Uncovering the Role of Anmeidan against Atherosclerosis from Integrated Network Pharmacology and Pharmacological Experiments.

BACKGROUND: Atherosclerosis (AS) is the leading cause of mortality in elderly individuals worldwide. Anmeidan (AMD) is a Traditional Chinese Medicine (TCM) formula composed of many herbs, many of which have been accepted for treating AS. This study aimed to explore whether AMD can inhibit the progress of AS and its possible mechanism. METHODS: ApoE-/- mice were used to establish the AS model and evaluate the therapeutic effect of AMD on AS. Based on network pharmacology technology, the potential mechanism of AMD for treating AS was explored, and lipid metabolism pathways related to AS were mainly studied. Next, the effects of AMD on liver lipid levels, antioxidant capacity, liver tissue morphology, and gene expression related to lipid metabolism in ApoE-/- mice were investigated. Cellular experiments were performed to confirm the lipid-lowering effect of AMD. Finally, the AMD composition was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: In ApoE-/- mice, AMD effectively alleviated AS by reducing serum total cholesterol, triglyceride, low-density lipoprotein levels, and plaque area, and increasing high-density lipoprotein levels. Network pharmacology indicated that AMD may suppress AS by regulating lipid metabolism pathways with multiple TCM components, which is consistent with the results of in vivo experiments and LC-MS/MS component identification. AMD significantly reduced liver lipid aggregation, intensified antioxidant enzyme activity, and upregulated the mRNA levels of ABCA1, ABCG1, and LDLR with increased cholesterol efflux. In addition, AMD decreased cholesterol levels in foam cells. CONCLUSIONS: This study confirmed that AMD could treat AS by regulating lipid metabolism and preliminarily explored the related mechanism. These findings provide new ideas for the treatment of AS with TCM.

Elucidation the mechanism of the active ingredient imperatorin promoting drug absorption in cell model.

Imperatorin (IMP) is the main bioactive furanocoumarin of Angelicae dahuricae radix, which is a well-known traditional Chinese medicine. The purpose of this study was to elucidate the role of IMP in promoting absorption and the possible mechanism on the compatible drugs of Angelicae dahuricae radix. The influence of IMP on drugs' intestinal absorption was conducted by the Caco-2 cell model. The mechanism was studied by investigating the transcellular transport mode of IMP and its influence on P-glycoprotein (P-gp)-mediated efflux, protein expression of P-gp and tight junction, and cell membrane potential. The result showed IMP promoted the uptake of osthole, daidzein, ferulic acid, and puerarin and improved the transport of ferulic acid and puerarin in Caco-2 cells. The absorption-promoting mechanism of IMP might involve the reduction of the cell membrane potential, decrease of P-gp-mediated drug efflux and inhibition of the P-gp expression level in the cellular pathway, and the loosening of the tight junction protein by the downregulation of the expression levels of occludin and claudin-1 in the paracellular pathway. This study provides new insights into the understanding of the improved bioavailability of Angelicae dahuricae radix with its compatible drugs.

Customizable Dual-Fluorescent Nanoparticles for Tracing and Quantifying of Cell Transport.

Purpose: Nanotechnology-based drug delivery systems (nano-DDS) have been developed to be a promising strategy to improve the efficacy, safety, physicochemical and pharmacokinetic/pharmacodynamics properties of drugs. It is very necessary to elucidate the delivery process in vivo or in cells for the rational design and accurate preparation of nano-DDS. The aim of this study was to construct a nano-DDS to visualize and quantify the intracellular behavior of the loaded cargo and carrier in such a system. Methods: A carboxyl-terminal end of poly(lactic-co-glycolic acid) polymer was fluorescently labeled with rhodamine B by conjugation of ethylenediamine. Dual-fluorescent nanoparticles (DFPs) were prepared from this fluorescently labeled polymer to encapsulate a fluorescent cargo, coumarin 6. The carrier and cargo of DFPs were monitored by confocal fluorescence microscopy during cellular uptake. Furthermore, the transcellular transportation of DFPs was evaluated quantitatively by measuring the fluorescence intensity. Results: The obtained fluorescent polymer showed stable and quantifiable characteristics. DFPs could be customized in terms of coumarin 6 content (97.7±1.0%), size (367.3±1.7 nm) and dual-emission fluorescence (green cargo and red carrier). DFPs did not significantly affect cell viability, the integrity of cell monolayers and the microscopic morphology at concentrations below 0.7 mg/mL within 3 h of co-incubation with Caco-2 cells. Multichannel fluorescence monitoring revealed that the fluorescence intensity of the carrier and cargo increased with time, but not synchronously. By calculating the residual, intracellular, and transport amounts of DFPs, the material balance between the total amount of cellular transport and the dose administered was obtained. Conclusion: Based on the advantages of dual fluorescent labeling, the differential behavior of cell trafficking can be visualized and quantitatively analyzed for the cargo and carrier of DFPs. These results provide insights into the cellular transport process of holistic nanoparticles and complement our understanding of the biological behaviors of nano-DDS.

Comparison of intestinal absorption characteristics between rhubarb traditional Chinese medicine preparation and activity ingredients using in situ and in vitro model.

Objective: The intestinal absorption characteristics of active ingredients are very important for oral administration of traditional Chinese medicine (TCM) to achieve the desired therapeutic effect. However, a deeper understanding about active ingredients absorption characteristics is still lack. The aim of this study was to investigate the absorption properties and mechanism of rhubarb active ingredients in TCM preparation and pure form. Methods: The intestinal absorption behavior of active ingredients in Shenkang extract (SKE) and rhubarb anthraquinone ingredients (RAI) were investigated by in situ single-pass intestinal perfusion model. And the bidirectional transport characteristics of these active ingredients were assessed by in vitro Caco-2 cell monolayer model. Results: In situ experiment on Sprague-Dawley rats, the effective permeability coefficient values of aloe-emodin, emodin and chrysophanol in RAI were higher than those in SKE, and the value of rhein in RAI was lower than that in SKE. But the easily absorbed segments of intestine were consistent for all ingredients, whether in SKE or in RAI. In vitro experiment, the apparent permeability coefficient values of rhein, emodin and chrysophanol in RAI were higher than those in SKE, and this value of aloe-emodin in RAI was lower than that in SKE. But their efflux ratio (ER) values in SKE and RAI were all similar. Conclusion: Four rhubarb anthraquinone ingredients in SKE and RAI have similar absorption mechanism and different absorption behavior, and the microenvironment of the study models influenced their absorption behavior. The results may provide an aid for understanding of the absorption characteristics of the TCM active ingredients in complex environments and the complementarities of different research models.

A Review on Increasing the Targeting of PAMAM as Carriers in Glioma Therapy.

Glioma is an invasive brain cancer, and it is difficult to achieve desired therapeutic effects due to the high postoperative recurrence rate and limited efficacy of drug therapy hindered by the biological barrier of brain tissue. Nanodrug delivery systems are of great interest, and many efforts have been made to utilize them for glioma treatment. Polyamidoamine (PAMAM), a starburst dendrimer, provides malleable molecular size, functionalized molecular structure and penetrable brain barrier characteristics. Therefore, PAMAM-based nanodrug delivery systems (PAMAM DDS) are preferred for glioma treatment research. In this review, experimental studies on PAMAM DDS for glioma therapy were focused on and summarized. Emphasis was given to three major topics: methods of drug loading, linkers between drug/ligand and PAMAM and ligands of modified PAMAM. A strategy for well-designed PAMAM DDS for glioma treatment was proposed. Purposefully understanding the physicochemical and structural characteristics of drugs is necessary for selecting drug loading methods and achieving high drug loading capacity. Additionally, functional ligands contribute to achieving the brain targeting, brain penetration and low toxicity of PAMAM DDS. Furthermore, a brilliant linker facilitates multidrug combination and multifunctional PAMAM DDS. PAMAM DDS show excellent promise as drug vehicles and will be further studied for product development and safety evaluation.

Potential antiviral activity of isorhamnetin against SARS-CoV-2 spike pseudotyped virus in vitro.

Coronavirus Disease 2019 (COVID-19) cases and deaths are still rising worldwide, there is currently no effective treatment for severe inflammation and acute lung injury caused by new coronavirus (SARS-COV-2) infection. Therapies to prevent or treat COVID-19, including antiviral drug and several vaccines, are still being development. Human angiotensin-converting enzyme 2 (ACE2), expressing in lung, has been confirmed to be a receptor for SARS-COV-2 infection, interventions for attachment of spike protein of SARS-CoV-2 to ACE2 may be a potential approach to prevent viral infections and it is considered as a potential target for drug development. In this study, we observed that seabuckthorn and its flavonoid compounds quercetin and isorhamnetin were shown strong retention to ACE2 overexpression HEK293 (ACE2h ) cells by CMC analysis. Based on drug receptor interaction analysis and viral entry studies in vitro, we evaluated the interaction of two flavonoid compounds and ACE2 as well as the inhibitory effect of the two compounds on viral entry. Surface plasmon resonance assay proved the effect that isorhamnetin bound to the ACE2, and its affinity (KD value) was at the micromolar level, that was, 2.51 ± 0.68 µM. Viral entry studies in vitro indicated that isorhamnetin inhibited SARS-CoV-2 spike pseudotyped virus entering ACE2h cells. Based on promising in vitro results, we proposed isorhamnetin to be a potential therapeutic candidate compound against COVID-19.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus.

BACKGROUND: The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating . The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PURPOSE: The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. METHODS: In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. RESULTS: Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD = (7.31 ± 0.62)e-7 M and (4.82 ± 0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. CONCLUSIONS: CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.