Metabolite of esculetin plays an important role in cytotoxic effects induced by chloroquine on porcine immature Sertoli cells.

Chloroquine (CQ) is widely used in the therapy against malarial, tumor and recently the COVID-19 pandemic, as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway. We previously reported that CQ (20 µM, 36 h) could reprogram transcriptome, and impair multiple signaling pathways vital to porcine immature Sertoli cells (iSCs). However, whether CQ treatment could affect the metabolomic compositions of porcine iSCs remains unclear. Here, we showed that CQ (20 µM, 36 h) treatment of porcine iSCs induced significant changes of 63 metabolites (11 up and 52 down) by the metabolomics method, which were involved in different metabolic pathways. Caffeic acid and esculetin, the top two up-regulated metabolites, were validated by ELISA. The combined analysis of metabolomics and transcriptome showed caffeic acid and esculetin to be highly correlated with multiple differentially expressed genes (DEGs), including Ndrg1, S100a8, Sqstm1, S100a12, S100a9, Ill1, Lif, Ntn4 and Peg10. Furthermore, esculetin treatment (53 nM, 36 h) significantly decreased the viability and proliferation, suppressed the mitochondrial function, whereas promoted the apoptosis of porcine iSCs, similar to those by CQ treatment (20 µM, 36 h). Collectively, our results showed that CQ treatment induces metabolic changes, and its effect on porcine iSCs could be partially mediated by esculetin.

HSP90AA1 promotes viability and lactate production but inhibits hormone secretion of porcine immature Sertoli cells.

Heat shock protein 90 (HSP90), as a molecular chaperone, regulates hundreds of protein clients under both physiological and stress conditions in eukaryotic cells. However, the functional role of HSP90 in mammalian male reproduction remains largely unknown. Here, we aimed to investigate the function and effect of HSP90AA1 on the basic and reproductive function of pig immature Sertoli cells (iSCs). We first confirmed that the transfection of pBI-CMV3-HSP90AA1 vector into porcine iSCs for 24 h significantly increased mRNA and protein levels of HSP90AA. Moreover, HSP90AA1 over-expression significantly increased cell viability and the PLK2 mRNA abundance, promoted lactate production via elevating the LDHA activity, and inhibited the secretion of anti-Mullerian hormone and estradiol. In comparison, HSP90AA inhibition by allylamino-17-demethoxygeldanamycin (17-AAG) (2 µM) treatment of pig iSCs for 36 h had a totally contrasting effect, i.e. significantly reduced cell viability, promoted cell apoptosis via modulating expression of genes related to cell cycle and apoptosis (CCNB1, CCN1, PLK2, PTMA, YBX3 and CASP3), suppressed lactate production via dropping LDHA activity, but increased the secretion of anti-Mullerian hormone and estradiol. Taken together, our findings demonstrated that HSP90AA1 could regulate positively cell viability and lactate production, but negatively the secretion of reproductive hormones (anti-Mullerian hormone and estradiol). However, the detailed molecular mechanism of HSP90AA1 remains to be investigated.

Melatonin mitigates Chloroquine-induced defects in porcine immature Sertoli cells.

Chloroquine (CQ) could function as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway, and is widely used on malarial, tumor and recently COVID-19. However, the effect of CQ treatment on porcine immature Sertoli cells (iSCs) remains unclear. Here we showed that CQ could reduce iSC viability in a dose-dependent manner. CQ treatment (20 µM) on iSCs for 36h could elevate oxidative stress, damage mitochondrial function and promote apoptosis, which could be partially rescued by melatonin (MT) (10 nM). Transcriptome profiling identified 1611 differentially expressed genes (DEGs) (776 up- and 835 down-regulated) (20 µM CQ vs. DMSO), mainly involved in MAPK cascade, cell proliferation/apoptosis, HIF-1, PI3K-Akt and lysosome signaling pathways. In contrast, only 467 (224 up- and 243 down-regulated) DEGs (CQ + MT vs. DMSO) could be found after MT (10 nM) addition, enriched in cell cycle, regulation of apoptotic process, lysosome and reproduction pathways. Therefore, the partial rescue effects of MT on CQ treatment were confirmed by multiple assays (cell viability, ROS level, mitochondrial function, apoptosis, and mRNA levels of selected genes). Collectively, CQ treatment could impair porcine iSC viability by deranging the signaling pathways related to apoptosis and autophagy, which could be partially rescued by MT supplementation.

Gossypol exposure induces mitochondrial dysfunction and oxidative stress during mouse oocyte in vitro maturation.

Gossypol is a yellow natural polyphenolic compound extracted from the seeds, leaves, stems, and flower buds of the cotton plant. Several studies have shown that exposure to gossypol impacts reproductive health in both humans and animals. However, whether gossypol exposure would influence oocyte quality has not yet been determined. Here, we studied the effects of gossypol on the meiotic maturation of mouse oocytes in vitro. The results revealed that gossypol exposure did not affect germinal vesicle breakdown (GVBD) but significantly reduced polar body extrusion (PBE) rates. Moreover, we observed meiotic spindle organization and chromosome alignment were entirely disturbed after gossypol exposure. Further, gossypol exposure also caused mitochondrial dysfunction and abruptly decreased the levels of cellular ATP, and diminished the mitochondrial membrane potential (MMP). Accordingly, gossypol-induced oxidative stress was confirmed through an increased level of reactive oxygen species (ROS). Early apoptosis incidence also increased as identified by positive Annexin-V signaling. Collectively, the above findings provide evidence that gossypol exposure impaired oocyte meiotic maturation, disturbed spindle structure and chromosome dynamics, disrupted mitochondrial function, induced oxidative stress, and triggered early apoptosis. These findings emphasize gossypol's adverse effects on oocyte maturation and thus on female fertility.

Long non-coding RNA MALAT1 targeting STING transcription promotes bronchopulmonary dysplasia through regulation of CREB.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non-coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up-regulated in the lung tissue from BPD neonates, hyperoxia-based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down-regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia-treated cells. Subsequently, qRT-PCR, Western blotting and dual-luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

Kupffer Cells Regulate Natural Killer Cells Via the NK group 2, Member D (NKG2D)/Retinoic Acid Early Inducible-1 (RAE-1) Interaction and Cytokines in a Primary Biliary Cholangitis Mouse Model.

BACKGROUND Kupffer cells and natural killer (NK) cells has been identified as contributing factors in the pathogenesis of hepatitis, but the detailed mechanism of these cell types in the pathogenesis of primary biliary cholangitis (PBC) is poorly understood. MATERIAL AND METHODS In this study, polyinosinic: polycytidylic acid (poly I: C), 2-octynoic acid-bovine serum albumin (2OA-BSA) and Freund's adjuvant (FA) were injected to establish a murine PBC model, from which NK cells and Kupffer cells were extracted and isolated. The cells were then co-cultivated in a designed culture system, and then NK group 2, member D (NKG2D), retinoic acid early inducible-1 (RAE-1), F4/80, and cytokine expression levels were detected. RESULTS The results showed close crosstalk between Kupffer cells and NK cells. PBC mice showed increased surface RAE-1 protein expression and Kupffer cell cytokine secretion, which subsequently activated NK cell-mediated target cell killing via NKG2D/RAE-1 recognition, and increased inflammation. NK cell-derived interferon-γ (IFN-γ) and Kupffer cell-derived tumor necrosis factor alpha (TNF-alpha) were found to synergistically regulate inflammation. Moreover, interleukin (IL)-12 and IL-10 improved the crosstalk between NK cells and Kupffer cells. CONCLUSIONS Our findings in mice are the first to suggest the involvement of the NKG2D/RAE-1 interaction and cytokines in the synergistic effects of NK and Kupffer cells in PBC.

Heat stress induces apoptosis through disruption of dynamic mitochondrial networks in dairy cow mammary epithelial cells.

Heat stress-induced reductions in milk yield and the dysfunction of mammary glands are economically important challenges that face the dairy industry, especially during summer. The aim of the present study is to investigate the effects of heat stress on mitochondrial function by using dairy cow mammary epithelial cells (DCMECs) as an in vitro model. Live cell imaging shows that the mitochondria continually change shape through fission and fusion. However, heat stress induces the fragmentation of mitochondria, as well as the decreased of ATP level, membrane potential, and anti-oxidant enzyme activity and the increased of respiratory chain complex I activity. In addition, the cytosolic Ca2+ concentration and cytochrome c expression (Cyto-c) were increased after heat stress treatment. Both qRT-PCR and western blot analysis indicate that mitofusin1/2 (Mfn1/2) and optic atrophy protein-1 (Opa-1) are downregulated after heat stress, whereas dynamin-related protein 1 (Drp1) and fission 1 (Fis-1) are upregulated, which explains the observed defect of mitochondrial network dynamics. Accordingly, the present study indicated that heat stress induced the dysfunction of DCMEC through disruption of the normal balance of mitochondrial fission and fusion.

Transcription factor E2F1 positively regulates interferon regulatory factor 5 expression in non-small cell lung cancer.

PURPOSE: Lung cancer is the most common malignant tumor in the world, and its incidence and mortality are very high. This study focuses on the mechanism of non-small cell lung cancer to find new therapeutic targets. METHODS: We used RT-PCR and Western blot to verify the linear relationship between E2F1 and IRF5 in normal lung tissue and lung cancer tissues. Secondly, we used overexpression and knock down E2F1 in cell lines to detect the expression of IRF5. The prime enzyme reporter plasmid verified that E2F1 binds to the core promoter region of IRF5; finally, CHIP experiments demonstrated that E2F1 binds directly to IRF5. RESULTS: We verified that E2F1 and IRF5 are decreased in patient tissues, and there is a strong linear relationship between E2F1 and IRF5. Secondly, we used overexpression of E2F1 or E2F1 siRNA transfected into HCC827 cells and found that E2F1 positively regulates the activity of the IRF5 promoter and the mRNA level of IRF5. Finally, the results of a chromatin immunoprecipitation assay demonstrated that E2F1 bound to the promoter region of IRF5 in vitro. These results suggested that the E2F1 transcription factor is the primary determinant for activating the basal transcription of the IRF5. CONCLUSION: The transcription factor E2F1 positively regulates IRF5 in non-small cell lung cancer.

Reduced nucleic acid methylation impairs meiotic maturation and developmental potency of pig oocytes.

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.

Primary familial brain calcifications linked with a novel SLC20A2 gene mutation in a Chinese family.

It has been recently reported that mutations in SLC20A2 gene are a major cause of primary familial brain calcifications, a rare neurodegenerative disorder characterized by symmetrical and bilateral intracranial calcification. We conducted a pedigree study by performing next Generation Sequencing in a Chinese family with three generations. Three members in this family developed Parkinsonism in their sixth decade, also, the proband presented with schizophrenia for 40 years. Next Generation Sequencing identified a novel nonsense heterozygous substitution c.1158C > A (p.Thr 386*) of SLC20A2 gene, introducing a stop codon in exon 10. The mutation was present in symptomatic and asymptomatic individuals with intracranial calcification, but absent in the individual without calcification, suggesting the mutation segregates with brain calcification. mRNA expression was decreased by 35% in the proband. We are the first to demonstrate a novel c.1158C > A mutation of SLC20A2 gene in a Chinese family with primary familial brain calcifications.

DMBA acts on cumulus cells to desynchronize nuclear and cytoplasmic maturation of pig oocytes.

As an environmental pollutant and carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) can destroy ovarian follicles at all developmental stages in rodents. However, the underlying molecular mechanism remains obscure. In the present study, we aim to address how DMBA affects the in vitro maturation and development of porcine oocytes. We discovered that for 20 µM DMBA-treated cumulus-oocyte complexes (COCs), the rate of oocyte germinal vesicle breakdown (GVBD) was significantly altered, and the extrusion rate of first polar body was increased. Moreover, oocytes from 20 µM DMBA-treated COCs had significant down-regulation of H3K9me3 and H3K27me3, up-regulation of H3K36me3, higher incidence of DNA double strand breaks (DSBs) and early apoptosis. In striking contrast, none of these changes happened to 20 µM DMBA-treated cumulus-denuded oocytes (CDOs). Furthermore, 20 µM DMBA treatment increased the reactive oxygen species (ROS) level, decreased mitochondrial membrane potential (Δ Ψm), and inhibited developmental competence for oocytes from both COC and CDO groups. Collectively, our data indicate DMBA could act on cumulus cells via the gap junction to disturb the synchronization of nuclear and ooplasmic maturation, and reduce the developmental competence of oocytes.

Multifunctional theranostic nanoplatform for cancer combined therapy based on gold nanorods.

Nanomaterials that integrate diagnostic and therapeutic functions within a single nanoplatform promise great advances in revolutionizing cancer therapy. A smart multifunctional theranostic drug-delivery system (DDS) based on gold nanorods (abbreviated as GNR/TSDOX) is designed for cancer-targeted imaging and imaging-guided therapy. In this intelligent theranostic DDS, the active targeting ligand biotin is introduced to track cancer sites in vivo. With the aid of photothermal/photoacoustic imaging, GNR/TSDOX can ablate cancer specifically and effectively. When stimulated with a single near-infrared (NIR) light source, this NIR light energy is effectively absorbed and converted into heat by GNR/TSDOX for localized photothermal therapy and the increase in temperature also further triggers the cascaded release of the anticancer drug for combined thermo-chemotherapy. More importantly, the in vivo cure effect can be well guided by regulating the irradiation time and intensity of the NIR light.

Bioinspired Nano-Prodrug with Enhanced Tumor Targeting and Increased Therapeutic Efficiency.

Nanotechnology-based drug delivery has a great potential to revolutionize cancer treatment by enhancing anticancer drug efficacy and reducing drug toxicity. Here, a bioinspired nano-prodrug (BiNp) assembled by an antineoplastic peptidic derivative (FA-KLA-Hy-DOX), a folate acid (FA)-incorporated proapoptotic peptide (KLAKLAK)(2) (KLA) to doxorubicin (DOX) via an acid-labile hydrozone bond (Hy) is constructed. The hydrophobic antineoplastic agent DOX is efficiently shielded in the core of nano-prodrug. With FA targeting moieties on the surface, the obtained BiNp shows significant tumor-targeting ability and enhances the specific uptake of cancer cells. Upon the trigger by the intracellular acidic microenvironment of endosomes, the antineoplastic agent DOX is released on-demand and promotes the apoptosis of cancer cells. Simultaneously, the liberated FA-KLA can induce the dysfunction of mitochondria and evoke mitochondria-dependent apoptosis. In vitro and in vivo results show that the nano-prodrug BiNp with integrated programmed functions exhibits remarkable inhibition of tumor and achieves a maximized therapeutic efficiency with a minimized side effect.

Expression of RNA-binding proteins DND1 and FXR1 in the porcine ovary, and during oocyte maturation and early embryo development.

The porcine oocyte and early embryo are transcriptionally quiescent following germinal vesicle breakdown in the oocyte and prior to activation of the embryonic genome, at approximately the 4-cell stage of development. Despite a lack of new transcription, mRNA and protein repertoires are subject to regulation during this time. One potential mechanism of regulation is through the functional activity of miRNAs and/or the presence of specific RNA-binding proteins. Both DND1 (dead end homolog 1) and FXR1 (fragile-X-mental retardation-related protein 1) are RNA-binding proteins that have been demonstrated to impact miRNA-mediated, post-transcriptional gene regulation. The objective was to characterize the presence and the expression changes in DND1 and FXR1 during pig oocyte maturation and early embryo development. DND1 and FXR1 expression were evaluated in oocytes and cumulus cells during meiotic progression and in 4-cell stage embryos using quantitative RT-PCR, Western blot analysis, and immunostaining. These data demonstrate DND1 and FXR1 mRNA are expressed in the maturing oocyte and early in vitro-fertilized embryos, with significantly less DND1 in 4-cell stage embryos as compared to germinal vesicle and metaphase II-arrested oocytes. Based on immunohistochemistry, DND1 protein abundance is greater in secondary follicles in comparison to primary and tertiary follicles. Using ribonucleoprotein immunoprecipitation from germinal vesicle-stage oocytes, DND1 was demonstrated to interact with several mRNAs associated with pluripotency. This work provides a better understanding of the biological relevance of DND1 and FXR1 during female gametogenesis and embryo development in pigs.

Nor-lignans and steroidal saponins from Asparagus gobicus.

From the roots of Asparagus gobicus, four new nor-lignans, 3'-methoxynyasin, iso-agatharesinol, gobicusins A, B and one new steroidal saponin, 3-O-[beta-D-xylopyranosyl(1-4)-beta-D-glucopyranosyl(1-2)-beta-D-glucopyranosyl]-(25S)-5beta-spirostan-3beta-ol (11) were isolated, together with twelve known compounds. The structures of the new compounds were established by spectroscopic methods including 2D-NMR techniques (1H-1H COSY, HMBC, HMQC) and chemical transformations. Nyasol (5) and 11 exhibited remarkable in vitro cytotoxic activity against cultured HO-8910 (human ovarian carcinoma) and Bel-7402 (human hepatoma) cells with IC50 vales of 30.6 and 29.4 microM, 5.2 and 5.2 microM, respectively.