Role of Ca2+/calmodulin and PI3K/AKT signaling pathways and active ingredients of BaoTaiYin in treatment of recurrent miscarriage

Abstract

Introduction: BaoTaiyin (BTY) is a traditional Chinese medicine decoction. It has been used to treat recurrent miscarriage (RM). However, there are no comprehensive systematic studies to identify the chemical compositions of BTY and molecular mechanisms on RM. Finding the chemical components of BTY and clarifying the underlying processes in the treatment of RM were the goals of the study.

Methods: We used ultra-high-performance liquid chromatography coupled with triple quadruple time-of-flight tandem mass spectrometry to analyze the chemical components of BTY, network analysis to predict the pharmacological effects of the identified active ingredients, and cell experiments to identify potential molecular mechanisms.

Results: We found 12 active ingredients among 61 components identified in BTY. These identified activities were linked to regulatory effects on 127 key signaling pathways, targeting 107 proteins. Through network analysis, we determined that insulin-like growth factor 1 receptor, matrix metalloproteinases, PI3K, and STAT3 may be the core targets of BTY's therapeutic effects on RM. We further explored this mechanism to find that aqueous extracts of BTY significantly enhanced IGFBP2 and CaMKK2 expression and trophoblast proliferation, whereas inhibitors of IGF1R/PI3K/AKT pathway or CaMKK2 blocked the effect of BTY on trophoblast proliferation. In addition, IGFBP2 siRNA suppressed BTY-induced CaMKK2 expression. Caffeic acid, as one of components of BTY, increased intracellular Ca²⁺ concentration and proliferation in trophoblast.

Conclusion: Our research showed that BTY may have therapeutic benefits on RM through multiple targets and pathways, such as the IGF1R/PI3K/AKT and Ca²⁺/calmodulin signaling pathways.

Keywords: BaoTaiYin; Ca2+ signal; mass spectrometry; proliferation; recurrent miscarriage; trophoblast proliferation.

FIGURE 1

UPLC high-resolution MS base peak chromatogram showing BaoTaiYin ion intensities in negative ion mode (A) and positive ion mode (B). (C). Ultraviolet chromatogram of BaoTaiYin using UPLC-ultraviolet at a wavelength of 254 nm.

FIGURE 2

Chemical structures for 12 active ingredients in BaoTaiYin.

FIGURE 3

Wayne diagram showing the intersection of the disease targets and drug targets. The pink section is the drug target, the blue section is the disease target, and their intersection contains the targets common to both.

FIGURE 4

Protein-protein interaction network. Nodes with darker colors and larger sizes are considered higher degree targets.

FIGURE 5

Top 10 enriched gene ontology terms for the biological processes, cellular components, and molecular functions of the potential targets.

FIGURE 6

Kyoto Encyclopedia of Genes and Genomes enrichment analysis of top 20 pathways. FDR represents false discovery rate.

FIGURE 7

Active ingredient–shared target–pathway network analysis. Orange triangles represent the disease (recurrent miscarriage, RM) and the drug (BaoTaiYin, BTY), yellow circles represent the herbs present in BTY, blue arrows represent pathways, red diamonds represent active ingredients, and green rectangles represent targets.

FIGURE 8

Effect of BaoTaiYin (BTY) on the expression of insulin-like growth factor-binding protein 2 (IGFBP2) and the impact of inhibitors targeting the insulin-like growth factor 1 receptor (IGF1R)-PI3K-AKT signaling pathway on BTY-induced proliferation of HTR-8/SVneo cells. Summary data illustrating cell viability (A and C), as assessed by the CCK8 assay, along with relative mRNA expression levels (B and D) for IGFBP2 (B) and proliferating cell nuclear antigen gene (PCNA, (D) in HTR-8/SVneo cells treated with solvent control, 10 μg/mL BTY, BTY combined with picropodophyllin (PPP, 0.2 μM), BTY combined with ZSTK474 (0.1 μM; a PI3K inhibitor), or BTY combined with afuresertib (0.2 μM; an Akt inhibitor) for a duration of 48 h. OD represents relative optical density. The mRNA levels were normalized to that of 18S rRNA. Data represent the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for the indicated comparison.

FIGURE 9

Effect of caffeic acid on proliferation and intracellular Ca²⁺ rise and role of CaMKK2 in BaoTaiYin (BTY)-induced proliferation in HTR-8/SVneo cells. (A, B), Representative traces and summary data showing 10 and 100 µM caffeic acid (CA)-induced intracellular Ca²⁺ concentration increase. (C), Summary data showing the proliferation of HTR-8/SVneo cells treated with solvent control (NC) and caffeic acid with 10, 30 and 100 µM concentration. (D-E), Representative images and summary data showing the expression levels of CaMKK2 in cells treated with or without BTY (10 μg/mL) (D), or transfected with control siRNA (siNC) or IGFBP2 siRNA and treated with BTY (10 μg/mL) (E). (F), Summary data showing the proliferation of HTR-8/SVneo cells treated with solvent control (NC), BTY (10 μg/mL) or BTY (10 μg/mL) + STO609 (5 μg/mL). Data represent the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for the indicated comparison.