OBJECTIVE To explore mecha-nisms of imperatorin on regulating P-glycoprotein(P-gp)in blood-brain barrier(BBB)based on net-work pharmacology combined with in vitro experi-ment.METHODS Drug targets were predicted using the Pharmapper and Swiss targets data-bases;disease targets were obtained through the Genecards database;intersections between drugs and disease targets were screened by Cytoscape software;the obtained core targets were used to construct protein-protein interaction(PPI)network,gene ontology(GO)functions,and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.The effects of imperatorin(20,50,100 μ mol·L-1)on P-gp activity were monitored in hCMEC/D3in vitro BBB model,and the effects of imperatorin on the expression of target proteins were verified using Western blot method.RESULTS 55 drug targets and 3102 disease targets were obtained from the network pharmacology screening,and 37 core targets were obtained after the combination.Enrichment analysis showed that core targets were closely related to chemical synaptic trans-mission regulation,neurotransmitter receptor activity,proteinkinaseregulationactivity,G protein-coupled receptor signaling pathway,neural active ligand receptor interaction pathway,PI3K-Akt sig-naling pathway,VEGF signaling pathway,etc..In vitro experimental validation suggested that all tested concentration groups of imperatorin signifi-cantly reduced the activity and expression of P-gp,which were achieved by significantly downregu-lating the phosphorylation levels of PI3K and Akt,and repressing the expression of VEGFR2 pro-tein.CONCLUSION Network pharmacology was used to predict the core targets and signaling pathways of imperatorin on regulating P-gp in BBB and relevant validation was conducted through in vitro experiments,providing a refer-ence basis for further exploration of the mecha-nisms of imperatorin on regulating P-gp in BBB.

Necrosis is a form of cell death, which is related to various serious diseases such as cardiovascular disease, cancer, and neurodegeneration. Necrosis-avid agents (NAAs) selectively accumulated in the necrotic tissues can be used for imaging and/or therapy of related diseases. The aim of this study was to preliminarily investigate necrosis avidity of I-evans blue (I-EB) and its mechanism. The biodistribution of I-EB at 24 h after intravenous administration showed that the radioactivity ratio of necrotic to viable tissue was 3.41 in the liver and 11.82 in the muscle as determined by counting in model rats. Autoradiography and histological staining displayed preferential uptake of I-EB in necrotic tissues. nuclear extracts from necrotic cells exhibited 82.3% of the uptake in nuclei at 15 min, as well as 79.2% of the uptake at 2 h after I-EB incubation. The DNA binding study demonstrated that evans blue (EB) has strong binding affinity with calf-thymus DNA (CT-DNA) (=5.08×10 L/(mol/L)). Furthermore, the accumulation of I-EB in necrotic muscle was efficiently blocked by an excess amount of unlabeled EB. In conclusion, I-EB can not only detect necrosis by binding the DNA released from necrotic cells, but also image necrotic tissues generated from the disease clinically.

Small interfering RNA (siRNA) has a promising future in the treatment of ocular diseases due to its high efficiency, specificity, and low toxicity in inhibiting the expression of target genes and proteins. However, due to the unique anatomical structure of the eye and various barriers, delivering nucleic acids to the retina remains a significant challenge. In this study, we rationally design PACD, an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block (A), a siRNA binding block (B) and a pH-responsive block (C). PACDs can self-assemble into nanosized polymeric micelles that compact siRNAs into polyplexes through simple mixing. By evaluating its pH-responsive activity, gene silencing efficiency in retinal cells, intraocular distribution, and anti-angiogenesis therapy in a mouse model of hypoxia-induced angiogenesis, we demonstrate the efficiency and safety of PACD in delivering siRNA in the retina. We are surprised to discover that, the PACD/siRNA polyplexes exhibit remarkable intracellular endosomal escape efficiency, excellent gene silencing, and inhibit retinal angiogenesis. Our study provides design guidance for developing efficient nonviral ocular nucleic acid delivery systems.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.

Metastasis is crucial for the mortality of non-small cell lung carcinoma (NSCLC) patients. The epithelial-mesenchymal transition (EMT) plays a critical role in regulating tumor metastasis. Glioma-associated oncogene 1 (Gli1) is aberrantly active in a series of tumor tissues. However, the molecular regulatory relationships between Gli1 and NSCLC metastasis have not yet been identified. Herein, we reported Gli1 promoted NSCLC metastasis. High Gli1 expression was associated with poor survival of NSCLC patients. Ectopic expression of Gli1 in low metastatic A549 and NCI-H460 cells enhanced their migration, invasion abilities and facilitated EMT process, whereas knock-down of Gli1 in high metastatic NCI-H1299 and NCI-H1703 cells showed an opposite effect. Notably, Gli1 overexpression accelerated the lung and liver metastasis of NSCLC in the intravenously injected metastasis model. Further research showed that Gli1 positively regulated Snail expression by binding to its promoter and enhancing its protein stability, thereby facilitating the migration, invasion and EMT of NSCLC. In addition, administration of GANT-61, a Gli1 inhibitor, obviously suppressed the metastasis of NSCLC. Collectively, our study reveals that Gli1 is a critical regulator for NSCLC metastasis and suggests that targeting Gli1 is a prospective therapy strategy for metastatic NSCLC.