Ethyl ferulate suppresses post-myocardial infarction myocardial fibrosis by inhibiting transforming growth factor receptor 1.

BACKGROUND: With an increasing number of myocardial infarction (MI) patients, myocardial fibrosis is becoming a widespread health concern. It's becoming more and more urgent to conduct additional research and investigations into efficient treatments. Ethyl ferulate (EF) is a naturally occurring substance with cardioprotective properties. However, the extent of its impact and the underlying mechanism of its treatment for myocardial fibrosis after MI remain unknown. PURPOSE: The goal of this study was to look into how EF affected the signaling of the TGF-receptor 1 (TGFBR1) in myocardial fibrosis after MI. METHODS: Echocardiography, hematoxylin-eosin (HE) and Masson trichrome staining were employed to assess the impact of EF on heart structure and function in MI-affected mice in vivo. Cell proliferation assay (MTS), 5-Ethynyl-2'-deoxyuridine (EdU), and western blot techniques were employed to examine the influence of EF on native cardiac fibroblast (CFs) proliferation and collagen deposition. Molecular simulation and surface plasmon resonance imaging (SPRi) were utilized to explore TGFBR1 and EF interaction. Cardiac-specific Tgfbr1 knockout mice (Tgfbr1ΔMCK) were utilized to testify to the impact of EF. RESULTS: In vivo experiments revealed that EF alleviated myocardial fibrosis, improved cardiac dysfunction after MI and downregulated the TGFBR1 signaling in a dose-dependent manner. Moreover, in vitro experiments revealed that EF significantly inhibited CFs proliferation, collagen deposition and TGFBR1 signaling followed by TGF-ß1 stimulation. More specifically, molecular simulation, molecular dynamics, and SPRi collectively showed that EF directly targeted TGFBR1. Lastly, knocking down of Tgfbr1 partially reversed the inhibitory activity of EF on myocardial fibrosis in MI mice. CONCLUSION: EF attenuated myocardial fibrosis post-MI by directly suppressing TGFBR1 and its downstream signaling pathway.

Calycosin protects against oxidative stress-induced cardiomyocyte apoptosis by activating aldehyde dehydrogenase 2.

Myocardial infarction (MI) is the leading cause of death worldwide, and oxidative stress is part of the process that causes MI. Calycosin, a naturally occurring substance with cardioprotective properties, is one of the major active constituents in Radix Astragali. In this study, effect of Calycosin was investigated in vivo and in vitro to determine whether it could alleviate oxidative stress and oxidative stress-induced cardiac apoptosis in neonatal cardiomyocytes (NCMs) via activation of aldehyde dehydrogenase 2 (ALDH2). Calycosin protected against oxidative stress and oxidative stress-induced apoptosis in NCMs. Molecular docking revealed that the ALDH2-Calycosin complex had a binding energy of -9.885 kcal/mol. In addition, molecular docking simulations demonstrated that the ALDH2-Calycosin complex was stable. Using BLI assays, we confirmed that Calycosin could interact with ALDH2 (KD  = 1.9 × 10-4 M). Furthermore, an ALDH2 kinase activity test revealed that Calycosin increased ALDH2 activity, exhibiting an EC50 of 91.79 µM. Pre-incubation with ALDH2 inhibitor (CVT-10216 or disulfiram) reduced the cardio-protective properties Calycosin. In mice with MI, Calycosin therapy substantially reduced myocardial apoptosis, oxidative stress, and activated ALDH2. Collectively, our findings clearly suggest that Calycosin reduces oxidative stress and oxidative stress-induced apoptosis via the regulation of ALDH2 signaling, which supports potential therapeutic use in MI.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. AIM OF THE STUDY: The purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). MATERIALS AND METHODS: A model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. RESULTS: We observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1ß. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10-7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. CONCLUSION: Our findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

[Construction and assessment of heart-specific green fluorescence zebrafish line].

Using the promoter for cardiac myosin light chain 2 (cmlc2) gene, an expression vector pTol2-cmlc2-IRES- EGFP for making heart-specific expression of exogenous gene in transgenic zebrafish was generated previously. Here, we reported the construction of a transgenic zebrafish line which stably expresses EGFP using this vector, and the effects of EGFP on the heart development and cardiac function of this transgenic zebrafish line were preliminarily analyzed. The results showed that the green fluorescence signal of cmlc2:EGFP line under fluorescence microscopy specifically expressed in heart and faithfully recapitulated both the spatial and temporal expression patterns of endogenous cmlc2 gene revealed by in situ hybridization in the early developmental stages. The cardiac morphology and development of this transgenic zebrafish line remained to be normal. Furthermore, the heart morphology and physiological function of this transgenic line have been analyzed using M-mode analysis. The results showed that there was no significant difference between the cmlc2:EGFP and the wild type lines with respect to heart period, heart rate, diastolic surface area and systolic surface area, and fractional area change. No tachyarrhythmia was observed in the embryos from either line. Thus, the excessive expression of EGFP in this transgenic line seemed to exert no detrimental effects on the function and development of zebrafish hearts during early stages. Our study laid a foundation for the construction of exogenous gene transgenic line using pTol2-cmlc2-IRES-EGFP vector to study the function of genes that expressed in heart.

Modulation of chemotactic and pro-inflammatory activities of endothelial progenitor cells by hepatocellular carcinoma.

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDI(TM) Culture-Inserts and µ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)-DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Antimutagenic constituents of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) with potential cancer chemopreventive activity.

Adlay has long been used in traditional Chinese medicine and as a nourishing food. The acetone extract of adlay hull had previously been demonstrated to possess potent antimutagenic activity. The aims of this study were to identify the antimutagenic constituents from adlay hull by using Ames antimutagenic activity-guide isolation procedures and to investigate their chemopreventive efficacies in cultured cells. The results demonstrated that six compounds showing great antimutagenic activity were identified by spectroscopic methods and by comparison with authentic samples to be p-hydroxybenzaldehyde, vanillin, syringaldehyde, trans-coniferylaldehyde, sinapaldehyde, and coixol. Two of them, trans-coniferylaldehyde and sinapaldehyde, exhibit relatively potent scavenging of DPPH radicals, inhibit TPA stimulated superoxide anion generation in neutrophil-like leukocytes, and induce Nrf2/ARE-driven luciferase activity in HSC-3 cells. Moreover, trans-coniferylaldehyde possesses cytoprotective efficacy against tert-butyl hydroperoxide-induced DNA double-strand breaks in cultured cells, and the chemopreventive potency induced by trans-coniferylaldehyde may be through the activation of kinase signals, including p38, ERK1/2, JNK, MEK1/2, and MSK1/2. In summary, we first identified six antimutagenic constituents from adlay hull. Among them, trans-coniferylaldehyde would be a highly promising agent for cancer chemoprevention and merits further investigation.

[Interventional effects of emodin on transforming growth factor-beta1/integrin-linked kinase signal way in interleukin-1beta-induced transdifferentiation of rat tubular epithelial-myofibroblasts].

OBJECTIVE: To study the effects of transforming growth factor-beta1/integrin-linked kinase (TGF-beta1/ILK) signal way in interleukin-1beta (IL-1beta)-induced rat tubular epithelial-myofibroblast transdifferentiation (TEMT), and to investigate whether emodin inhibits IL-1beta-induced TEMT through the TGF-beta1/ILK signal way-dependent mechanism. METHODS: Normal rat kidney epithelial cell line (NRK52E) was used in this study. NRK52E cells were divided into blank control group, emodin control group, IL-1beta-induced group, emodin-inhibited group, SB431542 (TGF-beta 1 type I receptor blocker)-inhibited group, emodin plus SB431542-inhibited group, emodin-pretreated group and emodin-reversed group. After 48-hour culture, morphological changes of the NRK52E cells were observed by an inverted phase contrast microscope. The expressions of alpha-smooth muscle actin (alpha-SMA) and E-cadherin were detected by two-color immunohistochemical staining, while the expressions of TGF-beta1 and ILK were detected by one-color immunohistochemical staining. We also performed the imaging analysis to quantitatively analyze the result of the immunohistochemical staining. The secretion of fibronectin (FN) was analyzed by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, IL-1beta might induce TEMT, which was showed in increasing expression of alpha-SMA, increasing secreting of FN and decreasing expression of E-cadherin, and at the same time the expressions of TGF-beta1 and ILK were enhanced (P<0.05). Emodin might inhibit all of those changes induced by IL-1beta (P<0.05). When TGF-beta1 signal way was intercepted, IL-1beta induced-TEMT was suppressed and the expression of ILK was decreased, however, there was no significant difference in expression of TGF-beta1 between the SB431542 group and the IL-1beta-induced group. Compared with emodin-inhibited group, emodin-pretreatment could not prevent IL-1beta induced-TEMT in a certain extent, but emodin could not revert IL-1beta-induced TEMT. Spearman correlation analysis showed that TGF-beta1 expression had positive correlation with expressions of alpha-SMA, FN, ILK and negative correlation with E-cadherin expression, and the expression of ILK was positively correlated with the expressions of alpha-SMA and FN and negatively correlated with E-cadherin expression. CONCLUSION: IL-1beta induces TEMT partly depending on TGF-beta1/ILK signal way, partly via which emodin inhibits the TEMT induced by IL-1beta.

[Interventional effect of emodin on the expression of intergern linked kinase in tubular epithelial-myofibroblast transdifferentiation].

AIM: To observe the change of ILK expression in interleukin-1beta(IL-1beta)-induced tubular epithelial-myofibroblast transdifferentiation, and to investigate whether emodin inhibit IL-1beta-induced tubular epithelial-myofibroblast transdifferentiation through an intergern linked kinase-dependent mechanism. METHODS: Normal rat kidney epithelial cell line (NRK52E) was cultured and then divided into blank group, emodin control group, IL-1beta-induced group and emodin-inhibited group. When the cells were cultured for 48 h, their morphological changes were observed by an inverted phase contrast microscope. The expression of a-smooth muscle actin (a-SMA) and E-cadherin were detected using a two-color immunohistochemistry staining technique, while the expression of integrin-linked kinase (ILK) was detected using a one-color immunohistochemistry staining technique. The secretion of fibronectin (FN) was analyzed by ELISA. RESULTS: NRK52E cells cultured with IL-1 became fibroblast-like in appearance. The expression of a-SMA was enhanced (65h5+/-1h7 vs 140h4+/-3h0, P<0h05), the expression of E-cadherin was decreased (82h5+/-1h0 vs 36h0+/-2h8, P<0h05), the expression of ILK was enhanced (36h1+/-3h1 vs 82h4+/-1h2, P<0h05), and the secretion of FN was increased (54h6+/-3h1 vs 124h8+/-3h2 mg/L, P<0h05). Emodin markedly inhibited all of those changes induced by IL-1beta. CONCLUSION: The expression of ILK is up-regulated in IL-1beta-induced tubular epithelial-myofibroblast transdifferentiation. Emodin might inhibit TEMT by a down-regulation the expression of ILK.

Eudragit E accelerated the diketopiperazine formation of enalapril maleate determined by thermal FTIR microspectroscopic technique.

PURPOSE: Enalapril may undergo the thermal-induced intramolecular interaction to cause an enalapril diketopiperazine (DKP) formation. It is interesting to study the influence of Eudragit E, as a coating polymer, on the stability of enalapril maleate. The reaction kinetics of the solid-state degradation process of pure enalapril maleate and Eudragit E/enalapril maleate mixture with different weight ratios were examined. The mechanism of solid-state interaction between Eudragit E and enalapril maleate was also discussed. METHODS: The cast samples of pure enalapril maleate or Eudragit E/enalapril maleate mixture after evaporating the solvent were prepared on an aluminum foil and also determined by reflectance Fourier transform infrared (FTIR) microspectroscopy equipped with thermal analyzer. RESULTS: The result indicates that the interaction might occur between enalapril maleate and Eudragit E in the solid state after evaporating the solvent. The thermal-dependent FTIR spectra show that not only the formation of DKP but also the six-membered cyclic anhydride occurred in the enalapril maleate/Eudragit E mixture in the heating process. Two pathways for solid-sate interaction were proposed. The stability of enalapril maleate was dependent on the weight ratio of enalapril maleate and Eudragit E. The activation energy (n = 3) of DKP formation for pure enalapril maleate was about 141.2+/-0.7 kJ/mol, but it was reduced significantly to 86.7+/-0.8 kJ/mol after interaction with Eudragit E (weight ratio: 1:1), suggesting Eudragit E might exacerbate the degradation of enalapril maleate. However, the degradation accelerated by Eudragit E was reduced in high content of Eudragit E. CONCLUSIONS: When the weight ratio of both components was 1:1, Eudragit E might interact with the carboxyl group of maleic acid to exacerbate the degradation of enalapril maleate. However, the excess amount of Eudragit E might somewhat reduce the degradation of enalapril, due to the interaction that occurred between Eudragit E and carboxyl group of enalapril.

Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit.

Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.

Intramolecular cyclization of diketopiperazine formation in solid-state enalapril maleate studied by thermal FT-IR microscopic system.

The pathway of diketopiperazine (DKP) formation of solid-state enalapril maleate has been studied by using a novel Fourier transform infrared microspectroscope equipped with a thermal analyzer (thermal FT-IR microscopic system). The thermogram of the conventional differential scanning calorimetry (DSC) method was also compared. The results show new evidence of IR peaks at 3250 cm(-1) (the broad O-H stretching mode of water), and at 1738 and 1672 cm(-1) (the carbonyl band of DKP), indicating DKP formation in enalapril maleate via intramolecular cyclization. Moreover, the disappearance of IR peaks from enalapril maleate at 3215 cm(-1) (the secondary amine), 1728 cm(-1) (the carbonyl group of carboxylic acid), and 1649 cm(-1) (the carbonyl stretching of tertiary amide) also confirmed the DKP formation. The thermal FT-IR microscopic system clearly evidenced that the DKP formation in enalapril maleate started from 129 degrees C, and reached a maximum at 137 degrees C. This result was also confirmed by the conventional DSC thermogram of the compressed mixture of KBr powder and enalapril maleate, in which an endothermic peak at 144 degrees C with an extrapolated onset temperature at 137 degrees C was observed. This strongly suggests that the thermal FT-IR microscopic system was able to qualitatively detect the formation of DKP derivatives in solid-state enalapril maleate via intramolecular cyclization.

Hydration-induced proton transfer in the solid state of norfloxacin.

Norfloxacin is a special compound. Its hydrate form seems to be more soluble in water than in the anhydrate form. To investigate the hydration behavior of norfloxacin, the moisture-sorption analysis of anhydrous norfloxacin in different humidities was determined by using differential scanning calorimetry and thermogravimetric analysis. The contents of free water and bound water in the moisture-equilibrated norfloxacin were estimated quantitatively by using a curve-fitting program. Fourier transform infrared microspectroscopy with or without thermal analyzer was used to examine the structural change and dehydration process of norfloxacin in different humidities. The result indicates that the water content sorbed to anhydrous norfloxacin changed lightly below 51% relative humidity (RH) but increased markedly beyond 51% RH. The content of free water in the moisture-equilibrated norfloxacin was nearly to zero below 55% RH, but increased dramatically in high humidity. The content of bound water also enhanced gradually with the external humidity and reached to a constant of one unit after > 75% RH. When norfloxacin anhydrate transformed to its hydrate, the infrared peak intensity at 1732 and 1253 cm(-1) assigned to the C=O and C-O groups of carboxylic acid decreased gradually with the increase of water content, but the infrared peak intensity at 1584 and 1339 cm(-1) corresponding to asymmetric and symmetric carboxylates increased. Furthermore, the peak at 2553 cm(-1) assigned to the NH(+)(2) also appeared clearly and shifted from 2558 cm(-1) in higher water content and humidity. The main functional groups of norfloxacin changed from COOH to COO(-) and NH to NH(+)(2), attributable to the proton transfer from COOH group to NH group. This suggests that the hydration can induce the interaction between norfloxacin molecules from hydrogen bonding to ionic bonding by a proton-transfer process in the solid state.