Rat Hepatic Stellate Cell Line Research Articles

Hyaluronic acid modified extracellular vesicles targeting hepatic stellate cells to attenuate hepatic fibrosis

RationaleTransforming growth factor-beta1 (TGF-β1) plays a pivotal role in promoting hepatic fibrosis, pirfenidone (PFD) could inhibit TGF-β1 signaling pathway to alleviate hepatic stellate cells (HSC) activation mediated hepatic fibrosis. The targeting delivery strategy of PFD to hepatic stellate cells is a challenge. Extracellular vesicles (EVs), cell-derived membranous particles are intraluminal nano-vesicles that play a vital role in intercellular communication, they also be considered as an ideal nano-carrier. MethodsIn this study, we developed a target strategy to deliver PFD to HSC with CD44 over-expression by EVs, hyaluronic acid (HA) modified DSPE-PEG2000 endows the active targeting ability of activated HSCs to PFD-loaded EVs. ResultsIn both rat hepatic stellate cell line HSC-T6 and rat hepatocyte cell line BRL, HA@EVs-PFD demonstrated the capacity to down-regulate the expression of collagen-synthesis-related proteins and showed superior inhibition efficacy of HSC-T6 activation compared to free PFD. In hepatic fibrosis model, 4 weeks of HA@EVs-PFD treatment resulted in a reduction in liver collagen fibers, significant improvement in hepatic cell morphology, and amelioration of hepatic fibrosis. ConclusionsHA@EVs-PFD, as a drug delivery system that effectively targets and inhibits activated HSCs to treat hepatic fibrosis, holds promise as a potential therapeutic agent against hepatic fibrosis.

Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 μg/L transforming growth factor β1 (TGF-β1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 μmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-β1 stimulation group, the TGF-β1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-β1 stimulation group, and the M2-type + TGF-β1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-β1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-β1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-β1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

The anti-liver fibrosis effect of Tibetan medicine (Qiwei Tiexie capsule) is related to the inhibition of NLRP3 inflammasome activation in vivo and in vitro

Ethnopharmacological relevanceQiwei Tiexie capsule (QWTX) is an improved form of a classical prescription of Tibetan medicine—Qiwei Tiexie pill. It has been employed in the treatment of a variety of chronic liver disorders, including liver fibrosis. Uncertainty still exists regarding the mechanism of QWTX action in liver fibrosis. Aim of the studyConfirm the anti-liver fibrosis effect of QWTX and reveal its mechanism from the perspective of NOD-like receptor protein 3 (NLRP3) inflammasome activation. Materials and methodsIn vivo experiment: A rat model of carbon tetrachloride -induced liver fibrosis was constructed. All rats were randomly divided into six groups: a control group, a model group, a group receiving the positive drug (Biejia Ruangan tablet), and three groups receiving QWTX at high, medium, and low doses. The contents of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBil) were detected in serum. Hematoxylin and eosin staining and Masson's staining were used to assess the histomorphological alteration of the liver. The levels of glutathione peroxidase, hydroxyproline, tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) in the liver were determined using the corresponding detection kits. Real-time polymerase chain reaction, immunofluorescence, and western blotting were used to determine the expression levels of NLRP3, adaptor protein (ASC), caspase-1, and alpha-smooth muscle actin (α-SMA). In vitro experiment: Four groups of rat hepatic stellate cell line (HSC-T6) cells were created: the control group, the low-dose QWTX group (0.05 mg/mL), the medium-dose QWTX group (0.1 mg/mL), and the high-dose QWTX group (0.2 mg/mL). Cell viability was assessed using a cell counting kit, and the amounts of collagen type I (Col I) and IL-1β in the cell lysate were measured using an enzyme-linked immunosorbent assay kit. The mRNA and protein expression of NLRP3, ASC, caspase-1, and α-SMA were also estimated. ResultsQWTX had an inhibitory effect on liver fibrosis and a negative effect on HSC activation, while it improved liver histopathological injury and abnormal liver function and increased hydroxyproline content and glutathione peroxidase activity in vivo. QWTX decreased the expression of α-SMA, NLRP3, caspase-1, ASC, and IL-1β both in vitro and in vivo. ConclusionsTibetan medicine QWTX had a significant anti-liver fibrosis effect that was related to the inhibition of NLRP3 inflammasome activation in vivo and in vitro.

Isorhamnetin Exerts Antifibrotic Effects by Attenuating Platelet-Derived Growth Factor-BB-induced HSC-T6 Cells Activation via Suppressing PI3K-AKT Signaling Pathway

Currently, liver fibrosis is growing worldwide; unfortunately, there is no definite cure for this disease. Hence, understanding the molecular pathways involved in the development of liver fibrosis can help to find a proper treatment. In this study, we aimed to evaluate the effects of isorhamnetin as an antifibrotic agent on platelet-derived growth factor (PDGF)-BB-activated hepatic stellate cells (HSC)-T6 cells in a concentration-dependent manner. We have also attempted to assess signaling pathways that may affect liver fibrosis. PDGF-BB was used to activate the HSC-T6 rat hepatic stellate cell line. The activated cells were treated with Isorhamnetin for 24 h. Finally, we compared the mRNA expression level of COLA1 and α-SMA and also the level of phosphorylated AKT protein with the control group. The obtained data revealed a significant increase in the expression level of the COLA1 and α-SMA genes (p > 0.05), as well as phosphorylated AKT protein, in the cells treated with PDGF-BB. In addition, 75 and 100 µM concentrations of Isorhamnetin markedly declined the COLA1 and α-SMA expression and also the phosphorylated AKT protein level in the HSC-T6 cells. Our findings suggest that Isorhamnetin decreases HSC-T6 activation, the expression of COLA1 and α-SMA, in vitro, which could act as an antifibrotic element to reduce and treat liver fibrosis disease.

Isorhamnetin Exerts Antifibrotic Effects by Attenuating Platelet-Derived Growth Factor-BB-induced HSC-T6 Cells Activation via Suppressing PI3K-AKT Signaling Pathway

Currently, liver fibrosis is growing worldwide; unfortunately, there is no definite cure for this disease. Hence, understanding the molecular pathways involved in the development of liver fibrosis can help to find a proper treatment. In this study, we aimed to evaluate the effects of isorhamnetin as an antifibrotic agent on platelet-derived growth factor (PDGF)-BB-activated hepatic stellate cells (HSC)-T6 cells in a concentration-dependent manner. We have also attempted to assess signaling pathways that may affect liver fibrosis. PDGF-BB was used to activate the HSC-T6 rat hepatic stellate cell line. The activated cells were treated with Isorhamnetin for 24 h. Finally, we compared the mRNA expression level of COLA1 and α-SMA and also the level of phosphorylated AKT protein with the control group. The obtained data revealed a significant increase in the expression level of the COLA1 and α-SMA genes (p > 0.05), as well as phosphorylated AKT protein, in the cells treated with PDGF-BB. In addition, 75 and 100 µM concentrations of Isorhamnetin markedly declined the COLA1 and α-SMA expression and also the phosphorylated AKT protein level in the HSC-T6 cells. Our findings suggest that Isorhamnetin decreases HSC-T6 activation, the expression of COLA1 and α-SMA, in vitro, which could act as an antifibrotic element to reduce and treat liver fibrosis disease.

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Ivermectin Attenuates CCl4-Induced Liver Fibrosis in Mice by Suppressing Hepatic Stellate Cell Activation.

Liver fibrosis, a common liver dysfunction with high morbidity and mortality rates, is the leading cause of cirrhosis and hepatocellular carcinoma, for which there are no effective therapies. Ivermectin is an antiparasitic drug that also has been showing therapeutic actions in many other diseases, including antiviral and anticancer actions, as well as treating metabolic diseases. Herein, we evaluated the function of ivermectin in regulating liver fibrosis. Firstly, carbon tetrachloride (CCl4)-injected Balb/c mice were used to assess the antifibrosis effects of ivermectin in vivo. Further, CFSC, a rat hepatic stellate cell (HSC) line, was used to explore the function of ivermectin in HSC activation in vitro. The in vivo data showed that ivermectin administration alleviated histopathological changes, improved liver function, reduced collagen deposition, and downregulated the expression of profibrotic genes. Mechanistically, the ivermectin treatment inhibited intrahepatic macrophage accumulation and suppressed the production of proinflammatory factors. Importantly, the ivermectin administration significantly decreased the protein levels of α-smooth muscle actin (α-SMA) both in vivo and in vitro, suggesting that the antifibrotic effects of ivermectin are mainly due to the promotion of HSC deactivation. The present study demonstrates that ivermectin may be a potential therapeutic agent for the prevention of hepatic fibrosis.

Cell-Permeable PROTAC Degraders against KEAP1 Efficiently Suppress Hepatic Stellate Cell Activation through the Antioxidant and Anti-Inflammatory Pathway.

Accumulating evidence indicates that oxidative stress and inflammation are involved in the physiopathology of liver fibrogenesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor, which regulates the expression of redox regulators to establish cellular redox homeostasis. The Nrf2 modulator can serve as a primary cellular defense against the cytotoxic effects of oxidative stress. We designed a chimeric Keap1-Keap1 peptide (KKP1) based on the proteolysis-targeting chimera technology. The KKP1 peptide not only can efficiently penetrate into the rat hepatic stellate cell line (HSC-T6) cells but also can induce Keap1 protein degradation by the ubiquitination-proteasome degradation pathway, which releases Nrf2 and promotes the transcriptional activity of the Nrf2/antioxidant response element pathway. It then activates the protein expression of the downstream antioxidant factors, the glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 (HO-1). Finally, Keap1 protein degradation inhibits the nuclear factor-kappaB inflammatory signal pathway, the downstream inflammatory factor tumor necrosis factor alpha, and the interleukin-1beta protein expression and further inhibits the expression of the fibrosis biomarker gene. The current research suggests that our designed KKP1 may provide a new avenue for the future treatment of liver fibrosis.

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Molecular mechanisms of Agardhiella subulata attenuates hepatic fibrosis by modulating hepatic stellate cell activation via the reduction of autophagy

In this study, we investigated the effects of Agardhiella subulata extract (ASE) and its underlying mechanisms in vivo and in vitro. Rats induced hepatic fibrosis with CCl4 and transforming growth factor-β1 (TGF-β1) activated rat hepatic stellate cell line (HSC-T6) was used. The results showed that ASE (oral, 10 or 50 mg/kg) effectively attenuated liver fibrogenesis in the rats. This was evidenced by decreases in serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels, histopathological changes, decreased collagen deposition, and extracellular matrix formation. ASE significantly reduced hepatocyte apoptosis, Kupffer cell activation, and the levels of fibrogenic molecules such as alpha-smooth muscle actin and collagen. Furthermore, ASE downregulated the TGF-β1/Smad signaling pathway and concomitantly inhibited the activation of hepatic stellate cells by reducing autophagy and inducing the regeneration of lipid droplets both in vivo and in vitro. Overall, ASE inhibited various mechanisms that are important targets in the prevention of hepatic fibrosis.

Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1/P53/SLC7A11 pathway.

Wogonoside (WG) is a flavonoid chemical component extracted from Scutellaria baicalensis, which exerts therapeutic effects on liver diseases. Ferroptosis, a novel form of programmed cell death, regulates diverse physiological/pathological processes. In this study, we attempted to investigate a novel mechanism by which WG mitigates liver fibrosis by inducing ferroptosis in hepatic stellate cells (HSCs). A CCl4 -induced mouse liver fibrosis model and a rat HSC line were employed for in vivo and in vitro experiments, both treated with WG. Firstly, the levels of the fibrotic markers α-smooth muscle actin (α-SMA) and α1(I)collagen (COL1α1) were effectively decreased by WG in CCl4 -induced mice and HSC-T6 cells. Additionally, mitochondrial condensation and mitochondrial ridge breakage were observed in WG-treated HSC-T6 cells. Furthermore, ferroptotic events including depletion of SLC7A11, GPX4 and GSH, and accumulation of iron, ROS and MDA were discovered in WG-treated HSC-T6 cells. Intriguingly, these ferroptotic events did not appear in hepatocytes or macrophages. WG-elicited HSC ferroptosis and ECM reduction were dramatically abrogated by ferrostatin-1 (Fer-1), a ferroptosis inhibitor. Importantly, our results confirm that SOCS1/P53/SLC7A11 is a signaling pathway which promotes WG attenuation of liver fibrosis. On the contrary, WG mitigated liver fibrosis and inducted HSC-T6 cell ferroptosis were hindered by SOCS1 siRNA and pifithrin-α (PFT-α). These findings demonstrate that SOCS1/P53/SLC7A11-mediated HSC ferroptosis is associated with WG alleviating liver fibrosis, which provides a new clue for the treatment of liver fibrosis.

MiR-146b-5p activation of hepatic stellate cells contributes to the progression of fibrosis by directly targeting HIPK1.

The present study aimed to explore the biological functions of microRNA (miR)-146b-5p and homeodomain interacting protein kinase 1 (HIPK1) in the progression of hepatic fibrosis (HF) and to identify the underlying mechanism. A rat HF model was established by administering a subcutaneous injection of carbon tetrachloride (CCl4). Relative levels of miR-146b-5p and HIPK1 in fibrotic rat liver tissues and the rat hepatic stellate cell (HSC) line HSC-T6 were measured by quantitative reverse transcription PCR, western blotting and immunohistochemistry. Following activation of HSC-T6 cells by lipopolysaccharide (LPS) induction, cell viability was examined by MTT assay. Transfection of miR-146b-5p mimic or inhibitor into HSC-T6 cells was performed, with the aim to identify the influence of miR-146b-5p on HSC-T6 cell behavior. The targeting relationship between miR-146b-5p and HIPK1 was predicted by TargetScan 7.2 and StarBase 3.0 and it was later verified by a dual-luciferase reporter assay. Through lentivirus transfection, the biological function of HIPK1 in regulating the progression of HF and the underlying mechanism were investigated. The results showed that miR-146b-5p was upregulated in liver tissues of rats with HF and activated HSC-T6 cells, while HIPK1 was downregulated in liver tissues of rats with HF and activated HSC-T6 cells. miR-146b-5p was able to upregulate the activation markers of LPS-induced HSC-T6 cells, upregulate COL1A1 and TGF-β, increase cell viability and contribute to fibrosis progression. HIPK1 was validated as the direct target of miR-146b-5p and its overexpression could effectively reduce the effect of miR-146b-5p in contribution to the progression of HF. In conclusion, miR-146b-5p was significantly upregulated during the progression of HF. By targeting and downregulating HIPK1, miR-146b-5p could significantly activate HSCs, upregulate COL1A1 and TGF-β and contribute to fibrosis progression. miR-146b-5p is a potential biomarker and therapeutic target for HF.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-β, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)—derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.

The in vitro effects of phospholipase D1-mTOR axis in liver fibrogenesis

AimsThe activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. Main methodsThe expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor β1 (TGFβ1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFβ1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). Key findingsSignificant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFβ1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFβ1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SignificanceThe PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.

Effect of downregulation of phosphatase and tensin homolog gene expression on p130crk- related substrate protein and paxillin signal transduction in activated hepatic stellate cells in vitro

Objective: To investigate the role of adenovirus-mediated short hairpin RNA (shRNA) in down-regulating the expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) on p130Crk-related substrates(p130Cas) and paxillin signal transduction to activate hepatic stellate cell (HSC) in vitro. Methods: The rat hepatic stellate cell line, HSC-T6 was cultured and activated in vitro. The adenovirus was used as a vector to transiently transfect shRNA targeting PTEN to activate HSC in vitro, and then PTEN low expression model of activated HSC in vitro was established. Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of PTEN, p130cas and paxillin in activated HSC. The experiment was divided into control group (HSC were transfected with DMEM medium instead of adenovirus), Ad-GFP group (HSC were infected with empty the adenovirus expressing green fluorescent protein (GFP) alone), and Ad-shRNA/PTEN group (HSC were infected with the recombinant adenovirus containing both shRNA targeting PTEN and GFP gene). One-way analysis of variance was used for comparison of multiple groups, and LSD test was used for inter-group comparison. Results: shRNA targeting PTEN was successfully transfected and significantly down-regulated the PTEN protein and mRNA expression of HSC in vitro (P < 0.05), and the PTEN low expression model of HSC in vitro was successfully constructed. Compared with the expression of p130cas mRNA in the three groups of HSC, the expression fold of p130cas mRNA in the Ad-GFP group and the Ad-shRNA / PTEN group was 1.01 times and 1.52 times, respectively. The expression of p130cas mRNA in HSC of the Ad-shRNA / PTEN group was significantly higher than control group and Ad-GFP group (P < 0.05), but there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of p130cas protein in the three groups was higher than that in the control group (0.74 ± 0.07) and the Ad-GFP group (0.72 ± 0.02); P < 0.05, but there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05). The expression of paxillin mRNA in the three groups of HSCs was compared with the expression of paxillin mRNA in the control group of HSC being 1, the expression folds of paxillin mRNA in the Ad-GFP group and Ad-shRNA / PTEN group were 0.97 times and 1.58 times, respectively. The expression of paxillin mRNA in the Ad-shRNA / PTEN group was higher than that in the control group and the Ad-GFP group (P < 0.05), and there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of paxillin protein in the three groups of HSCs was higher in the Ad-shRNA / PTEN group (0.91 ± 0.05) than control group (0.46 ± 0.03) and Ad-GFP group (0.50 ± 0.04), P < 0.05, and there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05). Conclusion: Down-regulation of PTEN expression can significantly boost p130cas and signal transduction activity of paxillin protein in activated HSC in vitro.

Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90

Gambogic acid (GA), a major ingredient of Garcinia hanburryi, is known to have diverse biological effects. The present study was designed to evaluate the anti-fibrotic effects of GA on hepatic fibrosis and reveal its underlying mechanism. We investigated the anti-fibrotic effect of GA on dimethylnitrosamine and bile duct ligation induced liver fibrosis in rats in vivo. The rat and human hepatic stellate cell lines (HSCs) lines were chose to evaluate the effect of GA in vitro. Our results indicated that GA could significantly ameliorate liver fibrosis associated with improving serum markers, decrease in extracellular matrix accumulation and HSCs activation in vivo. GA significantly inhibited the proliferation of HSC cells and induced the cell cycle arrest at the G1 phase. Moreover, GA triggered autophagy at early time point and subsequent initiates mitochondrial mediated apoptotic pathway resulting in HSC cell death. The mechanism of GA was related to inhibit heat shock protein 90 (HSP90) and degradation of the client proteins inducing PI3K/AKT and MAPK signaling pathways inhibition. This study demonstrated that GA effectively ameliorated liver fibrosis in vitro and in vivo, which provided new insights into the application of GA for liver fibrosis.

Inhibitory Effects of Novel Oridonin Analog CYD0617‐2 on Hepatic Stellate Cell Activation through NF‐κB and Stat3 Pathways

IntroductionInhibition of activated hepatic stellate cell (HSC) proliferation is recognized as a promising therapeutic strategy for liver fibrosis. Previously, we reported that natural compound oridonin exhibits potent anti‐hepatic fibrosis activity, and that NF‐κB and Stat3 signaling contribute to HSC activation. In this study, we determined the anti‐fibrotic effects of a novel oridonin analog CYD0617‐2 in activated HSCs and the underlying mechanism.MethodsThe activated human and rat HSC lines LX‐2 and HSC‐T6 were treated with CYD0617‐2. Cell viability was measured by alamarBlue assay. Apoptosis was determined by Cell death ELISA. Cellular proteins were analyzed with Western blot. Cytokines were determined with ELISA.ResultsCYD0617‐2 treatment significantly inhibited proliferation and induced apoptosis in both cell lines in a dose‐dependent manner approximately 10 fold more potent than the parent compound oridonin. CYD0617‐2 stimulated apoptotic proteins p21, p27, and cleaved‐PARP, while suppressing extracellular matrix proteins fibronectin and laminin. CYD0617‐2 blocked LPS‐induced NF‐κB p65 nuclear translocation and DNA binding activity, prevented LPS‐induced NF‐κB inhibitory protein IκBα phosphorylation and degradation, and suppressed LPS‐stimulated IKKα/β phosphorylation. LPS‐induced NF‐κB cytokines IL‐6, MCP‐1, and IL‐1β were attenuated by CYD0617‐2. The endogenous and LPS‐induced NF‐κB p65 S536 phosphorylation was inhibited by CYD0617‐2. Importantly, NF‐κB chemical inhibitor Bay‐11‐0782 was found to inhibit proliferation and promote apoptosis in both cell lines. Moreover, CYD0617‐2 attenuated Stat3 activation by suppressing pStat3/T705 phosphorylation and target gene c‐myc.ConclusionThe potent anti‐hepatic fibrogenetic effect of CYD0617‐2 may be through suppression of IKK/IκBα/NF‐κB and Stat3 pathway.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of SEPT6 on the biological behavior of hepatic stellate cells and liver fibrosis in rats and its mechanism

Hepatic stellate cells (HSCs) are key effectors during the development of liver fibrosis. Septin 6 (SEPT6) is a highly evolutionarily conserved GTP-binding protein that regulates various cell biological behaviors. The expression and function of SEPT6 in HSCs remain unknown. Here we demonstrate that SEPT6 expression is significantly elevated following the activation of primary rat HSCs, the human hepatic stellate cell line LX-2 and the rat hepatic stellate cell line HSC-T6, as well as in both human and rat fibrotic liver tissue. In vitro, the overexpression of SEPT6 promoted HSCs activation, proliferation, cell cycle progression and migration and inhibited HSCs apoptosis. In contrast, knockdown of SEPT6 exerted the opposite effects on HSCs. Mechanistically, SEPT6 exerted its pro-fibrogenic effect by promoting the expression of TGF-β1 and the phosphorylation of Smad2, Smad3, extracellular-signal-regulated kinase, c-Jun NH2-terminal kinase, stress-activated protein kinase-2, and protein kinase B. However, in HSC-T6 cells, blockade of the TGF-β1/Smad signaling pathway by SB431542 significantly decreased the expression of α-smooth muscle actin, cyclin D1, BCL2, and matrix metalloproteinase-2 and -9, which had been enhanced by SEPT6 overexpression. In vivo, adenovirus-mediated SEPT6 inhibition attenuated thioacetamide (TAA)-induced liver fibrosis in rats by decreasing the deposition of the extracellular matrix (ECM). SEPT6 inhibition decreased the proliferation capacity of HSCs and induced apoptosis of HSCs. Collectively, our results reveal that SEPT6 regulates various biological behaviors in HSCs through TGF-β1/Smad, mitogen-activated protein kinases and phosphatidylinositol-3-kinase/protein kinase B signaling pathways, thus promoting liver fibrosis.The cytoskeletal GTPase SEPT6 is elevated during hepatic stellate cell (HSC) activation and in liver fibrosis. SEPT6 promotes HSC activation, proliferation, cell cycle progression, survival and migration through the TGF-β1/Smad, MAPK and PI3K/AKT signaling pathways. Adenovirus-mediated SEPT6 inhibition attenuates thioacetamide-induced liver fibrosis.