To elucidate the mechanism of "Scrophulariae Radix-Fritillaria" in goiter by integrated metabolomics and serum pharmaco-chemistry

Abstract

The pharmacodynamic substances in "Scrophulariae Radix-Fritillaria" and the molecular mechanisms underlying its therapeutic effects against goiter were analyzed through metabolomics and serum pharmaco-chemistry. A rat model of goiter was established using propylthiouracil (PTU), and the animals were treated using "Scrophulariae Radix-Fritillaria." The efficacy of the drug pair was evaluated in terms of thyroid gland histopathology and blood biochemical indices. Serum and urine samples of the rats were analyzed by UPLC-Q-TOF/MS. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to screen potential biomarkers in urine and the corresponding metabolic pathways. The blood components of "Scrophulariae Radix-Fritillaria" were also identified, and their correlation with urine biomarkers was analyzed in order to screen for potential bioactive compounds. "Scrophulariae Radix-Fritillaria" mitigated injury to thyroid tissues and normalized the levels of the thyroid hormones FT3, FT4, and TSH. We also identified 22 urine biomarkers related to goiter, of which 19 were regulated by "Scrophulariae Radix-Fritillaria." Moreover, urine biomarkers are involved in tryptophan metabolism, steroid hormone biosynthesis, and beta-alanine metabolism, and these pathways may be targeted by the drug pair. In addition, 47 compounds of "Scrophulariae Radix-Fritillaria" were detected by serum pharmacochemistry, of which nine components, namely, syringic acid, paeonol, cedrol, and cis-ferulic acid, fetisinine, aucubigenin, linolenic acid, ussuriedine, and 5-(methylsulfanyl)pentanenitrile, were identified as potential effective substances against goiter. To summarize, we characterized the chemical components and mechanisms of "Scrophulariae Radix-Fritillaria" involved in the treatment of goiter, and our findings provide an experimental basis for its clinical application.

Keywords: UPLC-Q-TOF/MS; goiter; metabolomics; serum pharmacochemistry; “Scrophulariae Radix–Fritillaria” drug pair.

FIGURE 1

Comparison of weight and thyroid weight in each group. Note: compared with the control group, *p < 0.05 and **p < 0.01; compared with the model group, △p < 0.05 and △△p < 0.01; compared with the L-T4 group, #p < 0.05 and ##p < 0.01. (A): Weight (g) (B): Weight of thyroid (mg) (C): Relative thyroid weight (mg/100mg).

FIGURE 2

Representative images showing thyroid histopathology in each group (HE staining × 400). Note: (A) control group; (B) model group; (C) L-T4 group; and (D) “Scrophulariae Radix–Fritillaria” drug pair group.

FIGURE 3

Changes in FT3, FT4, and TSH contents in each group of rats. Note: compared with the control group, *p < 0.05 and **p < 0.01; compared with the model group, △p < 0.05 and △p < 0.01; and compared with the L-T4 group, #p < 0.05 and ##p < 0.01. (A): FT3 (pmol/L) (B): FT4 (pmol/L) (C): TSH (mIU/L).

FIGURE 4

Multivariate tests. Note: (A) PCA score chart (positive-ion mode). (B) PCA score chart (negative-ion mode). (C) OPLS-DA score chart (positive-ion mode). (D) OPLS-DA score chart (negative-ion mode).

FIGURE 5

S-plot and VIP-plot of the rat urine metabolic profile. (A): PCA score chart (positive-ion mode) (B): PCA score chart (negative-ion mode) (C): OPLS-DA score chart (positive-ion mode) (D): OPLS-DA score chart (negative-ion mode).

FIGURE 6

Metabolic pathway map.

FIGURE 7

Chromatographic diagram of BPI of the “Scrophulariae Radix–Fritillaria” drug pair.

FIGURE 8

Heatmap of correlation analysis between the serum components of the “Scrophulariae Radix–Fritillaria” drug pair and urine biomarkers in the goiter rat model.