Hepatoprotective Potency of Chrysophanol 8-O-Glucoside from Rheum palmatum L. against Hepatic Fibrosis via Regulation of the STAT3 Signaling Pathway

Rhubarb is a well-known herb worldwide and includes approximately 60 species of the Rheum genus. One of the representative plants is Rheum palmatum, which is prescribed as official rhubarb due to its pharmacological potential in the Korean and Chinese pharmacopoeia. In our bioactive screening, we found out that the EtOH extract of R. palmatum inhibited hepatic stellate cell (HSC) activation by transforming growth factor β1 (TGF-β1). Chemical investigation of the EtOH extract led to the isolation of chrysophanol 8-O-glucoside, which was determined by structural analysis using NMR spectroscopic techniques and electrospray ionization mass spectrometry (ESIMS). To elucidate the effects of chrysophanol 8-O-glucoside on HSC activation, activated LX-2 cells were treated for 48 h with chrysophanol 8-O-glucoside, and α-SMA and collagen, HSC activation markers, were measured by comparative quantitative real-time PCR (qPCR) and western blotting analysis. Chrysophanol 8-O-glucoside significantly inhibited the protein and mRNA expression of α-SMA and collagen compared with that in TGF-β1-treated LX-2 cells. Next, the expression of phosphorylated SMAD2 (p-SMAD2) and p-STAT3 was measured and the translocation of p-STAT3 to the nucleus was analyzed by western blotting analysis. The expression of p-SMAD2 and p-STAT3 showed that chrysophanol 8-O-glucoside strongly downregulated STAT3 phosphorylation by inhibiting the nuclear translocation of p-STAT3, which is an important mechanism in HSC activation. Moreover, chrysophanol 8-O-glucoside suppressed the expression of p-p38, not that of p-JNK or p-Erk, which can activate STAT3 phosphorylation and inhibit MMP2 expression, the downstream target of STAT3 signaling. These findings provided experimental evidence concerning the hepatoprotective effects of chrysophanol 8-O-glucoside against liver damage and revealed the molecular basis underlying its anti-fibrotic effects through the blocking of HSC activation.     

Honokiol Acts as a Potent Anti-Fibrotic Agent in the Liver through Inhibition of TGF-β1/SMAD Signaling and Autophagy in Hepatic Stellate Cells

Chronic liver injury may result in hepatic fibrosis, which can progress to cirrhosis and eventually liver failure. There are no drugs that are specifically approved for treating hepatic fibrosis. The natural product honokiol (HNK), a bioactive compound extracted from Magnolia grandiflora, represents a potential tool in the management of hepatic fibrosis. Though HNK has been reported to exhibit suppressive effects in a rat fibrosis model, the mechanisms accounting for this suppression remain unclear. In the present study, the anti-fibrotic effects of HNK on the liver were evaluated in vivo and in vitro. In vivo studies utilized a murine liver fibrosis model, in which fibrosis is induced by treatment with carbon tetrachloride (CCl₄). For in vitro studies, LX-2 human hepatic stellate cells (HSCs) were treated with HNK, and expression of markers of fibrosis, cell viability, the transforming growth factor-β (TGF-β1)/SMAD signaling pathway, and autophagy were analyzed. HNK was well tolerated and significantly attenuated CCl₄-induced liver fibrosis in vivo. Moreover, HNK decreased HSC activation and collagen expression by downregulating the TGF-β1/SMAD signaling pathway and autophagy. These results suggest that HNK is a new potential candidate for the treatment of hepatic fibrosis through suppressing both TGF-β1/SMAD signaling and autophagy in HSCs.     

Proteomic Changes of Activated Hepatic Stellate Cells

Hepatic stellate cells (HSC) are the major cellular drivers of liver fibrosis. Upon liver inflammation caused by a broad range of insults including non-alcoholic fatty liver, HSC transform from a quiescent into a proliferating, fibrotic phenotype. Although much is known about the pathophysiology of this process, exact cellular processes which occur in HSC and enable this transformation remain yet to be elucidated. In order to investigate this HSC transformation, we employed a simple, yet reliable model of HSC activation via an increase in growth medium serum concentration (serum activation). For that purpose, immortalized human LX-2 HSC were exposed to either 1% or 10% fetal bovine serum (FBS). Resulting quiescent (1% FBS) and activated (10% FBS) LX-2 cells were then subjected to in-depth mass spectrometry-based proteomics analysis as well as comprehensive phenotyping. Protein network analysis of activated LX-2 cells revealed an increase in the production of ribosomal proteins and proteins related to cell cycle control and migration, resulting in higher proliferation and faster migration phenotypes. Interestingly, we also observed a decrease in the expression of cholesterol and fatty acid biosynthesis proteins in accordance with a concomitant loss of cytosolic lipid droplets during activation. Overall, this work provides an update on HSC activation characteristics using contemporary proteomic and bioinformatic analyses and presents an accessible model for HSC activation. Data are available via ProteomeXchange with identifier PXD029121.     

Exosomes Secreted from Amniotic Membrane Contribute to Its Anti-Fibrotic Activity

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFβ1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFβ1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes.     

Role of the ROS-JNK Signaling Pathway in Hypoxia-Induced Atrial Fibrotic Responses in HL-1 Cardiomyocytes

By promoting atrial structural remodeling, atrial hypoxia contributes to the development of the atrial fibrillation substrate. Our study aimed to investigate the modulatory effect of hypoxia on profibrotic activity in cultured HL-1 cardiomyocytes and explore the possible signaling transduction mechanisms of profibrotic activity in vitro. Hypoxia (1% O₂) significantly and time-dependently increased the expression of hypoxia-inducible factor (HIF)-1α and fibrotic marker proteins collagen I and III (COL1A and COL3A), transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). Western blot or immunohistochemistry analysis showed that hypoxia-induced increase in COL1A and COL3A was significantly attenuated by the addition of SP600125 (a specific c-Jun N-terminal kinase [JNK] inhibitor) or expression of dominant-negative JNK before hypoxia treatment. The inhibition of hypoxia-activated phosphorylation of JNK signal components (JNK, MKK4, nuclear c-Jun and ATF-2) by pre-treatment with SP600125 could suppress hypoxia-stimulated HIF-1α upregulation and fibrotic marker proteins expression. Hypoxia significantly increased reactive oxygen species (ROS) production in cultured HL-1 atrial cells. Pre-treatment with N-acetylcysteine significantly abrogated the expression of nuclear HIF-1α, JNK transduction components and fibrotic marker proteins. Taken together, these findings indicated that the hypoxia-induced atrial profibrotic response occurs mainly via the ROS/JNK pathway, its downstream upregulation of HIF-1α and c-Jun/ATF2 phosphorylation and nuclear translocation to up-regulate the expression of fibrosis-related proteins (COL1A, COL3A, TGF-β1 and α-SMA). Our result suggests that suppression of ROS/JNK signaling pathway is a critical mechanism for developing a novel therapeutic strategy against atrial fibrillation.     

Differences between Mice and Humans in Regulation and the Molecular Network of Collagen,Type III,Alpha-1 at the Gene Expression Level: Obstacles that Translational Research Must Overcome

Collagen, type III, alpha-1 (COL3A1) is essential for normal collagen I fibrillogenesis in many organs. There are differences in phenotypes of mutations in the COL3A1 gene in humans and mutations in mice. In order to investigate whether the regulation and gene network of COL3A1 is the same in healthy populations of mice and humans, we compared the quantitative trait loci (QTL) that regulate the expression level of COL3A1 and the gene network of COL3A1 pathways between humans and mice using whole genome expression profiles. Our results showed that, for the regulation of expression of Col3a1 in mice, an eQTL on chromosome (Chr) 12 regulates the expression of Col3a1. However, expression of genes in the syntenic region on human Chr 7 has no association with the expression level of COL3A1. For the gene network comparison, we identified 44 top genes whose expression levels are strongly associated with that of Col3a1 in mice. We next identified 41 genes strongly associated with the expression level of COL3A1 in humans. There are a few but significant differences in the COL3A1 gene network between humans and mice. Several genes showed opposite association with expression of COL3A1. These genes are known to play important roles in development and function of the extracellular matrix of the lung. Difference in the molecular pathway of key genes in the COL3A1 gene network in humans and mice suggest caution should be used in extrapolating results from models of human lung diseases in mice to clinical lung diseases in humans. These differences may influence the efficacy of drugs in humans whose development employed mouse models.     

T-2 Toxin Induces Oxidative Stress at Low Doses via Atf3ΔZip2a/2b-Mediated Ubiquitination and Degradation of Nrf2

T-2 toxin is mainly produced by Fusarium species, which is an extremely toxic mycotoxin to humans and animals. It is well known that T-2 toxin induces oxidative stress, but the molecular mechanism is still unknown. In this study, we found that T-2 toxin significantly promoted reactive oxygen species (ROS) accumulation in MCF-7 cells at low doses which maintains cell viability at least 80%. Further analysis showed that T-2 toxin downregulated the expression of the master regulator of antioxidant defense gene, nuclear factor erythroid 2-related factor (Nrf2), and its targeted antioxidant genes. Overexpression of Nrf2 or its target gene heme oxygenase 1 (HO1) significantly blocked the ROS accumulation in MCF-7 cells under T-2 toxin treatment. Moreover, we found that T-2 toxin downregulated the antioxidant genes via inducing the expression of ATF3ΔZip2a/2b. Importantly, overexpression of ATF3ΔZip2a/2b promoted the ubiquitination and degradation of Nrf2. Altogether, our results demonstrated that T-2 toxin-induced ROS accumulation via ATF3ΔZip2a/2b mediated ubiquitination and degradation of Nrf2, which provided a new insight into the mechanism of T-2 toxin-induced oxidative stress.     

Design and Synthesis of a Series of Piperazine‐1‐carboxamidine Derivatives with Antifungal Activity Resulting from Accumulation of Endogenous Reactive Oxygen Species

In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure–activity relationship studies showed that piperazine‐1‐carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R³ and R⁵ positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine‐1‐carboxamidine derivatives to the accumulation of endogenous ROS.     

Neoagarooligosaccharide Protects against Hepatic Fibrosis via Inhibition of TGF-β/Smad Signaling Pathway

Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by β-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-β-treated LX-2 cells. Furthermore, downstream of TGF-β, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl₄)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl₄-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-β/Smad signaling pathway.     

β-Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3-L1 Cell Maturation into Adipocytes

Studies have elucidated that pyrethroids induce adipogenesis. It is also known that macrophages can affect the homeostasis of adipose tissue. However, whether and how the β-cypermethrin (β-CYP)-mediated inhibition of the macrophages affects adipogenesis remain unknown. To explore the effects of β-CYP on adipogenesis through modulating the function of macrophages, 3T3-L1 cells, a preadipocyte cell line, were exposed to culture medium from either RAW 264.7 cells, a macrophage cell line (RM), or β-CYP-treated RAW 264.7 cells (CRM). CRM decreased the inhibitory effects of RM treatment on cell proliferation and adipogenesis, as lipid accumulation, the CEBPA content, and Fasn and Acaca expression in 3T3-L1 cells were higher following CRM treatment than following RM treatment through the higher levels of the demethylated CEBPA promoter in 3T3-L1 cells. However, the medium from β-CYP- and N-acetyl-L-cysteine-cotreated RAW 264.7 cells (CNRM) partially restored the inhibitory effects of RAW 264.7 cells on 3T3-L1 cells that had been reduced by CRM, indicating that β-CYP might reduce the cytotoxicity and inhibitory effects of RAW 264.7 cells on the adipogenesis of 3T3-L1 cells through elevating ROS levels in RAW 264.7 cells. Moreover, exposure to β-CYP downregulated the TNF-α secretion in RAW 264.7 cells. In conclusion, these data demonstrated that β-CYP affected the function of RAW 264.7 cells, alleviating their inhibitory effects on adipogenesis and CEBPA demethylation in 3T3-L1 cells. β-CYP might achieve these effects through downregulating the secretion of TNF-α via elevating ROS levels in RAW 264.7 cells. Our experiments provide a new perspective on the obesogenic effect of pyrethroids.     

Phospholipids affect the intestinal absorption of carotenoids in mice

Previously, we have shown that uptake of carotenoids solubilized with mixed micelles by human intestinal Caco-2 cells is enhanced by lysophosphatidylcholine (lysoPC) and suppressed by PC. This study determined the effect of PC and lysoPC in mixed micelles on the accumulation of β-carotene and lutein in mice in order to elucidate the roles of micellar phospholipid in the intestinal uptake of carotenoids in vivo. Mixed micelles were composed of 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate, 200 μM carotenoid, and 3 mM phospholipid in PBS. The mice were fed single doses of β-carotene or lutein solubilized in PC (PC group), lysoPC (LPC group), and no phospholipid (NoPL group) micelles. The β-carotene responses in the plasma and liver of the PC group were markedly lower than those of the other two groups, whereas no differences were noticed between the LPC and NoPL groups. The average level of lutein in the plasma of the PC group after administration was significantly (P<0.05) lower than those of the other groups. Moreover, the average level of lutein in the liver was significantly (P<0.05) different among the groups in the order of LPC>NoPL>PC. Thus, the results clearly indicate that PC suppressed the accumulation of β-carotene and lutein in plasma and liver and that lysoPC enhanced the accumulation of lutein in liver. These results suggest that the hydrolysis of PC to lysoPC plays an important role in the intestinal uptake of carotenoids solubilized in mixed micelles.     

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein,Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.     

The Capillary Morphogenesis Gene 2 Triggers the Intracellular Hallmarks of Collagen VI-Related Muscular Dystrophy

Collagen VI-related disorders (COL6-RD) represent a severe form of congenital disease for which there is no treatment. Dominant-negative pathogenic variants in the genes encoding α chains of collagen VI are the main cause of COL6-RD. Here we report that patient-derived fibroblasts carrying a common single nucleotide variant mutation are unable to build the extracellular collagen VI network. This correlates with the intracellular accumulation of endosomes and lysosomes triggered by the increased phosphorylation of the collagen VI receptor CMG2. Notably, using a CRISPR-Cas9 gene-editing tool to silence the dominant-negative mutation in patients’ cells, we rescued the normal extracellular collagen VI network, CMG2 phosphorylation levels, and the accumulation of endosomes and lysosomes. Our findings reveal an unanticipated role of CMG2 in regulating endosomal and lysosomal homeostasis and suggest that mutated collagen VI dysregulates the intracellular environment in fibroblasts in collagen VI-related muscular dystrophy.     

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain

The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol^® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT–qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.     

Therapeutic Potential of Mesenchymal Stem Cells in a Pre-Clinical Model of Diabetic Kidney Disease and Obesity

Diabetic kidney disease (DKD) is a worldwide microvascular complication of type 2 diabetes mellitus (T2DM). From several pathological mechanisms involved in T2DM-DKD, we focused on mitochondria damage induced by hyperglycemia-driven reactive species oxygen (ROS) accumulation and verified whether mesenchymal stem cells (MSCs) anti-oxidative, anti-apoptotic, autophagy modulation, and pro-mitochondria homeostasis therapeutic potential curtailed T2DM-DKD progression. For that purpose, we grew immortalized glomerular mesangial cells (GMCs) in hyper glucose media containing hydrogen peroxide. MSCs prevented these cells from apoptosis-induced cell death, ROS accumulation, and mitochondria membrane potential impairment. Additionally, MSCs recovered GMCs’ biogenesis and mitophagy-related gene expression that were downregulated by stress media. In BTBR^ob/ob mice, a robust model of T2DM-DKD and obesity, MSC therapy (1 × 10⁶ cells, two doses 4-weeks apart, intra-peritoneal route) led to functional and structural kidney improvement in a time-dependent manner. Therefore, MSC-treated animals exhibited lower levels of urinary albumin-to-creatinine ratio, less mesangial expansion, higher number of podocytes, up-regulation of mitochondria-related survival genes, a decrease in autophagy hyper-activation, and a potential decrease in cleaved-caspase 3 expression. Collectively, these novel findings have important implications for the advancement of cell therapy and provide insights into cellular and molecular mechanisms of MSC-based therapy in T2DM-DKD setting.     

Pinolenic Acid Downregulates Lipid Anabolic Pathway in HepG2 Cells

Pine nut oil (PNO) was reported to reduce lipid accumulation in the liver. However, the specific effect of pinolenic acid (18:3, all‐cis‐Δ5,9,12), a unique component of PNO, on lipid metabolism has not been studied. We hypothesized that pinolenic acid downregulates the lipid anabolic pathway in HepG2 cells. HepG2 cells were incubated in serum‐free medium supplemented with 50 μM bovine serum albumin (BSA), palmitic acid, oleic acid, γ‐linolenic acid, pinolenic acid, eicosapentaenoic acid (EPA), or α‐linolenic acid for 24 h. Lipid accumulation was determined by Oil Red O (ORO) staining. The mRNA levels of genes related to fatty acid biosynthesis (SREBP1c, FAS, SCD1, and ACC1), fatty acid oxidation (ACC2, PPARα, CPT1A, and ACADL), cholesterol synthesis (SREBP2 and HMGCR), and lipoprotein uptake (LDLr) and of genes that may be involved in the downregulation of the lipogenic pathway (ACSL3, ACSL4, and ACSL5) were determined by qPCR. LDLR protein levels were measured by Western blot analysis. The mRNA levels of SREBP1c, FAS, and SCD1 were significantly downregulated by pinolenic acid treatment compared to BSA control (53, 54, and 38 % lower, respectively). In addition, the mRNA levels of HMGCR, ACSL3, and LDLr were significantly lower (30, 30, and 43 % lower, respectively), and ACSL4 tended to be lower in the pinolenic acid group (20 % lower, P = 0.082) relative to the control group. In conclusion, pinolenic acid downregulated the lipid anabolic pathway in HepG2 cells by reducing expression of genes related to lipid synthesis, lipoprotein uptake, and the regulation of the lipogenic pathway.