Accelerated and Severe Lupus Nephritis Benefits From M1, an Active Metabolite of Ginsenoside, by Regulating NLRP3 Inflammasome and T Cell Functions in Mice

Figure 1. Renal function, albuminuria, renal pathology, and anti-dsDNA antibodies. (A) Levels of serum BUN and (B) serum Cr. (C) Time-course studies of albuminuria levels. (D) Levels of serum anti-dsDNA autoantibody. (E) H&E staining. Arrows indicate mononuclear leukocyte infiltration. Original magnification x400. (F–J) Scoring of H&E. (K) Scoring of glomerulonephritis activity. The data are the means ± SEM for 8 mice per group. ASLN, accelerated and severe lupus nephritis. BUN, blood urea nitrogen. Cr, creatinine. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. #Not detectable. ns, no significant difference.

Figure 2. Serum inflammatory cytokine expression. Serum levels of (A) IL-1β, (B) IFN-γ, (C) IL-6, (D) TNF-α, (E) MCP-1 (F) IL-12 p70, and (G) IL-10. The horizontal dashed line indicates the mean of levels from normal control mice. The data are the means ± SEM for 8 mice per group. ASLN, accelerated and severe lupus nephritis. **p < 0.01, ***p < 0.005, and ****p < 0.001. ns, no significant difference.

Figure 3. T-cell function. (A) T-cell proliferation in splenocytes by thymidine uptake analysis. (B) CD3+CD69+ T cells; (C) CD4+CD69+ T cells; (D) CD4+IFNγ+ T cells; (E) CD4+IL-4+ T cells; (F) CD4+CD25+Foxp3+ Treg cells, in splenocytes, by flow cytometry. The data are the means ± SEM for 8 mice per group. ASLN, accelerated and severe lupus nephritis. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. ns, no significant difference.

Figure 4. ROS production. (A) ROS levels in BMDCs. The cells were incubated with M1 (10 μM) or NAC (10 mM; a ROS scavenger) for 30 min, then treated with or without 100 ng/ml LPS for 5.5 h and ATP (5 mM). (B) ROS levels in podocytes. The cells were incubated with M1 (10 μM) or NAC (10 mM; a ROS scavenger) for 30 min, then treated with or without 1 μg/ml LPS for 5.5 hr and ATP (5 mM). The ROS production were measured by 2′, 7′ -dichlorofluorescein diacetate after incubated with ATP. (C) NADPH oxidase p47phox (p47phox) and COX-2 protein levels in BMDCs by Western blotting and (D) semiquantitative analysis. BMDCs were incubated with M1 (10 μM) for 30 min, then treated with or without 100 ng/ml LPS for 6 h. (E) NADPH oxidase p47phox (p47phox) and COX-2 protein levels in podocytes by Western blotting and (F) semiquantitative analysis. The podocytes were incubated with M1 (10 μM) for 30 min, then treated with or without 1 μg/ml LPS for 6 h. The data are expressed as the means ± SEM for three separate experiments, and each experiment was performed in triplicate. (G) In situ renal ROS production with DHE staining and (H) scoring. Original magnification, 400×. ROS levels in (I) renal tissues and…

Figure 5. NLRP3 inflammasome activation. (A–F) BMDCs. (G–L) Podocytes. (M) Mouse J774A.1 macrophages. The BMDCs were incubated with the indicated concentrations of M1 for 30 min, and then incubated with 100 ng/ml LPS for 5.5 h and 5 mM ATP for 30 min. (A) IL-1β secretion in BMDCs was measured by ELISA, and (B) pro-caspase-1 (procasp-1) and secretion of caspase-1 (Casp-1) in supernatant of BMDCs were measured by Western blotting. (C,D) NLRP3 and proIL-1β protein levels and (E,F) p-IκB protein levels in BMDCs by Western blotting after incubated with M1 for 30 min, and then incubated with 100 ng/ml LPS for 6 h or the indicated times. (G) IL-1β secretion and (H) caspase-1 activity in podocytes were measured by ELISA after incubated with the indicated concentrations of M1 for 30 min, and then incubated with 100 ng/ml LPS for 5.5 h and 5 mM ATP for 30 min. (I,J) NLRP3 and proIL-1β and (K,L) p-IκB protein levels in podocytes by Western blotting after incubated with M1 for 30 min, and then incubated with 1 μg/ml LPS for 6 h or the indicated times. (M) IL-1β secretion in J774A.1 macrophages was measured by ELISA. The macrophages were incubated with the indicated concentrations of M1 for 30 min, and…

Figure 6. M1 inhibited the NLRP3 inflammasome via autophagy induction, and regulated adaptive immunity. (A,B,E,G,I–K) BMDCs. (C,D,F,H) Podocytes. Protein expression levels of LC3B, Atg5 and SIRT3 by Western blotting after incubated for 0–24 h with the indicated concentrations of M1 in (A,B) BMDCs and (C,D) podocytes. LC3B expression by immunofluorescence staining in (E) BMDCs and (F) podocytes. Both the cells incubated with M1 (10 μM) or vehicle (DMSO; 0.2%) for 6 h. Arrowhead indicates the positive staining. (G) Protein expression levels of caspase-1 (Casp-1) and IL-1β in supernatant of BMDCs by Western blotting, and (H) IL-1β secretion in podocytes by ELISA, after incubated for 5 mM 3-MA (autophagy inhibitor) for 30 min, then with 10 μM M1 for 30 min followed by 100 ng/ml (BMDCs) or 1 μg/ml (podocytes) LPS for 5.5 h, and 5 mM ATP for 30 min. (I) Expression levels of CD80+ (within gated CD11c+ cells) determined by flow cytometry in BMDCs from 8-week-old female NZB/WF1 mice, which were incubated with M1 (10 μM) 30 min, then 100 ng/ml LPS for 24 h and 5 mM ATP for 30 min. (J) OT-II antigen-specific T cell proliferation was measured by 3H-thymidine incorporation. The ratio of the total cell…

Figure 7. Schematic representation for the plausible mechanism of action for the therapeutic effects of M1 on ASLN. ASLN, accelerated and severe lupus nephritis.