Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional liquid chromatography-mass spectrometry based on a novel phthalic anhydride bonded stationary phase.

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional liquid chromatography-mass spectrometry was developed. In this two-dimensional liquid chromatography system, a novel phthalic anhydride-bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional liquid chromatography-mass spectrometry, this online two-dimensional liquid chromatography-mass spectrometry system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines.

Chromium Complexes Supported by Salen-Type Ligands for the Synthesis of Polyesters, Polycarbonates, and Their Copolymers through Chemoselective Catalysis.

Salen, Salan, and Salalen chromium (III) chloride complexes have been investigated as catalysts for the ring-opening copolymerization reactions of cyclohexene oxide (CHO) with CO2 and of phthalic anhydride (PA) with limonene oxide (LO) or cyclohexene oxide (CHO). In the production of polycarbonates, the more flexible skeleton of salalen and salan ancillary ligands favors high activity. Differently, in the copolymerization of phthalic anhydride with the epoxides, the salen complex showed the best performance. Diblock polycarbonate-polyester copolymers were selectively obtained by one-pot procedures from mixtures of CO2, cyclohexene oxide, and phthalic anhydride with all complexes. In addition, all chromium complexes were revealed to be very active in the chemical depolymerization of polycyclohexene carbonate producing cyclohexene oxide with high selectivity, thus offering the opportunity to close the loop on the life of these materials.

Zr(IV) Catalyst for the Ring-Opening Copolymerization of Anhydrides (A) with Epoxides (B), Oxetane (B), and Tetrahydrofurans (C) to Make ABB- and/or ABC-Poly(ester-alt-ethers).

Poly(ester-alt-ethers) can combine beneficial ether linkage flexibility and polarity with ester linkage hydrolysability, furnishing fully degradable polymers. Despite their promising properties, this class of polymers remains underexplored, in part due to difficulties in polymer synthesis. Here, a catalyzed copolymerization using commercially available monomers, butylene oxide (BO)/oxetane (OX), tetrahydrofuran (THF), and phthalic anhydride (PA), accesses a series of well-defined poly(ester-alt-ethers). A Zr(IV) catalyst is reported that yields polymer repeat units comprising a ring-opened PA (A), followed by two ring-opened cyclic ethers (B/C) (-ABB- or -ABC-). It operates with high polymerization control, good rate, and successfully enchains epoxides, oxetane, and/or tetrahydrofurans, providing a straightforward means to moderate the distance between ester linkages. Kinetic analysis of PA/BO copolymerization, with/without THF, reveals an overall second-order rate law: first order in both catalyst and butylene oxide concentrations but zero order in phthalic anhydride and, where it is present, zero order in THF. Poly(ester-alt-ethers) have lower glass-transition temperatures (-16 °C < Tg < 12 °C) than the analogous alternating polyesters, consistent with the greater backbone flexibility. They also show faster ester hydrolysis rates compared with the analogous AB polymers. The Zr(IV) catalyst furnishes poly(ester-alt-ethers) from a range of commercially available epoxides and anhydride; it presents a straightforward method to moderate degradable polymers' properties.

Biodegradable controlled-release polymer containing butylidenephthalide to treat a recurrent cervical spine glioblastoma with promising result: a compassionate trial report.

Intramedullary spinal glioblastoma multiforme (GBM) tends to recur within 11 months of surgical resection, even after adjuvant chemoradiation therapy. Treatment options for recurrent spinal GBM are often limited. (Z)-n-butylidenephthalide [(Z)-BP] is a natural compound that induces apoptosis, antiproliferation, anti-invasion and antistemness effects in GBM cells. The Cerebraca wafer consists of (Z)-BP within a biodegradable wafer that can be implanted in the parenchyma of the central nervous system to treat high-grade glioma. We present a 44-year-old woman with a recurrent spinal GBM who underwent microscopic surgical tumor excision under fluorescein sodium guidance and intraoperative neurophysiologic monitoring. Four Cerebraca wafers were implanted into the cord and intradural space during the operation. MRI revealed that both tumor volume and spinal cord edema had decreased 4 days after surgery; both had substantially decreased 16 months after surgery. Neurologic functions and quality of life were improved after salvage therapy. No adverse events were reported. Cerebraca wafer implantation during surgical re-excision of spinal GBM may be a novel therapeutic approach for reduction of the tumor size and subsequent spinal cord edema with no toxicity to the spinal cord.

Accelerated synthesis of phthalimide derivatives: Intrinsic reactivity of diamines towards phthalic anhydride evaluated by paper spray ionization mass spectrometry.

RATIONALE: Paper spray (PS) is a simple and innovative ambient ionization technique for mass spectrometry (MS) analysis. Under PS-MS conditions, chemical reactions, which usually occur slowly on a bulk scale, are accelerated. Moreover, the formation of products and transient species can be easily monitored. In this manuscript, reactions between phthalic anhydride and diamines were conducted and monitored using a PS-MS platform. The reaction products (phthalimides) have many pharmaceutical applications, but their traditional syntheses can take hours under reflux, requiring laborious purification steps. METHODS: In situ reactions were performed by dropping methanolic solutions of phthalic anhydride and diamines on a triangular paper. The analyses were achieved by positioning the triangle tip in front of the mass spectrometer entrance, whereas a metal clip was attached to the triangle base. After adding methanol to the paper, a high voltage was applied across the metal clip, and the mass spectra were acquired. RESULTS: The intrinsic reactivity of alkyl and aromatic diamines was evaluated. The carbon chain remarkably influenced the reactivity of aliphatic diamines. For aryl diamines, the ortho isomer was the most reactive. Moreover, for aryl amines with electron-withdrawing substituents, no reaction was noticed. CONCLUSIONS: Taking advantage of the unique characteristics of PS-MS, it was possible to investigate the intrinsic reactivity of model alkyl (ethylene versus propylene) and aryl (o-phenylene versus m-phenylene and p-phenylene) diamines towards phthalic anhydride. Some crucial parameters that affect the intrinsic reactivity of organic molecules, such as isomerism, intramolecular interaction, and conformation, were easily explored.

Butylidenephthalide Abrogates the Snail-Induced Cancer Stemness in Oral Carcinomas.

Oral cancer is one of the most common cancers worldwide, especially in South Central Asia. It has been suggested that cancer stem cells (CSC) play crucial roles in tumor relapse and metastasis, and approaches to target CSC may lead to promising results. Here, aldehyde dehydrogenase 1 (ALDH1) and CD44 were utilized to isolate CSCs of oral cancer. Butylidenephthalide, a bioactive phthalide compound from Angelica sinensis, was tested for its anti-CSC effects. MTT assay showed that a lower concentration of butylidenephthalide was sufficient to inhibit the proliferation of patient-derived ALDH1+/CD44+ cells without affecting normal cells. Administration of butylidenephthalide not only reduced ALDH1 activity and CD44 expression, it also suppressed the migration, invasion, and colony formation abilities of ALDH1+/CD44+ cells using a transwell system and clonogenic assay. A patient-derived xenograft mouse model supported our in vitro findings that butylidenephthalide possessed the capacity to retard tumor development. We found that butylidenephthalide dose-dependently downregulated the gene and protein expression of Sox2 and Snail. Our results demonstrated that overexpression of Snail in ALDH1-/CD44- (non-CSCs) cells induced the CSC phenotypes, whereas butylidenephthalide treatment successfully diminished the enhanced self-renewal and propagating properties. In summary, this study showed that butylidenephthalide may serve as an adjunctive for oral cancer therapy.

Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs.

Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (ATXN3), which encodes the mutant ATXN3 protein. The pathological defects of SCA3 such as the impaired aggresomes, autophagy, and the proteasome have been reported previously. To date, no effective treatment is available for SCA3 disease. This study aimed to study anti-excitotoxic effects of n-butylidenephthalide by chemically insulted Purkinje progenitor cells derived from SCA3 iPSCs. We successfully generated Purkinje progenitor cells (PPs) from SCA3 patient-derived iPSCs. The PPs, expressing both neural and Purkinje progenitor's markers, were acquired after 35 days of differentiation. In comparison with the PPs derived from control iPSCs, SCA3 iPSCs-derived PPs were more sensitive to the excitotoxicity induced by quinolinic acid (QA). The observations of QA-treated SCA3 PPs showing neural degeneration including neurite shrinkage and cell number decrease could be used to quickly and efficiently identify drug candidates. Given that the QA-induced neural cell death of SCA3 PPs was established, the activity of calpain in SCA3 PPs was revealed. Furthermore, the expression of cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptotic pathway, and the accumulation of ATXN3 proteolytic fragments were observed. When SCA3 PPs were treated with n-butylidenephthalide (n-BP), upregulated expression of calpain 2 and concurrent decreased level of calpastatin could be reversed, and the overall calpain activity was accordingly suppressed. Such findings reveal that n-BP could not only inhibit the cleavage of ATXN3 but also protect the QA-induced excitotoxicity from the Purkinje progenitor loss.

The Protective Effects of n-Butylidenephthalide on Retinal Ganglion Cells during Ischemic Injury.

Clinically, acute ischemic symptoms in the eyes are one of the main causes of vision loss, with the associated inflammatory response and oxidative stress being the key factors that cause injury. Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common type of ischemic optic neuropathy (ION); however, there are still no effective or safe treatment options to date. In this study, we investigated the neuroprotective effects of n-butylidenephthalide (BP) treatment in an experimental NAION rodent model (rAION). BP (10 mg/kg) or PBS (control group) were administered on seven consecutive days in the rAION model. Rats were evaluated for visual function by flash visual evoked potentials (FVEPs) at 4 weeks after NAION induction. The retina and optic nerve were removed for histological examination after the rats were euthanized. The molecular machinery of BP treatment in the rAION model was analyzed using Western blotting. We discovered that BP effectively improves retinal ganglion cell survival rates by preventing apoptotic processes after AION induction and reducing the inflammatory response through which blood-borne macrophages infiltrate the optic nerve. In addition, BP significantly preserved the integrity of the myelin sheath in the rAION model, demonstrating that BP can prevent the development of demyelination. Our immunoblotting results revealed the molecular mechanism through which BP mitigates the neuroinflammatory response through inhibition of the NF-κB signaling pathway. Taken together, these results demonstrate that BP can be used as an exceptional neuroprotective agent for ischemic injury.

Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach.

Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.

Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Identification of the metabolites of n-butylidenephthalide in rat and human liver microsomes by liquid chromatography-high-resolution mass spectrometry.

n-Butylidenephthalide (NBDP) is one of the bioactive constituents originally isolated from Ligusticum chuanxiong Hort. The aim of this study was to study the metabolic profiles of NBDP in rat and human liver microsomes. NBDP was individually incubated with liver microsomes of rat and human at 37°C for 1 h and the samples incubated were analyzed by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry. The identities of the metabolites were identified by accurate masses, product ions and retention times. Under the current conditions, a total of 14 metabolites were detected and identified. M12, M13 and M14 were biosynthesized and unambiguously characterized by nuclear magnetic resonance spectroscopy. All the metabolites can be detected in rat liver microsomes, whereas in human liver microsomes, M1, M3, M4, M5, M6 and M7 were not detected. Our results demonstrated that the metabolic pathways of NBDP included hydroxylation, hydration, hydrolysis and glutathione conjugation. This study provides an overview of the metabolic profiles of NBDP in vitro, which is helpful to understand the action of this compound.

IL-32γ suppressed atopic dermatitis through inhibition of miR-205 expression via inactivation of nuclear factor-kappa B.

BACKGROUND: IL-32 is a novel cytokine involved in many inflammatory diseases. However, the role of IL-32γ, an isotype of IL-32, in atopic dermatitis (AD) has not been reported. OBJECTIVE: We investigated the effects of IL-32γ on development of AD and its action mechanisms. METHODS: We used phthalic anhydride (PA) and an MC903-induced AD model using wild-type and IL-32γ transgenic mice. We conducted the therapy experiments by using recombinant IL-32γ protein in a reconstructed human skin model and PA-induced model. We conducted a receiver operating characteristic analysis of IL-32γ with new AD biomarkers, IL-31 and IL-33, in serum from patients with AD. RESULTS: Dermatitis severity and epidermal thickness were significantly reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. The concentration of AD-related cytokines was reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. Subsequent analysis showed that IL-32γ inhibits miR-205 expression in PA- and MC903-induced skin tissue samples and TNF-α/IFN-γ-treated HaCaT cells. IL-32γ reduced NF-κB activity in skin tissue samples from PA- and MC903-induced mice and TNF-α/IFN-γ-treated HaCaT cells. NF-κB inhibitor treatment with IL-32γ expression further suppressed expression of inflammatory mediators as well as miR-205 in TNF-α/IFN-γ-treated HaCaT cells. Furthermore, recombinant IL-32γ protein alleviated AD-like inflammation in in vivo and reconstructed human skin models. Spearman correlation analysis showed that serum levels of IL-32γ and miR-205 were significantly concordant in patients with AD. CONCLUSION: Our results indicate that IL-32γ reduces AD through the inhibition of miR-205 expression via inactivation of NF-κB.

Microbial Synthesis and Evaluation of Fungistatic Activity of 3-Butyl-3-hydroxyphthalide, the Mammalian Metabolite of 3-n-Butylidenephthalide.

Phthalides are bioactive compounds that naturally occur in the family Apiaceae. Considering their potentially versatile applications, it is desirable to determine their physical properties, activity and metabolic pathways. This study aimed to examine the utility of whole-cell biocatalysts for obtaining 3-butyl-3-hydroxyphthalide, which is the metabolite formulated during mammalian metabolism of 3-n-butylidenephthalide. We performed transformations using 10 strains of fungi, five of which efficiently produced 3-butyl-3-hydroxyphthalide. The product yield, determined by high-performance liquid chromatography, reached 97.6% when Aspergillus candidus AM 386 was used as the biocatalyst. Increasing the scale of the process resulted in isolation yields of 29-45% after purification via reversed-phase thin layer chromatography, depending on the strain of the microorganism used. We proposed different mechanisms for product formation; however, hydration of 3-n-butylidenephthalide seems to be the most probable. Additionally, all phthalides were tested against clinical strains of Candida albicans using the microdilution method. Two phthalides showed a minimum inhibitory concentration, required to inhibit the growth of 50% of organisms, below 50 µg/mL. The 3-n-butylidenephthalide metabolite was generally inactive, and this feature in combination with its low lipophilicity suggests its involvement in the detoxification pathway. The log P value of tested compounds was in the range of 2.09-3.38.

n-Butylidenephthalide Modulates Autophagy to Ameliorate Neuropathological Progress of Spinocerebellar Ataxia Type 3 through mTOR Pathway.

Spinocerebellar ataxia type 3 (SCA3), a hereditary and lethal neurodegenerative disease, is attributed to the abnormal accumulation of undegradable polyglutamine (polyQ), which is encoded by mutated ataxin-3 gene (ATXN3). The toxic fragments processed from mutant ATXN3 can induce neuronal death, leading to the muscular incoordination of the human body. Some treatment strategies of SCA3 are preferentially focused on depleting the abnormal aggregates, which led to the discovery of small molecule n-butylidenephthalide (n-BP). n-BP-promoted autophagy protected the loss of Purkinje cell in the cerebellum that regulates the network associated with motor functions. We report that the n-BP treatment may be effective in treating SCA3 disease. n-BP treatment led to the depletion of mutant ATXN3 with the expanded polyQ chain and the toxic fragments resulting in increased metabolic activity and alleviated atrophy of SCA3 murine cerebellum. Furthermore, n-BP treated animal and HEK-293GFP-ATXN3-84Q cell models could consistently show the depletion of aggregates through mTOR inhibition. With its unique mechanism, the two autophagic inhibitors Bafilomycin A1 and wortmannin could halt the n-BP-induced elimination of aggregates. Collectively, n-BP shows promising results for the treatment of SCA3.

Host pre-conditioning improves human adipose-derived stem cell transplantation in ageing rats after myocardial infarction: Role of NLRP3 inflammasome.

Functional decline of stem cell transplantation in ageing hosts is well documented. The mechanism for this is poorly understood, although it is known that advancing age does not provide an optimal milieu for exogenous stem cells to survive, engraft and differentiate. We showed that n-butylidenephthalide improved human adipose-derived stem cell (hADSC) engraftment via attenuating the production of reactive oxygen species (ROS). It remained unclear whether pre-treated hosts with n-butylidenephthalide can rejuvenate the ageing heart and improve hADSC engraftment by regulating the ROS/NLRP3 inflammasome-mediated cardiac fibrosis after myocardial infarction. One hour after coronary ligation, hADSCs were transplanted into the hearts of young and ageing Wistar rats that were pre-treated with or without n-butylidenephthalide for 3 days. At day 3 after infarction, myocardial infarction was associated with an increase in ROS levels and NLRP3 inflammasome activity with age. hADSC transplant effectively provided a significant decrease in ROS levels, NLRP3 inflammasome activity, IL-1ß levels and cardiac fibrosis in either young or old infarcted rats. However, the beneficial effects of hADSCs were greater in young compared with old rats in terms of NLRP3 inflammasome activity. The infarcted ageing rats pre-conditioned by n-butylidenephthalide improved engraftment and differentiation of hADSCs and additionally attenuated cardiac fibrosis compared with hADSCs alone. The anti-inflammation effects of n-butylidenephthalide were reversed by SIN-1. In conclusions, the increased NLRP3 inflammasome activity plays the pathogenesis of ageing-related functional hADSC decline in the ageing hosts. n-butylidenephthalide-pre-treated ageing hosts reversibly ameliorate the harsh microenvironments, improve stem cell engraftment and attenuate cardiac fibrosis after myocardial infarction.

Safety Assessment of Trimellitic Anhydride Copolymers as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 6 trimellitic anhydride copolymers as used in cosmetics. These ingredients are related as copolymers in that they all share trimellitic anhydride (ie, 1,2,4-benzenetricarboxylic acid anhydride) as a monomer, are reported to function as film formers in cosmetics, and are reported to be primarily used in nail products. Very limited safety data were available or submitted. The Panel concluded that Adipic Acid/Neopentyl Glycol/Trimellitic Anhydride Copolymer and Phthalic Anhydride/Trimellitic Anhydride/Glycols Copolymer are safe in nail product formulations in the present practices of use and concentration, but the data are insufficient to make a determination of safety on the use of these 2 ingredients in all other types of cosmetic formulations. The Panel also concluded that the available data are insufficient to make a determination that the remaining trimellitic anhydride copolymers are safe for use in cosmetic formulations.

Liposome Consolidated with Cyclodextrin Provides Prolonged Drug Retention Resulting in Increased Drug Bioavailability in Brain.

Although butylidenephthalide (BP) is an efficient anticancer drug, its poor bioavailability renders it ineffective for treating drug-resistant brain tumors. However, this problem is overcome through the use of noninvasive delivery systems, including intranasal administration. Herein, the bioavailability, drug stability, and encapsulation efficiency (EE, up to 95%) of BP were improved by using cyclodextrin-encapsulated BP in liposomal formulations (CDD1). The physical properties and EE of the CDD1 system were investigated via dynamic light scattering, transmission electron microscopy, UV-Vis spectroscopy, and nuclear magnetic resonance spectroscopy. The cytotoxicity was examined via MTT assay, and the cellular uptake was observed using fluorescence microscopy. The CDD1 system persisted for over 8 h in tumor cells, which was a considerable improvement in the retention of the BP-containing cyclodextrin or the BP-containing liposomes, thereby indicating a higher BP content in CDD1. Nanoscale CDD1 formulations were administered intranasally to nude mice that had been intracranially implanted with temozolomide-resistant glioblastoma multiforme cells, resulting in increased median survival time. Liquid chromatography-mass spectrometry revealed that drug biodistribution via intranasal delivery increased the accumulation of BP 10-fold compared to oral delivery methods. Therefore, BP/cyclodextrin/liposomal formulations have potential clinical applications for treating drug-resistant brain tumors.

N-Butylidenephthalide Inhibits the Phenotypic Switch of VSMCs through Activation of AMPK and Prevents Stenosis in an Arteriovenous Fistula Rat Model.

The phenotypic switch of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of vascular disorders, such as atherosclerosis, stenosis and restenosis, after vascular intervention. In our previous study, n-butylidenephthalide (BP) was reported to have anti-proliferating and apoptotic effects on VSMCs. The purpose of the current study is to further investigate its role in platelet-derived growth factor (PDGF)-induced VSMC phenotypic modulation in an arteriovenous fistula model. In vitro, we observed that BP inhibited the PDGF-induced cytoskeleton reorganization of the VSMCs. The enhanced expression of vimentin and collagen, as well as the migration ability induced by PDGF, were also inhibited by BP. By cell cycle analysis, we found that BP inhibited the PDGF-induced VSMCs proliferation and arrested the VSMCs in the G0/G1 phase. In an arteriovenous fistula rat model, the formation of stenosis, which was coupled with a thrombus, and the expression of vimentin and collagen in VSMCs, were also inhibited by administration of BP, indicating that BP inhibited the PDGF-induced phenotypic switch and the migration of VSMCs. Besides, the inhibitory effects of BP on the phenotypic switch were found to accompany the activated 5' AMP-activated protein kinase (AMPK) as well as the inhibited phosphorylation of mTOR. Knockdown of AMPK by gene silencing conflicted the effects of BP and further exacerbated the PDGF-induced VSMCs phenotypic switch, confirming the modulating effect that BP exerted on the VSMCs by this pathway. These findings suggest that BP may contribute to the vasculoprotective potential in vasculature.

Antitumor Effects of N-Butylidenephthalide Encapsulated in Lipopolyplexs in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common type of cancer and the second most common cause of cancer-related death in the world. N-Butylidenephthalide (BP), a natural compound, inhibits several cancers, such as hepatoma, brain tumor and colon cancer. However, due to the unstable structure, the activity of BP is quickly lost after dissolution in an aqueous solution. A polycationic liposomal polyethylenimine and polyethylene glycol complex (LPPC), a new drug carrier, encapsulates both hydrophobic and hydrophilic compounds, maintains the activity of the compound, and increases uptake of cancer cells. The purpose of this study is to investigate the antitumor effects and protection of BP encapsulated in LPPC in CRC cells. The LPPC encapsulation protected BP activity, increased the cytotoxicity of BP and enhanced cell uptake through clathrin-mediated endocytosis. Moreover, the BP/LPPC-regulated the expression of the p21 protein and cell cycle-related proteins (CDK4, Cyclin B1 and Cyclin D1), resulting in an increase in the population of cells in the G0/G1 and subG1 phases. BP/LPPC induced cell apoptosis by activating the extrinsic (Fas, Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways. Additionally, BP/LPPC combined with 5-FU synergistically inhibited the growth of HT-29 cells. In conclusion, LPPC enhanced the antitumor activity and cellular uptake of BP, and the BP/LPPC complex induced cell cycle arrest and apoptosis, thereby causing death. These findings suggest the putative use of BP/LPPC as an adjuvant cytotoxic agent for colorectal cancer.

Anti-inflammatory effect of bee venom in phthalic anhydride-induced atopic dermatitis animal model.

Globally, many people have been affected with atopic dermatitis (AD), a chronic inflammatory skin disease. AD is associated with multiple factors such as genetic, inflammatory, and immune factors. Bee venom (BV) is now widely used for the treatment of several inflammatory diseases. However, its effect on 5% phthalic anhydride (PA)-induced AD has not been reported yet. We investigated the anti-inflammatory and anti-AD effects of BV in a PA-induced animal model of AD. Balb/c mice were treated with topical application of 5% PA to the dorsal skin and ears for induction of AD. After 24 h, BV was applied on the back and ear skin of the mice three times a week for 4 weeks. BV treatment significantly reduced the PA-induced AD clinical score, back and ear epidermal thickness, as well as IgE level and infiltration of immune cells in the skin tissues compared to those of control mice. The levels of inflammatory cytokines in the serum were significantly decreased in BV-treated group compared to PA-treated group. In addition, BV inhibited the expression of iNOS and COX-2 as well as the activation of mitogen-activated protein kinase (MAPK) and NF-Ò¡B induced by PA in the skin tissues. We also found that BV abrogated the lipopolysaccharide or TNF-α/IFN-γ-induced NO production, expression of iNOS and COX-2, as well as MAPK and NF-Ò¡B signaling pathway in RAW 264.7 and HaCaT cells. These results suggest that BV may be a potential therapeutic macromolecule for the treatment of AD.