This study was supported by the general project of the Natural Science Foundation of Hebei Province, China (No. H2019423037).

Jiawei Shengjiang San to Address Diabetic Nephropathy Using Network Pharmacology

The authors have nothing to disclose.

Our study employed a combination of network pharmacology, molecular docking, and in vivo animal models. A critical step was the establishment of the &#34;drug-component-target&#34; network, which was crucial for identifying the potential mechanisms of JWSJS in treating DN, focusing particularly on its interaction with the EGFR/MAPK3/1 signaling pathway.
During this study, we made several modifications, particularly in the molecular docking process, to enhance the accuracy of our predi...

Diabetes mellitus (DM) is a chronic disease that affects multiple systems and can cause various complications due to continuous hyperglycemia, such as diabetic nephropathy (DN), retinopathy, and neuropathy1. DN is a serious complication of DM, accounting for about 30%-50% of end-stage renal disease (ESRD)2. Its clinical manifestation is microalbuminuria, which can progress to ESRD characterized by increased glomerular volume, mesangial stromal hyperplasia, and thickened glomerular basement membrane3. The pathogenesis of DN is complex and has not been fully elucidated. Clinical methods such as lowering blood glucose, regulating blood pressure, and reducing proteinuria are mostly used to delay its progress, but the effect is general. 
Currently, no specific drug has been found to treat DN4. For centuries, however, Chinese herbal medicines have been widely used in treating DM and its complications5 and have improved patients' clinical symptoms and delayed disease progression. Due to the advantages of multi-component, multi-target, and multi-pathway effects, Chinese herbal medicines are expected to be an innovative drug source for the treatment of DN6.
"Shengjiang san" originated from the "Wanbing Huichun" by the Ming Dynasty medical doctor Gong Tingxian. The book "Neifu Xianfang" describes the use of Bombyx Batryticatus, Cicadae Periostracum, Curcumaelongae Rhizoma, and Radix Rhei et Rhizome. Based on this, after adding Hedysarum Multijugum Maxim, Epimrdii Herba, and Smilacis Glabrae Rhixoma, it exerts the function of shengjiang san of increasing lucidity, decreasing turbidity, releasing stagnant "heat," and harmonizing "qi" and the blood7,8. It also increases the effect of strengthening the spleen and tonifying the kidneys. Its efficacy is consistent with the pathogenesis of DN's "qi" to rise and fall out of order due to deficiency of "vital energy," excessive dryness and "heat," and stagnation of "heat" caused by a triple energizer7,8.
Previous clinical studies have shown Chinese herbal medicines have been used to treat DM and its complications, and jiawei shengjiang san (JWSJS) has been shown to regulate blood glucose and lipids, reduce proteinuria, and significantly improve the clinical efficacy of patients with early DN7. The ability of JWSJS to reduce urinary protein and blood glucose levels in DN rats has been confirmed by previous studies. This probably happens by inhibiting the TXNIP/NLRP3 and RIP1/RIP3/MLKL signaling pathways, reducing podocyte pyroptosis, and preventing necrotic apoptosis in renal tissues of DN rats, thus achieving renal protection9. JWSJS can upregulate nephrin and podocin protein expression and reduce podocyte injury in DN rats, thus suggesting that JWSJS has an inhibitory effect on podocyte injury. JWSJS has a certain anti-DN effect with good safety profiles, but there is little research on it, and this work mostly focuses on pyroptosis and necrotic apoptosis. The literature is not sufficiently deep or systematic10. Our previous findings have confirmed that JWSJS can reduce proteinuria and alleviate kidney damage in DN rats7. However, there are only a few studies on the mechanism of JWSJS for DN treatment, and these lack depth and systematization. Thus, this study aims to analyze the molecular substances and mechanisms of action of JWSJS for DN treatment using network pharmacology and provide a solid foundation for future research.
Network pharmacology is an emerging method to study the mechanism of drug action, including cheminformatics, network biology, bioinformatics, and pharmacology11,12. Network pharmacology research design is quite similar to the holistic concept of traditional Chinese medicine13,14, and it is an important method to study the mechanism of Chinese herbal medicines. Molecular docking can study interactions between molecules and predict their binding patterns and affinity. Molecular docking has emerged as a critical technique in the field of computer-aided drug research15. Therefore, this study constructed a JWSJS-DN-target interaction network through network pharmacology and molecular docking methods that offers a reliable and theoretical basis for further exploration of DN treatment with JWSJS.

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		<tr>
			<td>2&#215;SYBR Green qPCR Master Mix&#160;</td>
			<td>Servicebio, Wuhan, China</td>
			<td>G3320-05</td>
			<td></td>
		</tr>
		<tr>
			<td>24-h urine protein quantification (UTP)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;-Diaminobenzidine</td>
			<td>Shanghai Huzheng Biotech, China</td>
			<td>91-95-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic biochemical analysis instrument</td>
			<td>Hitachi, Japan</td>
			<td>7170A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous Ethanol</td>
			<td>Biosharp, Tianjin, China</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BAX Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF0120</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>BCL-2 Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF6139</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>BX53 microscope</td>
			<td>Olympus, Japan</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform Substitute</td>
			<td>ECOTOP, Guangzhou, China</td>
			<td>ES-8522</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmond software&#160;</td>
			<td>New York, NY, USA</td>
			<td>Release 2019-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Constant Temperature Water Bath</td>
			<td>Changzhou Jintan Liangyou Instrument, China</td>
			<td>DK-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFR Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF6043</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Embed-812 RESIN</td>
			<td>Shell Chemical, USA</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fasting blood glucose (FBG)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>FC-type full-wavelength enzyme label analyser</td>
			<td>Multiskan; Thermo, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH&#160; Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF7021</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Glycated serum protein (GSP)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi, Japan</td>
			<td>H-7650</td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin/eosin (HE) staining solution</td>
			<td>Servicebio, USA</td>
			<td>G1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-Pro Plus</td>
			<td>MEDIA CYBERNETICS, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-Time PCR Amplification Instrument</td>
			<td>Applied Biosystems, USA</td>
			<td>iQ5&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Irbesartan tablets</td>
			<td>Hangzhou Sanofi Pharmaceuticals</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Biosharp, Tianjin, China</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;JWSJS granules</td>
			<td>Guangdong Yifang Pharmaceutical</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Kodak Image Station 2000 MM imaging system</td>
			<td>Kodak, USA</td>
			<td>IS2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-density cholesterol (LDL-C)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MAPK3/1Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF0155</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Medical Centrifuge</td>
			<td>Hunan Xiangyi Laboratory Instrument Development, China&#160;</td>
			<td>TGL-16K</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini trans-blot transfer system</td>
			<td>Bio-Rad, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN electrophoresis system</td>
			<td>Bio-Rad, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoVue Plus Spectrophotometer</td>
			<td>Healthcare Bio-Sciences AB, Sweden</td>
			<td>111765</td>
			<td></td>
		</tr>
		<tr>
			<td>p-EGFR Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF3044</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Periodic acid-Schiff (PAS) staining solution</td>
			<td>Servicebio, USA</td>
			<td>G1008</td>
			<td></td>
		</tr>
		<tr>
			<td>p-MAPK3/1 Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF1015</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td>&#160;Santa Cruz, USA</td>
			<td>sc-2357</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Streptozotocin</td>
			<td>Sigma, USA</td>
			<td>S0130</td>
			<td></td>
		</tr>
		<tr>
			<td>SureScript First-Strand cDNA Synthesis Kit</td>
			<td>GeneCopeia, USA</td>
			<td>QP056T</td>
			<td></td>
		</tr>
		<tr>
			<td>TriQuick Reagent</td>
			<td>Solarbio, Beijing, China</td>
			<td>R1100</td>
			<td></td>
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		<tr>
			<td>Ultra-Clean Workbench</td>
			<td>Suzhou Purification Equipment, China</td>
			<td>SW-CJ-1F&#160;</td>
			<td></td>
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antagonistic effect of jiawei shengjiang san on a rat model of diabetic nephropathy: related to egfr/mapk3/1 signaling pathway

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network pharmacology. Employing network pharmacology and molecular docking techniques, we predicted the active components and targets of JWSJS and constructed a meticulous "drug-component-target" network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were utilized to discern the therapeutic pathways and targets of JWSJS. Autodock Vina 1.2.0 was deployed for molecular docking verification, and a 100-ns molecular dynamics simulation was conducted to affirm the docking results, followed by in vivo animal verification. The findings revealed that JWSJS shared 227 intersecting targets with diabetic nephropathy, constructing a protein-protein interaction network topology. KEGG enrichment analysis denoted that JWSJS mitigates diabetic nephropathy by modulating lipids and atherosclerosis, the PI3K-Akt signaling pathway, apoptosis, and the HIF-1 signaling pathway, with mitogen-activated protein kinase 1 (MAPK1), MAPK3, epidermal growth factor receptor (EGFR), and serine/threonine-protein kinase 1 (AKT1) identified as collective targets of multiple pathways. Molecular docking asserted that the core components of JWSJS (quercetin, palmitoleic acid, and luteolin) could stabilize conformation with three pivotal targets (MAPK1, MAPK3, and EGFR) through hydrogen bonding. In vivo examinations indicated notable augmentation in body weight and reductions in glycated serum protein (GSP), low-density lipoprotein cholesterol (LDL-C), uridine triphosphate (UTP), and fasting blood glucose (FBG) levels due to JWSJS. Electron microscopy coupled with hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) staining highlighted the potential of each treatment group in alleviating kidney damage to diverse extents, exhibiting varied declines in p-EGFR, p-MAPK3/1, and BAX, and increments in BCL-2 expression in the kidney tissues of the treated rats. Conclusively, these insights suggest that the protective efficacy of JWSJS on diabetic nephropathy might be associated with suppressing the activation of the EGFR/MAPK3/1 signaling pathway and alleviating renal cell apoptosis.

Following the protocol, 90 active ingredients of JWSJS were finally obtained from the analysis after screening and deduplication according to the set standards of OB and DL. These included 20 kinds of Hedysarum Multijugum Maxim, 23 kinds of Epimrdii Herba, 15 kinds of Smilacis Glabrae Rhixoma, 16 kinds of Radix Rhei et Rhizome, four kinds of Curcumaelongae Rhizoma, 15 kinds of Cicadae Periostracum, and six kinds of Bombyx Batryticatus components. Because ther...

Read this Scientific Article Protocol about Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway at JoVE.com

Author Spotlight: Network Pharmacology and Molecular Docking to Decipher the Action of Jiawei Shengjiang San Against Diabetic Kidney Disease

Here, we present a protocol describing network pharmacology and molecular docking techniques to explore the mechanism of action of Jiawei Shengjiang San (JWSJS) in treating diabetic nephropathy.

Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network ...

antagonistic-effect-jiawei-shengjiang-san-on-rat-model-diabetic

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468 Views.  Hebei University of Chinese Medicine. Here, we present a protocol describing network pharmacology and molecular docking techniques to explore the mechanism of action of Jiawei Shengjiang San (JWSJS) in treating diabetic nephropathy.

Video: Author Spotlight: Network Pharmacology and Molecular Docking to Decipher the Action of Jiawei Shengjiang San Against Diabetic Kidney Disease

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...