The Flavonoid Baicalein Negatively Regulates Progesterone Target Genes in the Uterus in Vivo

Abstract

Baicalein is a flavonoid extracted from the root of Scutellaria baicalensis (Chinese skullcap) and is consumed as part of this botanical dietary supplement to reduce oxidative stress, pain, and inflammation. We previously reported that baicalein can also modify receptor signaling through the progesterone receptor (PR) and glucocorticoid receptor (GR) in vitro, which is interesting due to the well-established roles of both PR and GR in reducing inflammation. To understand the effects of baicalein on PR and GR signaling in vivo in the uterus, ovariectomized CD-1 mice were treated with DMSO, progesterone (P4), baicalein, P4 with baicalein, and P4 with RU486, a PR antagonist, for a week. The uteri were collected for histology and RNA sequencing. Our results showed that baicalein attenuated the antiproliferative effect of P4 on luminal epithelium as well as on the PR target genes HAND2 and ZBTB16. Baicalein did not change levels of PR or GR RNA or protein in the uterus. RNA sequencing data indicated that many transcripts significantly altered by baicalein were regulated in the opposite direction by P4. Similarly, a large portion of GO/KEGG terms and GSEA gene sets were altered in the opposite direction by baicalein as compared to P4 treatment. Treatment of baicalein did not change body weight, organ weight, or blood glucose level. In summary, baicalein functioned as a PR antagonist in vivo and therefore may oppose P4 action under certain conditions such as uterine hyperplasia, fibroids, and uterine cancers.

Figure 1.. Baicalein attenuates the inhibitory effect of progesterone on luminal and glandular epithelial cell proliferation.

Immunohistochemical staining against proliferation marker PCNA on uterine cross sections of 10% DMSO (A), 1 mg/kg progesterone (B), 25 mg/kg baicalein and 1 mg/kg progesterone (C), 1 mg/kg progesterone and 10 mg/kg RU486 (D) and 25 mg/kg baicalein (E) treated mice. (F) Percentage of proliferating luminal epithelial cells in each treatment group. Arrows indicate positive stains. Scale bar = 300 μm. N=5/group. Inset images were taken at 4x magnification. Asterisks indicate * p≤0.05, ** p≤0.01,*** p≤0.001.

Figure 2.. Baicalein decreased progesterone induced HAND2 expression in mouse uteri.

Immunohistochemical staining against progesterone receptor target HAND2 on uterine cross sections of 10% DMSO (A), 1 mg/kg progesterone (B), 25 mg/kg baicalein and 1 mg/kg progesterone (C), 1 mg/kg progesterone and 10 mg/kg RU486 (D) and 25 mg/kg baicalein (E) treated mice. Arrows indicate positive stains. Scale bar = 150 μm. Inset images were taken at 4x magnification.

Figure 3.. Baicalein decreased progesterone induced ZBTB16 expression in mouse uteri.

Immunohistochemical staining against progesterone receptor target ZBTB16 on uterine cross sections of 10% DMSO (A), 1 mg/kg progesterone (B), 25 mg/kg baicalein and 1 mg/kg progesterone (C), 1 mg/kg progesterone and 10 mg/kg RU486 (D) and 25 mg/kg baicalein (E) treated mice. Arrows indicate positive stains. Scale bar = 150 μm. Inset images were taken at 4x magnification.

Figure 4.. Baicalein did not alter PR protein levels in mouse uteri.

Immunohistochemical staining against progesterone receptor on uterine cross sections of 10% DMSO (A), 1 mg/kg progesterone (B), 25 mg/kg baicalein and 1 mg/kg progesterone (C), 1 mg/kg progesterone and 10 mg/kg RU486 (D) and 25 mg/kg baicalein (E) treated mice. Arrows indicate positive stains. Scale bar = 150 μm. Inset images were taken at 4x magnification.

Figure 5.. Baicalein’s effect on GR expression in mouse uteri.

Immunohistochemical staining against glucocorticoid receptor on uterine cross sections of 10% DMSO (A), 1 mg/kg progesterone (B), 25 mg/kg baicalein and 1 mg/kg progesterone (C), 1 mg/kg progesterone and 10 mg/kg RU486 (D) and 25 mg/kg baicalein (E) treated mice. Arrows indicate positive stains. Scale bar = 150 μm. Inset images were taken at 4x magnification.

Figure 6.. Common genes and pathways altered oppositely in the uteri of mice treated with progesterone and baicalein.

(A) 44 common genes were upregulated by progesterone but downregulated by baicalein treatment. (B) 18 common genes were downregulated by progesterone but upregulated by baicalein treatment. DAVID analysis revealed 24 GO terms (C) and 13 KEGG terms (D) that were altered oppositely by progesterone and baicalein treatment. (E) DAVID KEGG pathways downregulated by baicalein treatment. Full list of genes and GO terms can be found in Supplementary Info.

Figure 7.. Common GSEA genes sets altered oppositely in the uteri of mice treated with progesterone and baicalein.

(A) Two gene sets were upregulated by progesterone but downregulated by baicalein treatment (left) and three gene sets were downregulated by progesterone but upregulated by baicalein treatment (right). (B) Enrichment plots of the common gene sets altered oppositely by progesterone (top) and baicalein (bottom) treatment. NES: normalized enrichment score. FDR<0.05 is noted as significant.