Insulin resistance (IR) is likely to induce metabolic syndrome and type 2 diabetes mellitus (T2DM). Gluconeogenesis (GNG) is a complex metabolic process that may result in glucose generation from certain non-carbohydrate substrates. Chinese herbal medicine astragalus polysaccharides and berberine have been documented to ameliorate IR, and combined use of astragalus polysaccharide (AP) and berberine (BBR) are reported to synergistically produce an even better effect. However, what change may occur in the GNG signaling pathway of IR-HepG2 cells in this synergistic effect and whether AP-BBR attenuates IR by regulating the GNG signaling pathway remain unclear. For the first time, we discovered in this study that the optimal time of IR-HepG2 cell model formation was 48 h after insulin intervention. AP-BBR attenuated IR in HepG2 cells and the optimal concentration was 10 mg. AP-BBR reduced the intracellular H 2 O 2 content with no significant effect on apoptosis of IR-HepG2 cells. In addition, a rapid change was observed in intracellular calcium current of the IR-HepG2 cell model, and AP-BBR intervention attenuated this change markedly. The gene sequencing results showed that the GNG signaling pathway was one of the signaling pathways of AP-BBR to attenuate IR in IR-Hepg2 cells. The expression of p-FoxO1 Ser256 and PEPCK protein was increased, and the expression of GLUT2 protein was decreased significantly in the IR-HepG2 cell model, and both of these effects could be reversed by AP-BBR intervention. AP-BBR attenuated IR in IR-HepG2 cells, probably by regulating the GNG signaling Pathway.
Background/Aim. Erbuzhuyu decoction (EBZYD) is a traditional Chinese medicine (TCM) formula and has been used in infertility treatment. Meanwhile, acupuncture is also used to treat female infertility. However, it is unclear whether EBZYD combined with acupuncture has better therapeutic effect. The aim of this study was to explore the effect of EBZYD combined with acupuncture and investigate its mechanism in superovulation mice. Methods. The mice received the treatment of EBZYD, acupuncture, EBZYD combined with acupuncture, or miR-494-3p agomir combined with EBZYD and acupuncture. The blastocysts’ number, endometrial microstructure, and endometrial thickness were observed, followed by the detection of endometrial receptivity-related factors, PI3K/Akt/mTOR pathway-related proteins, and miR-494-3p expression using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Luciferase reporter assay was performed to confirm the targeting relationship between HOXA10 and miR-494-3p. Results. EBZYD combined with acupuncture treatment could increase the number of blastocysts, pinopodes, endometrial thickness, and the expression of endometrial receptivity-related factors, and the treatment effect of EBZYD combined with acupuncture was better than EBZYD or acupuncture alone. In addition, EBZYD combined with acupuncture treatment activated PI3K/Akt/mTOR pathway and inhibited the expression of miR-494-3p. HOXA10 is one of the target genes of miR-494-3p. Overexpression of miR-494-3p reversed the therapeutic effect of EBZYD combined with acupuncture and suppressed the expression of HOXA10 and the activity of PI3K/Akt/mTOR pathway. Conclusion. This study suggests that EBZYD combined with acupuncture could improve endometrial receptivity in superovulation mice via miR-494-3p/HOXA10 axis.
Successful implantation requires endometrial receptivity. To investigate the mechanisms of miR-494-3p on endometrial receptivity, GnRHa's superovulation scheme was designed to reduce endometrial receptivity, and the pregnant mice were injected with miR-494-3p antagomir. The regulatory role of miR-494-3p was identified by RT-qPCR, uterine blastocyst count, scanning electron microscopy, hematoxylin-eosin (HE) staining, and Western blot. Results indicated that miR-494-3p antagomir increased uterine blastocysts numbers, promoted the pinocytosis expressions, and increased endometrial thickness. Besides, miR-494-3p antagomir significantly increased leukemia inhibitory factor (LIF), Ang-2 and VEGF protein expressions, and up-regulated p-AKT/AKT and p-mTOR/mTOR protein ratios in endometrium. Luciferase assay confirmed that LIF was a potential target of miR-494-3p. Subsequently, human endometrial epithelial cells (hEECs) were transfected with miR-494-3p inhibitor and PI3K inhibitor (LY294002). The role of miR-494-3p was identified by RT-qPCR, CCK-8 assay, transwell assay and flow cytometry. Results indicated that miR-494-3p inhibitor significantly increased proliferation and invasion, and significantly inhibited apoptosis in hEECs, while LY294002 reversed its biological function. Overall, these results suggested that miR-494-3p is the key regulator of endometrial receptivity in mice, regulating this complex process through the PI3K/AKT/mTOR pathway. Understanding the role of miR-494-3p in endometrial receptivity is of great significance for exploring new targets for the diagnosis and treatment of early pregnancy failure, and improving the success rates of artificial reproduction.
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