The role of FGF21 in inhibition of heart failure with empagliflozin and related mechanisms / 药学学报

The aim of this study is to investigate the role of fibroblast growth factor 21 (FGF21) in empagliflozin (EMP) in treatment of heart failure and the related mechanisms. FGF21 knockout (FGF21 KO) and littermate wild-type (WT) mice induced by doxorubicin (Dox) were used to establish heart failure mouse model in vivo. The experiment process and animal welfare follow the regulations of Animal Ethics Committee of Hefei University of Technology strictly. The results suggest that Dox (5 mg·kg-1) induced typical heart failure symptoms in both WT and FGF21 KO mice. In WT mice, EMP (10 mg·kg-1) significantly improved Dox-induced cardiac atrophy, decreased myocardial systolic function, decreased left ventricular ejection fraction and shortened fraction; EMP treatment also significantly inhibited the increase of Dox-induced cardiotoxicity indexes (aspartate amino transferase, creatine kinase, hydroxybutyrate dehydrogenase, lactate dehydrogenase) in mice. Dox induced cardiac fibrosis, inflammation and oxidative stress were also significantly improved by EMP. However, in FGF21 KO mice, the therapeutic effects of EMP on heart failure was significantly inhibited. The results suggest that the function of EMP in treating heart failure partly depends on the presence of FGF21, and the mechanism may be related to the effect of FGF21 on improving fibrosis, inflammation and oxidative stress.

Preparation and characterization of linolenic acid-chitosan micelle for doxorubicin oral delivery and its in situ intestinal absorption in rats / 药学学报

In this study, a novel oral drug delivery system based on linolenic acid-modified chitosan (CS-LA) micelle was developed to improve the oral bioavailability of doxorubicin (DOX), which was proven by its in vivo intestinal absorption in rats. The DOX-loaded CS-LA micelles (CS-LA@DOX) were prepared by the dialysis method. The synthesized micelle material was identified by proton nuclear magnetic resonance spectroscopy (1H-NMR) and Fourier transform infrared spectroscopy (FT-IR). A series of the micelle properties, including particle size distribution, zeta potential, encapsulation efficiency (EE), drug loading (DL), micromorphology, polymorphy, and critical micelle concentration (CMC) were characterized or tested. The in vitro release of micelles was observed by the dialysis method, and the absorption-promoting effect of micelles was investigated by intestinal circulation experiments in rats. The animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Guilin Medical University. The results of 1H-NMR and FT-IR showed that CS and LA were covalently bound via an amide linkage. The DOX encapsulated in the micelle core was in an amorphous state. The as-prepared micelles in the transmission electron microscope (TEM) image showed regular spherical shapes and uniform sizes with a series of excellent characteristics including (119.2 ± 2.1) nm of mean particle size [polymer dispersity index (PDI), 0.190 ± 0.08], +12.1 mV of zeta potential, (70.23 ± 0.74) % of EE, (8.77 ± 0.02) % of DL and 51.75 μg·mL-1 of CMC. Compared with the reference, DOX hydrochloride, the proposed micelle drug delivery system showed an obvious sustained-release effect in vitro release; and enhanced drug absorption in the small intestine of rats.

Highly penetrable nanoparticles co-loading doxorubicin and IDO1 siRNA enhance tumor immunotherapy / 药学学报

There are two serious obstacles to tumor immunotherapy. Firstly, the immune response of the tumor is seriously reduced due to immunosuppressive tumor microenvironment (ITM) and low immunogenicity of tumor. The second obstacle is the dense and complex heterogeneous structures, which seriously prevent the nanoparticles (NPs) from penetrating deeper into tumor tissue. Immunogenic cell death (ICD) induced by doxorubicin (DOX) is an effective method to enhance tumor immune activity. However, interferon-γ (IFN-γ) secreted by cytotoxic T lymphocytes (CTL) after ICD induction would increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and enhance ITM. IDO1 siRNA would reduce the expression of IDO1 protein, regulate the tumor immunosuppressive microenvironment and regulate ITM, so as to enhance the ICD effect of DOX. In this paper, a novel charge conversional, particle size reduction and highly penetrable NPs based on a pH sensitive copolymer poly(ethylene glycol)-poly-L-lysine-2,3-dimethylmaleic anhydride (mPEG-PLL-DMA, PLD) and polyamidoamine (PAMAM) dendrimers to achieve deep delivery of tumor tissue. DOX and IDO1 siRNA were encapsulated to achieve efficient tumor immunotherapy. Preparation and cell level experiments showed that PLD material had significant pH sensitivity. Results of 3D tumor penetrable experiment in vitro showed that adding the pH sensitive material PLD significantly improved the permeability of the preparation. In addition, 4T1 tumor model was established for BALB/c mice and all animal experiments were displayed in according with the requirements of the Animal Experiment Ethics Committee of Shenyang Pharmaceutical University. The results of in vivo efficacy experiments and tissue experiments evaluated that IDO1 siRNA significantly improved the ICD effect owing to DOX, so as to significantly inhibit tumor growth.

Preparation of lipid membrane-wrapped nanoparticles loaded with metformin polymer and doxorubicin and evaluation of their therapeutic effect on breast cancer / 药学学报

In this study, the lipid membrane-wrapped nanoparticles loaded with metformin polymer (PolyMet) and doxorubicin (DOX) was prepared and then evaluated therapeutic effect on breast cancer. An anionic chain PGA-DOX based on γ-polyglutamic acid (PGA) with DOX was synthesized via amidation reaction and characterized by 1H NMR. The PGA-DOX and PolyMet were loaded via electrostatic attraction to prepare the co-delivery nanoparticles system (PolyMet-DOX-NPs). Then, PolyMet-DOX-NPs were coated with cationic liposome membrane to form the core-membrane structural system (PolyMet-DOX-lipid-nanoparticles, PolyMet-DOX-LNPs). The structure and morphology of PolyMet-DOX-LNPs were observed by transmission electron microscope. The particle size, zeta potential, encapsulation efficiency (EE), drug loading (DL), release behavior in vitro of PolyMet-DOX-LNPs were investigated. The MTT assay was used to examine the cytotoxicity of PolyMet combined with DOX on 4T-1 cells. The 4T1Fluc tumor-bearing mice model was used to evaluate the therapeutic efficacy of PolyMet-DOX-LNPs in vivo. All animal experiments were performed in line with ethical standards and approved by the Animal Experiments Ethical Committee of Zhejiang Chinese Medical University. 1H NMR spectrum showed that PGA-DOX was successfully synthesized with DOX grafting rate of (72.03 ± 1.29) %. The EE and DL of PolyMet-DOX-LNPs was (72.76 ± 1.92) % and (1.16 ± 0.12) %, respectively. PolyMet-DOX-LNPs exhibited a suitable size of (159.3 ± 7.4) nm and positive charge of (+36.3 ± 1.9) mV with good spheroidal morphology and dispersibility. The release profiles in vitro showed that PolyMet-DOX-LNPs exhibited a slowly and maintained release behavior at physiological pH value (pH 7.4) within 48 h. Further studies showed that PolyMet combined with DOX could synergistically enhance the cytotoxicity on 4T-1 cells. Bioluminescence imaging (BLI) result showed that the luminescence signal intensity of 4T-1Fluc cells was reduced after treatment with PolyMet-DOX-LNPs and the tumor volume growth was also inhibited. Additionally, the H&E staining and changes of body weight showed that PolyMet could reduce the toxicity of DOX. To sum up, PolyMet has a good synergistic effect with DOX in the treatment of breast cancer, which provide the foundation for this novel metformin polymer on the anti-tumor application.

Exploring the molecular mechanism of aqueous extract from Huang qi on doxorubicin induced nephropathy in rats using transcriptome sequencing technique / 药学学报

We studied the effect of aqueous extract from Huang qi on gene expression profile of doxorubicin induced nephropathy in rats, and explored the molecular mechanism of the intervention. The gene expression profiles of control group, model group and aqueous extract from Huang qi group were detected by using transcriptome sequencing technique. The differentially expressed genes (DEGs) were screened by STEM trend analysis software. GO function enrichment and KEGG pathway analysis were performed for DEGs, and the gene expression level was verified by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that, compared with the control group, 432 DEGs were obtained in doxorubicin nephropathy model group; compared with the model group, 811 DEGs were obtained due to aqueous extract of Huang qi. The results of GO function enrichment and KEGG enrichment analysis indicated that PI3K-AKT pathway (Col6a6, Nr4a1, Sgk1, Gng7) and lipid metabolism-related genes (Cpt1b, Pcsk9, Abca1, Ascm5) were the key pathways and genes in the treatment of doxorubicin induced nephropathy by aqueous extract from Huang qi, which played a protective role in kidney. In conclusion, the molecular mechanism of aqueous extract from Huang qi in protection against doxorubicin induced nephropathy rats is closely related to apoptosis-related genes and lipid metabolism-related genes, suggesting for the need of follow-up study for key gene validation and mechanism of action of aqueous extract from Huang qi for prevention of doxorubicin induced nephropathy.

Mechanisms of doxorubicin resistance of breast cancer MCF-7 cells mediated by exosomes / 药学学报

This study aimed to explore the roles of exosomes in doxorubicin-resistance in breast cancer cells. Using breast cancer parental cell line (MCF-7), doxorubicin-resistant cell line (MCF-7/ADR) and sensitive cell line co-cultured with doxorubicin-resistant supernatant (MCF-7/EXO) as models, the effects of doxorubicin on proliferation or apoptosis of MCF-7, MCF-7/EXO and MCF-7/ADR cells were detected by CCK8, and light or fluorescent microscopy. Exosomes in the supernatants of cell culture were extracted by ultracentrifugation, and the quantity of exosomes was determined by transmission electron microscopy, BCA and DiI labeling assay. Expression levels of exosome-specific biomarkers CD63 and Flotillin-1 were detected by Western blot. The uptake of MCF-7/ADR cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy. Western blot was used to detect the expression levels of multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1) in all three cell strains. Cell proliferation assays showed that IC50 of MCF-7/EXO cells to doxorubicin was 0.83 ± 0.09 μmol·L-1, which was significantly higher than 0.15 ± 0.05 μmol·L-1 (P<0.01) of MCF-7 cells, suggesting 5.5 times of increase in drug resistance. Apoptosis of MCF-7 cells was induced after doxorubicin treatment (P<0.001), but MCF-7/EXO cells were not significantly different (P>0.05). Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells (P<0.05). PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells. Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells. Taken together, these results indicate that exosomes of doxorubicin-resistant breast cancer cells can transmit drug resistance to sensitive cells, and the underlying mechanism may involve ABCB1 protein transport mediated by exosomes.

Smart fluorescent nano-delivery system for breast cancer cell tracing and growth inhibition / 药学学报

This study aimed to construct an intelligent fluorescent nanocarrier for tumor cell tracing. Doxorubicin (DOX) was used as a model drug, and the gene targeting siBcl-2 was loaded to achieve synergistic inhibition of tumor cells. Mesoporous silicon nanoparticles (MSN) were prepared by a sol-gel method, and acetaldehyde cystine (AC) and polyethyleneimine (PEI) were covalently modified. The prepared nanocarrier MSN-AC-PEI was uniformly dispersed, with a particle size of 235.53 nm and a potential of 14.63 mV. The carrier material MSN-AC-PEI could load siRNA with the mass ratio of 60∶1 (Wvectors∶WsiRNA) and protect siRNA from RNase I degradation. To simulate the microenvironment of tumor, DOX release in phosphate buffer (pH 5) including 10 mmol·L-1 glutathione (GSH) was investigated. The cumulative release rate of DOX at 120 h is 35 times that of the normal physiological environment, which lays the foundation for the intelligent release of DOX in tumor cells. The results of cell experiments showed that the carrier material MSN-AC-PEI had significant green fluorescence, and the traceability can be maintained for 24 h after taken up by MCF-7 cells. After 24 hours of administration of the nano drug delivery system MSN-AC-PEI@DOX/siBcl-2, the inhibition rate of tumor cell proliferation reached 40.91%, and the late apoptosis rate was 60.84%. The Western blot results showed that compared with free DOX and siBcl-2, the nano-delivery system MSN-AC-PEI@DOX/siBcl-2 can significantly reduce the expression of anti-apoptotic protein Bcl-2, thereby enhancing its anti-tumor ability.

Delivery of miR-375 and doxorubicin hydrochloride by lipid bilayer coated hollow mesoporous silica nanoparticles for liver cancer therapy / 药学学报

This study was designed to prepare a novel lipid bilayer coated hollow mesoporous silica nanocarrier for co-delivery of gene drugs and chemotherapeutic drugs to enhance the inhibitory activity of antitumor drugs in hepatoma cells. Hollow mesoporous silica was synthesized by modified StÖber method. Lipid-fusion principle was used to prepare lipid-hollow co-loaded doxorubicin (DOX) and miR-375 (LHMSN-DOX/miR-375). Meanwhile, the morphology, particle size, surface potential, drug loading and release were characterized in vitro. The inhibition of cell proliferation, cell migration and invasion was then evaluated. The results indicated that the core-shell structure of LHMSN-DOX/miR-375 was clear with an intact outer lipid membrane and an ordered internal HMSN mesoporous structure. The drug release amount was pH responsive while the drug was rapidly released under simulated intracellular acidic conditions relative to normal physiological environment. Compared with free DOX, LHMSN-DOX/miR-375 can deliver DOX and miR-375 to liver cancer cells and inhibit the proliferation, migration and invasion of cells more effectively.