) Spleen mitochondrial swelling was evaluated by monitoring the decreased absorbance of a mitochondrial suspension in response to calcium exposure. (C) Mitochondrial superoxide radical levels were detected by incubation with the DHE probe. (D) Mitochondrial membrane potential was evaluated using the JC-1 probe. (E) Splenic ATP content. (F) Splenic complex I activity. (G) Splenic complex Ⅱ activity. (H) Splenic complex Ⅲ activity. (I) Splenic complex Ⅳ activity. (J) Splenic ATP synthase. Results are expressed as the mean ± SEM (n = 6). A two-way analysis of variance using the general linear model was performed to assess significant differences for diet, LPS and their interaction. When the interaction effect was significant (p < 0.05), Tukey’s post hoc test was applied for multiple comparisons among groups. Columns labeled with different letters are significantly different (p < 0.05) based on Tukey’s test.