Andrographolide exerted anti-inflammatory effects thereby reducing sex hormone synthesis in LPS-induced female rats, but had no effect on hormone production in healthy ones

FIGURE 1. Chemical structure of Andrographolide (AG).

FIGURE 2. Schematic diagram of tests process.

FIGURE 3. Antipyretic effect of andrographolide on endotoxin-induced inflammation in rats (A) temperature figure; (B) relative basal temperature difference change figure); * p <0.05 or ** p <0.01 versus Normal group and # p <0.05 or ## p <0.01 versus Model group by ANOVA.

FIGURE 4. Overlaid representative UPLC-Exactive Plus Orbitrap Mass TIC chromatograms (positive ion) of the serum samples from the four groups, including normal (N), model (LPS 100 μg/kg, M), low dose group (LPS 100 μg/kg + AG 20 mg/kg, L) and high dose (LPS 100 μg/kg + AG 40 mg/kg, H) group.

FIGURE 5. Metabonomics pattern recognition, metabolite identification and pathway enrichment (A): PCA diagram; (B) PLS-DA diagram; (C) Opls-DA diagram; (D) cluster analysis diagram; (E) and (F) are Volcanic diagram of peak abundance of mass-to-charge ratio chromatography in normal vs. model group and high dose vs. model group, respectively). There are four groups including normal group(N), model group (LPS 100 μg/kg, M), low dose group (LPS 100 μg/kg + AG 20 mg/kg, L) and high dose (LPS 100 μg/kg + AG 40 mg/kg, H) group, and the number represents the ID in the group.

FIGURE 6. Metabolite biomarkers including structure, mz, retention time and its concentrion in four groups: normal (N), model (LPS 100 μg/kg, M), low dose group (LPS 100 μg/kg + AG 20 mg/kg, L) and high dose (LPS 100 μg/kg + AG 40 mg/kg, H) group. # p <0.05 or ## p <0.01 versus Model group by ANOVA.

FIGURE 7. The metabolic pathway enrichment map showed that after lipopolysaccharide induced female rats, glycerophosphate and steroid hormone pathways were more affected by AG.

FIGURE 8. The box plot of sex steroids including progesterone (A), testosterone (B), estradiol (C) and corticosterone (D), inflammatory cytokins including TNF-α (E), IL-6 (F) and IL-1β (G) concentrations in serums, and the sex hormone synthesis gene expression (H) and (I) in ovories in normal group (6, N), normal with AG administration group (6, AG 40 mg/kg, NAG), model group (6, LPS 100 μg/kg, M) and high dose (6, LPS 100 μg/kg + AG 40 mg/kg, AG) groups. *p < 0.05 or **p < 0.01 versus Normal group and #p < 0.05 or ##p < 0.01 versus Model group by ANOVA.

FIGURE 9. Correlation analysis of four sex steroids (progesterone, testosterone, estradiol and corticosterone), three inflmmatory cytokins (TNF-α, IL-6, and IL-1β) concentrations in serums, and the two sex hormone synthesis gene expression in ovories in verification experiment. The data name appears on the primary diagonal line. Data for each type is an aggregate of data of rats in all groups, including normal group (6, N), normal with AG administration group (6, AG 40 mg/kg, NAG), model group (6, LPS 100 μg/kg, M) and high dose (6, LPS 100 μg/kg + AG 40 mg/kg, AG) groups. The circles in the sub-boxes below the main diagonal indicate pairs of two kinds data of the same rat, and the red line depicts the correlation trend. The correlation coefficient is indicated in each box above the main diagonal.

FIGURE 10. The mechanism of andrographolide (AG) acting on sex hormone production pathways. Red arrows indicate stimulation and blue vertical bars indicate inhibition. AG acting on sex hormone production may be mediated by anti-inflammatory action, and is an indirect attributes.