The Baise City Scientific Research and Technology Development Program (20224128) supported this work. The authors thank Dr. Dev Sooranna of Imperial College London and YMUN for editing the manuscript. YYX and ZYW contributed equally to this study.

Therapeutic Effects of Xiaoyao Pills on Postmenopausal Osteoporosis in Mice

The authors declare there is no conflict of interest.

According to statistics, osteoporosis causes 1.5 million fractures each year in the United States, and the vast majority of these occur in postmenopausal women27. With an increase in the aging population, it is predicted that the majority of the world&#39;s future hip fractures will occur in Asia and that by 2050, the total number of these worldwide will reach 8.2 million28. The cost of preventing fractures is almost equal to that of treating them, and the use of medication...

The World Health Organization (WHO) defines osteoporosis (OP) as a disease characterized by a decrease in bone mass and deterioration of the microarchitecture of bone tissue, leading to an increase in bone brittleness and, thus an increased risk of fracture1. The clinical significance of osteoporosis is that it can lead to fractures, which are associated with high mortality, morbidity, and economic costs2. Postmenopausal osteoporosis (PMOP) is caused by a decrease in estrogen levels in women after menopause, which leads to an increase in osteoclast activity, resulting in bone loss and destruction of bone microstructure. This often causes osteoporosis with a serious impact on health3. Current therapies for PMOP include estrogen replacement therapy, bisphosphonates, and parathyroid hormone, but they can have varying degrees of adverse effects, insufficient long-term compliance, and high costs4. Therefore, affordable herbal medicine is a viable alternative for a large proportion of the population.
Xiaoyao pills are included in the Chinese Pharmacopoeia5, and these contain eight herbal components, including Chai Hu, Angelica sinensis, White paeonia lactiflora, Atractylodes macrocephala, Poria cocos, Menthae Herba, licorice, and fresh ginger. All these herbs are known to be effective in detoxifying the liver and strengthening the spleen, nourishing the blood, and regulating the menstrual cycle, and the mixture has also been used for treating depression6. However, the role of Xiaoyao pills in osteoporosis is unclear.
Early studies have suggested that inflammation can lead to bone loss7, and that the decline in bone density associated with this process may be accelerated by menopause. In addition, there is a strong relationship between the development of osteoporosis and inflammation. The inflammatory factor, interleukin-17 (IL-17), is a pro-inflammatory factor secreted by Th17 cells, a subset of CD4+ T lymphocytes. These cells are associated with several chronic inflammatory conditions, and they play an important role in the development of bone destruction in rheumatoid arthritis8. Additionally, IL-17 stimulates nuclear factor-&#954; B ligand receptor activator (RANKL), which regulates osteoclastogenesis, leading to greater bone resorption than bone formation9. IL-17 stimulates the expression of other osteoclastogenic cytokines such as TNF&#945;, IL-1, IL-6, and IL-8. It has the ability to synergize with other inflammatory factors, making it an important inflammatory effector10.
Studies have also shown a&#160;link between Xiaoyao pills and inflammation. Shi et al.11 and Fang et al.12 have recently confirmed that Xiaoyao pills can reduce the levels of IL-6 and TNF-&#945;, respectively. In another study of metabolism-associated steatohepatitis, it was reported that Xiaoyao pills could upregulate the expression of propionic acid, which in turn inhibits the expression of TNF-&#945; and exerts an anti-inflammatory effect13. However, at present, it is not known whether Xiaoyao pills can regulate the development of PMOM by mediating an inflammatory response through IL-17, which was the aim of this study.
This study predicted the intersection of the targets of Xiaoyao pills and osteoporosis-related genes through network pharmacology and bioinformatics analysis and analyzed the intersecting genes for protein interactions, GO, and KEGG. Based on the predicted results, the expression of Act1 and IL-6, which are key proteins in the IL-17 signaling pathway, can be observed14,15, as well as the bone turnover markers alkaline phosphatase (ALP) and collagen type I (COL-1), to observe the therapeutic efficacy of the Xiaoyao pills in the PMOM mice model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1ml Sampler</td>
			<td>Guangxi Beilunhe Medical Industrial Group Co.</td>
			<td>JYQ001</td>
			<td>For anesthesia in mice</td>
		</tr>
		<tr>
			<td>4%polyformaldehyde</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>P1110</td>
			<td>For tissue fixation</td>
		</tr>
		<tr>
			<td>6-0 absorbable suture (angled needle)</td>
			<td>Shanghai Pudong Jinhuan Medical Supplies Co.</td>
			<td>HZX-06</td>
			<td>For postoperative suturing</td>
		</tr>
		<tr>
			<td>Absorbent cotton ball</td>
			<td>Winner</td>
			<td>MIANQIU-500g</td>
			<td>For sterilization and hemostasis</td>
		</tr>
		<tr>
			<td>Adhesive slides</td>
			<td>Jiangsu Shitai Experimental Equipment Co.</td>
			<td>188105</td>
			<td>For tissue sectioning</td>
		</tr>
		<tr>
			<td>Amobarbital</td>
			<td>Sigma Aldrich (Shanghai) Trading Co.</td>
			<td>A-020-1ML</td>
			<td>For anesthesia in mice</td>
		</tr>
		<tr>
			<td>Bluing Solution</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>G1866</td>
			<td>Blue coloration of the nuclei of cells after the action of hematoxylin differentiation solution</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td>
			<td>(SCXK 2033-0063)</td>
			<td>For use in animal experiments</td>
		</tr>
		<tr>
			<td>Carbon steel surgical blades</td>
			<td>Premier Medical Equipment Co.</td>
			<td>SP239</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>CIKS/TRAF3IP2 Rabbit pAb</td>
			<td>BIOSS ANTIBODIES</td>
			<td>bs-6202R</td>
			<td>Binds to Act1 in tissues</td>
		</tr>
		<tr>
			<td>Cole&#39;s Hematoxylin Solution (For Conventional Stain)</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>G1140</td>
			<td>For staining paraffin sections</td>
		</tr>
		<tr>
			<td>Collagen Type I Polyclonal antibody</td>
			<td>proteintech</td>
			<td>14695-1-AP</td>
			<td>Binds to COL-1 in tissues</td>
		</tr>
		<tr>
			<td>DAB Substrate kit,20x</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>DA1010</td>
			<td>For tissue color development</td>
		</tr>
		<tr>
			<td>Disposable surgical sheet 50*60CM</td>
			<td>Nanchang Xuhui Medical Equipment Co.</td>
			<td>SP4529777</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>EDTA decalcification solution (pH 7.2)</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>E1171-500ml</td>
			<td>For tissue decalcification</td>
		</tr>
		<tr>
			<td>Enhanced Endogenous Peroxidase Bloching Buffer</td>
			<td>Beyotime Biotechnology</td>
			<td>P0100B</td>
			<td>Sequestration of tissue or cellular endogenous peroxidases</td>
		</tr>
		<tr>
			<td>Environmentally friendly dewaxing clear liquid</td>
			<td>Servicebio</td>
			<td>G1128-1L</td>
			<td>For dewaxing paraffin sections</td>
		</tr>
		<tr>
			<td>Ethyl Alcohol</td>
			<td>CHRON CHEMICALS</td>
			<td>64-17-5 (CAS)</td>
			<td>For dehydration of paraffin sections</td>
		</tr>
		<tr>
			<td>General Purpose Antibody Diluent</td>
			<td>Epizyme Biotech</td>
			<td>PS119L</td>
			<td>For antibody dilution</td>
		</tr>
		<tr>
			<td>Hematoxylin Differentiation Solution</td>
			<td>Servicebio</td>
			<td>G1039-500ML</td>
			<td>For differentiation after hematoxylin staining and removal of excessively bound and non-specifically adsorbed dye from tissues</td>
		</tr>
		<tr>
			<td>Hematoxylin Eosin (HE) Staining Kit</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>G1120-3*100ml</td>
			<td>For tissue staining</td>
		</tr>
		<tr>
			<td>High quality stainless steel surgical knife handle</td>
			<td>Premier Medical Equipment Co.</td>
			<td>SP0088</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td>
			<td>proteintech</td>
			<td>SA00001-2</td>
			<td>Binds to primary antibody and amplifies signal</td>
		</tr>
		<tr>
			<td>IL-17A Polyclonal antibody</td>
			<td>proteintech</td>
			<td>13082-1-AP</td>
			<td>Binds to IL-17 in tissues</td>
		</tr>
		<tr>
			<td>IL-6 Polyclonal antibody</td>
			<td>proteintech</td>
			<td>21865-1-AP</td>
			<td>Binds to IL-6 in tissues</td>
		</tr>
		<tr>
			<td>Immunohistochemistry pen</td>
			<td>Beijing Zhongshan Jinqiao Biotechnology Co.</td>
			<td>ZLI-9305 (YA0310)</td>
			<td>For drawing circles in immunohistochemistry</td>
		</tr>
		<tr>
			<td>Medical surgical suture Non-absorbent (ball) 5-0 3.5m</td>
			<td>Yangzhou Yuanlikang Medical Equipment Co.</td>
			<td>FHX-5-2</td>
			<td>For postoperative suturing</td>
		</tr>
		<tr>
			<td>Medical Suture Needles Angle Needles 4*10 3/8</td>
			<td>Chaohu Binxiong Medical Equipment Co.</td>
			<td>FHZ612-4</td>
			<td>For postoperative suturing</td>
		</tr>
		<tr>
			<td>Neutral Balsam</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.</td>
			<td>G8590</td>
			<td>As a slice sealer</td>
		</tr>
		<tr>
			<td>PBS(1X)</td>
			<td>Shenzhen Mohong Technology Co.,Ltd</td>
			<td>B0015</td>
			<td>Buffer for slide washing and partial solution dilution</td>
		</tr>
		<tr>
			<td>Protein Free Rapid Blocking Buffer (1X)</td>
			<td>Epizyme Biotech</td>
			<td>PS108P</td>
			<td>Avoiding non-specific binding of proteins</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Bone Alkaline Phosphatase antibody</td>
			<td>BIOSS ANTIBODIES</td>
			<td>bs-6292R</td>
			<td>Binds to alkaline phosphatase in tissues</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Affiliated Hospital Youjiang Medical University For Nationalities</td>
			<td>LHN500</td>
			<td>For animals by gavage</td>
		</tr>
		<tr>
			<td>Shaving/Electric clippers</td>
			<td>HANGZHOU HUAYUAN PET PRODUCTS CO., LTD.</td>
			<td>DTJ-002</td>
			<td>For shaving mice</td>
		</tr>
		<tr>
			<td>Stainless Steel Medical Needle Holder 14cm Coarse Needle</td>
			<td>Premier Medical Equipment Co.</td>
			<td>SP784</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>Stainless Steel Ophthalmic Forceps 10.5cm Curved (No Hook)</td>
			<td>Zhuoyouyue</td>
			<td>YKNWW-10.5</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>Stainless Steel Ophthalmic Scissors/Surgical Scissors 10CM Straight Tip</td>
			<td>Premier Medical Equipment Co.</td>
			<td>ZYJD-10-ZJ</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>Stainless Steel Tip Gastric Needle 12 Gauge 55mm Elbow</td>
			<td></td>
			<td>GWJ-12-55W</td>
			<td>For use in mice by gavage</td>
		</tr>
		<tr>
			<td>Tris-EDTA Antigen Repair Fluid (50x)</td>
			<td>proteintech</td>
			<td>PR30002</td>
			<td>For antigen repair of paraffin sections</td>
		</tr>
		<tr>
			<td>Wooden dissecting board 25*16cm</td>
			<td></td>
			<td>JP16*24</td>
			<td>For mouse surgery</td>
		</tr>
		<tr>
			<td>Xiaoyao pills</td>
			<td>Jiuzhitang Co.,Ltd.</td>
			<td>YPG-041</td>
			<td>For animal drug delivery</td>
		</tr>
		<tr>
			<td>Others</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R 4.3.1</td>
			<td></td>
			<td></td>
			<td>Data processing</td>
		</tr>
		<tr>
			<td>Cytoscape3.9.1</td>
			<td>National Resource for Network Biology</td>
			<td></td>
			<td>Building a regulatory network for traditional Chinese medicine</td>
		</tr>
		<tr>
			<td>ImageJ 1.54f</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>Image processing for immunohistochemistry results</td>
		</tr>
		<tr>
			<td>Adobe Photoshop 24.0.0</td>
			<td>Adobe</td>
			<td></td>
			<td>For image combination</td>
		</tr>
		<tr>
			<td>GraphpadPrism 9.5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Statistical analysis of data</td>
		</tr>
		<tr>
			<td>cellsens Dimension</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>For slicing and photographing</td>
		</tr>
		<tr>
			<td>OLYMPUS BX53</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>For HE staining and immunohistochemical section photography</td>
		</tr>
	</tbody>
</table>

studies on the anti-inflammatory effect of xiaoyao pills in the treatment of postmenopausal osteoporosis in mice

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of this condition, the patients are prone to fractures, and this can seriously affect their health and quality of life. The worldwide increase in life expectancy has made osteoporosis a global concern. The Xiaoyao pills were previously 
used in the treatment of depression. In addition, the drug appeared to have estrogen-like activity, which affected the expression of ALP, an early osteoblast-specific marker, and COL-1, a major component of bone extracellular matrix. Xiaoyao pills were assessed for their effects on postmenopausal osteoporosis (PMOM) in mice. The target information of each herbal component of Xiaoyao pills was accessed through the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Information from GeneCards, OMIM, PharmGkb, TTD, DrugBank, and other websites was used to construct the regulatory network of the herbal complex through Cytoscape and String network to assess the protein interactions. Mice were ovariectomized, and treated with high and low doses of Xiaoyao pills and these were compared to controls. Their symptoms were assessed by immunocytochemistry of bone tissues. The results suggested that Xiaoyao pills had the ability to alleviate the symptoms of PMOM in ovariectomized mice through the IL-17 signaling pathway. This drug has the potential to become a novel therapeutic agent for the treatment of osteoporosis.

Active ingredients and targets of action of Xiaoyao pills 
By searching the TCMSP database and screening according to the criteria of oral bioavailability (OB) &#8805; 30% and drug-like properties (DL) &#8805; 0.18, 125 active ingredients were found to exist in Xiaoyao pills. Among them, ingredients 6, 4, 9, 13, 2, 6, 83, and 2 were from White Paeonia lactiflora, Atractylodes macrocephala, Menthae herba, Chaihu, Angelica sinensis, Poria cocos, licorice an...

Author Spotlight: Integrating Traditional Chinese Medicine with Modern Pharmacology and Genomics for Assessing Postmenopausal Osteoporosis in Mice 

Postmenopausal osteoporosis has become a global public health problem. The aim of this study is to explore the therapeutic effects and related mechanisms of the traditional Chinese medicine Xiaoyao pills on this condition.

Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of ...

studies-on-anti-inflammatory-effect-xiaoyao-pills-treatment

Research

JoVE Journal

Biology

364 Views.  Youjiang Medical University for Nationalities. Postmenopausal osteoporosis has become a global public health problem. The aim of this study is to explore the therapeutic effects and related mechanisms of the traditional Chinese medicine Xiaoyao pills on this condition.

Video: Author Spotlight: Integrating Traditional Chinese Medicine with Modern Pharmacology and Genomics for Assessing Postmenopausal Osteoporosis in Mice 

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...