Topical Losartan Decreases Myofibroblast Generation But Not Corneal Opacity After Surface Blast-Simulating Irregular PTK in Rabbits.

Purpose: To evaluate the efficacy of topical losartan after blast injury-simulating irregular phototherapeutic keratectomy (PTK) in rabbits. Methods: Twelve NZW rabbits underwent 100 pulse 6.5 mm diameter PTK over a metal screen to generate severe surface irregularity and inhibit epithelial basement membrane regeneration. Corneas were treated with 0.8 mg/mL losartan in balanced salt solution (BSS) or BSS 50 µL six times per day for six weeks after PTK. All corneas had slit lamp photography, with and without 1% fluorescein at two, four, and six weeks after PTK, and were analyzed using immunohistochemistry for the myofibroblast marker α-smooth muscle actin (α-SMA), keratocyte marker keratocan, mesenchymal cell marker vimentin, transforming growth factor (TGF)-ß1, and collagen type IV. Results: Topical 0.8 mg/mL losartan six times a day significantly decreased anterior stromal α-SMA intensity units compared to BSS at six weeks after anterior stromal irregularity-inducing screened PTK (P = 0.009). Central corneal opacity, however, was not significantly different between the two groups. Keratocan, vimentin, TGF-ß1, or collagen type IV levels in the anterior stroma were not significantly different between the two groups. Conclusions: Topical losartan effectively decreased myofibroblast generation after surface blast simulation irregular PTK. However, these results suggest initial masking-smoothing PTK, along with adjuvant topical losartan therapy, may be needed to decrease corneal stromal opacity after traumatic injuries that produce severe surface irregularity. Translational Relevance: Topical losartan decreased scar-producing stromal myofibroblasts after irregular PTK over a metal screen but early smoothing of irregularity would also likely be needed to significantly decrease corneal opacity.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Olive leaf-derived PPAR agonist complex induces collagen IV synthesis in human skin models.

INTRODUCTION: Peroxisome proliferator-activated receptor (PPAR) agonists are known to modulate the synthesis of dermal lipids and proteins including collagens. Olive (Olea europaea) leaves have been reported to contain PPAR-binding ligands. Collagen IV, a major dermal-epidermal junction (DEJ) protein, degrades with both age and disease. Here, we report the formulation of a novel multi-ligand complex, Linefade, and its effects on collagen IV synthesis. METHODS: Linefade prepared from the leaves of Olea europaea contains 2% w/w plant extract solids dissolved in a mixture of glyceryl monoricinoleate and dimethyl isosorbide. In silico docking was performed with PPAR-α (PDB ID: 2P54). Linefade was evaluated for PPAR-α-dependent transcription in a luciferase reporter assay system. Cell viability and collagen IV levels in human dermal fibroblast cultures were measured using the MTT method and ELISA assay, respectively. Transcriptome analysis was conducted on a full-thickness reconstituted human skin (EpiDermFT) model. Ex vivo cell viability and collagen IV immunostaining were performed on human skin explants. RESULTS: In silico docking model of the major constituents (oleanolic acid and glyceryl monoricinoleate) produced a co-binding affinity of -6.7 Kcal/mole. Linefade significantly increased PPAR-α transcriptional activity in CHO cells and collagen IV synthesis in adult human dermal fibroblasts. Transcriptome analysis revealed that 1% Linefade modulated the expression of 280 genes with some related to epidermal differentiation, DEJ, PPAR, Nrf2 and retinoid pathways. An ex vivo human explant study showed that 1% Linefade, delivered via a triglycerides excipient, increased collagen IV levels along the dermal-epidermal junction by 52%. CONCLUSION: In silico modelling and in vitro and ex vivo analyses confirmed Linefade-mediated activation of PPAR-α and stimulation of collagen IV synthesis.

INTRODUCTION: Les agonistes du récepteur activé par les proliférateurs de peroxysomes (PPAR) sont connus pour moduler la synthèse des lipides cutanés et des protéines du derme, y compris des collagènes. Il a été signalé que les feuilles d'olivier (Olea europaea) contiennent des ligands de liaison aux PPAR. Le collagène IV, une protéine majeure de la jonction dermo-épidermique (DEJ), se dégrade avec l'âge et la maladie. Nous rapportons ici la formulation d'un nouveau complexe multi ligand, Linefade, et ses effets sur la synthèse du collagène IV. MÉTHODES: Le complexe Linefade préparé à partir des feuilles d'Olea europaea contient 2 % p/p de solides d'extraits végétaux dissous dans un mélange de monoricinoléate de glycéryle et d'isosorbide de diméthyle. Un docking in silico a été réalisé avec PPAR-α (PDB ID : 2P54). Linefade a été évalué pour la transcription dépendante du PPAR-α dans un système de test rapporteur à la luciférase. La viabilité cellulaire et les niveaux de collagène IV dans les cultures de fibroblastes dermiques humains ont été respectivement mesurés en utilisant la méthode MTT et le test ELISA. L'analyse du transcriptome a été réalisée sur un modèle de peau humaine reconstitué sur toute son épaisseur (EpiDermFT). La viabilité cellulaire ex vivo et l'immunomarquage du collagène IV ont été réalisés sur des explants de peau humaine. RÉSULTATS: Le modèle de docking in silico des principaux constituants (acide oléanolique et monoricinoléate de glycéryle) a produit une affinité de liaison conjointe de -6,7 Kcal/mole. Linefade a augmenté de manière significative l'activité transcriptionnelle du PPAR-α dans les cellules CHO et la synthèse du collagène IV dans les fibroblastes dermiques humains chez les personnes adultes. L'analyse du transcriptome a révélé que 1% de Linefade modulait l'expression de 280 gènes dont certains étaient liés à la différenciation épidermique, à la DEJ, au PPAR, à la voie Nrf2 et aux voies rétinoïdes. Une étude ex vivo sur des explants humains a montré que 1% de Linefade, délivré via un excipient de triglycérides, augmentait de 52% les niveaux de collagène IV le long de la jonction dermo-épidermique. CONCLUSION: La modélisation in silico et les analyses in vitro et ex vivo ont confirmé l'activation du PPAR-- α et la stimulation de la synthèse du collagène IV par Linefade.

Characterization of a New Full-Thickness In Vitro Skin Model.

Since 30 years, bioengineering allowed to reconstruct human tissues using normal human cells. Skin is one of the first organ to be reconstructed thanks to the development of specific cell culture media and supports favoring the culture of human skin cells, such as fibroblasts, keratinocytes, or melanocytes. Skin models have evolved from epidermis to complex models including a dermis. The purpose of the present study was to design a reconstructed full-thickness (FT) skin suitable to perform in vitro testing of both molecules and plant extracts. First, we reconstructed epidermis with normal human keratinocytes displaying the expected multilayered morphology and expressing specific epidermal proteins (e-cadherin, claudin-1, p63, Ki67, Keratin 10, filaggrin, and loricrin). Then, a dermal equivalent was developed using a collagen matrix allowing the growth of fibroblasts. The functionality of the dermis was demonstrated by the measurement of skin parameters such as rigidity or elasticity with Ballistometer® and other parameters such as the contraction over time and the expression of dermal proteins. The combination of these two compartments (dermis and epidermis) allowed to reconstruct an FT model. This study model allowed to study the communication between compartments and with the establishment of a dermoepidermal junction showing the expression of specific proteins (collagen XVII, laminin, and collagen IV). Impact statement The objective of our research project was to design a three-dimensional human full-thickness (FT) skin suitable to perform in vitro testing of molecules and plant ingredients. The combination of these two reconstructed compartments (dermis and epidermis) allowed to reconstruct an FT model. This study model allowed to study the communication between compartments and with the establishment of a dermoepidermal junction showing the expression of specific proteins (collagen XVII, laminin, and collagen IV). This in vitro model can be use by cosmetic and pharmaceutical industries to study the effect of chemical or natural compounds on the skin.

Curative effects of traditional Chinese medicine on liver fibrosis: A protocol for a systematic review and meta-analysis.

BACKGROUND: Assessing the effectiveness and safety of traditional Chinese medicine on liver fibrosis is the main purpose of this systematic review protocol. METHODS: The following electronic databases will be searched from their respective inception dates to 1st December 2021: PubMed, MEDLINE, the Cochrane Library, Embase, WorldSciNet, Ovid, the Allied and Complementary Medicine Database, the Web of Science, Chinese Biomedical Literature Database, China National Knowledge Infrastructure, the Chongqing VIP Chinese Science and Technology Periodical Database, the Wanfang Database, and the China Biology Medicine Disc. All published randomized controlled trials in English or Chinese related to curative effects of Traditional Chinese medicine on liver fibrosis will be included. The primary outcome is the levels of serum hyaluronic acid, laminin, type III procollagen, and type IV procollagen. There is no secondary outcomes. Two reviewers will conduct the study selection, data extraction, and assessment independently. The assessment of risk of bias and data synthesis will be conducted with Review Manager Software V.5.2. RESULTS: The results will provide a high-quality synthesis of current evidence for researchers in this subject area. CONCLUSION: The conclusion of our study will provide an evidence to judge whether traditional Chinese medicine is an effective intervention for patients with liver fibrosis. REGISTRATION NUMBER: INPLASY202110017.

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

Decreased Expression of Canstatin in Rat Model of Monocrotaline-Induced Pulmonary Arterial Hypertension: Protective Effect of Canstatin on Right Ventricular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease which causes right ventricular (RV) failure. Canstatin, a C-terminal fragment of type IV collagen α2 chain, is expressed in various rat organs. However, the expression level of canstatin in plasma and organs during PAH is still unclear. We aimed to clarify it and further investigated the protective effects of canstatin in a rat model of monocrotaline-induced PAH. Cardiac functions were assessed by echocardiography. Expression levels of canstatin in plasma and organs were evaluated by enzyme-linked immunosorbent assay and Western blotting, respectively. PAH was evaluated by catheterization. RV remodeling was evaluated by histological analyses. Real-time polymerase chain reaction was performed to evaluate RV remodeling-related genes. The plasma concentration of canstatin in PAH rats was decreased, which was correlated with a reduction in acceleration time/ejection time ratio and an increase in RV weight/body weight ratio. The protein expression of canstatin in RV, lung and kidney was decreased in PAH rats. While recombinant canstatin had no effect on PAH, it significantly improved RV remodeling, including hypertrophy and fibrosis, and prevented the increase in RV remodeling-related genes. We demonstrated that plasma canstatin is decreased in PAH rats and that administration of canstatin exerts cardioprotective effects.

Transcriptomic Analysis Reveals the Protection of Astragaloside IV against Diabetic Nephropathy by Modulating Inflammation.

BACKGROUND: Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease. Recently, there is no specific drug available to block the kidney damage. Astragaloside IV (AS-IV) is a major active component of Astragalus membranaceus (Fisch) Bge and has been demonstrated to benefit the kidney functions. This study explores the potential pharmacological action of AS-IV in DN of rats. METHODS: Male Sprague-Dawley rats were fed with high-fat diet and injected with streptozotocin to induce diabetes. The diabetic rats were randomized and treated with vehicle or AS-IV (80 mg/kg) daily by gavage for 12 weeks as the DN or AS-IV group, respectively. The normal control rats were fed with normal chow and injected with vehicles (n = 8 per group). These rats were monitored for diabetes- and kidney function-related measures. The expression profiles of gene mRNA transcripts in the kidney tissues were analyzed by RNA-seq and quantitative RT-PCR. The levels of advanced glycation end products (AGEs), IL-1ß, and IL-18 in the serum samples and kidney tissues were quantified by ELISA. The levels of collagen IV (COL-4) and fibronectin (FN) expression in kidney tissues were examined by immunohistochemistry and Western blot. RESULTS: In comparison with the DN group, AS-IV treatment significantly reduced blood glucose levels, food and water consumption, 24 h urine, renal index values, 24 h urine total proteins, blood urea nitrogen (BUN) levels, and creatinine clearance rates (CCR), accompanied by minimizing the DN-induced early kidney damages, fibrosis, and microstructural changes. Furthermore, AS-IV treatment significantly modulated the DN-altered gene transcription profiles in the kidney of rats, particularly for inflammation-related genes, including the nucleotide-binding oligomerization domain-like receptor signaling, which was validated by quantitative RT-PCR. AS-IV treatment significantly decreased the levels of serum and kidney AGEs, IL-1ß, and IL-18 expression and fibrosis indexes in the kidney of rats. CONCLUSION: AS-IV treatment ameliorated the severity of DN by inhibiting inflammation-related gene expression in the kidney of rats.

Olive oil supplementation prevents extracellular matrix deposition and reduces prooxidant markers and apoptosis in the offspring´s heart of diabetic rats.

Maternal diabetes induces fetal programming of cardiovascular diseases. Diabetes induced-cardiac fibrosis is a process that may start in utero and may be related to the prooxidant/proinflammatory environment. The aim of this study was to investigate the effect of a maternal diet enriched in olive oil on the levels of components and regulators of the extracellular matrix, on prooxidant markers and on apoptosis rate in the heart of 21-day-old offspring of diabetic rats. Maternal diabetes was induced by neonatal administration of streptozotocin. During pregnancy, diabetic and control rats were fed with diets supplemented or not with 6% olive oil. The heart of the offspring was studied at 21 days of age. We found increased deposition of collagen IV and fibronectin in the offspring´s heart of diabetic rats, which was prevented by the maternal diets enriched in olive oil. Increases in connective tissue growth factor were also prevented by the maternal diets enriched in olive oil. Prooxidant markers as well as apoptosis, which were increased in the heart of the offspring of diabetic rats, were prevented by the maternal olive oil dietary treatment. Our findings identified powerful effects of a maternal diet enriched in olive oil on the prevention of increased extracellular matrix deposition and increased prooxidant markers in the heart of 21-day-old offspring of diabetic rats.

Selenium-mediated gga-miR-29a-3p regulates LMH cell proliferation, invasion, and migration by targeting COL4A2.

Selenium (Se) is an essential trace element that has several functions in cellular processes related to cancer prevention. While the cancericidal effect of Se has been reported in liver cancer, the mechanism has not been clarified. MiR-29a has widely been reported as a tumor suppressor; however, it also acts as a carcinogenic agent by increasing cell invasion in human epithelial cancer cells and hepatoma cells. In a previous study, we found that miR-29a-3p is a Se-sensitive miRNA. However, its effect in the chicken hepatocellular carcinoma cell line (LMH) is still unknown. In the present study, we found that the expression of miR-29a-3p in LMH cells was decreased by Se supplementation and increased under Se-deficient conditions. Flow cytometry and CCK-8 results suggested that Se decreased LMH cell proliferation induced by miR-29a-3p overexpression. Transwell and gap-closure assays implied that Se mediated LMH cell invasion and migration by downregulating miR-29a-3p. Quantitative real-time polymerase chain reaction and Western blotting results suggested that Se mitigated miR-29a-3p overexpression-induced LMH cell proliferation by downregulating CDK2, cyclin-D1, CDK6, and cyclin-E1. We further demonstrated that collagen type IV alpha 2 (COL4A2) is a target gene of miR-29a-3p. COL4A2 activates the RhoA/ROCK pathway to promote LMH cell invasion and migration. In conclusion, Se mediated miR-29a-3p overexpression induced LMH cell invasion and migration by targeting COL4A2 to inactivate the RhoA/ROCK pathway.

Ferric citrate reduces fibroblast growth factor 23 levels and improves renal and cardiac function in a mouse model of chronic kidney disease.

Iron deficiency, anemia, hyperphosphatemia, and increased fibroblast growth factor 23 (FGF23) are common and interrelated complications of chronic kidney disease (CKD) that are linked to CKD progression, cardiovascular disease and death. Ferric citrate is an oral phosphate binder that decreases dietary phosphate absorption and serum FGF23 concentrations while increasing iron stores and hemoglobin in patients with CKD. Here we compared the effects of ferric citrate administration versus a mineral sufficient control diet using the Col4a3 knockout mouse model of progressive CKD and age-matched wild-type mice. Ferric citrate was given to knockout mice for four weeks beginning at six weeks of age when they had overt CKD, or for six weeks beginning at four weeks of age when they had early CKD. Ten-week-old knockout mice on the control diet showed overt iron deficiency, anemia, hyperphosphatemia, increased serum FGF23, hypertension, decreased kidney function, and left ventricular systolic dysfunction. Ferric citrate rescued iron deficiency and anemia in knockout mice regardless of the timing of treatment initiation. Circulating levels and bone expression of FGF23 were reduced in knockout mice given ferric citrate with more pronounced reductions observed when ferric citrate was initiated in early CKD. Ferric citrate decreased serum phosphate only when it was initiated in early CKD. While ferric citrate mitigated systolic dysfunction in knockout mice regardless of timing of treatment initiation, early initiation of ferric citrate also reduced renal fibrosis and proteinuria, improved kidney function, and prolonged life span. Thus, initiation of ferric citrate treatment early in the course of murine CKD lowered FGF23, slowed CKD progression, improved cardiac function and significantly improved survival.

Ameliorative effect of zinc supplementation on compromised small intestinal health in streptozotocin-induced diabetic rats.

Zinc depletion during diabetes postulates a role for zinc nutrition in the management of associated complications. The present study evaluated zinc supplementation for countering the compromised intestinal integrity through moderation of oxidative stress and suppression of stress-stimulated inflammatory proliferation in streptozotocin-induced diabetic rats. Diabetic rats were provided with supplemental zinc for six weeks (5 and 10-times of normal level). Supplemental zinc nurtured diabetic groups evidenced a significant reversal of the disruption of intestinal ultra structure. While the brush border membrane (BBM) of diabetic animals showed decreased fluidity with increased cholesterol: phospholipid ratio and altered polyunsaturated to saturated fatty acid ratio, the same was countered in zinc supplementation. A stimulated activity of BBM-bound enzymes suggested a modulation in membrane dynamics in diabetic condition which was moderated in zinc treatment. Higher expression of the lipid oxidative markers, oxidative stress markers, concomitant inflammatory markers, cytokines, fibrosis factors and apoptotic regulatory proteins in the intestines were curbed by zinc supplementation. The pathological aberrations of the intestinal architecture in diabetic animals were similarly reverted. Thus, supplemental zinc has a favourable consequence in restricting the compromised intestinal health in diabetes which was exerted through a defensive stimulus on oxidative stress induced cytokines, inflammatory propagation, and subsequent injury.

Dianthus superbus Improves Glomerular Fibrosis and Renal Dysfunction in Diabetic Nephropathy Model.

Glomerular fibrosis is caused by an accumulation of intercellular spaces containing mesangial matrix proteins through either diffused or nodular changes. Dianthus superbus has been used in traditional medicine as a diuretic, a contraceptive, and an anti-inflammatory agent. The aim of this study was to investigate the effects of Dianthus superbus-EtOAc soluble fraction (DS-EA) on glomerular fibrosis and renal dysfunction, which has been implicated in diabetic nephropathy in human renal mesangial cells and db/db mice. DS-EA was administered to db/db mice at 10 or 50 mg/kg/day for 8 weeks. DS-EA treatment significantly ameliorated blood glucose, insulin, the homeostasis model assessment of insulin resistance (HOMA-IR) index, and HbA1c in diabetic mice. DS-EA decreased albumin excretion, creatinine clearance (Ccr), and plasma creatinine levels. DS-EA also ameliorated the levels of kidney injury molecules-1 (KIM-1) and C-reactive protein. DS-EA reduced the periodic acid-Schiff (PAS) staining intensity and basement membrane thickening in glomeruli of the diabetic nephropathy model. In addition, DS-EA suppressed transforming growth factor-ß (TGF-ß)/Smad signaling. Collagen type IV, a glomerular fibrosis biomarker, was significantly decreased upon DS-EA administration. DS-EA pretreatment attenuated levels of inflammation factors such as intracellular cell adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1). DS-EA inhibited the translocation of nuclear factor kappa B (NF-κB) in Angiotensin II (Ang II)-stimulated mesangial cells. These findings suggest that DS-EA has a protective effect against renal inflammation and fibrosis. Therefore, DS-EA may serve as a potential therapeutic agent targeting glomerulonephritis and glomerulosclerosis, which lead to diabetic nephropathy.

Lonicera japonica Attenuates Carbon Tetrachloride-Induced Liver Fibrosis in Mice: Molecular Mechanisms of Action.

Liver fibrosis is a worldwide clinical issue that generally causes hepatic cirrhosis. Lonicerae Japonicae Flos (dried flower buds of Lonicera japonica Thunb) is a traditional heat-clearing and detoxifying herbal medicine in China. This study aims to observe the protection of the water extract of Lonicerae Japonicae Flos (FL) from carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Liver fibrosis was induced in mice by intraperitoneal injection of 2 ml/kg CCl4 twice a week for 4 weeks. FL's attenuation of CCl4-induced liver fibrosis in mice was evidenced by the results of Masson's trichrome and Sirius red staining, liver hydroxyproline content and serum amount of collagen IV. FL reduced hepatic stellate cells (HSCs) activation and reversed the epithelial-mesenchymal transition (EMT) process in mice treated with CCl4. FL also alleviated liver oxidative stress injury and enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) anti-oxidant signaling pathway in mice treated with CCl4. Additionally, the main phenolic acids in FL including chlorogenic acid (CGA) and caffeic acid (CA) both reduced HSCs activation in vitro. In summary, FL attenuates CCl4-induced liver fibrosis in mice by inhibiting HSCs activation, reversing EMT and reducing liver oxidative stress injury via inducing Nrf2 activation. CGA may be the main active compound contributing to the antifibrotic activity of FL.

Astragaloside IV ameliorates high glucose­induced renal tubular epithelial­mesenchymal transition by blocking mTORC1/p70S6K signaling in HK­2 cells.

Astragaloside IV (AST) is the major active saponin in Astragalus membranaceus and, reportedly, has a variety of pharmacological activities. However, the potential of AST to ameliorate high glucose­mediated renal tubular epithelial­mesenchymal transition (EMT) remains undetermined. The aim of the present research was to explore the effect and mechanism of AST in EMT of renal tubular epithelial cells, as an underlying mechanism of renal fibrosis and a vital feature involved in diabetic nephropathy. The effect of AST on the EMT of renal tubular epithelial cells (HK­2) stimulated by high glucose was investigated and it was attempted to elucidate the potential underlying mechanism. The expression of E­cadherin and α­smooth muscle actin were determined by western blotting and immunofluorescence assays. The expression of the mammalian target of rapamycin complex 1 (mTORC1)/ ribosomal protein S6 kinase ß­1 (p70S6K) signaling pathway and protein levels of four transcriptional factors (snail, slug, twist and zinc finger E­box­binding homeobox 1) were also determined by western blotting. Additionally, extracellular matrix components, including fibronectin (FN) and collagen type IV (Col IV) were detected by ELISA. The results suggested that the EMT of HK­2 cells and the mTORC1/p70S6K pathway were activated by high glucose. The expression of snail and twist in HK­2 cells was elevated by high glucose. Furthermore, extracellular matrix components, FN and Col IV, were increased in HK­2 cells cultured with high glucose. In turn, treatment with AST reduced EMT features in HK­2 cells, inhibited mTORC1/p70S6K pathway activation, downregulated expression of snail and twist, and reduced secretion of FN and Col IV. In summary, the findings suggested that AST ameliorates high glucose­mediated renal tubular EMT by blocking the mTORC1/p70S6K signaling pathway in HK­2 cells.

Effects of Resveratrol on the Renin-Angiotensin System in the Aging Kidney.

The renin-angiotensin system (RAS), especially the angiotensin II (Ang II)/angiotensin II type 1 receptor (AT1R) axis, plays an important role in the aging process of the kidney, through increased tissue reactive oxygen species production and progressively increased oxidative stress. In contrast, the angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axis, which counteracts the effects of Ang II, is protective for end-organ damage. To evaluate the ability of resveratrol (RSV) to modulate the RAS in aging kidneys, eighteen-month-old male C57BL/6 mice were divided into two groups that received either normal mouse chow or chow containing resveratrol, for six months. Renal expressions of RAS components, as well as pro- and antioxidant enzymes, were measured and mouse kidneys were isolated for histopathology. Resveratrol-treated mice demonstrated better renal function and reduced albuminuria, with improved renal histologic findings. Resveratrol suppressed the Ang II/AT1R axis and enhanced the AT2R/Ang 1-7/MasR axis. Additionally, the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, 8-hydroxy-2'-deoxyguanosine, 3-nitrotyrosine, collagen IV, and fibronectin was decreased, while the expression of endothelial nitric oxide synthase and superoxide dismutase 2 was increased by resveratrol treatment. These findings demonstrate that resveratrol exerts protective effects on aging kidneys by reducing oxidative stress, inflammation, and fibrosis, through Ang II suppression and MasR activation.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Disrupting interaction of PSD-95 with nNOS attenuates hemorrhage-induced thalamic pain.

Hemorrhages occurring within the thalamus lead to a pain syndrome. Clinical treatment of thalamic pain is ineffective, at least in part, due to the elusive mechanisms that underlie the induction and maintenance of thalamic pain. The present study investigated the possible contribution of a protein-protein interaction between postsynaptic density protein 95 (PSD-95) and neuronal nitric oxide synthase (nNOS) to thalamic pain in mice. Thalamic hemorrhage was induced by microinjection of type IV collagenase into unilateral ventral posterior medial/lateral nuclei of the thalamus. Pain hypersensitivities, including mechanical allodynia, heat hyperalgesia, and cold allodynia, appeared at day 1 post-microinjection, reached a peak 5-7 days post-microinjection, and persisted for at least 28 days post-microinjection on the contralateral side. Systemic pre-treatment (but not post-treatment) of ZL006, a small molecule that disrupts PSD-95-nNOS interaction, alleviated these pain hypersensitivities. This effect is dose-dependent. Mechanistically, ZL006 blocked the hemorrhage-induced increase of binding of PSD-95 with nNOS and membrane translocation of nNOS in thalamic neurons. Our findings suggest that the protein-protein interaction between PSD-95 and nNOS in the thalamus plays a significant role in the induction of thalamic pain. This interaction may be a promising therapeutic target in the clinical management of hemorrhage-induced thalamic pain.

[Effects of centellaasiatica granule on the expression of Smad 2/3, Smad 7 and collagen â £ in the mesangial cells stably expressed TGF-ß1].

OBJECTIVES: Stably expressed transforming growth factor -beta 1(TGF-ß1)MCs were obtained and the effects of centellaasiatica (CA) granule on the expressions of Smad 2/3, Smad 7 and collagen Ⅳ and the level of Smad 2/3 phosphorylation were observed. METHODS: Lipofectin method was used to transfect TGF-ß1 vector into MC, and the stably expressed TGF-ß1 cell lines were selected by G418. The cells were divided into three groups. Control group:normal MC + RPMI 1640 + 10% normal rat serum; TGF-ß1 group:stably expressed TGF-ß1 MC + RPMI 1640 + 10% normal rat serum; CA group:stably expressed TGF-ß1 MC + RPMI 1640 + 10% rat serum containing high CA. The experiments were repeated for five times. The contents of TGF-ß1 and collagen Ⅳ in the culture medium were detected with ELISA, the expressions of mRNA and protein of TGF-ß1, Smad 2/3, Smad 7 and the level of Smad 2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot. RESULTS: The contents of TGF-ß1 and collagen Ⅳ in the culture medium of stably-expressed TGF-ß1 MC were increased significantly, and the CA could reverse the effects of TGF-ß1. The expressions of mRNA and protein of TGF-ß1, Smad 2/3 and the level of Smad 2/3 phosphorylation were increased significantly in TGF-ß1 transfected MC, and CA could dramatically reduce the expressions of mRNA and protein of TGF-ß1, Smad 2/3 and the level of Smad 2/3 phosphorylation. The high expression of TGF-ß1 decreased the expression of Smad 7 mRNA and protein, and the CA could antagonize the effect of mRNA expression. CONCLUSIONS: The MCs stably-expressed TGF-ß1 can activate the TGF-ß1/Smad signal pathway and increase the expression of collagen Ⅳ. CA can decrease the occurrence of diabetic nephropathy(DN) by reducing the production of collagen Ⅳ through inhibiting the TGF-ß1/Smad signal pathway.