Autophagy-inhibiting biomimetic nanodrugs enhance photothermal therapy and boost antitumor immunity.

The instinctive protective stress responses of tumor cells hamper low-temperature photothermal therapy (LTPTT), resulting in tumor recurrence and metastasis. The rapid blood clearance and low-efficiency tumor enrichment of nanomedicines also decrease the efficacy of LTPTT. In this study, we fabricated coassembled photothermal agents (indocyanine green, ICG) and autophagy inhibitors (chloroquine, CQ) and red blood cell and cancer cell hybrid membrane (RCm)-camouflaged ICGCQ@RCm nanoparticles (ICGCQ@RCm NPs) to enhance tumor LTPTT. The ICGCQ@RCm NPs exhibited prolonged blood drug circulation and markedly enhanced drug accumulation in tumor tissues. The ICGCQ@RCm NPs reduced the thermal tolerance of tumor cells to sensitize ICG-mediated LTPTT by inhibiting protective autophagy. The ICGCQ@RCm NPs exerted strong immunogenic cell death (ICD) after efficient LTPTT to activate antitumor immunity. In addition, ICGCQ@RCms optimized the therapeutic efficacy by imaging-guided LTPTT, taking advantage of the near-infrared (NIR) fluorescence of ICG. Consequently, the ICGCQ@RCm NPs effectively inhibited tumors under mild LTPTT, significantly suppressed tumor metastasis and prolonged the survival time of tumor-bearing mice. Furthermore, the ICGCQ@RCm NPs showed high biosafety in vitro and in vivo. The ICGCQ@RCm NPs demonstrated tumor-targeting and imaging-guided autophagy inhibition-sensitized LTPTT using two Food and Drug Administration (FDA)-approved drugs, which have great potential for clinical application.

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases.

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 µM to 16.81 µM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 µM to 11.51 µM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Effects of Biejia Ruangan Tablet-containing serum on matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in cultured renal interstitial fibroblasts.

OBJECTIVE: To investigate the effects of Biejia Ruangan Tablet ([symbol in text], BRT)-containing serum on the expression of matrix metalloproteinase (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) in cultured renal interstitial fibroblasts. METHODS: Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose (7 g/kg), mid-dose (3.5 g/kg), and low-dose (1.75 g/kg) BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-ß1 (recombined human TGF-ß1), and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1. RESULTS: The high dose BRT-containing serum could decrease the type I and III procollagen gene expression which boosted by TGF-ß1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF-ß1 (P <0.05). Treatment of cells with recombined human TGF-ß1 had no significant effect on MMP-9 expression and BRT-containing serum also had no effect on MMP-9 expression. CONCLUSIONS: High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type I and III procollagen and TIMP-1 expression.

Bioactive metabolites produced by the endophytic fungus Phomopsis sp. YM355364.

A new compound, 16-acetoxycytosporone B (1), along with four known ones, dankasterone A (2), dankasterone B (3), 3beta,5alpha,9alpha-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (4), and cyclonerodiol oxide (5), were isolated from Phomopsis sp. YM355364, an endophytic fungus of Aconitum carmichaeli. Their structures were characterized by spectral analysis. Compound 2 exhibited significant inhibitory activity against influenza A/Thailand/Kan353/2004(H5N1) pseudovirus with all IC50 value of 3.56 microM. Compounds 1, 2, and 4 showed either moderate or weak antifungal activities against four pathogenic fungi.

Inhibition of the tubular epithelial-to-mesenchymal transition in vivo and in vitro by the Uremic Clearance Granule ().

OBJECTIVE: To investigate the effect of the Uremic Clearance Granule (UCG, ), a Chinese patent medicine, on tubular epithelial-to-mesenchymal transition (EMT) in a unilateral ureteral obstruction (UUO) model in vivo and transforming growth factor (TGF)-ß1 induced EMT of HK-2 cells in vitro. METHODS: In vivo study, 50 Sprague Dawley rats were divided into three groups: a sham operation group (n=10), a UUO group (n=20), and a UUO with UCG treatment group (n=20). The UCG was given at a dose of 4.5 g/kg body weight per day by gavage after surgery. In vitro study, HK-2 cells were cultured in 10% fetal bovine serum (FBS), 10% healthy rat serum, 10% FBS and TGF-ß1 (10 ng/mL), 10% healthy rat serum and TGF-ß1, or 10% rat serum containing the uremic clearance granule and TGF-ß1. The expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in kidney tissues and HK-2 cells were investigated by Western blot analysis and immunofluorescence staining. RESULTS: The rats of the UUO group showed obvious tubulointerstitial fibrosis, compared with the sham operation group rats. Tubulointerstitial fibrosis score was reduced by 17.5%±1.1% at day 7 and by 20.0%±1.2% at day 14 in the UCG-treated group, compared with the UUO group. The UCG could maintained expression of E-cadherin and suppressed expression of vimentin and α-SMA in kidney tissues of UUO rats at days 7 and 14, as determined by Western blot analysis and immunofluorescence staining. Rat serum containing the UCG partially inhibited TGF-ß1-induced fibroblast phenotype of HK-2 cells and maintained the epithelial morphology of HK-2 cells in vitro. This occurred partially through a reduction of vimentin expression and an increase of E-cadherin expression. CONCLUSION: These results suggest that the UCG prevents tubular EMT and may be a promising agent for treating tubulointerstitial fibrosis.

Protective and antioxidant properties of wasp (Vespa magnifica) honeycomb extract: a potential inhibitor against acidified ethanol-induced gastric lesions.

OBJECTIVE: To examine the protective effects of wasp (Vespa magnifica) honeycomb extract (WCE) against gastric lesions in rats induced by 60% acidified ethanol, and evaluate its capacity to suppress oxidative stress in the gastric tissue. METHODS: Wistar rats were subjected to intragastric administration of 60% acidified ethanol to induce gastric lesions following an 8-day oral pretreatment with WCE at 0, 25, 100 and 150 mg/kg or with saline. The levels of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, myeloperoxidase (MPO) activity and total antioxidant capacity in the gastric tissues were determined. RESULTS: Oral administration of 25, 100 and 150 mg/kg WCE prior to 60% acidified ethanol administration significantly inhibited the formation of gastric lesions (with a reduction by 44.2%-87.1%), decreased the mucosal MPO activity (by 16.4%-56.6%) and increased the total antioxidant capacity of the gastric tissue (by 0.5, 1.47 and 1.83 folds, respectively) in a dose-dependent manner. At a high concentration (above 1 mg/ml), WCE also exhibited a stronger DPPH radical scavenging activity than butylated hydroxytoluene (BHT). CONCLUSION: The ethanol extract of wasp honeycombs can suppress the formation of acidified ethanol-induced gastric lesions by reducing free radical oxidation and neutrophils infiltration in the gastric tissue in rats.

Strategies for efficient repetitive production of succinate using metabolically engineered Escherichia coli.

Escherichia coli AFP111, a pflB, ldhA, ptsG triple mutant of E. coli W1485, can be recovered for additional succinate production in fresh medium after two-stage fermentation (an aerobic growth stage followed by an anaerobic production stage). However, the specific productivity is lower than that of two-stage fermentation. In this study, three strategies were compared for reusing the cells. It was found when cells were aerobically cultivated at the end of two-stage fermentation without supplementing any carbon source, metabolites (mainly succinate and acetate) could be consumed. As a result, enzyme activities involved in the reductive arm of tricarboxylic acid cycle and the glyoxylate shunt were enhanced, yielding a succinate specific productivity above g⁻¹(DCW)h⁻¹ and a mass yield above 0.90 g g⁻¹ in the subsequent anaerobic fermentation. In addition, the intracellular NADH of cells subjected to aerobic cultivation with metabolites increased by more than 3.6 times and the ratio of NADH to NAD+ increased from 0.4 to 1.3, which were both favorable for driving the TCA branch to succinate.

Succinic acid production with metabolically engineered E. coli recovered from two-stage fermentation.

Escherichia coli AFP111 cells recovered from spent two-stage fermentation broth were investigated for additional production of succinic acid under anaerobic conditions. Recovered cells produced succinic acid in an aqueous environment with no nutrient supplementation except for glucose and MgCO(3). In addition, initial glucose concentration and cell density had a significant influence on succinic acid mass yield and productivity. Although the final concentration of succinic acid from recovered cells was lower than from two-stage fermentation, an average succinic acid mass yield of 0.85 g/g was achieved with an average productivity of 1.81 g/l h after three rounds of recycling, which was comparable to two-stage fermentation. These results suggested that recovered cells might be reused for the efficient production of succinic acid.

[A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins].

OBJECTIVE: To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors. METHODS: HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method. RESULTS: No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner. CONCLUSION: We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.

[Study of the mechanism of caffeoyl glucopyranoses in inhibiting HIV-1 entry using pseudotyped virus system].

OBJECTIVE: To investigate the inhibitory activities of caffeoyl glucopyranoses purified from Balanophora japonica Makino on HIV entry and their mechanism. METHODS: HIV-1 Env pseudovirus was used to evaluate the anti-HIV-1 activity of those compounds. ELISA and molecular docking were used to study the mechanism of the actions of the active compounds. RESULTS: We used the HIV-1 Env pseudovirus to test the anti-HIV-1 activity of the six phenolic compounds (final concentration 25 microg/ml), and found that only 1,2,6-Tri-O-caffeoyl-beta-D-glucopyranose (TCGP) and 1,3-Di-O-caffeoyl-4-O-galloyl-beta-D- glucopyranose (DCGGP) could effectively inhibit the entry of HIV-1 Env pseudovirus into the target cells in a dose-dependent manner, with IC(50) values of 5.5-/+0.2 and 5.3-/+0.1 microg/ml, respectively. These two compounds could also blocked the gp41 six-helix bundle formation. Molecular docking analysis suggested that they might bind to the hydrophobic cavity of the gp41 N-trimeric coiled-coil. CONCLUSION: TCGP and DCGGP are potent HIV-1 entry inhibitors targeting gp41 and can serve as lead compounds for developing novel anti-HIV-1 microbicides for prevention of sexual HIV-1 transmission.

[Isolation and characterization of the anti-HIV active component from Eucommia ulmoides].

OBJECTIVE: To investigate the anti-HIV effects of Eucommia ulmoides Oliver, so as to provide experimental basis for searching a new efficacious drug for treatment of AIDS. METHODS: Using phytochemistry to isolate compounds from Eucommia ulmoides Oliver, the inhibitory activity of Samples on the HIV gp41 six-helix bundle formation was determined by a modified sandwich ELISA and PAGE. RESULTS: The Samples from Eucommia ulmoides Oliver had potent inhibitory activity on the HIV gp41 six-helix bundle formation. CONCLUSION: Eucommia uloides Oliver can inhibit HIV by targeting HIV gp41.