Long-circulation zwitterionic dendrimer nanodrugs for phototherapy of tumors.

The development of stealth and effective antitumor nanodrugs has been drawing great attention. Herein, generation five poly(amide amine) dendrimer (G5 PAMAM) was modified by zwitterionic material carboxybetaine methacrylamide (CBMAA) on its surface to prepare zwitterionic dendrimer (G5-CBMAAn). The results showed that G5-CBMAA30 had the longest blood circulation time due to its thickest zwitterionic layer, and its residual rate after injection into mice at 2 and 12 h was as high as 47.22 % and 14.37 %, respectively. Nanodrug G5-CBMAA30-ICG was prepared by containing indocyanine green (ICG) in the cavity of G5-CBMAA30. G5-CBMAA30-ICG had better tumor targeting ability and antitumor effect than free ICG in mice after laser irradiation, and the tumor inhibition rate was 96.6 % after 14 days' treatment. The prepared G5-CBMAA30-ICG has great potential applications in the field of antitumor by phototherapy.

Doxorubicin-loaded natural daptomycin micelles with enhanced targeting and anti-tumor effect in vivo.

Development of a simple method to enhance targeting and anti-tumor effect of the chemotherapeutic agents in vivo is a major problem. Amphipathic and natural daptomycin is biocompatible antibacterial polypeptide used in clinical practice. Herein, doxorubicin (DOX) was stabilized by zwitterionic daptomycin (Dap) micelles in aqueous solution to form a zwitterionic nanodrug (Dap-DOX micelles). The hydrodynamic size and zeta potential of Dap-DOX micelles were 85 nm and -10 mV, respectively. The study on the controlled release showed that more DOX molecules were released from Dap-DOX micelles at acidic condition of tumor tissue than that at neutral condition of normal tissue which was due to pH responsiveness of Dap-DOX micelles. Dap-DOX micelles exhibited good stability in fibrinogen solution. Moreover, MTT studies showed that Dap-DOX micelles had higher cytotoxicity than free DOX. Notably, the results of flow cytometry indicated that the average fluorescence intensity of Dap-DOX micelle-treated cells was higher than that of free DOX-treated cells, and acidic conditions were more favorable for Dap-DOX micelles than normal pH in cell uptake assay. More importantly, Dap-DOX micelles were biocompatible in vivo based on the changes of weight and blood indexes of mice. Dap-DOX micelles were selectively accumulated at tumor sites in vivo through EPR effect, which reduced the toxicity of free DOX and achieved excellent tumor inhibition effect. The tumor inhibition rate of Dap-DOX micelles reached 96%. Dap-DOX micelles also effectively inhibited the growth of bacterial. Taken together, Dap-based drug delivery systems are promising and effective in cancer therapy.

Pterostilbene Ameliorates DSS-Induced Intestinal Epithelial Barrier Loss in Mice via Suppression of the NF-κB-Mediated MLCK-MLC Signaling Pathway.

The integrity of the intestinal barrier is critical for homeostasis. In this study, we investigated the protective effect of pterostilbene (PTE) on the intestinal epithelium barrier. In vitro results of transepithelial electrical resistance (TEER) in Caco-2 cells indicated that PTE counteracted tumor necrosis factor α (TNFα)-induced barrier damage. In vivo PTE pretreatment markedly ameliorated intestinal barrier dysfunction induced by dextran sulfate sodium (DSS). Notably, intestinal epithelial tight junction (TJ) molecules were restored by PTE in mice exposed to DSS. The mechanism study revealed that PTE prevented myosin light-chain kinase (MLCK) from driving phosphorylation of MLC (p-MLC), which is crucial for maintaining intestinal TJ stability. Furthermore, PTE blunted translocation of NF-κB subunit p65 into the nucleus to downregulate MLCK expression and then to safeguard TJs and barrier integrity. These findings suggest that PTE protected the intestinal epithelial barrier through the NF-κB- MLCK/p-MLC signal pathway.

Nobiletin Attenuates DSS-Induced Intestinal Barrier Damage through the HNF4α-Claudin-7 Signaling Pathway.

The intestinal epithelium barrier functions to protect human bodies from damages such as harmful microorganisms, antigens, and toxins. In this study, we evaluated the protective effect and molecular mechanism of a dominant polymethoxyflavone nobiletin (NOB) from tangerine peels on intestinal epithelial integrity. The results from transepithelial electrical resistance (TEER) suggested that NOB pretreatment counteracts epithelial injury induced by inflammatory cytokines (TEER value in 48 h: vehicle, 135.6 ± 3.9 Ω/cm2; TNF-α + IL-1ß, 90.7 ± 0.5 Ω/cm2; 10 µM NOB + TNF-α + IL-1ß, 126.1 ± 0.8 Ω/cm2; 100 µM NOB + TNF-α + IL-1ß, 125.3 ± 0.5 Ω/cm2. P < 0.001). Clinical and pathological test results suggested that administration of NOB effectively alleviates intestinal barrier injury induced by dextran sulfate sodium (DSS) as evidenced by the length of colon villi on day 7 (control, 253.7 ± 4.8 µm, DSS 131.6 ± 4.6 µm, NOB + DSS, 234.5 ± 5.1 µm. P < 0.001). Interestingly, when screening tight junction molecules for intestinal barrier integrity, we observed that independent treatment with NOB sharply increased claudin-7 levels (ratio of claudin-7 over GAPDH: control, 1.0 ± 0.06; DSS, 0.02 ± 0.001; NOB + DSS, 0.3 ± 0.07. P < 0.001), which was previously suppressed upon DSS stimulation. Furthermore, hepatocyte nuclear factor 4α (HNF-4α) transcriptional regulation of claudin-7 contributed to intestinal barrier homeostasis. Therefore, our study suggests potential intestinal protective strategies based on polymethoxyflavones of aged tangerine peels.

Therapeutic effects of conditioned medium from bone marrow-derived mesenchymal stem cells on epithelial-mesenchymal transition in A549 cells.

Pulmonary fibrosis (PF) is a chronic lung disease. The transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be a modulator of the molecular aspects of the fibrosis pathway. However, it is still unknown as to whether the conditioned medium from BMSCs (BMSCs-CM) inhibits the epithelial-mesenchymal transition (EMT) process. This study confirmed the hypothesis that BMSCs-CM exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549) by suppressing the phosphorylation of Smad3. We used the A549 cells in vitro to detect morphological evidence of EMT by phase-contrast microscopy. These cells were randomly divided into 4 groups as follows: the control group, the TGF-ß1 group, the SIS3 (specific inhibitor of Smad3) group and the BMSCs-CM group. The immunofluorescence method was used to determined the location of E-cadherin (E-calcium mucins; E-cad), α-smooth muscle actin (α-SMA) and p-Smad3. The expression levels of E-cad, CK8, α-SMA, vimentin, p-Smad3, Snail1, collagen I (COLI) and collagen III (COLIII) were detected by western blot analysis. Following exposure to TGF-ß1, the A549 cells displayed a spindle-shaped fibroblast-like morphology. In accordance with these morphological changes, the expression levels of E-cad and CK8 were downregulated, while the expression levels of α-SMA and vimentin were upregulated. Along with this process, the expression levels of p-Smad3, Snail1, COLI and COLIII were increased. However, the cells in the BMSCs-CM group and SIS3 group exhibited a decrease in the levels of α-SMA and vimentin (which had been upregulated by TGF-ß1), and an increase in the levels of E-cad and CK8 expression (which had been downregulated by TGF-ß1). On the whole, these results indicated that BMSCs-CM suppressed the EMT which might be associated with TGF-ß1/Smad3. This study provides the theoretical basis for the research of the mechanisms responsible for pulmonary disease.

[Inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline on myofibroblast differentiation by regulating acetylated tubulin α in silicotic rat model].

OBJECTIVE: To explore the inhibition effect and mechanism of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP)on myofibroblast differentiation via regulating acetylated tubulin α (Ac-Tub α)in vivo and in vitro. METHODS: Silicotic model were made by SiO2 douched and divided into 6 groups as follows: control (4w, 8w)group, silicotic model (4w, 8w)group and post-or pre-treatment by Ac-SDKP group. Pulmonary fibroblasts were divided into 5 groups: (1) control; (2) Ang II; (3) Ang II+Ac-SDKP; (4) Ang II+Valsartan; (5) Ang II+TCS histone deacetylase (HDAC)6 20b. The localization of Ac-Tub α and α-smooth muscle actin (SMA) were observed by immunohistochemical (IHC) and immunofluorescence staining. The protein levels of Ac-Tub α, α-SMA, collagen type I (col I) and HDAC6 were measured by western blot. RESULTS: In silicotic nodules and interstitial fibrosis area, positive expression of α-SMA, a classical marker of myofibroblast, was ob-served by IHC, accompanied with absence expression of Ac-Tub α. Furthermore, Ac-SDKP post-treatment could attenuate the levels of col I, α-SMA and HDAC6 to 48.39%, 52.63% and 70.18% compared with the silicotic 8w group respectively. And in Ac-SDKP pre-treatment group, compared with the silicotic 8w group, these protein levels were decreased to 32.26%, 64.91% and 54.39% respectively (P<0.05). The up-regulation of Ac-Tub α was found in Ac-SDKP post-and pre-treatment and increased to 3.00 and 2.90 folds compared with the silicotic 8w group. Compared with control group, the levels of α-SMA, HDAC6 and col I in Ang II group were up-regulated to 1.66, 3.56 and 4.00 folds accompanied with down-regulation of Ac-Tub by 44.44% (P<0.05). Pre-treatment with Valsartan, TCS HDAC6 20b or Ac-SDKP could inhibited all this changes induced by Ang II in vitro. CONCLUSION: Ac-SDKP can inhibit the myofibroblast differentiation and collagen deposition via sup-press HDAC6 and up-regulate the expression of Ac-Tub α in vivo and in vitro.

[Inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline on myofibroblast differentiation of MRC-5 human fetal lung fibroblasts inuced by Ang II].

OBJECTIVE: To explore the inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation of MRC-5 human fetal lung fibroblasts induced by angiotensin (Ang) II. METHODS: The study was divided into 2 step: (1) MRC-5 human fetal lung fibroblasts was induced for 48 h at different dose of Ang II and at different time point by 100 nmol/L Ang II. Then the expression of collagen type I and α-smooth muscle actin (α-SMA) were mesaured by western blot. (2) MRC-5 human fetal lung fibroblasts were divided into 4 group: (1) control, (2) Ang II, (3) Ang II+Ac-SDKP, (4) Ang II+8-Me-cAMP (a specific activator of Epac). The α-SMA expression was observed by immnocytochemical stain. The protein expression of collagen type I, α-SMA, serum response factor (SRF), myocardin-related transcription factor (MRTF)-A, exchange protein directly activated by cAMP (Epac) 1, 2 were measured by Westen blot. RESULTS: Myofibroblast differentiation could be induced by Ang II from MRC-5 cells with a dose- and time-dependent manner. The up-regulation of SRF and MRTF-A were observed in MRC-5 cells induced by Ang II and accompanied with collagen I and α-SMA increased. Pre-treatment with 8-Me-cAMP or Ac-SDKP could attenuated all this changes induced by Ang II, and promoted the expression of Epac1. CONCLUSION: Ac-SDKP can inhibit the myofibroblast differentiation of MRC-5 cells induced by Ang II via Epac1 activating.

[Effect of N-acetyl-seryl-aspartyl-lysyl-proline on regulation of expression of ras-raf-ERK1/2 signal transduction pathway in lung of rats with silicosis].

OBJECTIVE: to investigate the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on the expressions of c-Raf, ERK1/2 and TGF-ß1 in the lung of rats with silicosis, thus to investigate the regulating of AcSDKP on the Ras-Raf-ERK1/2 signal transduction pathway. METHODS: rats were instilled with silica through trachea as silicotic models and administered AcSDKP in the experiment. Rats were divided into 6 groups randomly, 10 rats in each group: Control 1 and 2 of silicotic model: each rat was intratracheally instilled with 1.0 ml normal sodium and was killed after 4 or 8 weeks; Silicotic model 1 and Silicotic model 2: each rat was intratracheally instilled with 1ml silica suspension and was killed after 4 or 8 weeks; Anti-fibrosis treatment of AcSDKP: after each rat was intratracheally instilled with 1ml silica suspension for 4 weeks, AcSDKP 800 microg × kg(-1) × d(-1) was administered into every rat and rats were killed at the eighth week; Preventing fibrosis treatment of AcSDKP: after AcSDKP 800 microg × kg(-1) × d(-1) was administered into every rat for 48 hours, each rat was intratracheally instilled with 1.0 ml silica suspension and rats were killed at the eighth week. The expression of c-Raf, phospho-c-Raf, ERK1/2, phospho-ERK1/2 and TGF-ß1 was measured by immunohistochemistry and western blot assay. RESULTS: compared with the corresponding control groups, the expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-ß1 increased in the lung tissue of the silicotic models. Compared with the corresponding model groups, after administration AcSDKP, the expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-ß1 in the lung tissue reduced obviously. In anti-fibrosis treatment of AcSDKP group, expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-ß1 decreased to 52.25%, 51.72% and 67.74% compared with those of the silicotic model 1, and expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-ß1 decreased to 49.37%, 55.76%, 65.63% compared with those of the silicotic model 2; In preventing fibrosis treatment of AcSDKP group, expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-ß1 decreased to 54.64%, 55.76% and 78.91% compared with those of the silicotic model 2 (P < 0.05) while the expressions of c-Raf and ERK1/2 were not different significantly among each groups. CONCLUSION: AcSDKP possibly plays an important role in anti-silicotic fibrosis by blocking the TGF-ß-induced Ras-Raf-ERK1/2 signal transduction pathway.