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><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 &amp;amp; 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 &amp;amp; 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&#039;s Hematoxylin Solution (For Conventional Stain)</td><td>Beijing Solarbio Science &amp;amp; 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 &amp;amp; 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 &amp;amp; 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 &amp;amp; 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 &amp;amp; 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>&lt;strong&gt;Others&lt;/strong&gt;</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

393 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 

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Biochemistry

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

Behavior

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.

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  ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, 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 ...

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17&#946;-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, &#946;-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley ...

Medicine

Osteoporosis Management in Rats Through Lingnan Fire-Needle Therapy

Operation Procedure and Precautions of Lingnan Fire-Needle Therapy in Osteoporosis Model Rats

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected effects on some chronic diseases and incurable diseases. Osteoporosis (OP) has a high incidence in postmenopausal women and middle-aged and elderly men, and the treatment cycle is long. According to Traditional Chinese Medicine (TCM), Lingnan fire-needle therapy has shown potential in treating osteoporosis. However, there is still a long way to go before it can be widely used. This article focuses on the application of Lingnan fire-needle therapy in the intervention of OP in rats. It covers the selection of needle tools, acupuncture point selection, positioning of rats' bodies, and fixation methods. We also outline the steps and precautions to be taken during and after needling with fire needles. The experiment was done with three groups: a normal group, a model group, and a fire-needle group, each containing 10 rats. The rats in the fire-needle group were treated with fire-needle intervention for six sessions. After the intervention period, we collected femoral specimens and performed micro-CT scans. The results suggest that fire needling can enhance bone morphology and mineral density in OP rats. This information can serve as a methodological basis for conducting basic research on fire-needle therapy.

Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention

Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected ...

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Operation Procedure and Precautions of Lingnan Fire-Needle Therapy in Osteoporosis Model Rats