Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

OBJECTIVE: The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. METHODS: Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. RESULTS: The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. CONCLUSION: Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Exploring the synergic mechanism of Ligusticum striatum DC. and borneol in attenuating BMECs injury and maintaining tight junctions against cerebral ischaemia based on the HIF-1α/VEGF signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum striatum DC., also known as Ligusticum chuanxiong Hort. (LCH), is widely used in China for its excellent effect in ischaemic stroke (IS) patients, and borneol (BO) has been confirmed to maintain the blood‒brain barrier (BBB) after stroke. They are often used as a combination in the prescriptions of IS patients. Although the advantage of their combined treatment in improving brain ischaemia has been verified, their synergistic mechanism on BBB maintenance is still unclear. AIM OF THE STUDY: This study was designed to evaluate the synergistic effect of maintaining the BBB between LCH and BO against IS and to further explore the potential mechanism. MATERIALS AND METHODS: After primary mouse brain microvascular endothelial cells (BMECs) were extracted and identified, the duration of oxygen-glucose deprivation (OGD) and the doses of LCH and BO were optimized. Then, the cells were divided into five groups: control, model, LCH, BO, and LCH + BO. Cell viability, injury degree, proliferation and migration were detected by CCK-8, LDH, EdU and wound-healing assays, respectively. Hoechst 33342 staining was adopted to detect the apoptosis rate, and western blotting was employed to observe the expressions of Bax, Bcl-2, caspase-3 and cleaved caspase-3. The TEER value and NaF permeability were measured to assess tight junction (TJ) function, while ZO-1, occludin and claudin-5 were also probed by western blotting. Moreover, the HIF-1α/VEGF pathway was observed to explore the underlying mechanism of BBB maintenance. In vivo, global cerebral ischaemia/reperfusion (GCIR) surgery was performed to establish an IS model. After treatment with LCH (200 mg/kg) and/or BO (160 mg/kg), histopathological structure and BMECs repair were observed by HE staining and immunohistochemistry of vWF. Meanwhile, TJ-associated proteins in vivo were also detected by western blotting. RESULTS: Basically, LCH and BO had different emphases. LCH significantly attenuated the vacuolar structure, nuclear pyknosis and neuronal loss of GCIR mice, while BO focused on promoting BMECs proliferation and angiogenesis and inhibiting the degradation of TJ-associated proteins in vivo after IS. Interestingly, their combination further enhanced these effects. OGD injury markedly reduced the viability, proliferation and migration of primary BMECs; decreased the ratio of Bcl-2/Bax, TEER value, and the expressions of ZO-1, occludin and claudin-5; induced LDH release and apoptosis; and increased the cleaved caspase-3/caspase-3 ratio and NaF permeability. Meanwhile, BO might be the main contributor to the combinative treatment in ameliorating OGD-induced damage of BMECs and degradation of TJ-related proteins, and the potential mechanism might be involved in upregulating the HIF-1α/VEGF signalling pathway. Although LCH showed no obvious improvement, it could enhance the therapeutic effect of BO. Interestingly, their combination even produced some new improvements, including the reduction of cleaved caspase-3 and increase in TEER value, none of which were exhibited in their monotherapies. CONCLUSIONS: LCH and BO exhibited complementary therapeutic features in alleviating cerebral ischaemic injury by inhibiting BMECs apoptosis, maintaining the BBB and attenuating the loss of neurons. LCH preferred to protect ischaemic neurons, while BO played a key role in protecting BMECs, maintaining the BBB and TJs by activating the HIF-1α/VEGF signalling pathway.

Catalpol ameliorates endothelial dysfunction and inflammation in diabetic nephropathy via suppression of RAGE/RhoA/ROCK signaling pathway.

Catalpol is an iridoid glycoside compound isolated from the root of Rehmannia glutinosa, which has been reported to be a promising candidate for the treatment of diabetic diseases. The present study aimed at investigating the effects and potential mechanism of catalpol on endothelial dysfunction and inflammation in diabetic nephropathy (DN). We constructed DN mice and advanced glycation end products (AGEs)-induced mouse glomerular endothelial cells (mGECs) injury model. The results demonstrated that catalpol effectively improved renal pathology and decreased levels of urine protein, serum creatinine, and blood urea nitrogen in DN mice. Catalpol significantly reduced endothelial dysfunction and inflammatory infiltration of macrophages in DN mice and AGEs-induced mGECs. To further study the protective mechanism of catalpol, we transfected DN mice with recombinant adeno-associated virus expressing receptor of AGEs (RAGE) and intervened AGEs-induced mGECs with inhibitors. Catalpol reversed endothelial dysfunction and inflammation aggravated by RAGE overexpression in DN mice. Meanwhile, catalpol significantly inhibited the RAGE/Ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) pathway in DN mice with RAGE overexpression. Moreover, the combination of catalpol with inhibitors of RAGE, RhoA and ROCK exerted stronger anti-endothelial dysfunction and anti-macrophage infiltration effects on AGEs-induced mGECs compared with catalpol alone. In short, this study indicated that catalpol could ameliorate endothelial dysfunction and inflammation via suppression of RAGE/RhoA/ROCK pathway, hereby delaying the progression of DN.

Synergic Neuroprotection Between Ligusticum Chuanxiong Hort and Borneol Against Ischemic Stroke by Neurogenesis via Modulating Reactive Astrogliosis and Maintaining the Blood-Brain Barrier.

Background: Ligusticum chuanxiong Hort (LCH) is a famous ethnomedicine in Asia known for its excellent output on stroke treatment, and borneol usually acts as an assistant for its reducing permeability of the blood-brain barrier (BBB) after stroke. Although their synergy against brain ischemia was verified in previous studies, the potential mechanism is still unknown. Methods: The research aimed to explore the exact synergic mechanisms between LCH and borneol on neurogenesis within the areas of the dentate gyrus and subventricular zone. After treating middle cerebral artery occlusion rats with LCH (0.1 g/kg) and/or borneol (0.08 g/kg), the neurological severity score, brain infarct ratio, Nissl staining, Evans blue permeability, BBB ultrastructure, and expressions of von Willebrand factor and tight junction-associated proteins were measured. Co-localizations of Nestin+/BrdU+ and doublecortin+/BrdU+, and expressions of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) were observed under a fluorescence microscope. Moreover, astrocyte polarization markers of complement component 3 and pentraxin 3, and relevant neurotrophins were also detected by immunoblotting. Results: Basically, LCH and borneol had different focuses, although both of them decreased infarct areas, and increased quantity of Nissl bodies and expression of brain-derived neurotrophic factor. LCH increased the neurological severity score, NeuN+ cells, and the ratios of Nestin+/BrdU+ and doublecortin+/BrdU+, and decreased GFAP+ cells and ciliary neurotrophic factor expression. Additionally, it regulated the expressions of complement component 3 and pentraxin 3 to transform astrocyte phenotypes. Borneol improved BBB ultrastructure and increased the expressions of von Willebrand factor, tight junction-associated proteins, vascular endothelial growth factor, and vascular endothelial growth factor receptor 2. Unexpectedly, their combined therapy showed more obvious regulations on the Nissl score, Evans blue permeability, doublecortin+/BrdU+, NeuN+ cells, brain-derived neurotrophic factor, and vascular endothelial growth factor than both of their monotherapies. Conclusions: The results indicated that LCH and borneol were complementary to each other in attenuating brain ischemia by and large. LCH mainly promoted neural stem cell proliferation, neurogenesis, and mature neuron preservation, which was probably related to the transformation of reactive astrocytes from A1 subtype to A2, while borneol preferred to maintain the integrity of the BBB, which provided neurogenesis with a homeostatic environment.

Catalpol ameliorates diabetes-induced testicular injury and modulates gut microbiota.

AIMS: To explore the mechanisms of diabetes mellitus (DM)-induced testicular injury caused by modulation of testicular glycolysis and gut microbiota (GM), and evaluation of the efficacy of catalpol in reversing testicular morbidity. MAIN METHODS: A model of DM-induced testicular injury was established using a high-fat diet in KK-Ay mice. Microbial communities in the feces of mice in normal, model and catalpol (Cat) groups were analyzed by 16S gene sequencing. Correlations between the GM and lactate metabolism levels, lactate dehydrogenase activity, and indicators of testicular injury were analyzed. KEY FINDINGS: Cat significantly reduced general indicators of diabetes in mice with DM-induced reproductive injury, mitigated damage to the testicular tissue, and increased sperm count and motility. Additionally, the levels of products of glycolysis metabolism (e.g. lactate) increased following Cat treatment compared with the Model group. Disorders in the GM were also reversed in the Cat group. SIGNIFICANCE: Cat ameliorated DM-induced testicular injury in KK-Ay mice by increasing the energy available to germ cells through glycolysis, principally through modulation of the GM and a reduction in the quantities of associated pathogenic bacteria.

Loganin alleviates macrophage infiltration and activation by inhibiting the MCP-1/CCR2 axis in diabetic nephropathy.

BACKGROUND/AIMS: The theory of inflammation is one of the important theories in the pathogenesis of diabetic nephropathy (DN). We herein aimed to explore whether loganin affected macrophage infiltration and activation upon diabetic nephropathy (DN) by a spontaneous DN mice and a co-culture system of glomerular mesangial cells (GMCs) and macrophage cells (RAW264.7) which was induced by advanced glycation end products (AGEs). METHODS AND KEY FINDINGS: Loganin showed remarkable capacity on protecting renal from damage by mitigating diabetic symptoms, improving the histomorphology of the kidney, decreasing the expression of extracellular matrix such as FN, COL-IV and TGF-ß, reversing the production of IL-12 and IL-10 and decreasing the number of infiltrating macrophages in the kidney. Moreover, loganin showed markedly effects by suppressing iNOS and CD16/32 expressions (M1 markers), increasing Arg-1 and CD206 expressions (M2 markers), which were the phenotypic transformation of macrophage. These effects may be attributed to the inhibition of the receptor for AGEs (RAGE) /monocyte chemotactic protein-1 (MCP-1)/CC chemokine receptor 2 (CCR2) signaling pathway, with significantly down-regulated expressions of RAGE, MCP-1 and CCR2 by loganin. Loganin further decreased MCP-1 secretion when RAGE was silenced, which means other target was involved in regulating the MCP-1 expression. While loganin combinated with the inhibitor of CCR2 exerted stronger anti-inhibition effects of iNOS expression, suggesting that CCR2 was the target of loganin in regulating the activation of macrophages. SIGNIFICANCE: Loganin could ameliorate DN kidney damage by inhibiting macrophage infiltration and activation via the MCP-1/CCR2 signaling pathway in DN.

Radix Rehmanniae and Corni Fructus against Diabetic Nephropathy via AGE-RAGE Signaling Pathway.

BACKGROUND AND AIMS: Radix Rehmanniae and Corni Fructus (RC) have been widely applied to treat diabetic nephropathy (DN) for centuries. But the mechanism of how RC plays the therapeutic role against DN is unclear as yet. METHODS: The information about RC was obtained from a public database. The active compounds of RC were screened by oral bioavailability (OB) and drug-likeness (DL). Gene ontology (GO) analysis was performed to realize the key targets of RC, and an active compound-potential target network was created. The therapeutic effects of RC active compounds and their key signal pathways were preliminarily probed via network pharmacology analysis and animal experiments. RESULTS: In this study, 29 active compounds from RC and 64 key targets related to DN were collected using the network pharmacology method. The pathway enrichment analysis showed that RC regulated advanced glycosylation end product (AGE-) RAGE and IL-17 signaling pathways to treat DN. The animal experiments revealed that RC significantly improved metabolic parameters, inflammation renal structure, and function to protect the kidney against DN. CONCLUSIONS: The results revealed the relationship between multicomponents and multitargets of RC. The administratiom of RC might remit the DM-induced renal damage through the AGE-RAGE signaling pathway to improve metabolic parameters and protect renal structure and function.

Protective effects of catalpol on diabetes mellitus-induced male reproductive damage via suppression of the AGEs/RAGE/Nox4 signaling pathway.

AIMS: Diabetes mellitus (DM)-induced reproductive damage is an important cause of infertility for male DM patients, we herein evaluated the effects of catalpol on diabetic reproductive damage through the suppression of the AGEs/RAGE/Nox4 signaling pathway. METHODS: KK-Ay diabetic reproductive damage mice were administered with catalpol for 8 weeks, the testis/body weight ratio, testicular histopathology, the levels of endogenous hormone and the activity of testicular marker enzymes were determined. In vitro, the GC-2 cell injury model was induced by advanced glycation end-products (AGEs) and pretreated with catalpol. Cell viability, apoptosis, and oxidative stress markers were detected and the mechanism based on the AGEs/RAGE/Nox4 pathway was explored. KEY FINDINGS: Catalpol showed remarkable capacity on protecting diabetic reproductive damage by improving the histomorphology of the testes, increasing the testis/body weight ratio and activity of acid phosphatase (ACP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (γ-GT). The reduced testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in DM mice were also reversed with catalpol intervention. Moreover, catalpol showed markedly effects of anti-oxidative in vivo and in vitro, which significantly down-regulated reactive oxygen species (ROS) levels and restored superoxide dismutase (SOD) activity, meanwhile decreased GC-2 cell apoptosis and Bax/Bcl-2 ratio. Moreover, the over-expression of receptors for AGEs (RAGE), NADPH oxidase type 4 (Nox4) and phosphorylation of nuclear transcription factor-κB p65 (NF-κB p65) were suppressed by catalpol. SIGNIFICANCE: Catalpol could alleviate DM-induced male reproductive damage by inhibiting oxidative stress-induced apoptosis and inflammation mediated by AGEs/RAGE/Nox4 signaling pathway.

3D spheroids generated on carbon nanotube-functionalized fibrous scaffolds for drug metabolism and toxicity screening.

The mechanical and electrical stimuli have a profound effect on the cellular behavior and function. In this study, a series of conductive nanofibrous scaffolds are developed by blend electrospinning of poly(styrene-co-maleic acid) (PSMA) and multiwalled-carbon nanotubes (CNTs), followed by grafting galactose as cell adhesion cues. When the mass ratios of CNTs to PSMA increase up to 5%, the alignment, Young's modulus and conductivity of fibrous scaffolds increase, whereas the average diameter, pore size and elongation at break decrease. Primary hepatocytes cultured on the scaffolds are self-assembled into 3D spheroids, which restores the hepatocyte polarity and sufficient expression of drug metabolism enzymes over an extended period of time. Among these conductive scaffolds, hepatocytes cultured on fibers containing 3% of CNTs (F3) show the highest clearance rates of model drugs, offering a better prediction of the in vivo data with a high correlation value. Moreover, the drug metabolism capability is maintained over 15 days and is more sensitive towards the inducers and inhibitors of metabolizing enzymes, demonstrating the applicability for drug-drug interaction studies. Thus, this culture system has been demonstrated as a reliable in vitro model for high-throughput screening of metabolism and toxicity in the early phases of drug development.

Synergistic protection of tetramethylpyrazine phosphate and borneol on brain microvascular endothelium cells injured by hypoxia.

The combination of tetramethylpyrazine phosphate (TMPP) and borneol (BO) protects against cerebral ischemia. However, the mechanism for their synergistic effect is unclear. In this study, an oxygen-glucose deprivation (OGD) injured brain model was induced in microvascular endothelium cells (BMECs). TMPP and BO concentrations were optimized according to an MTT assay. Cells were divided into five groups: control, model, TMPP, BO, and TMPP+BO. Subsequently, oxidative stress was evaluated based on the levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS). Intracellular calcium ([Ca2+]i) was detected using a laser confocal microscope. Cellular apoptosis was examined via Hoechst 33342 staining, flow cytometry, and expression of p53, B-cell lymphoma 2 (BCL-2), BCL-2-like protein 4 (BAX), and caspase-3 mRNA. Angiogenesis was evaluated based on expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), fibroblast growth factor receptor 1 (FGFR1), Vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2. Results showed that 5.0 µM TMPP and 0.5 µM BO were optimal. Monotherapy significantly enhanced CAT, BCL-2, and VEGF, and also reduced [Ca2+]i, apoptosis, and BAX. TMPP increased SOD, GSH-Px, and bFGF, and reduced MDA, ROS, p53, and caspase-3 levels. BO reduced VEGFR1 expression. TMPP+BO combination exhibited synergistic effects in decreasing apoptosis, and modulating expression of BCL-2, BAX, and VEGFR1. These results indicate that protection of OGD-injured BMECs by TMPP+BO combination involves anti-oxidation, apoptosis inhibition, and angiogenesis. Moreover, their synergistic mechanism was mainly related to the regulation of apoptosis and angiogenesis.

Catalpol ameliorates advanced glycation end product-induced dysfunction of glomerular endothelial cells via regulating nitric oxide synthesis by inducible nitric oxide synthase and endothelial nitric oxide synthase.

Catalpol (Cat.) is an iridoid glucoside extracted from the root of Rehmannia glutinosa Libosch. In this study, we investigated whether Cat. could protect the mouse glomerular endothelial cells against the deleterious effect induced by advanced glycation end products (AGEs) and explored potential mechanisms. We found that 10 µM Cat. showed a protective effect on dead cells stimulated by AGEs. Cat. significantly decreased the expression of p-NF-κBp65 and inducible nitric oxide synthase (iNOS) and increased the expression of phosphorylated-endothelial nitric oxide synthase (p-eNOS; Ser1177), PI3K, p-Akt (Thr308), and total-Akt. Moreover, Cat. restored the integrity of glomerular endothelial barrier by increasing endothelial tight gap junction protein and ameliorated the endothelial hyperpermeability induced by AGEs via modulating the nitric oxide (NO) production. Additionally, Cat. attenuated the massive release of NO induced by AGEs, inhibiting the macrophage infiltration by modulating the NO production, accompanied by the decrease in the release of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1 in vitro. Therefore, Cat. ameliorated AGEs-induced endothelial dysfunction via inhibiting the NF-κB/iNOS pathway and activating the PI3K/Akt/eNOS pathway. © 2019 IUBMB Life, 71(9):1268-1283, 2019.

Tuning the conductivity and inner structure of electrospun fibers to promote cardiomyocyte elongation and synchronous beating.

The key to addressing the challenges facing cardiac tissue engineering is the integration of physical, chemical, and electrical cues into scaffolds. Aligned and conductive scaffolds have been fabricated as synthetic microenvironments to improve the function of cardiomyocytes. However, up to now, the influence of conductive capability and inner structure of fibrous scaffolds have not been determined on the cardiomyocyte morphologies and beating patterns. In the current study, highly aligned fibers were fabricated with loaded up to 6% of carbon nanotubes (CNTs) to modulate the electrical conductivity, while blend and coaxial electrospinning were utilized to create a bulk distribution of CNTs in fiber matrices and a spatial embedment in fiber cores, respectively. Conductive networks were formed in the fibrous scaffolds after the inoculation of over 3% CNTs, and the increase in the conductivity could maintain the cell viabilities, induce the cell elongation, enhance the production of sarcomeric α-actinin and troponin I, and promote the synchronous beating of cardiomyocytes. Although the conductivity of blend fibers is slightly higher than that of coaxial fibers with the same CNT loadings, the lower exposures to CNTs resulted in higher cell viability, elongation, extracellular matrix secretion and beating rates for cardiomyocytes on coaxial fibers. Taken altogether, core-sheath fibers with loaded 5% of CNTs in the fiber cores facilitated the cardiomyocyte growth with a production of organized contractile proteins and a pulsation frequency close to that of the atrium. It is suggested that electrospun scaffolds that couple conductivity and fibrous structure considerations may provide optimal stimuli to foster cell morphology and functions for myocardial regeneration or establishment of in vitro cardiomyocyte culture platform for drug screening.

Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism.

The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5 days and maintained throughout 15 days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15 days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism.

Spheroid culture of primary hepatocytes with short fibers as a predictable in vitro model for drug screening.

The establishment of a reliable in vitro liver model for drug screening remains challenging with respect to tethering the growth of hepatocyte spheroids and adapting to the current high-throughput system. In the current study, short fibers are utilized as scaffolds for the generation of size-controlled hepatocyte spheroids that recapitulate in vivo hepatic phenotypes and functions. The spheroid formation is modulated by the length and galactose/RGD grafts of short fibers, and short 50 µm long fibers motivate the spheroid formation with optimal hepatic function. Short fibers distribute throughout the entire spheroid for tethering hepatocyte growth to form compact spheroids. Compared with scaffold-free spheroid culture on agarose-coated plates, the spheroid culture with short fibers achieves higher clearance rates of model drugs and provides a better prediction of the in vivo drug clearance rate with a correlation value of 0.886. In addition, the drug metabolism capability is highly sensitive to the inducers and inhibitors of metabolizing enzymes, and the responsiveness is maintained during 20 days of culture, exhibiting an efficient in vitro model for determining drug-drug interactions. Therefore, the spheroid culture with short fibers provides an easily manipulated strategy to maintain hepatocyte functions for a prolonged period and enable ready deployment in conventional multiwell plates and diverse organ-on-a-chip devices for high-throughput drug screening.

Spatial distribution and antitumor activities after intratumoral injection of fragmented fibers with loaded hydroxycamptothecin.

There was only a small percentage of drug delivered to tumors after systemic administration, and solid tumors also have many barriers to prevent drug penetration within tumors. In the current study, intratumoral injection of drug-loaded fiber fragments was proposed to overcome these barriers, allowing drug accumulation at the target site to realize the therapeutic efficacy. Fragmented fibers with hydroxycamptothecin (HCPT) loaded were constructed by cryocutting of aligned electrospun fibers, and the fiber lengths of 5 (FF-5), 20 (FF-20), and 50µm (FF-50) could be easily controlled by adjusting the slice thickness. Fragmented fibers were homogeneously dispersed into 2% sodium alginate solution, and could be smoothly injected through 26G1/2 syringe needles. FF-5, FF-20 and FF-50 fiber fragments indicated similar release profiles except a lower burst release from FF-50. In vitro viability tests showed that FF-5 and FF-20 fiber fragments caused higher cytotoxicity and apoptosis rates than FF-50. After intratumoral injection into murine H22 subcutaneous tumors, fragmented fibers with longer lengths indicated a higher accumulation into tumors and a better retention at the injection site, but showed less apparent diffusion within tumor tissues. In addition to the elimination of invasive surgery, HCPT-loaded fiber fragments showed superior in vivo antitumor activities and fewer side effects than intratumoral implantation of drug-loaded fiber mats. Compared with FF-5 and FF-50, FF-20 fiber fragments indicated optimal spatial distribution of HCPT within tumors and achieved the most significant effects on the animal survival, tumor growth inhibition and tumor cell apoptosis induction. It is suggested that the intratumoral injection of drug-loaded fiber fragments provided an efficient strategy to improve patient compliance, allow the retention of fragmented fibers and spatial distribution of drugs within tumor tissues to achieve a low systemic toxicity and an optimal therapeutic efficacy.

Engineering blood vessels through micropatterned co-culture of vascular endothelial and smooth muscle cells on bilayered electrospun fibrous mats with pDNA inoculation.

Although engineered blood vessels have seen important advances during recent years, proper mechanical strength and vasoactivity remain unsolved problems. In the current study, micropatterned fibrous mats were created to load smooth muscle cells (SMC), and a co-culture with endothelial cells (EC) was established through overlaying on an EC-loaded flat fibrous mat to mimic the layered structure of a blood vessel. A preferential distribution of SMC was determined in the patterned regions throughout the fibrous scaffolds, and aligned fibers in the patterned regions provided topological cues to guide the orientation of SMC with intense actin filaments and extracellular matrix (ECM) production in a circumferential direction. Plasmid DNA encoding basic fibroblast growth factors and vascular endothelial growth factor were integrated into electrospun fibers as biological cues to promote SMC infiltration into fibrous mats, and the viability and ECM production of both EC and SMC. The layered fibrous mats with loaded EC and SMC were wrapped into a cylinder, and engineered vessels were obtained with compact EC and SMC layers after co-culture for 3 months. Randomly oriented ECM productions of EC formed a continuous endothelium covering the entire lumenal surface, and a high alignment of ECM was shown in the circumferential direction of SMC layers. The tensile strength, strain at failure and suture retention strength were higher than those of the human femoral artery, and the burst pressure and radial compliance were in the same range as the human saphenous vein, indicating potential as blood vessel substitutes for transplantation in vivo. Thus, the establishment of topographical cues and biochemical signals in fibrous scaffolds demonstrates advantages in modulating cellular behavior and organization found in complex multicellular tissues.

Micropatterned coculture of vascular endothelial and smooth muscle cells on layered electrospun fibrous mats toward blood vessel engineering.

A major challenge in vascular engineering is the establishment of proper microenvironment to guide the spatial organization, growth, and extracellular matrix (ECM) productions of cells found in blood vessels. In the current study, micropatterned fibrous mats with distinct ridges and grooves of different width were created to load smooth muscle cells (SMCs), which were assembled by stacking on vascular endothelial cell (EC)-loaded flat fibrous mats to mimic the in vivo-like organized structure of blood vessels. SMCs were mainly distributed in the ridges, and aligned fibers in the patterned regions led to the formation of elongated cell bodies, intense actin filaments, and expressions of collagen I and α-smooth muscle actin in a parallel direction with fibers. ECs spread over the flat fibrous mats and expressed collagen IV and laminin with a cobblestone-like feature. A z-stack scanning of fluorescently stained fibrous mats indicated that SMCs effectively infiltrated into fibrous scaffolds at the depth of around 200 µm. Compared with SMCs cultured alone, the coculture with ECs enhanced the proliferation, infiltration, and cytoskeleton elongation of SMCs on patterned fibrous mats. Although the coculture of SMCs made no significant difference in the EC growth, the coculture system on patterned fibrous scaffolds promoted ECM productions of both ECs and SMCs. Thus, this patterned fibrous configuration not only offers a promising technology in the design of tissue engineering scaffolds to construct blood vessels with durable mechanical properties, but also provides a platform for patterned coculture to investigate cell-matrix and cell-cell interactions in highly organized tissues.

Effects of continuous haemofiltration on serum enzyme concentrations, endotoxemia, homeostasis and survival in dogs with severe heat stroke.

AIM: To examine the effectiveness of continuous haemofiltration as a treatment for severe heat stroke in dogs. METHODS: Dogs were randomly allocated to a control or continuous haemofiltration group (both n=8). Heat stroke was induced by placing anaesthetised dogs in a high temperature cabin simulator. Upon confirmation of heat stroke (rectal temperature>42 °C, mean arterial pressure (MAP) decrease>25 mmHg), dogs were removed from the chamber and continuous haemofiltration was initiated and continued for 3h for dogs in the continuous haemofiltration group. Dogs in the control group were observed at room temperature. RESULTS: Rectal temperature, haemodynamics, pH, blood gases and electrolyte concentrations rapidly returned to baseline in the continuous haemofiltration group, but not the control group. After 3h, rectal temperature was 36.68±0.51 °C in the continuous haemofiltration group and 39.83±1.10 °C in the control group (P<0.05). Continuous haemofiltration prevented endotoxin and all serum enzyme concentrations from increasing and caused malondialdehyde concentrations to decrease. After 3h, endotoxin concentrations were 0.14±0.02 EU ml(-1) in the continuous haemofiltration group and 0.23±0.05 EU ml(-1) in the control group (P=0.003), while malondialdehyde concentrations were 4.86±0.61 mmol l(-1) in the continuous haemofiltration group and 8.63±0.66 mmol l(-1) in the control group (P<0.001). Five dogs died in the control group within 3h, whereas no dogs died in the continuous haemofiltration group. CONCLUSIONS: Continuous haemofiltration rapidly reduced body temperature, normalised haemodynamics and electrolytes, improved serum enzyme concentrations and increased survival in dogs with heat stroke. Continuous haemofiltration may be an effective treatment for heat stroke.

[Influence of electroacupuncture plus intragastric administration of extract of hypericum perforatum L on ethology and brain microcirculation in depression rats].

OBJECTIVE: To observe the effect of combined administration of intragastric perfusion of extract of Hypericum Perforatum L (HP-L) and electroacupuncture (EA) of "Baihui" (GV 20) and "Yamen" (GV 15) on behavior and brain microcirculation in depression rats. METHODS: Female SD rats were randomized into control, model, lower-dose of HP-L (lower-dose in short, 10 mg/kg), lower-dose+ EA, higher-dose (20 mg/kg) and higher-dose+ EA groups (n = 10/group). Depression model was established by lonely raising and chronic unpredictable mild stress (tail cramping, water-deprivation, fasting, electrical shock stimulation, etc. ) for 21 days. EA (2 Hz, 1 mA) was applied to "Baihui"(GV 20) and "Yamen"(GV 15) for 20 min, once daily for 14 days. Changes of ethology including glucose-consumption during 1 h, crossing and rearing scores of open-field test during 3 min (for assessing the rats' locomoto)and laser Doppler flowmetry values of cortical regional cerebral bloodflow (r CBF) were detected, and Morris water maze test (for assessing the rats' learning-memory ability) was conducted. RESULTS: In comparison with the control group, the sucrose consumption, crossing and rearing scores of open-field test, the average swimming velocity (ASV). the ratios of path length and swimming duration near the hidden-platform and the path length and swimming duration far from the platform of Morris water maze test during 70 seconds, and the cortical r CBF value in the model group were decreased significantly (P < 0.01), while the total swimming distance and escape latency in the model group increased apparently (P < 0.01). Compared to the model group, the average sucrose consumption, crossing and rearing scores of open-field test, the ASV, and the ratios of path length and swimming duration near the platform and those far from the platform in the lower-dose. lower-dose + EA, higher-dose and higher-dose + EA groups, and the cortical r CBF in the lower-dose + EA and higher-dose + EA groups were increased considerably (P < 0.05, P < 0.01). The total swimming distances and escape latencies of lower-dose, lower-dose + EA, higher-dose and higher-dose + EA groups were significantly shortened in comparison with the model group (P < 0.05, P < 0.01). The sucrose consumption and crossing score were significantly higher in the higher-dose + EA group than the lower-dose group (P < 0.05). The escape latency was significantly shorter in the higher-dose + EA group than in the lower-dose group (P < 0.05). No significant differences were found among the lower-dose, lower-dose + EA and higher-dose groups the sucrose consumption, crossing score and escape latency: among the lower-dose, lower-dose + EA, higher-dose and higher-dose + EA groups in the rearing score and ASV; among the lower-dose, higher-dose and model groups in the cortical r CBF (P > 0.05). CONCLUSION: EA can enhance the effect of extract of HP-L in increasing sucrose consumption, crossing score and cerebral blood flow, and in shortening escape latency in depression rats, which may contribute to their effect in improving depression. But HP-L itself has no effect on cortical microcirculation.