Chlorogenic acid promotes angiogenesis and attenuates apoptosis following cerebral ischaemia-reperfusion injury by regulating the PI3K-Akt signalling.

CONTEXT: Chlorogenic acid (CGA) has good antioxidant effects, but its explicit mechanism in cerebral ischaemia-reperfusion injury is still uncertain. OBJECTIVE: We studied the effect of CGA in human brain microvascular endothelial cells (HBMECs) under OGD/R damage. MATERIALS AND METHODS: HBMECs in 4 groups were treated with oxygen-glucose deprivation/re-oxygenation (OGD/R) (4 + 24 h), normal no CGA treatment and different concentrations (20, 40 or 80 µM) of CGA. Male C57BL/6J mice were classified as sham, middle cerebral artery occlusion (MCAO), and MCAO + CGA (30 mg/kg/day) groups. Mice in the sham group were not subjected to MCAO. Cell viability, apoptosis, angiogenesis and related protein levels were investigated by CCK-8, flow cytometry, TUNEL staining, tube formation and western blot assays. Infarct volume of brain tissues was analyzed by TTC staining. RESULTS: CGA curbed apoptosis (from 32.87% to 13.12% in flow cytometry; from 34.46% to 17.8% in TUNEL assay) but accelerated cell angiogenesis of HBMECs with OGD/R treatment. Moreover, CGA augmented activation of the PI3K-Akt signalling (p-PI3K/PI3K level, from 0.39 to 0.49; p-Akt/Akt level, from 0.52 to 0.81), and the effect of CGA on apoptosis and angiogenesis was abolished by an inhibitor of PI3K-Akt signalling. Furthermore, CGA attenuated infarct (from 41.26% to 22.21%) and apoptosis and promoted angiogenesis and activation of the PI3K/Akt signalling in MCAO-induced mice. CONCLUSIONS: CGA effectively repressed apoptosis and promoted angiogenesis in OGD/R-treated HBMECs and MCAO-treated mice by modulating PI3K-Akt signalling. Our research provides a theoretical basis for the use of CGA in the treatment of ischaemic stroke.

Activation of NF-κB signaling pathway during HCG-induced VEGF expression in luteal cells.

Vascular endothelial growth factor (VEGF) plays an essential role in luteal angiogenesis, the present study therefore utilized luteal cells cultured in vitro to further investigate the activation and contribution of nuclear factor (NF)-κB to VEGF expression induced by human chorionic gonadotrophin (HCG). The present results showed HCG induced VEGF expression as well as hypoxia-inducible factor (HIF)-1α mRNA and protein expressions, which was blocked by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Further analysis found that these increases of VEGF and HIF-1α mRNA induced by HCG were also blocked by NF-κB siRNA transfection, which was consistent with PDTC treatment. However, HIF-1α siRNA treatment significantly decreased HCG induced-VEGF expression with no effect on NF-κB mRNA expression. Furthermore, combination of HIF-1α siRNA and PDTC treatment did not further decrease VEGF mRNA expression, and the result of chromatin immunoprecipitation indicated NF-κB may regulate HIF-1α transcription through binding with its promoter. Taken together, the present results clearly demonstrated that NF-κB was activated to regulate VEGF expression by increasing HIF-1α transcription in luteal cells treated with HCG. Therefore, the present study provided a new and important mechanism of luteal angiogenesis during the formation of corpus luteum in mammals.

The effect of AQP4 on tau protein aggregation in neurodegeneration and persistent neuroinflammation after cerebral microinfarcts.

This study aimed to investigate the effect of aquaporin-4 (AQP4) on tau protein aggregation in neurodegeneration and persistent neuroinflammation after cerebral microinfarcts. A model of diffuse ischemic brain injury was established, and adenovirus was injected stereotactically through the lateral ventricle of mice. The water content of the brain tissue was measured. The co-expression of glial fibrillary acidic protein (GFAP) and AQP4 and the aggregation of p-tau and neuronal marker were detected through immunofluorescence double staining. The expression of phosphorylated microtubule-associated protein tau (p-tau, Ser202/Thr205, Thr205, Ser396, Ser404), interleukin(IL)-6, IL-1ß, tumor necrosis factor (TNF)-a, growth associated protein43 (GAP43), GFAP, and ionized calcium-binding adapter molecule 1 (Iba1) was detected through Western blot. It was found that the brain water content in the model group was increased and decreased after the AQP4 interference. Compared with the sham group, the expression of GFAP, p-tau, IL-1ß, TNF-a, Iba1, and p-tau was increased in the model group (p < 0.05). Compared with the model group, the expression of p-tau, IL-6, IL-1ß, TNF-a, GFAP, and Iba1 was decreased after AQP4 interference (p < 0.05). It is indicated that AQP4 positively regulates neurodegeneration and persistent neuroinflammation caused by tau protein aggregation after cerebral microinfarcts.

Rolling circle amplification (RCA) -based biosensor system for the fluorescent detection of miR-129-2-3p miRNA.

Herein, a versatile fluorescent bioanalysis platform for sensitive and specific screening of target miRNA (miR-129-2-3p) was innovatively designed by applying target-induced rolling circle amplification (RCA) for efficient signal amplification. Specifically, miR-129-2-3p was used as a ligation template to facilitate its ligation with padlock probes, followed by an RCA reaction in the presence of phi29 DNA polymerase. The dsDNA fragments and products were stained by SYBR Green I and then detected by fluorescence spectrophotometry. As a result, miR-129-2-3p concentrations as low as 50 nM could be detected. Furthermore, the expression of miR-129-2-3p in breast cancer patients was about twice that in healthy people. Therefore, the results indicated that the RCA-based biosensor system could be a valuable platform for miRNA detection in clinical diagnosis and biomedical study.

Chlorogenic Acid Prevents Microglia-Induced Neuronal Apoptosis and Oxidative Stress under Hypoxia-Ischemia Environment by Regulating the MIR497HG/miR-29b-3p/SIRT1 Axis.

Background: Chlorogenic acid (CGA) is a polyphenolic compound with antioxidant and anti-inflammatory properties. CGA has been shown to improve neuroinflammation. This study is aimed at elucidating the exact mechanism by which CGA reduces neuroinflammation. Methods: Oxygen and glucose deprivation (OGD) was utilized to treat BV2 microglia and HT-22 hippocampal neurons to engineer an in vitro model of hypoxic ischemia reperfusion. The levels of inflammatory factors (IL-1ß, IL-6, TNF-α, IL-4, and IL-10) and oxidative stress factors (MDA, SOD, and GSH-PX) in microglia were determined by ELISA kits. The neuron proliferation was assessed by CCK-8 assay, and LDH kit was used to determine LDH release in neurons. The fluorescent dye DCF-DA was employed to measure ROS levels in neurons. Correlation of MIR497HG, miR-29b-3p, and SIRT1/NF-κB in neurons and microglia was determined by qRT-PCR. Expressions of inflammatory proteins (COX2, iNOS), oxidative stress pathways (Nrf2, HO-1), and apoptosis-related proteins (Bcl-2, Bax, caspase3, caspase8, and caspase9) in microglia or neurons were determined by western blot. The interactions between MIR497HG and miR-29b-3p, as well as between miR-29b-3p and SIRT1, were determined by dual luciferase assay and RIP assay. Results: CGA attenuated OGD-mediated inflammation and oxidative stress in microglia and inhibited microglia-mediated neuronal apoptosis. CGA increased the levels of MIR497HG and SIRT1 and suppressed the levels of miR-29b-3p in BV2 and HT-22 cells. MIR497HG knockdown, miR-29b-3p upregulation, and SIRT1 inhibition inhibited CGA-mediated anti-inflammatory and neuronal protective functions. There is a targeting correlation between MIR497HG, miR-29b-3p, and Sirt1. MIR497HG sponges miR-29b-3p to regulate SIRT1 expression in an indirect manner. Conclusion: CGA upregulates MIR497HG to curb miR-29b-3p expression, hence initiating the SIRT1/NF-κB signaling pathway and repressing OGD-elicited inflammation, oxidative stress, and neuron apoptosis.

HIF-1α Activation Promotes Luteolysis by Enhancing ROS Levels in the Corpus Luteum of Pseudopregnant Rats.

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α -induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy.

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

HIF-1α/BNIP3-Mediated Autophagy Contributes to the Luteinization of Granulosa Cells During the Formation of Corpus Luteum.

During the luteinization after ovulation in mammalian ovary, the containing cells undergo an energy consuming function re-determination process to differentiate into luteal cells under avascular environment. Previous evidences have delineated the contribution of autophagy to the cell differentiation and the catabolic homeostasis in various types of mammalian cells, whereas few interest had been focused on the involvement of autophagy in the luteinization of granulosa cells during the formation of early corpus luteum. Herein, the present study investigated that expression and contribution of autophagy during granulosa cell luteinization and early luteal development through in vivo and in vitro experiments. The results clearly demonstrated that HIF-1α/BNIP3-mediated autophagy plays a vital role in the luteinization of granulosa cells during the early luteal formation in vivo and in vitro. In the neonatal corpus luteum, HIF-1α up-regulated BNIP3 expressions, which contributed to the autophagic initiation by disrupting beclin1 from Bcl-2/beclin1 complex and protected cells from apoptosis by curbing the skew of mitochondria balance under avascular niche. Notably, Inhibition of HIF-1α activity by echinomycin enhanced the levels of cytoplasmic cytochrome c and cell apoptosis in the nascent corpus luteum. These findings revealed that HIF-1α/BNIP3-mediated autophagy enabled the process of granulosa cell luteinization and protected the granulosa-lutein cells from further apoptosis under hypoxia niche. To our knowledge, the present study firstly clarified that HIF-1α/BNIP3-mediated autophagy contributes to the luteinization of granulosa cells during the formation of pregnant corpus luteum, which will help us further understanding the luteal biology and provide us new clues for the treatment of luteal insufficiency.

Defective insulin signaling and the protective effects of dimethyldiguanide during follicular development in the ovaries of polycystic ovary syndrome.

It is established that the physiological effects of insulin are primarily mediated by the insulin signaling pathway. However, a defective insulin signaling is closely associated with the clinical manifestations of polycystic ovary syndrome (PCOS), which include excess androgen levels, insulin resistance and anovulation, and is involved in the pathophysiology of PCOS at the molecular level. Dimethyldiguanide (DMBG) has been widely employed to alleviate reproduction dysfunction in women with PCOS, however, the exact mechanism of this effect remains unclear. The objective of the present study was to investigate the effects of DMBG on the expression of the insulin signaling pathway in the ovaries of rats with PCOS, and to identify the potential underlying molecular mechanisms of these effects in PCOS. In the present study, a PCOS rat model was induced by letrozole, and successful establishment of the model was confirmed by examining ovarian histology and determining serum testosterone levels, by hematoxylin and eosin staining and ELISA, respectively. Subsequently, the expression of two key elements of insulin signaling, insulin receptor substrate (IRS)­2 and phosphatidylinositol 3­kinase (PI3K), was determined by immunohistochemistry and western blot analysis. The results demonstrated that IRS­2 and PI3K expression was markedly decreased in PCOS ovaries, which was rescued by DMBG treatment. These results indicate that IRS­2/PI3K signaling may be involved in the development of PCOS and the therapeutic effects of DMBG on PCOS. To further confirm the effects of DMBG on insulin signaling expression during this process, the expression of an additional two downstream proteins, phosphoinositide­dependent kinase­1 (PDK­1) and the mammalian target of rapamycin (mTOR), was also investigated in the present study, and the results demonstrated that the expression of PDK­1 and mTOR was significantly reduced in PCOS ovaries and increased following DMBG treatment, further indicating that altered insulin signaling may have an important role in the development and treatment of PCOS. In conclusion, the results of the present study indicate that the reduced expression of proteins involved in insulin signaling may contribute to the development of the clinical features of PCOS, and DMBG reverses reduced expression of insulin signaling components, by a mechanism that is yet to be determined, to attenuate certain symptoms of PCOS, such as obesity. To the best of our knowledge, the present study is the first to provide data regarding the detailed changes of insulin signaling during the development and treatment of PCOS, and may provide an important reference for clinical PCOS treatment.