[A glass micropipette vacuum technique of cerebrospinal fluid sampling in C57BL/6 mice].

The purpose of this study was to establish a suitable method for extracting cerebrospinal fluid (CSF) from C57BL/6 mice. A patch clamp electrode puller was used to draw a glass micropipette, and a brain stereotaxic device was used to fix the mouse's head at an angle of 135° from the body. Under a stereoscopic microscope, the skin and muscle tissue on the back of the mouse's head were separated, and the dura mater at the cerebellomedullary cistern was exposed. The glass micropipette (with an angle of 20° to 30° from the dura mater) was used to puncture at a point 1 mm inboard of Y-shaped dorsal vertebral artery for CSF sampling. After the first extraction, the glass micropipette was connected with a 1 mL sterile syringe to form a negative pressure device for the second extraction. The results showed that the successful rate of CSF extraction was 83.33% (30/36). Average CSF extraction amount was (7.16 ± 0.43) µL per mouse. In addition, C57BL/6 mice were given intranasally ferric ammonium citrate (FAC) to establish a model of brain iron accumulation, and the CSF extraction technique established in the present study was used for sampling. The results showed that iron content in the CSF from the normal saline control group was not detected, while the iron content in the CSF from FAC-treated group was (76.24 ± 38.53) µmol/L, and the difference was significant. These results suggest that glass micropipette vacuum technique of CSF sampling established in the present study has the advantages of simplicity, high success rate, large extraction volume, and low bleeding rate, and is suitable for the research on C57BL/6 mouse neurological disease models.

[A Review of Chronic Stress and the Initiation and Evolution of Cancer].

Chronic stress activates the typical neuroendocrine system, hypothalamus pituitary adrenal axis and sympathetic nervous system, and leads to a sustained non-specific adaptive response. It has been proved that chronic stress can promote tumor initiation and induce tumor evolution, especially in immune function and remodeling of tumor microenvironment. However, due to the complex mechanism of chronic stress and the great difference in individual tolerance, the research evidence of chronic stress in tumor genesis and progression is still unclear. Therefore, in this paper, we review the research on the relationship between chronic stress and tumor initiation and evolution, focusing on the molecular mechanism of chronic stress promoting tumor occurrence and development, inhibiting immune response and remodeling tumor immune microenvironment, and exploring the stress management program of healthy people and cancer patients, so as to provide clues for exploring new strategies of cancer prevention and treatment. In our opinion, targeting the cAMP/PKA/CREB signaling pathway to reverse tumor treatment strategy, the relationship between the tumor and stress, inflammation, immunity, the suppressor activity of ß receptor antagonist and its mechanism as well as associated with different treatment options, still need to be further explored. A healthy lifestyle, positive life attitudes and professional stress management guidance are essential for the prevention and treatment of cancer.

Over-Expression of Inhibitor of Differentiation 2 Attenuates Post-Infarct Cardiac Fibrosis Through Inhibition of TGF-ß1/Smad3/HIF-1α/IL-11 Signaling Pathway.

Background: Cardiac fibrosis after myocardial infarction mainly causes cardiac diastolic and systolic dysfunction, which results in fatal arrhythmias or even sudden death. Id2, a transcriptional repressor, has been shown to play an important role in the development of fibrosis in various organs, but its effects on cardiac fibrosis remain unclear. This study aimed to explore the effects of Id2 on cardiac fibrosis after myocardial infarction and its possible mechanisms. Methods: This study was performed in four experimental groups: control group, treatment group (including TGF-ß1, hypoxia or MI), treatment+GFP group and treatment+Id2 group. In vitro anoxic and fibrotic models were established by subjecting CFs or NRVMs to a three-gas incubator or TGF-ß1, respectively. An animal myocardial infarction model was established by ligating of the left anterior descending coronary artery followed by directly injecting of Id2 adenovirus into the myocardial infarct's marginal zone. Results: The results showed that Id2 significantly improved cardiac EF and attenuated cardiac hypertrophy. The mRNA and protein levels of α-SMA, Collagen I, Collagen III, MMP2 and TIMP1 were higher in treatment+Id2 group than those in treatment group as well as in treatment+GFP group both in vivo and in vitro. Immunofluorescence revealed that both α-SMA and vimentin were co-expressed in the treatment group and GFP group, but the co-expression were not detected in the control group and Id2 group. Additionally, our findings illustrated that Id2 had protective effects demonstrated by its ability to inhibit the TGF-ß1/Smad3/HIF-1α/IL-11 signaling pathways. Besides, over-expression of Id2 reduced cardiomyocytes apoptosis. Conclusion: In conclusion, this study demonstrated that over-expression of Id2 preserved cardiac function and ameliorated adverse cardiac remodeling, which might be a promising treatment target for cardiac fibrosis and apoptosis.

Transcription Factor prrx1 Promotes Brown Adipose-Derived Stem Cells Differentiation to Sinus Node-Like Cells.

This study investigated whether overexpression of paired-related homeobox 1 (prrx1) can successfully induce differentiation of brown adipose-derived stem cells (BADSCs) into sinus node-like cells. The experiments were performed in two groups: adenovirus-green fluorescent protein (Ad-GFP) group and Ad-prrx1 group. After 5-7 days of adenoviral transfection, the expression levels of sinus node cell-associated pacing protein (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 [HCN4]) and ion channel (calcium channel, voltage-dependent, T type, alpha 1G subunit [Cacna1g]), as well as transcription factors (T-box 18 [TBX18], insulin gene enhancer binding protein 1 [ISL-1], paired-like homeodomain transcription factor 2 [pitx2], short stature homeobox 2 [shox2]), were detected by western blot and reverse transcription-quantitative polymerase chain reaction. Immunofluorescence assay was carried out to detect whether prrx1 was coexpressed with HCN4, TBX18, and ISL-1. Finally, whole-cell patch-clamp technique was used to record pacing current hyperpolarization-activated inward current (If). The isolated cells were CD90+, CD29+, and CD45-, indicating that pure BADSCs were successfully isolated. After 5-7 days of Ad transfection into cells, the mRNA levels and protein levels of pacing-related factors (TBX18, ISL-1, HCN4, shox2, and Cacna1g) in Ad-prrx1 group were significantly higher than those in Ad-GFP group. However, the expression level of pitx2 was decreased. Immunofluorescence analysis showed that prrx1 was coexpressed with TBX18, ISL-1, and HCN4 in the Ad-prrx1 group, which did not appear in the Ad-GFP group. Whole-cell patch clamps were able to record the If current in the experimental group rather than in the Ad-GFP group. Overexpression of prrx1 can successfully induce sinus node-like cells.

Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction.

Cardiac remodeling predisposes to heart failure if the burden is unresolved, and heart failure is an important cause of mortality in humans. The aim of the present study was to identify the key genes involved in cardiac pathological remodeling induced by pressure overload. Gene expression profiles of the GSE5500, GSE18224, GSE36074 and GSE56348 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs), defined as |log2FC|>1 (FC, fold change) and an adjusted P­value of <0.05, were screened using the R software with the limma package. Gene ontology enrichment analysis was performed and a protein­protein interaction (PPI) network of the DEGs was constructed. A cardiac remodeling model induced by transverse aortic constriction (TAC) was established. Furthermore, consistent DEGs were further validated using reverse transcription­quantitative polymerase chain reaction (RT­PCR) analysis, western blotting and immunohistochemistry in the ventricular tissue samples after TAC or sham operation. A total of 24 common DEGs were identified (23 significantly upregulated and 1 downregulated), of which 9 genes had been previously confirmed to be directly involved in cardiac remodeling. Hence, the level of expression of the other 15 genes was detected in subsequent studies via RT­PCR. Based on the results of the PPI network analysis and RT­PCR, we further detected the protein levels of Itgbl1 and Asporin, which were consistent with the results of bioinformatics analysis and RT­PCR. The expression of Itgbl1, Aspn, Fstl1, Mfap5, Col8a1, Ltbp2, Mfap4, Pamr1, Cnksr1, Aqp8, Meox1, Gdf15 and Srpx was found to be upregulated in a mouse model of cardiac remodeling, while that of Retnla was downregulated. Therefore, the present study identified the key genes implicated in cardiac remodeling, aiming to provide new insight into the underlying mechanism.

MicroRNA-155 inhibition attenuates endoplasmic reticulum stress-induced cardiomyocyte apoptosis following myocardial infarction via reducing macrophage inflammation.

Endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis plays an important role in the pathological process following myocardial infarction (MI). Macrophages that express microRNA-155 (miR-155) mediate cardiac inflammation, fibrosis, and hypertrophy. Therefore, we investigated if miR-155 regulates ERS-induced cardiomyocyte apoptosis after MI using a mouse model, lipopolysaccharide (LPS)-induced rat bone marrow derived macrophages (BMDMs)and hypoxia-induced neonatal rat cardiomyocytes (NRCMs). In vivo, miR-155 levelswere significantly higher in the MI group compared to the sham group. MI increasedmacrophage infiltration, nuclear factor-κB (NF-κB) activation, ERS induced-apoptosis, and SOCS1 expression, all of which were attenuated by the miR-155 antagomir, with the exception of SOCS1 expression. Additionally, post-MI cardiac dysfunction was significantly improved by miR-155 inhibition. In vitro, LPS upregulated miR-155 expression in BMDMs, and the miR-155 antagomir decreased LPS-induced macrophage inflammation and NF-κB pathway activation, but increased expression of SOCS1. Hypoxia increased NF-κB pathway activation, ERS marker expression, and apoptosis in NRCMs. Interestingly, conditioned medium from LPS-induced macrophages in combination with the miR-155 antagomir decreased, while the miR-155 agomir increased, the hypoxia-induced effects in NRCM's. The miR-155 agomir effects were reversed by inhibiting the NF-κB pathway in cardiomyocytes. Moreover, SOCS1 knockdown in LPS-induced macrophages promoted NF-κB pathway activation and ERS-induced cardiomyocyte apoptosis in the hypoxia-induced NRCMs, but the SOCS1-siRNA-induced effects were markedly decreased by miR-155 antagomir treatment. These data suggest that miR-155 inhibition attenuates ERS-induced cardiomyocyte apoptosis after MI via reducing macrophage inflammation through the SOCS1/NF-κB pathway.

[Exosomes and their roles in neurodegenerative diseases: from pathogenic, diagnostic and therapeutical perspectives].

Exosomes are extracellular membranous vesicles with a diameter of 30-100 nm derived from a variety of eukaryocytes. The cargo of exosomes includes proteins, lipids, nucleic acids, and substances of the cells from which they originate. They can transfer functional cargo to neighboring and distal cells, therefore contributing to intercellular communication in both physiological and pathological processes. In recent years, it was shown that exosomes in several neurodegenerative diseases are closely related to the transmission of disease-related misfolded proteins (such as α-synuclein, tau, amyloid ß-protein, etc). These proteins are transported by exosomes, thus promoting the propagation to unaffected cells or areas and accelerating the progression of neurodegenerative diseases. This review focuses on the origin and composition, biological synthesis, secretion, function of exosomes, as well as their roles in the pathogenesis and progression of neurodegenerative diseases. In addition, we also discuss that exosomes can serve as biomarkers and drug delivery vehicles, and play a role in the diagnosis and treatment of neurodegenerative diseases.

C1q/TNF-related protein-9 attenuates retinal inflammation and protects blood-retinal barrier in db/db mice.

C1q/TNF-related protein-9(CTRP9) is an adipose cytokine, a closest adiponectin paralog, which has anti-inflammatory, antioxidant, vasodilation and anti-atherosclerosis effects. In addition, it can increase insulin sensitivity, decrease blood glucose level and inhibit the apoptosis of endothelial cells. However, it remains unclear whether CTRP9 has beneficial effects on diabetic retinopathy (DR). An adenoviral vector expressing CTRP9 was intravenously injected into db/db mice, aged 12 weeks, at day 15 post injection, and the process was repeated. The transfection efficiency of CTRP9 was assessed by enzyme linked immunosorbent assay. We used RT-PCR, immunofluorescence, and Western blot to determine proinflammatory cytokines, adhesion molecules and tight-junction proteins. The breakdown of blood-retinal barrier (BRB) was evaluated using Evans blue and retinal staining. CTRP9 suppresses the expression of interleukin-1 beta, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and adhesion molecules in the retina of db/db mice. CTRP9 can balance the expression of pigment epithelium-derived factor and vascular endothelial growth factor. CTRP9 can also inhibit the activation of nuclear factor Kappa B in the retina of db/db mouse. In addition, CTRP9 can prevent the breakdown of BRB and downregulation of tight-junction proteins in the retina of db/db mice. Evans blue assay revealed the breakdown of BRB and vascular leakage in the retinas of diabetic mice. CTRP9 can both qualitatively and quantitatively alleviate the vascular leakage in the early stage of diabetic retinas. CTRP9 can inhibit the inflammation of diabetic retinopathy and protect blood-retinal barrier via decreasing proinflammatory cytokines and preventing the downregulation of tight-junction proteins.

Inhibition of microRNA-155 attenuates sympathetic neural remodeling following myocardial infarction via reducing M1 macrophage polarization and inflammatory responses in mice.

Inflammation plays an important role in sympathetic neural remodeling induced by myocardial infarction (MI). MiR-155 is a vital regulator of inflammatory responses, and macrophage-secreted miR-155 promotes cardiac fibrosis and hypertrophy. However, whether miR-155 influences MI-induced sympathetic neural remodeling is not clear. Therefore, we examined the role of miR-155 in MI-induced sympathetic neural remodeling and the related mechanisms in both an mouse model and in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs). Our data showed that miR-155 expression was significantly enhanced in the myocardial tissues of MI mice compared to sham mice. Also, MI up-regulated the electrophysiological parameters, M1 macrophage polarization, inflammatory responses, and suppressor of cytokine signaling 1 (SOCS1) expression, which coincided with the increased expression of sympathetic nerve remodeling markers(nerve growth factor, tyrosine hydroxylase and growth-associated protein 43). Except for SOCS1, these proteins were attenuated by miR-155 antagomir. In vitro, LPS-stimulation promoted miR-155 expression in BMDMs. Consistent with the in vivo findings, miR-155 antagomir diminished the LPS-induced M1 macrophage polarization, nuclear factor (NF)-κB activation, and the expression of pro-inflammatory factors and nerve growth factor; but it increased the expression of SOCS1. Inversely, miR-155 agomir significantly potentiated LPS-induced pathophysiological effects in BMDMs. MiR-155 agomir-induced effects were reversed by the NF-κB inhibitor. Mechanistically, treatment with siRNA against SOCS1 augmented the aforementioned LPS-mediated activities, which were antagonized by the addition of miR-155 antagomir. In conclusion, miR-155 inhibition downregulated NGF expression via decreasing M1 macrophage polarization and inflammatory responses dependent on the SOCS1/NF-κB pathway, subsequently diminishing MI-induced sympathetic neural remodeling and ventricular arrhythmias (VAs).

Galangin ameliorates cardiac remodeling via the MEK1/2-ERK1/2 and PI3K-AKT pathways.

Cardiac remodeling is associated with inflammation and apoptosis. Galangin, as a natural flavonol, has the potent function of regulating inflammation and apoptosis, which are factors related to cardiac remodeling. Beginning 3 days after aortic banding (AB) or Sham surgery, mice were treated with galangin for 4 weeks. Cardiac remodeling was assessed according to echocardiographic parameters, histological analyses, and hypertrophy and fibrosis markers. Our results showed that galangin administration attenuated cardiac hypertrophy, dysfunction, and fibrosis response in AB mice and angiotensin II-treated H9c2 cells. The inhibitory action of galangin in cardiac remodeling was mediated by MEK1/2-extracellular-regulated protein kinases 1/2 (ERK1/2)-GATA4 and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3ß (GSK3ß) activation. Furthermore, we found that galangin inhibited inflammatory response and apoptosis. Our findings suggest that galangin protects against cardiac remodeling through decreasing inflammatory responses and apoptosis, which are associated with inhibition of the MEK1/2-ERK1/2-GATA4 and PI3K-AKT-GSK3ß signals.

Chlorogenic Acid Functions as a Novel Agonist of PPARγ2 during the Differentiation of Mouse 3T3-L1 Preadipocytes.

Rosiglitazone (RG) is a well-known activator of peroxisome proliferator-activated receptor-gamma (PPARγ) and used to treat hyperglycemia and type 2 diabetes; however, its clinical application has been confounded by adverse side effects. Here, we assessed the roles of chlorogenic acid (CGA), a phenolic secondary metabolite found in many fruits and vegetables, on the differentiation and lipolysis of mouse 3T3-L1 preadipocytes. The results showed that CGA promoted differentiation in vitro according to oil red O staining and quantitative polymerase chain reaction assays. As a potential molecular mechanism, CGA downregulated mRNA levels of the adipocyte differentiation-inhibitor gene Pref1 and upregulated those of major adipogenic transcriptional factors (Cebpb and Srebp1). Additionally, CGA upregulated the expression of the differentiation-related transcriptional factor PPARγ2 at both the mRNA and protein levels. However, following CGA intervention, the accumulation of intracellular triacylglycerides following preadipocyte differentiation was significantly lower than that in the RG group. Consistent with this, our data indicated that CGA treatment significantly upregulated the expression of lipogenic pathway-related genes Plin and Srebp1 during the differentiation stage, although the influence of CGA was weaker than that of RG. Notably, CGA upregulated the expression of the lipolysis-related gene Hsl, whereas it did not increase the expression of the lipid synthesis-related gene Dgat1. These results demonstrated that CGA might function as a potential PPARγ agonist similar to RG; however, the impact of CGA on lipolysis in 3T3-L1 preadipocytes differed from that of RG.

[Capacity of extensive green roof to retain rainwater runoff in hot and humid region.] / 湿热地区简单式屋顶绿化的截流雨水效应.

The water logging has become the environmental problem of major cities with the sharp increase of impermeable urban pavement as the contributing cause. Abroad, the green roof has been widely used as a practical measure to intercept rainwater, yet the capacity of green roof to retain rainwater varies with climate conditions. As the hot and humid climate zone features high temperature, humidity and precipitation, it is meaningful to study the capacity of green roof to retain rainwater under such climatic condition. In this research, 3 plat forms were set up in Guangzhou in rainy and hot summer to test the capability of simple green roof to retain rainwater runoff, and the efficiency of green roof to retain rainwater under local climate conditions was worked out based on the meteorological observation and data measurement during the 13-month test period. The results showed that the simple green roof with a substrate thickness of 30, 50 and 70 mm could retain 27.2%, 30.9% and 32.1% of precipitation and reduce the average peak value by 18.9%, 26.2% and 27.7%, respectively. Given an urban built-up area of 1035.01 km2 in Guangzhou and a roof area percentage of approximately 37.3% and assuming the green roofs with 30 mm-thick substrate were applied within the area, the light, medium and heavy rain could be delayed at 72.8%, 22.6% and 17.4%, respectively. Accordingly, the rainwater retained could reach up to 14317×104 m3. It suggested the great potential of the simple green roof in retaining rainwater. The research could serve as reference for the hot and humid climate zone to alleviate water logging and visualize sponge city construction.

Microvascular decompression surgery: surgical principles and technical nuances based on 4000 cases.

BACKGROUND: As an etiological treatment of trigeminal neuralgia (TN) and hemifacial spasm (HFS), microvascular decompression (MVD) has been popularized around the world. However, as a functional operation in the cerebellopontine angle (CPA), this process can be risky and the postoperative outcomes might not be good enough sometimes. OBJECTIVE: In order to obtain a better result with less complication, this surgery should be further addressed. METHODS: With experience of more than 4000 MVDs, we have gained knowledge about the operative technique. Through abundant intraoperative photos, each step of the procedure was demonstrated in detail and the surgical strategy was focused. RESULTS: The principle of MVD is to separate the nerve-vessel confliction rather than isolate it with prostheses. A prompt identification of the conflict site is important, which hinges on a good exposure. A satisfactory working space can be established by an appropriate positioning of the patient's head and a proper craniectomy as well as a rational approach. A sharp dissection of arachnoids leads to a maximal visualization of the entire intracranial course of the nerve root. All the vessels contacting the trigeminal or facial nerve should be treated. Intraoperative electrophysiological mentoring is helpful to distinguish the offending artery for hemifacial cases. CONCLUSION: MVD is an effective treatment for the patient with TN or HFS. Immediate relief can be achieved by an experienced neurosurgeon with good knowledge of regional anatomy. A safe surgery is the tenet of MVD, and accordingly, no single step of the procedure should be ignored.

Apelin-13-induced proliferation and migration induced of rat vascular smooth muscle cells is mediated by the upregulation of Egr-1.

Apelin-13 plays an important role in the migration and proliferation of vascular smooth muscle cells (VSMCs); however, the underlying mechanisms are still unclear. Egr-1 is a nuclear transcription factor, which is considered to be the critical initiating factor of the processes of VSMC proliferation and migration. Egr-1 is known to regulate the expression of osteopontin (OPN), which is a marker of the phenotypic modulation that is a necessary condition of VSMC proliferation and migration. We hypothesized that the role of Apelin-13 is mediated via upregulation of Egr-1. To test this hypothesis, we analyzed the effects of Apelin-13 treatment on Egr-1 mRNA and protein expression in A10 rat aortic VSMCs by RT-PCR and Western blotting, respectively. Results showed that, Apelin-13 upregulated the expression of Egr-1. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor, PD98059, inhibited the upregulation of Egr-1 by Apelin-13. In addition, this upregulation was inhibited by treatment of VSMCs with the Egr-1 specific deoxyribozyme ED5 (DNAenzyme/10-23 DRz). Furthermore, ED5 treatment was found to significantly inhibit Apelin-13-induced migration and proliferation of VSMCs using transwell and MTT assays, respectively. The evaluation of OPN mRNA and protein expression levels by RT-PCR and Western blot analyses revealed that ED5 treatment also inhibited Apelin-13-induced OPN upregulation. The results of this study indicated that Apelin-13 upregulates Egr-1 via ERK. Furthermore, Apelin-13 induced the proliferation and migration of VSMCs as well as the upregulation of OPN via the upregulation of Egr-1. These results will provide an important theoretical and experimental basis for the control of inappropriate remodeling of vessel walls, and will hopefully lead to the prevention and treatment of vascular remodeling diseases.

[Study on the names of Muzhi and Pinzhi before the Tang dynasty].

Muzhi (male hemorrhoid) and Pinzhi (female hemorrhoid) recorded in bamboo slips found in the Han Tomb of Zhanjiashan were named after the shape of genitalia of males and females, which conserved the original meaning of Mu (male) and Pin (female). In Wu shi er Bingfang (Formularies for 52 Kinds of Disorders), a silk manuscript found in the Han Tombs of Mawangdui, Pinzhi and Muzhi were defined as hemorrhoid inside and outside the anus, which used the extended meaning of Mu (male) and Pin (female). In Sui and Tang dynasties, Muzhi and Pinzhi were named after morphology or location of the hemorrhoid. The famous doctor of the Tang dynasty, XU Zeren, made a more perfect classification of hemorrhoids. In all, the names of Muzhi and Pinzhi were related to location, morphology and features of hemorrhoids and the influence of yin-yang thought to medicine.

Efficacy and safety of intra-aortic balloon pump-assisted interventional therapy in different age groups of patients with acute coronary syndrome.

BACKGROUND: Currently intra-aortic balloon pump (IABP) has been widely used in patients with acute coronary syndrome (ACS) who undergo percutaneous coronary intervention (PCI). However, few studies have been done with regard to the clinical outcome and safety of IABP in assisting PCI in aged patients with ACS. The purpose of the present study was to evaluate the safety of IABP in different age groups of patients with ACS. METHODS: Data on 292 ACS patients who received IABP-assisted PCI in Shenyang General Hospital of People's Liberation Army were retrospectively analyzed. More specifically, the successful rate, mortality and complications associated with the treatment were compared between the senior (>/= 60 years old) and the non-senior (< 60 years old) groups of patients. RESULTS: The attack rate of non-ST segment elevation ACS was significantly higher in the senior group than in the non-senior group (38.8% vs 21.1%, P < 0.01). The incidence of the IABP-associated complications was not significantly different between both groups (P > 0.05). CONCLUSION: The clinical outcome and safety of IABP-assisted PCI in the elderly patients were comparable to that for the non-elderly patients.