The molecular mechanism of naringin improving endometrial receptivity of OHSS rats.

Controlling ovarian hyperstimulation syndrome (OHSS) in the controlled ovarian hyperstimulation treatment is necessary to increase the implantation success rate. This study aimed to explore the effect of naringin on the endometrial receptivity of OHSS rats. Female rats were randomly assigned to six groups: Blank, model, low-dose naringin (100 mg/kg/day), medium-dose naringin (200 mg/kg/day), high-dose naringin (400 mg/kg/day), and positive (0.18 mg/kg/day estradiol valerate) groups. Except for the blank group, rats established the OHSS model on Day 7, and their treatments were from Day 0 to 14, separately. Hematoxylin and eosin, immunohistochemical, and scanning electron microscopy were performed to detect the naringin effects on the endometrial receptivity of the OHSS model. Next, circRNAs transcriptome analysis was performed to screen circRNAs. Western blot analysis and real-time quantitative PCR were used to verify it. Our study showed that naringin treatments increased embryo number, endometrial thickness, pinopodes number, and Ki67 expression in the OHSS rats. Moreover, the result of circRNAs transcriptome sequencing showed that naringin significantly inhibited the rnocirc_008140 expression in the OHSS rats and significantly inhibited the changes of 28 gene ontology terms and three Kyoto Encyclopedia of Genes and Genomes pathways which were induced by OHSS. Abcc4 and Rps6ka5 genes were the enriched genes of those pathways. Finally, 24 miRNA target genes of rnocirc_008140 were predicted. Our study showed that naringin significantly improved the endometrial receptivity of OHSS rats to increase the embryo implantation success by reducing rnocirc_008140-adsorbed miRNAs to regulate Abcc4 and Rps6ka5 expression.

Regulation mechanism of miR-494-3p on endometrial receptivity in mice via PI3K/AKT/mTOR pathway.

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.

Effects of Erbuzhuyu Decoction Combined with Acupuncture on Endometrial Receptivity Are Associated with the Expression of miR-494-3p.

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.

Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.