Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Bidirectional sensitivity of CALHM1 channel to protons from both sides of plasma membrane.

Calcium homeostasis modulator 1 (CALHM1), a newly discovered voltage-dependent nonselective ion channel, has drawn attention for its role in neuronal activity and taste sensation. Its sluggish voltage-dependent activation is facilitated by lowering extracellular Ca2+ concentration ([Ca2+]e). Here, we investigated the effects of extracellular and intracellular pH (pHe and pHi) on human CALHM1. When normalized to the amplitude of the CALHM1 current (ICALHM1) under whole cell patch clamp at symmetrical pH 7.4, ICALHM1 decreased at acidic pHe or pHi, whereas it sharply increased at alkaline pHe or pHi. The effects of pH were preserved in the inside-out configuration. The voltage dependence of ICALHM1 showed leftward and rightward shifts at alkaline and acidic pHe and pHi, respectively. Site-directed mutagenesis of the water-accessible charged residues of the pore and nearby domains revealed that E17, K229, E233, D257, and E259 are nonadditively responsible for facilitation at alkaline pHi. Identification of the pHe-sensing residue was not possible because mutation of putative residues impaired membrane expression, resulting in undetectable ICALHM1. Alkaline pHe-dependent facilitation appeared gradually with depolarization, suggesting that the sensitivity to pHe might be due to H+ diffusion through the open-state CALHM1. At pHe 6.2, decreased [Ca2+]e could not recover the inhibited ICALHM1 but further augmented the increased ICALHM1 at pHe 8.6, suggesting that unidentified common residues might contribute to the [Ca2+]e and acidic pHe. This study is the first, to our knowledge, to demonstrate the remarkable pH sensitivity of CALHM1, which might contribute to the pH-dependent modulation of neuronal excitability or taste sensation.

Lower troponin expression in the right ventricle of rats explains interventricular differences in E-C coupling.

Despite distinctive functional and anatomic differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, the IonOptix system, and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single-cell contractility, and cytosolic Ca2+ ([Ca2+]i), respectively. Myofilament proteins were analyzed by immunoblotting. RVCM showed significantly shorter action potential duration (APD) and higher density of transient outward K+ current (Ito). However, the triggered [Ca2+]i change (Ca2+ transient) was not different, while the decay rate of the Ca2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was ∼60% lower in RVCM, which is partly responsible for the slower decay of the Ca2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca2+ buffering capacity (κS), which was proved by in situ analysis. The introduction of these new levels of cTn, Ito, and SERCA into a mathematical model of rat LVCM reproduced the similar Ca2+ transient, slower Ca2+ decay, shorter APD, and smaller ΔSL of RVCM. Taken together, these data show reduced expression of cTn proteins in the RVCM, which provides an explanation for the interventricular difference in the E-C coupling kinetics.

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (ICALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as ICALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect ICALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both ICALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Thermosensitivity of the voltage-dependent activation of calcium homeostasis modulator 1 (calhm1) ion channel.

Calcium homeostasis modulator 1 (calhm1) proteins form an outwardly rectifying nonselective ion channel having exceedingly slow kinetics and low sensitivity to voltage that is shifted by lowering extracellular Ca2+ ([Ca2+]e). Here we found that physiological temperature dramatically facilitates the voltage-dependent activation of the calhm1 current (Icalhm1); increased amplitude (Q10, 7-15) and fastened speed of activation. Also, the leftward shift of the half-activation voltage (V1/2) was similary observed in the normal and lower [Ca2+]e. Since calhm1 is highly expressed in the brain and taste cells, the thermosensitivity should be considered in their electrophysiology.

Auricularia auricula increases an apoptosis in human hepatocellular carcinoma cells via a regulation of the peroxiredoxin1.

Auricularia auricula (A. auricula) has been reported to have positive health effects. Therefore, this study was conducted to explore possible mechanisms of A. auricula-induced anticancer activity in hepatocellular carcinoma (HCC) cells. First, using proliferative assay including MTT assay and real-time cell electronic sensing technique, we founded that A. auricula has an antiproliferative effect on various cancer cell lines. Among five cancer cell lines, we focused on huh-7 cell line, HCC cell line, due to that A. auricula has most dramatic antiproliferative effects on huh-7 cell line. Following experiments, we founded that its antiproliferative effects was related with apoptosis-inducing activities. For more investigation, a two-dimensional electrophoresis based-proteomic analysis (2DE-GE) was employed for identification of possible target-related proteins of A. auricula-induced apoptosis. Among seven identified proteins, we focused on peroxiredoxin1 (PRDX1), which has been known as an anti-oxidative enzyme. We confirmed downregulation of expression of PRDX1 following A. auricula treatment in mRNA and protein level. In order to obtain a more validation of the correlation of A. auricula-induced cell death and anti-oxidative enzyme. We investigated the level of anti-oxidative enzymes, total glutathione (GSSG/GSH), and superoxide dismutase (SOD) levels in treated cells and PRDX1 gene-silenced cells. GSH and SOD levels were decreased in the treated cells and PRDX1 gene-silenced cells. Our findings suggest that A. auricula is a potent inducer of apoptosis in HCC cells via PRDX1-inhibition pathways. PRACTICAL APPLICATIONS: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality. The principal treatment is surgical resection or liver transplantation. However, in most patients with HCC the diagnosis is often late, thereby excluding the patients from definitive surgical resection. Chemotherapy and radiotherapy are generally ineffective. Newer treatments are needed with several being in development. In this research article, we provide regulation mechanism of PRDX1 in HCC. PRDX1 has a proliferative effect and play a role in cancer development or progression. Overexpression of PRDX1 in cancer cells implies the role of PRDX1 in the cancer therapy. PRDX1 is currently being investigated as a new target for gene therapy in cancer. A. auricula is an apoptotic inducer of HCC cells through PRDX1 pathway. Regulation of PRDX1 in HCC may contribute to cancer treatment. Therefore, the potentials of targeting apoptosis would be a viable therapeutic strategy to improve the outcome of HCC patients.

Teaching cardiac excitation-contraction coupling using a mathematical computer simulation model of human ventricular myocytes.

To understand the excitation-contraction (E-C) coupling of cardiomyocytes, including the electrophysiological mechanism of their characteristically long action potential duration, is one of the major learning goals in medical physiology. However, the integrative interpretation of the responses occurring during the contraction-relaxation cycle is challenging due to the dynamic interaction of underlying factors. Starting in 2017, we adopted the mathematical computer simulation model of human ventricular myocyte (Cardiac E-C_Sim), hypothesizing that this educational technology may facilitate students' learning of cardiac physiology. Here, we describe the overall process for the educational application of Cardiac E-C_Sim in the human physiology practicum of Seoul National University College of Medicine. We also report the results from questionnaires covering detailed assessment of the practicum class. The analysis of results and feedback opinions enabled us to understand how the students had approached the problem-solving process. As a whole, the students could better accomplish the learning goals using Cardiac E-C_Sim, followed by constructive discussions on the complex and dynamic mechanisms of cardiac E-C coupling. We suggest that the combined approach of lecture-based teaching and computer simulations guided by a manual containing clinical context would be broadly applicable in physiology education.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4.

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Triple arginine residues in the proximal C-terminus of TREK K+ channels are critical for biphasic regulation by phosphatidylinositol 4,5-bisphosphate.

TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

Suppression of hERG K+ current and cardiac action potential prolongation by 4-hydroxynonenal via dual mechanisms.

Oxidative stress under pathological conditions, such as ischemia/reperfusion and inflammation, results in the production of various reactive chemicals. Of these chemicals, 4-hydroxynonenal (4-HNE), a peroxidation product of ω6-polyunsaturated fatty acid, has garnered significant attention. However, the effect of 4-HNE on cardiac electrophysiology has not yet been reported. In the present study, we investigated the effects of 4-HNE on several cardiac ion channels, including human ether-a-go-go-related (hERG) channels, using the whole-cell patch clamp technique. Short-term exposure to 100 µM 4-HNE (4-HNE100S), which mimics local levels under oxidative stress, decreased the amplitudes of rapidly activating delayed rectifier K+ current (IKr) in guinea pig ventricular myocytes (GPVMs) and HEK293T cells overexpressing hERG (IhERG). MS analysis revealed the formation of 4-HNE-hERG adduct on specific amino acid residues, including C276, K595, H70, and H687. Long-term treatment (1-3 h) with 10 µM 4-HNE (4-HNE10L), suppressed IKr and IhERG, but not IKs and ICa,L. Action potential duration (APD) of GPVMs was prolonged by 37% and 64% by 4-HNE100S and 4-HNE10L, respectively. Western blot analysis using surface biotinylation revealed a reduction in mature membrane hERG protein after treatment with 4-HNE10L. Proteasomal degradation inhibitors, such as bortezomib, prevented the 4-HNE10L-induced decrease in mature hERG, suggesting a retrograde degradation of membrane hERG due to 4-HNE. Taken together, 4-HNE100S and 4-HNE10L suppressed IhERG via functional inhibition and downregulation of membrane expression of hERG, respectively. The exposure of 4-HNE under pathological oxidative stress may increase the risk of proarrhythmic events via APD prolongation.

Daidzein causes cytochrome c-mediated apoptosis via the Bcl-2 family in human hepatic cancer cells.

Daidzein, which belongs to the group of isoflavones from soybeans, has been extensively researched prostate, cervix, brain, breast, and colon cancer cell lines. However, daidzein has not been thoroughly investigated in human hepatic cancer cells; therefore, we investigated whether it inhibits hepatic cancer cell growth. Decreased cell proliferation was measured in daidzein-treated hepatic cancer cells (SK-HEP-1) upon real-time cell electronic sensing analysis however, it was not affected on normal human hepatocytes (Chang). Daidzein-induced apoptosis was demonstrated by comet and TUNEL assay. Moreover, we conducted two-dimensional electrophoresis to study the mechanism of daidzein-induced apoptosis in daidzein-treated SK-HEP-1 cells. Expression of peroxiredoxin-3 (Prdx-3), which modulates redox homeostasis of cells, was increased in protein analysis. Additionally, we measured the levels of reactive oxygen species and it was decreased in daidzein-treated SKHEP-1 cells. Daidzein-induced apoptosis in SK-HEP-1 cells was also associated with the up-regulation of Bak and down-regulation of Bcl-2 and Bcl-xL proteins. Moreover, daidzein treatment increased in the release of mitochondrial cytochrome c and activation of APAF-1, caspase 9 and caspase 3. Overall, these result indicate that daidzein is a potent inducer of apoptosis in hepatic cancer cells via mitochondrial pathway.

Apoptosis by aloe-emodin is mediated through down-regulation of calpain-2 and ubiquitin-protein ligase E3A in human hepatoma Huh-7 cells.

Natural flavonoids are associated with anti-proliferation of cancer growth. However, the antioxidant and anti-proliferation effects of AE (aloe-emodin) have not been well studied. We have investigated how AE affects the proliferation of hepatic hepatocellular carcinoma cells and exerts an anti-cancer effect. The cytotoxic effect of AE was demonstrated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Huh-7 cells were inhibited by AE treatment in both dose- and time-dependent manners. The IC(50) level of AE was ∼75 µM. AE also has anti-proliferative effects via induction of DNA damage and apoptosis. 2-DE (two-dimensional electrophoresis) revealed that several proteins were related to the anti-cancer effects of AE. CAPN2 (calpain-2) and UBE3A (ubiquitin-protein ligase E3A), which are associated with the apoptosis signalling pathway, were verified by Western blotting. AE exhibited potent anti-proliferative effects on Huh-7 cells via down-regulation of CAPN2 and UBE3A. The findings support the possibility of AE being a chemopreventative agent.

Galangin induces apoptosis in gastric cancer cells via regulation of ubiquitin carboxy-terminal hydrolase isozyme L1 and glutathione S-transferase P.

Galangin has been shown to have anti-cancer property against several types of cancer cells. Many studies have described the anti-oxidant and apoptotic effects of galangin. However, the mechanism of galangin-induced apoptosis has not yet been studied for human gastric cancer cells. We investigated galangin-induced apoptosis of human gastric cancer SNU-484 cells. Galangin inhibited proliferation of SNU-484 cells in a dose- and time-dependent manner. The results showed that galangin significantly decreased the viability of SNU-484 cells at 50-200 µM for 24 h and 48 h. Galangin-induced cell death was characterized with the changes in cell morphology, DNA fragmentation, cell cycle, activation of caspase-3/-9, poly (ADP-ribose) polymerase (PARP) cleavage, and expression of MAP kinase such as ERK1/2 and JNK. For identification of proteins potentially involved in apoptosis, a two-dimensional electrophoresis was employed. Proteomic analysis showed that several proteins were associated with anti-cancer properties of galangin. Of particular interest, these proteins included ubiquitin carboxy-terminal hydrolase isozyme L1 (Uch-L1) and glutathione S-transferase P (GSTP), which are involved in apoptosis of SNU-484 cells. Western blot analysis confirmed up-regulation of Uch-L1 and down-regulation of GSTP following galangin treatment. Our results suggest that Uch-L1 and GSTP be involved in galangin-induced apoptosis in human gastric cancer SNU-484 cells.

Sulforaphane induces apoptosis in human hepatic cancer cells through inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4, mediated by hypoxia inducible factor-1-dependent pathway.

The anti-cancer activity of sulforaphane (SFN) has recently been investigated in several cancer cell lines, including human hepatic cancers. However, the mechanism of SFN-induced cell death in human hepatic cancer cells is still not well understood. The aim of the present work is to explore the possible mechanisms of SFN-induced apoptosis in hepatocellular carcinoma cells using proteomic analysis. A two-dimensional electrophoresis (2-DE)-based-proteomic analysis was employed for identification of possible target-related proteins of SFN-induced apoptosis. Among eleven proteins identified as regulated, we focused on the down-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4 (PFKFB4) protein, which has been known as a key modulator of glycolysis. We also showed that SFN down-regulated the expression of the transcriptional factor, hypoxia inducible factor-1α (HIF-1α), which strongly regulates PFKFB4 expression. In order to obtain a broad understanding of the correlation of HIF-1α and SFN, we observed the inhibition of the activity of mitogen-activated protein kinases, regulators of HIF-1α activity. Our findings suggest that SFN is a potent inducer of apoptosis in hepatocellular carcinoma cells via PFKFB4-inhibition pathways. HIF-1 pathway inhibition may be mediated by the inhibition of mitogen-activated protein kinases.

A mechanism of apigenin-induced apoptosis is potentially related to anti-angiogenesis and anti-migration in human hepatocellular carcinoma cells.

Apigenin (APG) has been shown to have a strong anti-cancer effect on various cancer models via a programmed cell death, apoptosis. However, the fundamental mechanisms of these effects are still unclear. In the present study, we examined the question of whether or not APG can inhibit proliferation of hepatocellular carcinoma (HCC), huh-7 cells, resulting in apoptosis. In APG-treated cells, we observed typical features of apoptosis. To identify the proteins related to APG-induced apoptosis, we performed two-dimensional electrophoresis analysis and identified differentially expressed proteins. Among these proteins, we focused on vimentin, which plays a physiological role, such as cell migration and adhesion. We validated expression of vimentin in both mRNA and protein levels, verifying its decrease. In addition, we observed that APG down-regulated the expression levels of type I collagen, which collaborated with vimentin in cell migration and decreased the releasing amounts of VEGF and MMP-8, which are closely relevant to angiogenic activity. Finally, we confirmed the decreased capacity of cell migration due to down-regulation of vimentin, type I collagen, VEGF, and MMP-8 induced by APG. Based on the overall results, we suggested that vimentin was potentially associated with APG-induced apoptosis, as a key regulator in angiogenesis and migration.

Mesenchymal stem cells' interaction with skin: wound-healing effect on fibroblast cells and skin tissue.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to secrete growth factors. Because wound healing is associated with fibroblast cells and extracellular matrix (ECM) in the dermis and epidermis, we used fibroblast cells to resolve the question of whether or not MSCs regulate wound healing in vitro via a regenerative function. Using a cell proliferation assay, we demonstrated that conditioned media (CM) obtained from MSCs significantly enhanced the cell survival ability of fibroblast cells. Moreover, by measurement of mRNA and protein, we observed that CM also promoted the production or secretion of collagen, elastin, and fibronectin. To better understand the effects of ECM-related wound healing, we measured the level of collagen-degradative enzyme (matrix metalloprotease-1), and observed that CM suppressed matrix metalloprotease-1 expression. For the determination of oxidative stress, which has an influence on wound healing, we performed the superoxide dismutase and glutathione peroxidase assays; our results suggested that CM inhibited the oxidative stress of fibroblast cells. In order to widely investigate the wound-healing effects of MSCs, we performed in vivo experiments, and observed that MSCs stimulated wound healing. In summary, the results of this study suggest that MSCs inhibit the loss of fibroblast cells and ECM, and accumulation of oxidative stress. We found that MSCs stimulate wound healing in vitro and in vivo, suggesting that MSCs have the potential to enhance wound healing.

Fisetin induces apoptosis in Huh-7 cells via downregulation of BIRC8 and Bcl2L2.

This study aimed to investigate the effects of fisetin, a common dietary natural flavonoid, on apoptosis of Huh-7 cells. The MTT assay was used to evaluate the reduction of cell viability. In the DNA fragmentation assay and comet assay, fisetin-induced DNA damage was visible as a formation of DNA fragmentation and comet tails. Fisetin also induced intracellular accumulation of reactive oxygen species. Two-dimensional gel electrophoresis was performed to evaluate protein expression in fisetin-treated and control cells, and Baculoviral IAP repeat-containing protein 8 (BIRC8) and apoptosis regulator Bcl-W (Bcl2L2) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their expression was confirmed by western blotting. The anticancer effect of fisetin in Huh-7 cells may result from the down regulation of BIRC8 and the Bcl2L2.