Electrospun Chitosan-Polyvinyl Alcohol Nanofiber Dressings Loaded with Bioactive Ursolic Acid Promoting Diabetic Wound Healing.

The design and development of novel dressing materials are urgently required for the treatment of chronic wounds caused by diabetic ulcers in clinics. In this study, ursolic acid (UA) extracted from Chinese herbal plants was encapsulated into electrospun nanofibers made from a blend of chitosan (CS) and polyvinyl alcohol (PVA) to generate innovative CS-PVA-UA dressings for diabetic wound treatment. The as-prepared CS-PVA-UA nanofiber mats exhibited randomly aligned fiber morphology with the mean fiber diameters in the range of 100-200 nm, possessing great morphological resemblance to the collagen fibrils which exist in the native skin extracellular matrix (ECM). In addition, the CS-PVA-UA nanofiber mats were found to possess good surface hydrophilicity and wettability, and sustained UA release behavior. The in vitro biological tests showed that the high concentration of UA could lead to slight cytotoxicity. It was also found that the CS-PVA-UA nanofiber dressings could significantly reduce the M1 phenotypic transition of macrophages that was even stimulated by lipopolysaccharide (LPS) and could effectively restore the M2 polarization of macrophages to shorten the inflammatory period. Moreover, the appropriate introduction of UA into CS-PVA nanofibers decreased the release levels of TNF-α and IL-6 inflammatory factors, and suppressed oxidative stress responses by reducing the generation of reactive oxygen species (ROS) as well. The results from mouse hepatic hemorrhage displayed that CS-PVA-UA nanofiber dressing possessed excellent hemostatic performance. The in vivo animal experiments displayed that the CS-PVA-UA nanofiber dressing could improve the closure rate, and also promote the revascularization and re-epithelization, as well as the deposition and remodeling of collagen matrix and the regeneration of hair follicles for diabetic wounds. Specifically, the mean contraction rate of diabetic wounds using CS-PVA-UA nanofiber dressing could reach 99.8% after 18 days of treatment. In summary, our present study offers a promising nanofibrous dressing candidate with multiple biological functions, including anti-inflammation, antioxidation, pro-angiogenesis, and hemostasis functions, for the treatment of hard-to-heal diabetic wounds.

Electrospun multifunctional nanofibrous mats loaded with bioactive anemoside B4 for accelerated wound healing in diabetic mice.

With the worldwide prevalence of diabetes and considering the complicated microenvironment of diabetic wounds, the design and development of innovative multifunctional wound dressing materials are much wanted for the treatment of hard-to-heal wounds in diabetic patients. In the present study, anti-inflammatory ingredients loaded with nanofibrous wound dressing materials were manufactured by a promising blend-electrospinning strategy, and their capability for treating the diabetic wound was also systematically explored. A polymer blend consisting of Chitosan (CS) and polyvinyl alcohol (PVA) was electrospun into CS-PVA nanofibrous mats as control groups. In the meanwhile, a bioactive ingredient of Chinese medicine Pulsatilla, anemoside B4(ANE), with different contents were loaded into the electrospinning solution to construct CS-PVA-ANE nanofibrous mats. The developed CS-PVA-ANE wound dressing materials exhibited multifunctional properties including prominent water absorption, biomimetic elastic mechanical properties, and sustained ANE releasing behavior, as well as outstanding hemostatic properties. The in vitro studies showed that the CS-PVA-ANE nanofiber mats could significantly suppress lipopolysaccharide (LPS)-stimulated differentiation of pro-inflammatory (M1) macrophage subsets, and notably reduce the reactive oxygen species (ROS) generation, as well as obviously decrease inflammatory cytokine release. The in vivo animal studies showed that the CS-PVA-ANE nanofiber mats promoted the healing of diabetic wounds by significantly enhancing wound closure rates, accelerating excellent angiogenesis, promoting re-epithelization and collagen matrix deposition throughout all stages of wound healing. The present study demonstrated that CS-PVA-ANE nanofiber mats could effectively shorten the wound-healing time by inhibiting inflammatory activity, which makes them promising candidates for the treatment of hard-to-heal wounds caused by diabetes.

Cryptotanshinone protects dextran sulfate sodium-induced experimental ulcerative colitis in mice by inhibiting intestinal inflammation.

The incidence of ulcerative colitis (UC) is increasing in recent years. The protective effect of cryptotanshinone, a natural compound from Salvia miltiorrhiza Bunge, on UC was investigated both in vivo and in vitro models. UC model was established by dextran sulfate sodium administration in drinking water and cryptotanshinone was orally administrated. RAW264.7 cells were stimulated by lipopolysaccharide (LPS) with or without cryptotanshinone pretreatment. The body weights and disease activity index (DAI) were recorded. The pathological alterations were evaluated by H&E staining. The levels of pro-inflammatory cytokines in colon tissues and cell culture medium were determined with enzyme-linked immune sorbent assay (ELISA) kits. The protein expression was detected by Western blotting and immunohistochemistry. Results showed that cryptotanshinone significantly increased the body weight and colon length, reduced the score of DAI, and improved pathological changes. Furthermore, the expression of inducible nitric oxide synthase, cyclooxygenase-2, receptor-interacting protein kinase 3, NF-κB p65 and the secretion of tumor necrosis factor-α, IL-6 in colon tissues and LPS-stimulated cells were significantly inhibited by cryptotanshinone. Besides, cryptotanshinone significantly inhibited LPS-triggered toll-like receptor 4 luciferase reporter activity with an IC50 at 7.2 µM. In conclusion, cryptotanshinone ameliorated experimental UC possibly by inhibiting intestinal inflammation.

Preventive effects of a natural anti-inflammatory agent Salvianolic acid A on acute kidney injury in mice.

Acute kidney injury (AKI) is an abrupt loss of kidney function with high mortality. Inflammatory is considered driving the progression of AKI. Salvianolic acid A (SA), one of the major ingredients of Salvia miltiorrhiza Bunge, displays plenty of biological effects. Herein, the effect of SA on lipopolysaccharide (LPS)-induced AKI in mice and further related mechanism in inflammatory cells were explored. In vivo experiments demonstrated that SA significantly ameliorated LPS-challenged AKI by preventing glomerulus atrophy and decreasing plasma creatinine and blood urea nitrogen (BUN) levels. Meanwhile, SA significantly decreased the release of serum inflammatory cytokines and blocked macrophage infiltration in damaged renal tissue. In in vitro studies, SA significantly decreased TNF-α and IL-6 release levels and altered the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-stimulated macrophages, which were consistent with the results from in vivo experiments. Furthermore, SA that bound to Toll-Like Receptor 4 (TLR4) was able to reduce endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation in response to LPS stimulation. All silence of TLR4 gene, ROS scavenger and Ca2+ chelator decreased inflammatory cytokines releases. Taken together, SA could be used as a potential therapeutic agent for preventing AKI by suppressing inflammatory responses.

Structural characterization and in vitro-in vivo evaluation of effect of a polysaccharide from Sanguisorba officinalis on acute kidney injury.

We report here an acidic polysaccharide, namely RSP-3, which ameliorates acute kidney injury and is obtained from Sanguisorba officinalis. We extracted and purified two polysaccharides from this herb based on the acidity and screened them for their effect in regulating the immunological activity of macrophages. Among them, RSP-3 exhibited significant anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated macrophages by decreasing TNF-α and IL-6 levels. Subsequently, we found that RSP-3 suppressed ER stress, reduced ROS production and blocked NF-κBp65 translocation. After fully characterizing RSP-3 with a series of analytical technologies, we tested its anti-acute kidney injury (AKI) effect in vivo. In a murine AKI model induced by LPS, treatment with RSP-3 effectively ameliorated renal function. Besides, it decreased the levels of TNF-α and IL-6 in serum and reduced macrophage infiltration in injured kidney tissue. In sum, RSP-3, with a significant protective effect against AKI by showing anti-inflammatory activity, may become a meaningful drug candidate for treatment of AKI.

Aqueous extract of Salvia miltiorrhiza Bunge-Radix Puerariae herb pair ameliorates diabetic vascular injury by inhibiting oxidative stress in streptozotocin-induced diabetic rats.

Vascular diabetic complications are the leading cause of mortality and morbidity for diabetes. The present study was designed to investigate the protective effect of herb pair Salvia miltiorrhiza Bunge-Radix Puerariae (DG) on diabetic vascular injury induced by streptozotocin. The protective effect of DG was determined by oral administration of DG (50 and 200 mg/kg) in rats and on high glucose (HG)-induced endothelial injury. DG showed no effect on body weight, fasting blood glucose (FBG) but decreased the serum levels of insulin, nitric oxide (NO), hydrogen peroxide (H2O2), malondialdehyde (MDA), soluble intercellular cell adhesion molecule-1 (s-ICAM-1) and vascular cell adhesion molecule-1 (s-VCAM-1), and increased superoxide dismutase (SOD) and catalase (CAT) levels. The pathological alterations of aorta was improved by DG. Furthermore, the increased expression of ICAM-1,VCAM-1, NOX2, and NOX4 in aorta were inhibited by DG. HG-induced endothelial ROS formation, ICAM-1,VCAM-1, NOX4 expression and monocyte-endothelial adhesion were dramatically suppressed by DG as well. In addition, both GKT137831, a NOX4 inhibitor, and PDTC, a NF-κB inhibitor, could significantly inhibited HG-induced ICAM-1, VCAM-1 expression and monocyte-endothelial adhesion. These results suggested that DG improved diabetic vascular injury possibly by reducing oxidative stress, which provides scientific evidence for the application of DG for diabetic vascular therapy.

Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation.

Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin-induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 µmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg-1·d-1, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Upregulation of miR-124 by physcion 8-O-ß-glucopyranoside inhibits proliferation and invasion of malignant melanoma cells via repressing RLIP76.

Melanoma is the most malignant type of skin cancer. In recent years, mounting studies have evidenced the involvement of miRNAs in melanoma. One of these miRNAs, miR-124 has been found aberrantly downregulated in a variety of human malignancies. In this study, our results showed that the expression of miR-124 was significantly lower in malignant melanoma tissues and cell lines and miR-124 functioned as a tumor suppressor in melanoma. Moreover, our findings showed that miR-124 exerted anti-tumor effect by directly targeting RLIP76, a stress-inducible non-ABC transporter that plays a crucial role in the development of melanoma. Furthermore, our study also showed that physcion 8-O-ß-glucopyranoside, a natural compound from medicinal plant, could inhibit the proliferation and invasion of melanoma cells by targeting miR-124/RLIP76 signaling.

l-carnitine protects human hepatocytes from oxidative stress-induced toxicity through Akt-mediated activation of Nrf2 signaling pathway.

In our previous study, l-carnitine was shown to have cytoprotective effect against hydrogen peroxide (H2O2)-induced injury in human normal HL7702 hepatocytes. The aim of this study was to investigate whether the protective effect of l-carnitine was associated with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway. Our results showed that pretreatment with l-carnitine augmented Nrf2 nuclear translocation, DNA binding activity and heme oxygenase-1 (HO-1) expression in H2O2-treated HL7702 cells, although l-carnitine treatment alone had no effect on them. Analysis using Nrf2 siRNA demonstrated that Nrf2 activation was involved in l-carnitine-induced HO-1 expression. In addition, l-carnitine-mediated protection against H2O2 toxicity was abrogated by Nrf2 siRNA, indicating the important role of Nrf2 in l-carnitine-induced cytoprotection. Further experiments revealed that l-carnitine pretreatment enhanced the phosphorylation of Akt in H2O2-treated cells. Blocking Akt pathway with inhibitor partly abrogated the protective effect of l-carnitine. Moreover, our finding demonstrated that the induction of Nrf2 translocation and HO-1 expression by l-carnitine directly correlated with the Akt pathway because Akt inhibitor showed inhibitory effects on the Nrf2 translocation and HO-1 expression. Altogether, these results demonstrate that l-carnitine protects HL7702 cells against H2O2-induced cell damage through Akt-mediated activation of Nrf2 signaling pathway.

Modulation of oxidized-LDL receptor-1 (LOX1) contributes to the antiatherosclerosis effect of oleanolic acid.

Oleanolic acid (OA) is a bioactive pentacyclic triterpenoid. The current work studied the effects and possible mechanisms of OA in atherosclerosis. Quails (Coturnix coturnix) were treated with high fat diet with or without OA. Atherosclerosis was assessed by examining lipid profile, antioxidant status and histology in serum and aorta. Human umbilical vein endothelial cells (HUVECs) were exposed to 200µg/mL ox-LDL for 24h, then cell viability was assessed with MTT assay; reactive oxygen species (ROS) was assessed with DCFDA staining. Expression levels of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were measured with real time PCR and western blotting. Furthermore, LOX-1 was silenced with lentivirus and the expression levels assessment was repeated. OA treatment improved the lipid profile and antioxidant status in quails fed with high fat diet. Histology showed decreased atherosclerosis in OA treated animals. Ox-LDL exposure decreased viability and induced ROS generation in HUVECs, and this progression was alleviated by OA pretreatment. Moreover, elevated expression of LOX-1, NADPH oxidase subunits, nrf2 and ho-1 were observed in ox-LDL exposed HUVECs. OA pretreatment prevented ox-LDL induced increase of LOX-1 and NADPH oxidase subunits expression, while further increased nrf2 and ho-1 expression. Silencing of LOX-1 abolished ox-LDL induced effects in cell viability, ROS generation and gene expression. OA could alleviate high fat diet induced atherosclerosis in quail and ox-LDL induced cytotoxicity in HUVECs; the potential mechanism involves modulation of LOX-1 activity, including inhibition of expression of NADPH oxidase subunits and increase of the expression of nrf2 and ho-1.

Tegillarca granosa extract Haishengsu induces apoptosis in human hepatocellular carcinoma cell line BEL-7402 via Fas-signaling pathways.

This study was designed to investigate the apoptosis-inducing properties of Tegillarca granosa extract Haishengsu (HSS) in human hepatocellular carcinoma cell line BEL-7402. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells was determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. Apoptosis of BEL-7402 cells was demonstrated by fluorescence microscope with flow cytometry with Annexin V-FITC/PI double staining and Hoechst 33258 staining. Western blot analysis and RT-PCR were used to determine the expression levels of Fas. Expressions of caspase-8 and caspase-3 were examined by caspase activity assay and western blot analysis. HSS inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in a dose- and time-dependent manner. Our results showed HSS had positive effect on apoptosis through flow cytometry assay and fluorescence microscope. The expressions of Fas protein and mRNA were up-regulated following the treatment. Caspase-8 and caspase-3 were activated in the cells cultured with HSS. In conclusion, HSS induced apoptosis of human hepatocellular carcinoma BEL-7402 cells. The apoptosis was associated with the up-regulation of Fas and the activations of caspase-8 and caspase-3.

Effects of Ginkgo biloba extract EGb761 on human colon adenocarcinoma cells.

AIMS: To investigate the effect of Ginkgo biloba extract (EGb761) on cell proliferation and apoptosis in human colon cancer cells. METHODS: Human colon cancer cell lines (HT-29) were cultured and incubated with various concentrations (0-320 mg/l) of EGb 761 solution for up to 72 h. Cell viability, cell apoptosis, cell cycle, expression of caspase-3, the mRNA levels of p53, and Bcl-2 were assessed. RESULTS: EGb 761 inhibited the growth of HT-29 cells in a time-dose-dependent manner. At 80 and 320 mg/L, EGb 761 increased the number of cells in the G0/G1 phase and reduced cells in the G2/M and S phase. EGb 761 treatment also increased the apoptosis ratio of the HT-29 cells. EGb 761 treatment was associated with an increase in caspase-3 activities, reduction in bcl-2 mRNA expression and elevation in p53 mRNA expression. CONCLUSION: EGb 761 inhibits the progression of human colon cancer cells. Its therapeutic effect may be related to enhanced caspase-3 activities, up-regulation of p53 and down-regulation of bcl-2 genes.

Inhibitory effect of polypeptide from Chlamys farreri on ultraviolet A-induced oxidative damage on human skin fibroblasts in vitro.

We have previously reported that polypeptide from Chlamys farreri (PCF) inhibits the oxidative damage of ultraviolet A (UVA) on HeLa cells in vitro [Acta Pharm. Sin. 23 (2002) 961]. To further elucidate a possible role for PCF on UVA-damaged normal human cells, we established the oxidative damage models of normal human dermal fibroblasts (NHDF) exposed to UVA to study the protective effect of PCF on human dermal fibroblasts in vitro. In this study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cell viability. The intracellular superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT), xanthine oxidase (XOD), malondialdehyde (MDA), reactive oxygen species (ROS), total antioxidative capacity (T-AOC), and anti-superoxide anion capacity (A-ASC) were measured. The effect of PCF on UVA-induced apoptosis were investigated by Annexin V-FITC assay. Intracellular calcium was determined with the calcium-sensitive fluorochrome Fluo-3, and mitochondrial transmembrane potential with rhodamine 123. Comet assay was employed to detect the UVA-induced DNA damage. The ultrastructure of cell was observed under transmission electron microscope. The results indicated that PCF could greatly enhance the viability of NHDF and markedly promote SOD, GSH-px, T-AOC, and A-ASC, while the amounts of MDA and ROS, the activity of XOD were decreased. PCF could inhibit UVA-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium was decreased and the mitochondrial transmembrane potential was increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease UVA-induced damage, especially membrane. Our results suggest that the supplementation of PCF appears to reduce the UVA-induced normal human dermal fibroblasts damage efficiently. It may be involved in the PCF's abilities of scavenging oxygen free radical, inhibiting lipid peroxidation, increasing antioxidative enzymes, decreasing intracellular calcium and protection of membrane structure in NHDF irradiated by UVA.

Protective effect of polypeptide from Chlamys farreri on mitochondria in human dermal fibroblasts irradiated by ultraviolet B.

AIM: To study the effect of polypeptide from Chlamys farreri (PCF) on mitochondria of human dermal fibroblasts irradiated by ultraviolet B (UVB) in vitro. METHODS: Malondialdehyde (MDA) and antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined by biochemical methods. Mitochondrial transmembrane potential was measured by flow cytometry. Ultrastructure of fibroblasts was observed with transmission electron microscope. RESULTS: UVB (1.176 x 10(-4) J/cm(2)) induced mitochondria damage in dermal fibroblast and PCF (0.25%-1%) reduced the damage in a concentration-dependent manner. Furthermore, PCF also concentration-dependently maintained the stability of mitochondrial transmembrane potential. PCF was able to reduce the MDA formation caused by UVB, meanwhile increased the activities of SOD and GSH-PX. The differences among the PCF groups and UVB model group were significant (P<0.05, P<0.01). CONCLUSION: The UVB-induced mitochondria damage was alleviated by PCF in human dermal fibroblasts.