Immunostimulatory activity and structure-activity relationship of epimedin B from Epimedium brevicornu Maxim

Abstract

Epimedii Folium (EF, Epimedium brevicornu Maxim.), a traditional botanical drug, is famous for treating bone fractures, joint diseases, and several chronic illnesses. However, some studies indicated that EF could induce idiosyncratic drug-induced liver injury (IDILI) in the clinic. The NLRP3 inflammasome plays a crucial role in the pathogenesis of various human diseases, including IDILI. In the present study, we showed that epimedin B could specifically facilitate nigericin- or ATP-induced NLRP3 inflammasome activation under synergistic induction of mitochondrial reactive oxygen species. Moreover, epimedin B resulted in activation of Caspase-1 and IL-1β secretion in a lipopolysaccharide (LPS)-mediated susceptibility mouse model. MCC950 pretreatment completely abrogated activation of the NLRP3 inflammasome and prevented liver injury. Importantly, several studies have confirmed that some active constituents of EF could enhance activation of the NLRP3 inflammasome and may be involved in the pathogenesis of EF-IDILI. No reports are available on whether the structure-activity relationship associated with the immunostimulatory activity in EF contributes to the pathogenesis of EF-IDILI. These findings have changed our conventional understanding about the more glycogen, the more immunostimulatory activity.

Keywords: NLRP3 inflammasome; epimedii folium; epimedin B; idiosyncratic drug-induced liver injury; mitochondrial ROS.

FIGURE 1

Effect of the main constituents of Epimedii folium on NLRP3 inflammasome activation. (A) Western blot analysis of supernatants (Sup) and whole-cell lysates (Lys) derived from lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) subjected to treatment with the main constituents of EF (40 μM) and stimulated with nigericin (10 μmol/L). (B–D) Caspase-1 activity (B) ELISA of IL-1β (C), and TNF-α (D) in Sup derived from the samples described in (A). Data are expressed as the mean ± SD from at least three biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant, RLUs; relative light units.

FIGURE 2

The relationship between the chemical structure and NLRP3 inflammasome activity of the main active constituents of Epimedii folium. Epimedin B, Icariside I, and Icariside II could promote NLRP3 inflammasome activation. Six other major active constituents of Epimedii folium (EF) (epimedin A, epimedin A1, epimedin C, icariin, icaritin, and anhydroicaritin) could not induce NLRP3 inflammasome activation.

FIGURE 3

Epimedin B promotes NLRP3 inflammasome activation in bone marrow-derived macrophages and THP1 cells stimulated by nigericin or ATP. (A) Western blot analysis of supernatants (Sup) and whole-cell lysates (Lys) derived from lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) subjected to treatment with various doses of epimedin B prior to nigericin stimulation. (B–E) Caspase-1 activity (B), the release of lactate dehydrogenase (LDH) (C), ELISA of IL-1β (D) and TNF-α (E) levels in supernatants (Sup) from samples described in (A) (F,G) Caspase-1 activity (F) and ELISA of IL-1β (G) of Sup and Lys derived from LPS-primed BMDMs subjected to treatment with various doses of epimedin B prior to ATP stimulation. (H) Western blot analysis of Sup and Lys derived from PMA-primed THP1 cells subjected to treatment with various doses of epimedin B prior to nigericin stimulation. (I–K) Caspase-1 activity (I), release of LDH (J), and ELISA of IL-1β (K) in Sup from samples described in (H). Data are presented as the mean ± SD from at least three biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant, RLUs; the relative light units.

FIGURE 4

Epimedin B does not affect NLRP3 inflammasome activation induced by SiO2, poly (I:C), and intracellular lipopolysaccharide, as well as AIM2 and NLRC4 inflammasome. (A,B) Western blot analysis of supernatants (Sup) and whole-cell lysates (Lys) derived from lipopolysaccharide (LPS)/Pam3CSK4-primed bone marrow-derived macrophages (BMDMs) subjected to treatment with epimedin B and stimulated with nigericin, ATP, SiO2, poly (I:C), poly (dA:dT), Salmonella, or intracellular LPS. (C–H) Caspase-1 activity (C,F), ELISA of IL-1β (D,G), and TNF-α (E,H) in Sup derived from samples described in A and (B). RLUs, relative light units. Data are presented as the mean ± SD from at least three biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

FIGURE 5

Epimedin B promotes ATP or nigericin-induced ASC oligomerization but does not block K⁺ efflux and Ca²⁺ flux. (A) Western blot analysis of whole-cell lysates from bone marrow-derived macrophages (BMDMs) subjected to treatment with epimedin B for 1 h, and stimulated with lipopolysaccharide (LPS) (50 ng/ml) for 3 h or BMDMs were stimulated with LPS (50 ng/ml) for 3 h and then subjected to treatment with epimedin B for 1 h (B,C) ELISA of TNF-α (B) and IL-6 (C) in Sup derived from samples described in (A) (D) Western blot analysis of ASC oligomerization from LPS-primed BMDMs subjected to treatment with various doses of epimedin B prior to nigericin stimulation. (E) Western blot analysis of ASC oligomerization from LPS-primed BMDMs subjected to treatment with epimedin B and stimulated with nigericin, ATP, SiO2, and poly (I:C). (F) Quantification of potassium efflux in LPS-primed BMDMs subjected to treatment with various doses of epimedin B and stimulated with nigericin. (G) A trace of ATP-induced Ca²⁺ flux was measured using the FLIPRTETRA system in LPS-primed BMDMs subjected to treatment with epimedin (B). Data are presented as the mean ± SD from at least three biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

FIGURE 6

Epimedin B facilitates NLRP3 inflammasome activation by increasing mitochondrial reactive oxygen species (mtROS) production. (A–D) Percentage of ROS-positive cells in lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) subjected to treatment with epimedin B that were either not stimulated (A) or stimulated with nigericin (B), ATP (C) or SiO2 (D), followed by staining with MitoSox. After completion of the staining and washing procedures, flow cytometry was conducted to determine the level of mtROS production. (E) Percentage of reactive oxygen species (ROS)-positive cells in LPS-primed BMDMs subjected to treatment with epimedin B that were either not stimulated or stimulated with nigericin, ATP, or SiO2. (F) Western blot analysis of supernatants and whole-cell lysates derived from LPS-primed BMDMs subjected to treatment with epimedin B, NAC, or epimedin B plus NAC prior to stimulation with nigericin or without stimulation. (G) Caspase-1 activity in samples described in (F). Data are presented as the mean ± SD derived from at least three biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

FIGURE 7

Epimedin B promotes early liver injury and inflammatory mediator production in vivo. (A–H) Female C57BL/6 mice (age: 6–8 weeks) subjected to starvation for 24 h were administered with 2 mg/kg of lipopolysaccharide (LPS) or its saline vehicle via the tail vein (i.v.). After an observation period of 2 h, various doses of epimedin B (20 mg/kg, 40 mg/kg, 80 mg/kg) or its vehicle were administered through intraperitoneal injection for 6 h (A,B) Serum levels of ALT (A) and AST (B) and (C,D) Serum levels of IL-1β (C) and TNF-α (D) determined by ELISA. (E,F) PCR of IL-1β (E) and IL-18 (F) mRNA levels. (G,H) Representative micrographs of H&E staining (G) and F4/80 staining (H). Data are presented as the mean ± SD. The significance of the differences was analyzed using unpaired Student’s t-test: ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

FIGURE 8

Epimedin B induces IDILI by promoting NLRP3 inflammasome activation in vivo. (A–I) Female C57BL/6 mice (age: 6–8 weeks) were administered MCC950 (40 mg/kg) or its saline vehicle through intraperitoneal injection. After 1 h, lipopolysaccharide (LPS) (2 mg/kg) or its saline vehicle was administered via the tail vein for 2 h. Subsequently, epimedin B (40 mg/kg) was administered via intraperitoneal injection for 6 h (A,B) Serum levels of ALT (A) and AST (B), and (C,D) Serum levels of IL-1β (C) and TNF-α (D) determined by ELISA. (E–G) PCR of IL-1β (E), IL-18 (F), and TNF-α (G) mRNA levels. (H) Western blot analysis of pro-caspase-1 and cleaved caspase-1 expression in liver tissue. (I) Representative micrographs of H&E staining. Data are presented as the mean ± SD. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.