Dracocephalum moldavica Ethanol Extract Suppresses LPS-Induced Inflammatory Responses through Inhibition of the JNK/ERK/NF-κB Signaling Pathway and IL-6 Production in RAW 264.7 Macrophages and in Endotoxic-Treated Mice

Abstract

The excessive synthesis of interleukin-6 (IL-6) is related to cytokine storm in COVID-19 patients. Moreover, blocking IL-6 has been suggested as a treatment strategy for inflammatory diseases such as sepsis. Sepsis is a severe systemic inflammatory response syndrome with high mortality. In the present study, we investigated the anti-inflammatory and anti-septic effects and the underlying mechanisms of Dracocephalum moldavica ethanol extract (DMEE) on lipopolysaccharide (LPS)-induced inflammatory stimulation in RAW 264.7 macrophages along with septic mouse models. We found that DMEE suppressed the release of inflammatory mediators NO and PGE₂ and inhibited both the mRNA and protein expression levels of iNOS and COX-2, respectively. In addition, DMEE reduced the release of proinflammatory cytokines, mainly IL-6 and IL-1β, in RAW 264.7 cells by inhibiting the phosphorylation of JNK, ERK and p65. Furthermore, treatment with DMEE increased the survival rate and decreased the level of IL-6 in plasma in LPS-induced septic shock mice. Our findings suggest that DMEE elicits an anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophages and an anti-septic effect on septic mouse model through the inhibition of the ERK/JNK/NF-κB signaling cascades and production of IL-6.

Keywords: Dracocephalum moldavica; inflammation; interleukin-6; lipopolysaccharide; sepsis.

Figure 1

HPLC-UV chromatogram analysis of oleanolic acid in D. moldavica with detector responses at 210 nm.

Figure 2

Effects of D. moldavica on LPS-induced inflammatory response in RAW 264.7 cells. Cells were pretreated with D. moldavica for 1 h and then treated with LPS (1 μg/mL) for 24 h. Cell viability was determined by MTT assay (n = 5) (A,B). The production of NO was measured by Griess reaction (n = 3) (C). The level of PGE₂ was measured by PGE₂ ELISA kit (n = 3) (D). The mRNA expression of iNOS and COX-2 was determined by RT-PCR (n = 3) (E,F). The protein levels of iNOS and COX-2 were measured by Western blotting and the quantification of iNOS and COX-2 was normalized to the control (n = 3) (G,H). The data shown are representative of three independent experiments and indicate mean ± S.E.M. ^### p < 0.001 versus the vehicle-treated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the LPS-treated group.

Figure 3

Effects of D. moldavica on LPS-induced proinflammatory cytokine expression in RAW 264.7 cells. Cells were pretreated with D. moldavica for 1 h and then treated with LPS (1 μg/mL) for 24 h. The mRNA expression of IL-6 and IL-1β was determined by RT-PCR (n = 3) (A,B) and the secretion of IL-6 and IL-1β was measured by IL-6 and IL-1β ELISA kit (n = 3) (C,D). The data shown are representative of three independent experiments and indicate mean ± S.E.M. ^# p < 0.05, ^### p < 0.001 versus the vehicle-treated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the LPS-treated group.

Figure 4

Effects of D. moldavica on the MAPK/NF-κB pathway in RAW 264.7 cells. Cells were pretreated with D. moldavica for 1 h and then treated with LPS (1 μg/mL) for 30 min. The phosphorylation activity was normalized to the untreated control group. The expression of phospho-JNK, JNK, phospho-ERK, ERK, phosphor-p65, p65 and β-actin was determined by Western blotting (n = 3) (A–C). The data shown are representative of three independent experiments and indicate mean ± S.E.M. ^### p < 0.001 versus the vehicle-treated controls; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the LPS-treated group.

Figure 5

Effect of D. moldavica on the survival rate and level of IL-6 in plasma in LPS-induced septic shock mice. Mice were administered with D. moldavica (50, 100 and 200 mg/kg p.o.) or vehicle (0.9% saline) for 7 days and then injected with LPS (25 mg/kg, i.p.). The survival rate was measured for 3 days and blood samples were collected 12 h after LPS injection. Timetable of the LPS-induced septic shock mouse model (A). Survival rate of the group injected with D. moldavica or LPS (control D. moldavica 100 mpk, 100%; LPS 25 mpk, 38%; D. moldavica 50 mpk + LPS, 42%; D. moldavica 100 mpk + LPS, 58%; D. moldavica 200 mpk + LPS, 75%) (n = 8/group) (B). The levels of IL-6 and IL-1β in plasma were determined by ELISA kit (n = 4/group) (C,D). The data shown are representative of three independent experiments and indicate mean ± S.E.M. ^# p < 0.05 and ^### p < 0.001 versus the vehicle-treated controls; * p < 0.05 versus the LPS-treated group.

Figure 6

The anti-inflammatory pathways of D. moldavica in LPS-stimulated RAW 264.7 macrophages.