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.

Roles of SP/KLF transcription factors in odontoblast differentiation: From development to diseases.

OBJECTIVES: A zinc-finger transcription factor family comprising specificity proteins (SPs) and Krüppel-like factor proteins (KLFs) plays an important role in dentin development and regeneration. However, a systematic regulatory network involving SPs/KLFs in odontoblast differentiation has not yet been described. This review examined the expression patterns of SP/KLF gene family members and their current known functions and mechanisms in odontoblast differentiation, and discussed prospective research directions for further exploration of mechanisms involving the SP/KLF gene family in dentin development. MATERIALS AND METHODS: Relevant literature on SP/KLF gene family members and dentin development was acquired from PubMed and Web of Science. RESULTS: We discuss the expression patterns, functions, and related mechanisms of eight members of the SP/KLF gene family in dentin development and genetic disorders with dental problems. We also summarize current knowledge about their complementary or synergistic actions. Finally, we propose future research directions for investigating the mechanisms of dentin development. CONCLUSIONS: The SP/KLF gene family plays a vital role in tooth development. Studying the complex complementary or synergistic interactions between SPs/KLFs is helpful for understanding the process of odontoblast differentiation. Applications of single-cell and spatial multi-omics may provide a more complete investigation of the mechanism involved in dentin development.

Qiangxin recipe improves doxorubicin-induced chronic heart failure by enhancing KLF5-mediated glucose metabolism.

BACKGROUND: Qiangxin recipe (QXF) is a well-known Chinese herbal medicine commonly used in Asia for thousands of years to treat cardiovascular diseases, but its underlying mechanism remains unclear. PURPOSE: This study aimed to illustrate whether Qiangxin Recipe (QXF) induce glucose metabolism and inhibit cardiomyocyte apoptosis by promoting the activation of the transcription factor Krüppel like factor 5 (KLF5). MATERIAL AND METHODS: In vitro experiments, we constructed an H9C2 cardiomyocyte injury model using doxorubicin and used RNA-seq data analysis to detect the mechanism of QXF. In in vivo experiments, C57 BL/6 mice injected with doxorubicin (4 mg/kg every 6 days, for 30 days) to construct a CHF mouse model and randomly divided into to the normal control group, Dox group and Dox+QXF group (2.12 g/kg/day, 4.24 g/kg/day, for 30 days). Using Echocardiography, serum biochemical indices BNP, cTnl; and histopathological tests involving HE staining, Tunel staining and Immuno-dual fluorescence colocalization to analyze the therapeutic mechanism of QXF. RESULTS: We verified that the Qiangxin recipe could reverse cardiomyocyte dying through enhancing glucose metabolism and reducing apoptosis to improve CHF. Mechanistically, we discovered that the Qiangxin recipe promoted the activation of transcription factor Krüppel-like factor 5 (KLF5) to induce glucose metabolism and inhibit apoptosis in cardiomyocytes. Further, we identified that KLF5 increased the promoter activity of hexokinase 2 (HK2) and B-cell CLL/lymphoma 2 (BCL2) genes, which further enhanced glucose metabolism and inhibited apoptosis of cardiomyocytes. CONCLUSIONS: We highlighted the importance of KLF5-mediated signaling pathways in the treatment of CHF as shown by their participation in glucose metabolism and apoptosis in a doxorubicin-induced model of cardiomyocyte injury, as well as show that Qiangxin recipe can be used as a novel targeted therapy for the treatment of CHF. Compared with previous studies, we provide new ideas for the treatment of Doxorubicin-induced CHF from the perspective of energy metabolism.

Kruppel-like Factors 3 Regulates Migration and Invasion of Gastric Cancer Cells Through NF-κB Pathway.

Context: The poorly understood regulatory mechanisms impede gastric cancer therapy. Kruppel-like factors (KLFs) are associated with the development of various tumors, The studies on the role of the KLF transcription factor 13 (KLF13) in gastric cancer progression haven't been studied. Objective: The current study aimed to investigate the role of KLF13 in the migration and invasion of gastric cancer and the regulatory mechanism of KLF13 in gastric cancer progression. Design: The research team performed a laboratory study. Setting: The study took place at the Zengcheng District People's Hospital of Guangzhou in Zengcheng, China. Participants: In addition to using normal gastric cells, GES1, and seven gastric cancer cell lines, the research team compared the fresh, gastric cancer tissues (T) and paired, adjacent, noncancerous gastric tissues (ANT) from eight patients undergoing surgical resection at the hospital. The research team also downloaded the data for 33 gastric cancer tissues and adjacent, normal gastric tissues from the Cancer Genome Atlas' TCGA database. Intervention: The research team used: (1) short hairpin RNAs (shRNAs) to knock down KLF13, (2) wound healing and transwell invasion analyses to determine the effects of KLF13 on the migration and invasion of gastric cancer, and (3) a Luciferase reporter assay to determine the effects of KLF13 on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Results: KLF13 was upregulated in gastric cancer cells and tissues, and the patients with a high KLF13 expression had poor outcome. Downregulation of KLF13 significantly inhibited the migration and invasion of gastric cancer cells. Mechanistically, downregulation of KLF13 significantly inhibited NF-κB activity, and its targets such as: (1) snail family transcriptional repressor 1 (SNAI1 or Snail), (2) snail family transcriptional repressor 2 (SNAI2 or Slug), (3) zinc finger e-box binding homeobox 1 (ZEB1), (4) Smad interacting protein 1 (Sip1), (5) twist family basic helix-loop-helix (BHLH) transcription factor (Twist), (6) matrix metallopeptidase 2 (MMP2), and (7) MMP9. Tumor necrosis factor alpha (TNF-α) can activate NF-κB. Treating with TNF-α can reverse the effects of KLF13 downregulation on migration and invasion, confirming that KLF13 promotes the migration and invasion of gastric cancer cells through activating the NF-κB pathway. Conclusions: KLF13 promoted the migration and invasion of gastric cancer cells through activating the NF-κB pathway, providing a new target for gastric cancer therapy.

Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression.

The overexpansion of adipose tissues leads to obesity and eventually results in metabolic disorders. Garcinia cambogia (G. cambogia) has been used as an antiobesity supplement. However, the molecular mechanisms underlying the effects of G. cambogia on cellular processes have yet to be fully understood. Here, we discovered that G. cambogia attenuated the expression of CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), an important adipogenic factor, suppressing its transcription in differentiated cells. In addition, G. cambogia inhibited macroautophagic/autophagic flux by decreasing autophagy-related gene expression and autophagosome formation. Notably, G. cambogia markedly elevated the expression of KLF3 (Kruppel-like factor 3 (basic)), a negative regulator of adipogenesis, by reducing SQSTM1/p62-mediated selective autophagic degradation. Furthermore, increased KLF3 induced by G. cambogia interacted with CTBP2 (C-terminal binding protein 2) to form a transcriptional repressor complex and inhibited Cebpa and Pparg transcription. Importantly, we found that RPS6KA1 and STAT3 were involved in the G. cambogia-mediated regulation of CEBPB and autophagic flux. In an obese animal model, G. cambogia reduced high-fat diet (HFD)-induced obesity by suppressing epididymal and inguinal subcutaneous white adipose tissue mass and adipocyte size, which were attributed to the regulation of targets that had been consistently identified in vitro. These findings provide new insight into the mechanism of G. cambogia-mediated regulation of adipogenesis and suggest molecular links to therapeutic targets for the treatment of obesity.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG: autophagy-related; Baf: bafilomycin A1; BECN1: beclin 1; CEBP: CCAAT/enhancer binding protein (C/EBP); CHX: cycloheximide; CREB: cAMP response element binding protein; CTBP: C-terminal binding protein; EGCG: (-)-epigallocatechin gallate; eWAT: epididymal white; G. cambogia: Garcinia cambogia; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HFD: high-fat diet; iWAT: inguinal subcutaneous white; KLF: Kruppel-like factor; LAP: liver-enriched transcriptional activating proteins; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; PPARG: peroxisome proliferator activated receptor gamma; qPCR: quantitative real-time PCR; RFP: red fluorescent protein; RPS6KA1: ribosomal protein S6 kinase A1; siRNA: small-interfering RNA; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TEM: transmission electron microscopy.

Insight into susceptibility genes associated with bipolar disorder: a systematic review.

OBJECTIVE: Bipolar disorder (BD) is a severe disorder, and it is associated with an increased risk of mortality. About 25% of patients with BD have attempted and 11% have died by suicide. All these characteristics suggest that the disorders within the bipolar spectrum are a crucial public health problem. With the development of molecular genetics in recent decades, it was possible to more easily detect risk genes associated with this disorder. This study aimed at summarizing the findings of systematic reviews and meta-analyses on the topic and assessing the quality of the available evidence. MATERIALS AND METHODS: PubMed/Medline and Web of Science were searched to identify systematic reviews and meta-analyses published during 2013-2019. Standard methodology was applied to synthesize and assess the retrieved literature. RESULTS: This systematic review identifies a number of potential risk genes associated with bipolar disorder whose mechanism of action has yet to be confirmed. They are divided into several groups: 1) a list of the most significant susceptibility genetic factors associated with BD; 2) the implication of the ZNF804A gene in BD; 3) the role of genes involved in calcium signaling in BD; 4) DNA methylation in BD; 5) BD and risk suicide genes; 6) susceptibility genes for early-onset BD; 7) candidate genes common to both BD and schizophrenia; 8) genes involved in cognitive status in BD cases; 9) genes involved in structural alteration in BD brain tissue; 10) genes involved in lithium response in BD. CONCLUSIONS: Future research should concentrate on molecular mechanisms by which genetic variants play a major role in BD. Supplemental research is needed to replicate the applicable results.

A high level of KLF12 causes folic acid-resistant neural tube defects by activating the Shh signaling pathway in mice†.

Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Krüppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing fetuses died in utero at approximately 10.5 days post-coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant fetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signaling pathway, leading to the upregulation of patched 1, GLI-Krüppel family member GLI1, hedgehog-interacting protein, etc., whereas FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signaling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future.

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.

Azilsartan ameliorates ox-LDL-induced endothelial dysfunction via promoting the expression of KLF2.

BACKGROUND: Oxidized LDL(Ox-LDL) mediated endothelial dysfunction is involved in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Azilsartan is a potent agent for the treatment of hypertension as the antagonist of the angiotensin II receptor. This study will investigate whether Azilsartan possesses a beneficial effect against endothelial cell dysfunction induced by ox-LDL and explore the underlying preliminary mechanism. METHODS: Ox-LDL was applied to construct an in vitro endothelial dysfunction model in human umbilical vascular endothelial cells (HUVECs). The expression of lectin-type oxidized LDL receptor 1 (LOX-1), endothelial nitric oxide synthase (eNOS), tight junction protein occludin, and transcriptional factor Krüppel-like factor 2 (KLF2) was detected using qRT-PCR and Western blot. ELISA and qRT-PCR were utilized to evaluate the production of chemokine monocyte chemotactic protein 1 (MCP-1) and chemokine (C-X-C motif) Ligand 1 Protein (CXCL1) in treated HUVECs. The generation of nitro oxide (NO) was determined using DAF-FM DA staining assay. KLF2 was silenced by transfecting the cells with specific Small interfering RNA (siRNA). FITC-dextran permeation assay was used to check the endothelial monolayer permeability of treated HUVECs. RESULTS: Firstly, the elevated expressions of LOX-1, MCP-1, and CXCL-1 induced by stimulation with ox-LDL were significantly suppressed by Azilsartan. The downregulated eNOS and reduced production of NO induced by ox-LDL were reversed by the introduction of Azilsartan. Secondly, enlarged endothelial monolayer permeability and decreased expression of occludin stimulated with ox-LDL were greatly reversed by treatment with Azilsartan but were abolished by silencing the expression of KLF2. Lastly, the inhibited expression of KLF2 induced by ox-LDL was significantly elevated by the introduction of Azilsartan. CONCLUSION: Azilsartan might ameliorate ox-LDL-induced endothelial damage via elevating the expression of KLF2.

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.

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.

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.

Tanshinone II A attenuates vascular remodeling through klf4 mediated smooth muscle cell phenotypic switching.

The aim of this study is to investigate the therapeutic role of Tanshinone II A, a key integrant from salvia miltiorrhiza, against pathological vascular remodeling. Completed ligation of mouse left common carotid arteries animal model and rat smooth muscle cells used to investigate the role of Tanshinone II A in regulating pathological vascular remodeling through hematoxylin and eosin staining, immunohistochemistry staining, immunofluorescence staining, adenovirus infection, real time PCR and western blotting. Our data demonstrated that Tanshinone II A treatment suppresses vascular injury-induced neointima formation. In vitro studies on rat smooth muscle cell indicated that Tanshinone II A treatment attenuates PDGF-BB induced cell growth, and promotes smooth muscle cell differentiated marker genes expression that induced by rapamycin treatment. Tanshinone II A treatment significant inhibits rat smooth muscle cell proliferation and migration. Tanshinone II A promotes KLF4 expression during smooth muscle phenotypic switching. Overexpression of KLF4 exacerbates Tanshinone II A mediated smooth muscle cell growth inhibition. Tanshinone II A plays a pivotal role in regulating pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. This study demonstrated that Tanshinone II A is a potential therapeutic agent for vascular diseases.

Generation of induced pluripotent stem cells from large domestic animals.

BACKGROUND: Induced pluripotent stem cells (iPSCs) have enormous potential in developmental biology studies and in cellular therapies. Although extensively studied and characterized in human and murine models, iPSCs from animals other than mice lack reproducible results. METHODS: Herein, we describe the generation of robust iPSCs from equine and bovine cells through lentiviral transduction of murine or human transcription factors Oct4, Sox2, Klf4, and c-Myc and from human and murine cells using similar protocols, even when different supplementations were used. The iPSCs were analyzed regarding morphology, gene and protein expression of pluripotency factors, alkaline phosphatase detection, and spontaneous and induced differentiation. RESULTS: Although embryonic-derived stem cells are yet not well characterized in domestic animals, generation of iPS cells from these species is possible through similar protocols used for mouse or human cells, enabling the use of pluripotent cells from large animals for basic or applied purposes. Herein, we also infer that bovine iPS (biPSCs) exhibit similarity to mouse iPSCs (miPSCs), whereas equine iPSs (eiPSCs) to human (hiPSCs). CONCLUSIONS: The generation of reproducible protocols in different animal species will provide an informative tool for producing in vitro autologous pluripotent cells from domestic animals. These cells will create new opportunities in animal breeding through transgenic technology and will support a new era of translational medicine with large animal models.

The Use of RNAi Technology to Interfere with Zfx Gene Increases the Male Rates of Red Deer (Cervus elaphus) Offspring.

Zinc finger protein X-linked (Zfx) was regarded to be a sex determination factor and plays a critical role in spermatogenesis. RNAi is an effective method of silencing Zfx mRNA expression. However, there has been little research on the use of RNAi technology to control the sex of the offspring of red deer (Cervus elaphus). The objective of this study was first to explore an efficient method to alter the red deer offspring sex-ratio by silencing the gene Zfx during spermatogenesis. Three recombinant expression vectors pLL3.7/A, pLL3.7/B, and pLL3.7/C were constructed to interrupt the Zfx gene. The results showed that the expression of Zfx mRNA was significantly silenced by pLL3.7/A (P < 0.01), compared with the control group. The group injected with pLL3.7/A produced 94 red deer, including 68 males and 26 females. The male rates (72.34%) were significantly higher than the control groups (P < 0.01). Our result suggests that Zfx siRNA is a useful approach to control offspring sex in red deer. This study further confirms that the Zfx gene plays a significant role in the process of X spermatogenesis.

Enterococcus faecium L-15 Extract Enhances the Self-Renewal and Proliferation of Mouse Skin-Derived Precursor Cells.

Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.

Ginkgo biloba Induces Thrombomodulin Expression and Tissue-Type Plasminogen Activator Secretion via the Activation of Krüppel-Like Factor 2 within Endothelial Cells.

The effects of thrombo-prevention, such as antiplatelet and anticoagulant activity, have been reported with the usage of Ginkgo biloba extract (GbE); however, the detailed mechanism has not yet been fully investigated, especially the role of Krüppel-like factor 2 (KLF2). This study aimed to investigate whether GbE can activate KLF2 and then induce thrombomodulin (TM) and tissue-type plasminogen activator (t-PA) secretion to enhance the effects of thrombo-prevention. Different concentrations of GbE were incubated with human umbilical vein endothelial cells (HUVECs) to evaluate its effect on endothelial cells. We found that KLF2 expression is correlated to the risk of atherosclerosis and venous thromboembolism in clinical practice. In the HUVEC cell model, GbE stimulated the expression of KLF2 in a dose-dependent manner. Moreover, TM and t-PA secretion increased when the cells were cultured with GbE. Both the expressions and activities of TM and t-PA in the GbE-treated cells declined after KLF2 was blocked by shKLF2. In sum, with GbE treatment, KLF2 expression in human endothelial cells was significantly activated, which in turn induced an increase in the protein expression and activity of TM and t-PA. After shRNA inhibited the KLF2 expression, GbE stopped inducing the expression and activity of TM and t-PA. These findings suggest that GbE exerts an antithrombotic effect on endothelial cells by increasing the TM expression and t-PA secretion; further, KLF2 is a key factor in this mechanism.

Low-dose phloretin alleviates diabetic atherosclerosis through endothelial KLF2 restoration.

We investigated whether low-dose phloretin served as daily dietary supplements could ameliorate diabetic atherosclerosis and the role of kruppel-like factor 2 (KLF2). HUVECs cultured in high glucose medium were treated with different concentrations of phloretin and KLF2 mRNA, and protein level was detected. Diabetes was induced using streptozotocin in Apoe-/- mice after which they were fed a high-cholesterol diet for 8 weeks. Diabetic mice injected with KLF2 shRNA-lentivirus or control virus were treated with 20 mg/kg phloretin. Glucose, lipid profile, aortic atheroma, and endothelial nitric oxide synthase (eNOS) expression were detected. Phloretin retained endothelial function by KLF2-eNOS activation under hyperglycemia. Low-dose phloretin helped with lipid metabolism, and blocked the acceleration of atherosclerosis in STZ-induced diabetic mice since the early stage, which was diminished by KLF2 knockdown. Low-dose phloretin exhibited athero-protective effect in diabetic Apoe-/- mice dependent on KLF2 activation. This finding makes phloretin for diabetic atherosclerosis.