Electroacupuncture Promotes Angiogenesis in Mice with Cerebral Ischemia by Inhibiting miR-7.

OBJECTIVE: To observe the angiogenesis effect of electroacupuncture (EA) at Shuigou acupoint (GV 26) in the treatment of cerebral ischemia, and explore the value of miRNA-7 (miR-7) in it. METHODS: First, 48 mice were randomly divided into sham operation, middle cerebral artery occlusion (MCAO) model, and EA treatment groups. Then 9 mice were divided into carrier control group, miR-7 knockout group and miR-7 overexpression group (n=3 each group). Finally, 20 mice were divided into model and carrier control group, model and miR-7 knockout group, EA treatment and carrier control group and EA treatment and miR-7 overexpression group, with 3-6 mice in each group. The MCAO model was established in the MCAO and EA groups. Neurological deficit score and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the severity of cerebral ischemia. Hematoxylin-eosin staining was used to describe basic pathological changes. Immunohistochemistry was used to quantify cerebral microvessel density. Real-time PCR and Western blot were used to detect the expression of miR-7 and its downstream target genes Krüppel-like factor 4/vascular endothelial growth factor (KLF4/VEGF) and angiopoietin-2 (ANG-2) in the ischemic cerebral cortex. RESULTS: After EA, neurological deficit scores and infarction volumes decreased, and the density of cerebral microvessels increased. In the MCAO group, miR-7 expression was higher than that in the sham group (P<0.01). After EA at GV 26, miR-7 expression decreased (P<0.01) and the expression of downstream target genes KLF4/VEGF and ANG-2 increased as compared with the MCAO group (P<0.01). After EA combined with overexpression of miR-7, the expression of downstream target genes KLF4/VEGF and ANG-2 decreased compared to the control EA group (P<0.01). After miR-7 knockdown, the expression of KLF4/VEGF and ANG-2 increased (P<0.05 or P<0.01). CONCLUSIONS: EA could promote angiogenesis in MCAO mice likely by inhibiting the expression of miR-7 and relieving inhibition of downstream target genes KLF4/VEGF and ANG-2.

Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPß pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated. AIM OF THE STUDY: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinß (C/EBPß) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. MATERIALS AND METHODS: Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPß in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPß using siRNAs, the relationship between KLF4 and C/EBPß in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPß pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPß pathway. CONCLUSIONS: ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPß were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPß pathway. Furthermore, we confirmed that KLF4 and C/EBPß regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.

Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Decreased Expression of KLF4 Leading to Functional Deficit in Pediatric Patients with Intestinal Failure and Potential Therapeutic Strategy Using Decanoic Acid.

Pediatric intestinal failure (IF) is the reduction in gut function to below the minimum necessary for the absorption of macronutrients and/or water and electrolytes, such that intravenous supplementation is required to maintain health and/or growth. The overall goal in treating IF is to achieve intestinal adaptation; however, the underlying mechanisms have not been fully understood. In this study, by performing single-cell RNA sequencing in pediatric IF patients, we found that decreased Kruppel-Like Factor 4 (KLF4) may serve as the hub gene responsible for the functional deficit in mature enterocytes in IF patients, leading to the downregulation of solute carrier (SLC) family transporters (e.g., SLC7A9) and, consequently, nutrient malabsorption. We also found that inducible KLF4 was highly sensitive to the loss of certain enteral nutrients: in a rodent model of total parenteral nutrition mimicking the deprivation of enteral nutrition, the expression of KLF4 dramatically decreased only at the tip of the villus and not at the bottom of crypts. By using IF patient-derived intestinal organoids and Caco-2 cells as in vitro models, we demonstrated that the supplementation of decanoic acid (DA) could significantly induce the expression of KLF4 along with SLC6A4 and SLC7A9, suggesting that DA may function as a potential therapeutic strategy to promote cell maturation and functional improvement. In summary, this study provides new insights into the mechanism of intestinal adaptation depending on KLF4, and proposed potential strategies for nutritional management using DA.

Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

BACKGROUND: Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence. METHODS: We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O2/5% CO2) or air (21% O2/5% CO2) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O2) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed. RESULTS: Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence. CONCLUSION: Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung.

Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Molecular mechanism of anti-adipogenic effect of vitexin in differentiating hMSCs.

In this study, we evaluated a detailed molecular mechanism of anti-adipogenic activity of vitexin, apigenin flavone glucoside, present in germinated fenugreek seeds, in differentiating human mesenchymal stem cells (hMSCs). The lipid content of differentiated adipocytes was estimated by ORO staining. Effect on mitotic clonal expansion was checked by cell cycle analysis. Expression of early and terminal adipocyte differentiation markers, anti- and pro-adipogenic transcription factors and signalling intermediates regulating them was evaluated at RNA and protein level. We found vitexin to be non-cytotoxic up to 20 µM at which intracellular lipid accumulation was significantly decreased. Cell cycle analysis suggested that vitexin does not affect mitotic clonal expansion. Expression of early and late differentiation markers, such as CEBPα, CEBPß, PPARγ, FABP4, perilipin, adiponectin and Glut4 was significantly reduced in the presence of vitexin. Expression of KLF4 and KLF15, positive regulators of PPARγ, was decreased, whereas that of negative regulators, namely KLF2, GATA2, miR20a, miR27a, miR27b, miR128, miR130a, miR130b, miR182 and miR548 increased with vitexin treatment. This effect was mediated by the activation of the AMP-activated protein kinase (AMPK) pathway via the activation of LepR and additionally by inhibiting ROS. Thus, our results showed that vitexin regulates the expression of PPARγ and inhibits adipogenesis of hMSCs at an early stage of differentiation.

Magnolol attenuates depressive-like behaviors by polarizing microglia towards the M2 phenotype through the regulation of Nrf2/HO-1/NLRP3 signaling pathway.

PURPOSE: Magnolol (MA) exhibits anti-depressant effect by inhibiting inflammation. However, its effect on microglia polarization remains not fully understood. Herein, our study was performed to evaluate the effect of MA on microglia polarization in chronic unpredictable mild stress (CUMS)-induced depression and explore its potential mechanism. STUDY DESIGN: The CUMS procedure was conducted, and the mice were intragastrically treated with MA. BV2 cells were pretreated with MA prior to LPS/ATP challenge. METHODS: The levels of TNF-α, IL-1ß, IL-6 and IL-4, IL-10 in brain and BV2 cells were examined by ELISA. The mRNA expressions of Arg1, Ym1, Fizz1 and Klf4 in brains were measured. ROS content was determined using flow cytometry. Immunofluorescence was employed to evaluate Iba-1 level, Nrf2 nuclear translocation, Iba-1+CD16/32+ and Iba-1+CD206+ cell population. The protein expressions of Nrf2, HO-1, NLRP3, caspase-1 p20 and IL-1ß in brains and BV2 cells were investigated by western blot. Nrf2 siRNA was induced in experiments to explore the role of Nrf2 in MA-mediated microglia polarization. The ubiquitination of Nrf2 was visualized by Co-IP. RESULTS: The treatment with MA notably relieved depressive like behaviors, suppressed pro-inflammatory cytokines, promoted anti-inflammatory cytokines and the transcription of M2 phenotype microglia-specific indicators. MA upregulated the expression of Nrf2, HO-1, downregulated the expression of NLRP3, caspase-1 p20, IL-1ß both in vivo and in vitro. MA also reduced ROS concentration, promoted Nrf2 nucleus translocation and prevented Nrf2 ubiquitination. Nrf2 Knockdown by siRNA abolished the MA-mediated microglia polarization. CONCLUSION: The present research demonstrated that MA attenuated CUMS-stimulated depression by inhibiting M1 polarization and inducing M2 polarization via Nrf2/HO-1/NLRP3 signaling.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

Baihui (DU20)-penetrating-Qubin (GB7) acupuncture regulates microglia polarization through miR-34a-5p/Klf4 signaling in intracerebral hemorrhage rats.

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Krüppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.

Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites.

Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.

The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation.

Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.

Berberine improves dietary-induced cardiac remodeling by upregulating Kruppel-like factor 4-dependent mitochondrial function.

Multiple studies have showed that berberine protects against heart diseases, including obesity-associated cardiomyopathy. However, it is not fully disclosed the potential molecular mechanisms of berberine on controlling cardiac remodeling. Kruppel-like factor (KLF) 4, identified as a critical transcriptional factor, participates in multiple cardiac injuries. The present study was to explore whether KLF4 determined the cardioprotective benefits of berberine in dietary-induced obese mice. High fat diet-induced obese mice were treated with berberine with or without lentivirus encoding Klf4 siRNA, and cardiac parameters were analyzed by multiple biological approaches. In dietary-induced obese mouse model, administration of berberine obviously increased cardiac level of KLF4, which closely correlated with improvement of cardiac functional parameters. Co-treatment of lentivirus encoding Klf4 siRNA abolished cardioprotective benefits of berberine, including induction of cardiac hypertrophy, fibrosis, functional disorders, inflammatory response and oxidative stress. Mechanistically, we found berberine improved cardiac mitochondrial biogenesis and activities, whereas silencing Klf4 decreased berberine-upregulated mitochondrial quality, ATP production and oxygen consumption. Our present study demonstrated that berberine protected against dietary-induced cardiac structural disorders and mitochondrial dysfunction dependent on cardiac KLF4 signaling. Cardiac KLF4 was one of potential therapeutic targets for obesity-induced cardiac injuries.

Piperlongumine attenuates angiotensin-II-induced cardiac hypertrophy and fibrosis by inhibiting Akt-FoxO1 signalling.

BACKGROUND: Cardiac hypertrophy and fibrosis are closely related to cardiac dysfunction, especially diastolic dysfunction. Limited medications can be used to simultaneously delay cardiac hypertrophy and fibrosis in clinical practice. Piperlongumine (PLG) is an amide alkaloid extracted from Piper longum and has been shown to have multiple biological effects, including anticancer and antioxidant effects. However, the role of PLG in cardiac hypertrophy and fibrosis is not clear. PURPOSE: The aim of this study was to reveal the role of PLG in cardiac hypertrophy and fibrosis and the associated mechanism. METHODS: Cardiac hypertrophy and fibrosis were induced by angiotensin II (Ang II) in vivo and in vitro. The effect of PLG in vivo, in vitro and its mechanism were investigated by proliferation and apoptosis assays, western blot, real-time PCR, immunofluorescence, histochemistry, echocardiography, flow cytometry and chromatin immunoprecipitation. RESULTS: Proliferation and apoptosis assays showed that 2.5 µM PLG slightly inhibited proliferation and did not promote apoptosis. Treatment with 5 mg/kg PLG obviously inhibited Ang II-induced cardiac hypertrophy and fibrosis in vivo. In vitro studies of neonatal rat cardiomyocytes (NRCMs) showed that the anti-hypertrophic effect of PLG was mediated by reducing the phosphorylation of Akt and thereby preserving the level of Forkhead box transcription factor O1 (FoxO1), since knockdown of FoxO1 by siRNA reversed the protective effect of PLG on NRCMs. In addition, PLG significantly decreased the Ang II-induced expression of profibrotic proteins in neonatal cardiac fibroblasts by reducing the expression of Krüppel-like factor 4 (KLF4) and the recruitment of KLF4 to the promoter regions of transforming growth factor-ß and connective tissue growth factor. CONCLUSION: We demonstrate the cardioprotective effects of PLG in both cardiac hypertrophy and fibrosis and the potential value of PLG for developing novel medications for pathological cardiac hypertrophy and heart failure.

Anti-perimenopausal osteoporosis effects of Erzhi formula via regulation of bone resorption through osteoclast differentiation: A network pharmacology-integrated experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases. AIM OF THE STUDY: In this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis. MATERIALS AND METHODS: The active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis. RESULTS: The results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL-a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis-was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05). CONCLUSIONS: Our present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation.

KLF4 Exerts Sedative Effects in Pentobarbital-Treated Mice.

KLF4 is a zinc-finger transcription factor that plays an essential role in many biological processes, including neuroinflammation, neuron regeneration, cell proliferation, and apoptosis. Through effects on these processes, KLF4 has likely roles in Alzheimer's disease, Parkinson's disease, and traumatic brain injury. However, little is known about the role of KLF4 in more immediate behavioral processes that similarly depend upon broad changes in brain excitability, such as the sleep process. Here, behavioral approaches, western blot, and immunohistochemical experiments were used to explore the role of KLF4 on sedation and the potential mechanisms of those effects. The results showed that overexpression of KLF4 prolonged loss of righting reflex (LORR) duration in pentobarbital-treated mice and increased c-Fos expression in the lateral hypothalamus (LH) and the ventrolateral preoptic nucleus (VLPO), while it decreased c-Fos expression in the tuberomammillary nucleus (TMN). Moreover, overexpression of KLF4 reduced the expression of p53 in the hypothalamus and increased the expression of STAT3 in the hypothalamus. Therefore, these results suggest that KLF4 exerts sedative effects through the regulation of p53 and STAT3 expression, and it indicates a role of KLF4 ligands in the treatment of sleep disorders.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Effects of Sesame Oil Aroma on Mice after Exposure to Water Immersion Stress: Analysis of Behavior and Gene Expression in the Brain.

(1) Background: Sesame has been popular as a healthy food since ancient times, and effects of the aroma component of roasted sesame are also expected. However, little research has been reported on its scent; (2) Methods: Jcl:ICR male mice were housed under water immersion stress for 24 h. Then, the scent of saline or sesame oil was inhaled to stress groups for 90 min. We investigated the effects of sesame oil aroma on the behavior and brains of mice; (3) Results: In an elevated plus maze test, the rate of entering to open arm and the staying time were decreased by the stress. These decrements were significantly enhanced by sesame oil aroma. Stress had a tendency to increase the serum corticosterone concentration, which was slightly decreased by the aroma. Expression of Kruppel-like factor-4 (Klf-4) and Dual-specificity phosphatase-1 (Dusp-1) in the striatum were increased by water immersion stress, and the level of Klf-4 and Dusp-1 in the striatum and hippocampus were significantly attenuated by sesame oil aroma (4) Conclusions: The present results strongly suggest that the odor component of sesame oil may have stress suppressing effects. Moreover, Klf-4 and Dusp-1 may be sensitive stress-responsive biomarkers.

Interplay among p21Waf1/Cip1, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury.

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.