Impact of follicular size categories on oocyte quality at trigger day in young and advanced-age patients undergoing GnRH-ant therapy.

Aim: To study the effect of follicle sizes of different proportions on oocyte and embryo quality in young and advanced-age patients, and provide evidence for personalized protocol adjustment. Methods: This was a retrospective real-world data study including a total of 11,462 patients who had started their first in vitro fertilization cycle with a gonadotropin-releasing hormone antagonist (GnRH-ant) protocol during 2018-2021. We classified patients into groups according to the size of the dominant proportion of follicles on the human chorionic gonadotropin (hCG) trigger day: Large, Medium, Small, and Equal (containing equivalent proportions of all three size categories). The Cochran-Mantel-Haenszel test by different Anti-Mullerian Hormone (AMH) and antral follicle count (AFC) was used to compare factors such as the metaphase II (MII) oocyte rate, normal fertilization rate, and two pronuclei (2PN) cleavage rate between groups. General linear model (GLM) analysis was performed for inter-group comparison of the oocyte and embryo quality. Results: In patients aged < 35 years and with AMH ≥ 1.2µg/L, the MII oocyte percentages in the Large and Medium groups were significantly higher than in the Small group (P < 0.001). The germinal vesicle (GV) oocyte and unavailable oocyte percentages in the Large and Medium groups were lower than in the Small group (P < 0.001). Among patients aged ≥ 35 years with AFC < 5 and AMH ≥ 1.2µg/L, the GV oocyte percentage in the Large group was significantly lower than in the Medium group (2.54% vs. 4.46%, P < 0.001). In patients < 35 years, the GLM demonstrated that the Large and Medium groups had positively impacted on the development of MII oocyte and live birth rate(LBR) of first embryo transfer(ET)(ß>0, all P value < 0.05);and had less likely to develop into unavailable oocyte, degenerated oocyte, GV oocyte and MI oocyte rates relative to the Small group(ß<0, all P value < 0.05). And among patients ≥ 35 years, the Medium group had positively impacted on the development of MII oocyte and 2PN rates relative to the Small group(ß>0, all P value < 0.05); and had less likely to develop into MI oocytes relative to the Small group(ß<0, all P value < 0.05). The GLM indicated that AMH, along with Gn total dose, start dose, and Gn days, had significant impact on oocyte and embryo quality. For young patients, age was not a significant influencing factor, but for advanced-age patients, age influenced the outcomes. Conclusion: Our analysis suggests that for young patients (< 35 years), triggering when there is a high proportion of large or medium follicles results in better quality oocytes, while for older patients (≥ 35 years), it is better to trigger when the proportion of medium follicles is no less than that of small follicles. Further research is required to confirm these findings.

M1 macrophage-derived exosomes containing miR-150 inhibit glioma progression by targeting MMP16.

A large amount of clinical and experimental evidence indicates that M1 macrophages can inhibit tumor progression and expansion; however, the molecular mechanism by which macrophage-derived exosomes inhibit the proliferation of glioblastoma cells has not yet been elucidated. Here, we used M1 macrophage exosomes encapsulating microRNAs to inhibit the proliferation of glioma cells. Exosomes derived from M1 macrophages exhibited high expression levels of miR-150, and the inhibition of glioma cell proliferation mediated by exosomes derived from M1 macrophages was dependent on this microRNA. Mechanistically, miR-150 is transferred to glioblastoma cells through M1 macrophages and binds to MMP16, downregulating its expression and inhibiting glioma progression. Overall, these findings indicate that M1 macrophage-derived exosomes carrying miR-150 inhibit the proliferation of glioblastoma cells through targeted binding to MMP16. This dynamic mutual influence between glioblastoma cells and M1 macrophages provides new opportunities for the treatment of glioma.

High-frequency repetitive transcranial magnetic stimulation protects against cerebral ischemia/reperfusion injury in rats: Involving the mitigation of ferroptosis and inflammation.

BACKGROUND AND AIM: Repetitive transcranial magnetic stimulation (rTMS) has been found to attenuate cerebral ischemia/reperfusion (I/R) injury. However, its effects and mechanism of action have not yet been clarified. It has been reported that cerebral I/R injury is closely associated not only with ferroptosis but also with inflammation. Hence, the current study aimed to investigate whether high-frequency rTMS attenuates middle cerebral artery occlusion (MCAO)-induced cerebral I/R injury and further to elucidate the mediatory role of ferroptosis and inflammation. METHODS: The protective effects of rTMS on experimental cerebral I/R injury were investigated using transient MCAO model rats. Neurological scores and pathological changes of cerebral ischemic cortex were assessed to evaluate the effects of rTMS on cerebral I/R injury. The involvement of ferroptosis and that of inflammation were examined to investigate the mechanism underlying the effects of rTMS. RESULTS: High-frequency rTMS remarkably rescued the MCAO-induced neurological deficits and morphological damage. rTMS treatment also increased the mRNA and protein expression of glutathione-dependent peroxidase 4, decreased the mRNA and protein levels of acyl-CoA synthetase long-chain family member 4 and transferrin receptor in the cortex. Moreover, rTMS administration reduced the cerebrospinal fluid IL-1ß, IL-6, and TNF-α concentrations. CONCLUSION: These findings implicated that high-frequency rTMS alleviates MCAO-induced cerebral I/R injury, and the underlying mechanism could involve the inhibition of ferroptosis and inflammation. Our study identifies rTMS as a promising therapeutic agent for the treatment of cerebral I/R injury. Moreover, the mechanistic insights into ferroptosis and inflammation advance our understanding of it as a potential therapeutic target for diseases beyond cerebral ischemia stroke.

The effects of chronic unpredicted mild stress on maternal negative emotions and gut microbiota and metabolites in pregnant rats.

Background: Chronic long-term stress is associated with a range of disorders, including depression and a variety of other chronic illnesses. It is well known that maternal exposure to psychosocial stress during pregnancy significantly increases the likelihood of adverse pregnancy outcomes. The gut microbiota has been a popular topic, it is a key mediator of the gut-brain axis and plays an important role in human health; changes in the gut microbiota have been related to chronic stress-induced health impairment, however, the relationship between maternal negative emotions and abnormal gut microbiota and its metabolites during maternal exposure to chronic stress during pregnancy remains unclear. Methods: Pregnant rats were subjected to chronic unpredicted mild stress (CUMS) to establish the rat model of chronic stress during pregnancy. The behavioral changes were recorded using sucrose preference test (SPT) and open-field test (OFT), plasma corticosterone levels were determined by radioimmunoassay, and a comprehensive method combining 16S rRNA gene sequencing and gas chromatography-mass spectrometry (GC-MS) metabolomics was used to study the effects of stress during pregnancy on the function of intestinal microbiota and its metabolites. Results: Chronic stress during pregnancy not only increased maternal plasma corticosterone (P < 0.05), but also caused maternal depression-like behaviors (P < 0.05). Chronic stress during pregnancy changed the species composition at the family level of maternal gut microbiota, the species abundance of Ruminococcaceae in the stress group (23.45%) was lower than the control group (32.67%) and the species abundance of Prevotellaceae in the stress group (10.45%) was higher than the control group (0.03%) (P < 0.05). Vertical locomotion and 1% sucrose preference percentage in pregnant rats were negatively correlated with Prevotellaceae (r =  - 0.90, P < 0.05). Principal component analysis with partial least squares discriminant analysis showed that the integration points of metabolic components in the stress and control groups were completely separated, indicating that there were significant differences in the metabolic patterns of the two groups, and there were seven endogenous metabolites that differed (P < 0.05). Conclusions: The negative emotional behaviors that occur in pregnant rats as a result of prenatal chronic stress may be associated with alterations in the gut microbiota and its metabolites. These findings provide a basis for future targeted metabolomics and gut flora studies on the effects of chronic stress during pregnancy on gut flora.

Prenatal chronic stress impairs the learning and memory ability via inhibition of the NO/cGMP/PKG pathway in the Hippocampus of offspring.

Numerous clinical and animal studies have found that antenatal chronic stress can lead to pathological changes the hippocampal development from embryos to adult, but the mechanisms are not well understood. Proteomic analyses provide a new insight to explore the potential mechanisms of this impairment. In this study, gestating rats were subjected to chronic unpredictable mild stress (CUMS) during pregnant days using nine different stimulations, and the changes of the learning and memory performance and the expression of proteins in the hippocampus of offspring were measured. It was found that prenatal chronic stress led to growth retardation, impaired spatial learning and memory ability in the offspring. Furthermore, prenatal stress caused various degrees of damage to neurons, Nissl body, mitochondria and synaptic structures in hippocampal CA3 region of offspring. In addition, 26 significantly different expressed proteins (DEPs) were found between the two groups by using isoquantitative tag-based relative and absolute quantification (iTRAQ) proteomics analysis. Further analyses of these DEPs showed that involved with different molecular functions and several biological processes, such as biological regulation and metabolic processes. Among these, the KEGG pathway enrichment showed that learning and memory impairment was mainly associated with the cyclic guanosine monophosphate protein kinase G (cGMP-PKG) pathway. At the same time, compared with OPC group, the NO, nNOS and cGMP level were significantly decreased, and the expression of PKG protein was also dropped. All of these results suggested that pregnant rats exposed to chronic psychological stress might impair spatial learning and memory ability of offspring, by disturbing the NO/cGMP/PKG signaling pathway.

Contribution of hippocampal BDNF/CREB signaling pathway and gut microbiota to emotional behavior impairment induced by chronic unpredictable mild stress during pregnancy in rats offspring.

Background: Numerous studies have shown that exposure to prenatal maternal stress (PMS) is associated with various psychopathological outcomes of offspring. The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota-gut-brain axis) has been aconsensus; however, there is a lack of research on the involvement mechanism of gut microbiota in the regulation of the BDNF/CREB signaling pathway in the hippocampus of prenatally stressed offspring. Methods: Pregnant rats were subjected to chronic unpredictable mild stress (CUMS) to establish the prenatal maternal stress model. The body weight was measured and the behavioral changes were recorded. Offspring were tested to determine emotional state using sucrose preference test (SPT), open-field test (OFT) and suspended tail test (STT). Gut microbiota was evaluated by sequencing the microbial 16S rRNA V3-V4 region, and the interactive analysis of bacterial community structure and diversity was carried out. The expression of hippocampal BDNF, TrkB and CREB mRNA and proteins were respectively measured using RT-PCR and Western blotting. Results: Prenatal maternal stress increased maternal plasma corticosterone levels, slowed maternal weight gain and caused depression-like behaviors (all P < 0.05). In offspring, prenatal maternal stress increased plasma corticosterone levels (P < 0.05) and emotional behavior changes (depression-like state) were observed (P < 0.05). The species abundance, diversity and composition of the offspring's gut microbiota changed after the maternal stress during pregnancy (P < 0.05). Compared with the control group's offspring, the species abundance of Lactobacillaceae was dropped, while the abundance of the Muribaculaceae species abundance was risen. Concurrent, changes in the hippocampal structure of the offspring and decreases in expression of BDNF/CREB signaling were noted (P < 0.05). Conclusions: Prenatal maternal stress leads to high corticosterone status and abnormal emotion behavior of offspring, which may be associated with the abnormal BDNF/CREB signaling in hippocampus of offspring caused by the change of gut microbiota composition.

The mechanism of enriched environment repairing the learning and memory impairment in offspring of prenatal stress by regulating the expression of activity-regulated cytoskeletal-associated and insulin-like growth factor-2 in hippocampus.

BACKGROUND: Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring. METHODS: Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting. RESULTS: There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment. CONCLUSIONS: The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.

Hypoxic Glioma Stem Cell-Derived Exosomes Containing Linc01060 Promote Progression of Glioma by Regulating the MZF1/c-Myc/HIF1α Axis.

Glioma stem cells (GSC) are a subpopulation of tumor cells with special abilities to proliferate and differentiate in gliomas. They are one of the main causes of tumor recurrence, especially under hypoxic conditions. Although long noncoding RNAs (lncRNA) are known to be involved in numerous biological processes and are implied in the occurrence of certain diseases, their role in tumor development and progression remains poorly understood. Here we explored the mechanisms by which lncRNA derived from hypoxic GSCs (H-GSC) cause glioma progression. Isolation and identification of the Linc01060 gene, the exosomes containing them, and the proteins from tumor cells regulating the gene allowed for studying the effects of Linc01060 on proliferation and glycometabolism. H-GSC exerted their effects by transferring exosomes to glioma cells, resulting in a significant increase in Linc01060 levels. Mechanistically, Linc01060 directly interacted with the transcription factor myeloid zinc finger 1 (MZF1) and enhanced its stability. Linc01060 facilitated nuclear translocation of MZF1 and promoted MZF1-mediated c-Myc transcriptional activities. In addition, c-Myc enhanced the accumulation of the hypoxia-inducible factor-1 alpha (HIF1α) at the posttranscriptional level. HIF1α bound the hormone response elements of the Linc01060 promoter, upregulating the transcription of Linc01060 gene. Clinically, Linc01060 was upregulated in glioma and was significantly correlated with tumor grade and poor clinical prognosis. Overall, these data show that secretion of Linc01060-containing exosomes from H-GSCs activates prooncogenic signaling pathways in glioma cells to promote disease progression. SIGNIFICANCE: These findings suggest that inhibition of Linc01060-containing exosomes or targeting the Linc01060/MZF1/c-Myc/HIF1α axis may be an effective therapeutic strategy in glioma.

Silencing of EPCAM suppresses hepatic fibrosis and hepatic stellate cell proliferation in mice with alcoholic hepatitis via the PI3K/Akt/mTOR signaling pathway.

Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Epithelial cell adhesion molecule (EPCAM) plays a role in hepatitis. Therefore, the current study aimed to explore the effect of EPCAM and its potential mechanism in AH. Bioinformatic analysis was performed to screen differentially expressed genes associated with AH. AH mouse models were established through a Lieber-DeCarli liquid diet containing 4% ethanol, which were co-treated with siRNA against EPCAM or the PI3K/Akt/mTOR signaling pathway inhibitor in order to investigate the effects of EPCAM and the PI3K/Akt/mTOR signaling pathway on hepatic fibrosis, hepatic stellate cell (HSC) proliferation and apoptosis. The relationship between EPCAM and the PI3K/Akt/mTOR signaling pathway was investigated for the purposes of elucidating the potential mechanism of EPCAM in AH. EPCAM was predicted to regulate AH progression through the PI3K/Akt/mTOR signaling pathway. Silencing EPCAM or inhibition of the PI3K/Akt/mTOR signaling pathway inhibited the hepatic fibrosis and HSC proliferation yet induced HSC apoptosis. Moreover, silencing EPCAM was found to repress the PI3K/Akt/mTOR signaling pathway as evidenced by decreased levels of Bcl2 yet increased levels of caspase-3. Collectively, silencing EPCAM could hinder AH progression by inhibiting the PI3K/Akt/mTOR signaling pathway, which might serve as a potential therapeutic target for AH treatment.

Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.

HSV-2 increases TLR4-dependent phosphorylated IRFs and IFN-ß induction in cervical epithelial cells.

Our previous studies demonstrated that HSV-2 infection up-regulates TLR4 expression and induces NF-kB activity, thereby facilitating innate immune response in human cervical epithelial cells. This process requires involvement of TLR4 adaptors, Mal and MyD88. In the current study, we found that HSV-2 infection increases levels of phosphoryalted IRF3 and IRF7, then regulating expression of type I IFN. As expected, these changes induced by HSV-2 infection depended upon TLR4. Knockdown of TRIF and/or TRAM by siRNAs indicated that TRIF/TRAM might be involved in expression of IFN-ß. Our results demonstrate for the first time that IRF3 and IRF7 are both involved in inducing TLR4-dependent IFN-ß expression in response to HSV-2 in its primary infected genital epithelial cells. Thus, TLR4-Mal/MyD88 and TLR4-TRIF/TRAM signaling may synergize and/or cooperate in innate immune response of cervical epithelial cells to HSV-2 infection.

[Simultaneous determination of six ingredients in Huoxiang Zhengqi oral liquid by UPLC].

OBJECTIVE: To develop a UPLC method for the simultaneous determination of liquiritin, narirutin, hesperidin, ammonium glycyrrhetate, honokiol and magnolol in Huoxiang Zhengqi oral liquid. METHOD: A Zorbax Eclipse C18 column was used with the mobile phase of acetonitrile and 0. 05% phosphate acid by gradient elution at the detection wavelength of 220 nm. The flow rate was 0.42 mL x min(-1) and the column temperature was 30 degrees C. RESULT: The calibration curves were linear in the ranges of 0.001 7-0.034, 0.003 4-0.068, 0.006 4-0.128, 0.012 8-0.256, 0.003 2-0.064, 0.006 4-0.128 microg, respectively. The average recoveries were 103.3%, 98.39%, 98.29%, 102.1%, 98.45%, 102.2% with RSDs of 2.1%,1.0%, 0.50%, 2.3%, 0.9%, 2.0%, respectively. CONCLUSION: The UPLC method was simple, rapid and accurate, it could be used for quality control of Huoxiang Zhengqi oral liquid.

TLR4-MyD88/Mal-NF-kB axis is involved in infection of HSV-2 in human cervical epithelial cells.

We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced activity of NF-kB reporter and expression of cytokines are TLR4-dependent using approaches with shRNA and TLR4 antagonist. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathway are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-α) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-ß by ELISA analysis. Taken together, our results from the in vitro infection model reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in human cervical epithelial cells in response to HSV-2 infection.

[In vitro balanced sustained-release of Panax notoginseng saponins controlled with various matrix materials].

To explore the influence of matrix materials in complicate ingredients on traditional Chinese medicine and investigate the excipients selection model based on balanced release characteristics of multicomponents, the influence of HPMC (K4M, K15M, K100M) and Carbomer (934P, 971P, 974P) was illustrated by testing in vitro release of ginsenoside-Rg1, ginsenoside-Rb1 and notoginsenoside-R1 in Panax notoginseng saponins (model drug, PNS). According to in vitro release results of PNS matrix tablets in water and artificial intestinal juice, the release curves were analyzed with Peppas equation and simulating factor (f). Significant differences in k value and n value among ginsenoside-Rg1, ginsenoside-Rb1 and notoginsenoside-R1 existed in various formulations. The release behaviors from various excipients could be described with Non-Fickian transport or super Case II transport pattern. The f2 values for ginsenoside-Rg1, ginsenoside-Rb1 and notoginsenoside-R1 in 971P matrix tablet containing 30% Carbomer 971P were 74.91, 53.45, 57.89 in water and 79.35, 55.51, 51.89 in artificial intestinal juice, respectively. The release profiles fit for the regulation of FDA. The result revealed that the balanced release rates of Rg1, Rb1 and R1 in 971P matrix tablet were obtained.