Xiaochaihutang Inhibits the Activation of Hepatic Stellate Cell Line T6 Through the Nrf2 Pathway.
ABSTRACT: Xiaochaihutang (XCHT) is one of classic prescriptions in Treatise on Febrile Diseases in China which was reported to have the effect of anti-hepatic fibrosis in vivo. Activation of hepatic stellate cells (HSCs) is now well established as a central driver of fibrosis in liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important element for anti-oxidative damage which is one of the key factors responsible for occurrence. This study was to investigate the effect of XCHT compound serum on HSCs activation and focus on the Nrf2 pathway. Rats in treatment groups were given the appropriate doses of XCHT granules (5 g/kg) and Silybin (50 mg/kg) for 6 days, and the serum were obtained. The compound serum was used to intervene HSCs. The results found that XCHT compound serum significantly inhibited the proliferation of HSCT6 cells. The number of ?-SMA positive stained cells in HSCT6 cells and the content of Collagen type I (collagen-I) in supernatant were significantly decreased indicating suppression of activated HSCs. Compared with the control group, the nuclear transcription of Nrf2 and the expressions of Nqo1, GCLC, and GCLM were significantly increased in XCHT group. However, the effects of XCHT were inhibited in Nrf2-siRNA transfected HSCT6 cells. These studies demonstrated that XCHT could inhibit HSCT6 cell proliferation and activation. The mechanism might be related to up-regulation of the Nrf2 pathway against oxidative stress.
Project description:Oxidative stress induced by high ammonia, which leads to astrocyte edema, is the key to acute hepatic encephalopathy (AHE). Nuclear factor erythroid 2-related factor 2 (NRF2) has been implicated in oxidative stress, but the mechanism of NRF2 against ammonia-induced astrocytes edema has not been fully studied. We confirmed that the NRF2 pathway is related to brain edema caused by AHE and found that Xiaochaihutang (XCHT) could effectively activate the NRF2 pathway to treat AHE. The model of AHE was established with thioacetamide (TAA) in rats. Rat behaviors were observed, brain water content, blood ammonia levels, glutamine synthetase (GS), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) were determined after XCHT treatment. Furthermore, the expression of NRF2 pathway proteins and mRNA, glial fibrillary acidic protein (GFAP) and aquaporins 4 (AQP4) were examined. In order to determine whether XCHT has a direct effect on cerebral edema caused by high ammonia, we examined the effect of XCHT compound serum on cortical astrocytes in the presence of ammonia, through microscopic observation and immunofluorescence (IF). Results showed that AHE induced by TAA changed the behavior of the rats, and increased brain water content, blood ammonia levels, GS and MDA content meanwhile decreasing T-SOD, but these symptoms were improved by treatment with XCHT. XCHT protected brain edema by activating the NRF2 pathway and increasing the expression of downstream proteins and genes. Astrocytes treated with 5 mM ammonia also showed an increase in the AQP4 protein expression but a decrease in XCHT compound serum and ammonia-induced cell edema groups. This study demonstrates that the NRF2 pathway is involved in the brain edema in AHE, and XCHT may represent a useful prescription for the treatment of AHE.
Project description:Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.
Project description:Xiao Chai Hu Tang (XCHT), a traditional herbal formula, is widely administered as a cancer treatment. However, the underlying molecular mechanisms of its anticancer effects are not fully understood. In the present study, a computational pharmacological model that combined chemical space mapping, molecular docking and network analysis was employed to predict which chemical compounds in XCHT are potential inhibitors of cancer-associated targets, and to establish a compound-target (C-T) network and compound-compound (C-C) association network. The identified compounds from XCHT demonstrated diversity in chemical space. Furthermore, they occupied regions of chemical space that were the same, or close to, those occupied by drug or drug-like compounds that are associated with cancer, according to the Therapeutic Targets Database. The analysis of the molecular docking and the C-T network demonstrated that the potential inhibitors possessed the properties of promiscuous drugs and combination therapies. The C-C network was classified into four clusters and the different clusters contained various multi-compound combinations that acted on different targets. The study indicated that XCHT has a polypharmacological role in treating cancer and the potential inhibitory components of XCHT require further investigation as potential therapeutic strategies for cancer patients.
Project description:This study explored the effects of a classical Chinese medicine formula- Xiao-Chai-Hu Tang(XCHT) on the model mice with D-galactosamine -induced liver injury. Sixty male imprinting control region (ICR) mice were used in the present study, and they were separated randomly into 6 groups: a normal control group (Group A, n=10), a model control (Group B, n=10), a positive control (Group C, n=10), a low dose of XCHT group (Group D, n=10), a medium dose of XCHT group (Group E, n=10), and a high dose of XCHT group (Group F, n=10). ELISA was used to detect the IL-6 and TNF-? levels in the serum. Real-time PCR was performed to assess the expression of FasmRNA, Fas-LmRNA, Bcl-2mRNA of the liver tissues. Western blotting was used to detect the Bax protein expression of the liver tissues. The serum IL-6 and TNF-? levels of Group B were significantly higher than the other groups (P<0.05). The expression of Fas mRNA, Fas-LmRNA, and Bax protein of the liver tissues of Group B were significantly higher than those of the other groups (P<0.05). The expression of Bcl-2 mRNA of the liver tissues of Group B was significantly lower than other groups (P<0.05). Both of XCHT and biphenyl dicarboxylate significantly decreased the serum IL-6 and TNF-? levels and FasmRNA, FasLmRNA, Bax protein expression and increased the Bcl-2 mRNA expression of the liver tissues of model mice (P<0.05). It may be through decreasing the serum IL-6 and TNF-? levels and FasmRNA, FasLmRNA, Bax protein expression and increasing the Bcl-2 mRNA expression of the liver tissues that XCHT significantly relieved the D-galactosamine -induced liver injury.
Project description:The transcription factor Nrf2 is an important modulator of antioxidant and drug metabolism, carbohydrate and lipid metabolism, as well as heme and iron metabolism. Regulation of Nrf2 expression occurs transcriptionally and post-transcriptionally. Post-transcriptional regulation entails ubiquitination followed by proteasome-dependent degradation. Additionally, Nrf2-mediated gene expression is subject to negative regulation by ATF3, Bach1 and cMyc. Nrf2-mediated gene expression is an important regulator of a cell's response to radiation. Although a majority of studies have shown that Nrf2 deficient cells are radiosensitized and Nrf2 over expression confers radioresistance, Nrf2's role in mediating the radiation response of crypt cells is controversial. The Nrf2 activator CDDO attenuates radiation-mediated crypt injury, whereas intestinal crypts in Nrf2 null mice are radiation resistant. Further investigation is needed in order to define the relationship between Nrf2 and radiation sensitivity in Lgr5+ and Bmi1+ cells that regulate regeneration of crypt stem cells. In hematopoietic compartments Nrf2 promotes the survival of irradiated osteoblasts that support long-term hematopoietic stem cell (LT-HSC) niches. Loss of Nrf2 in LT-HSCs increases stem cell intrinsic radiosensitivity, with the consequence of lowering the LD5030. An Nrf2 deficiency drives LT-HSCs from a quiescent to a proliferative state. This results in hematopoietic exhaustion and reduced engraftment after myoablative irradiation. The question of whether induction of Nrf2 in LT-HSC enhances hematopoietic reconstitution after bone marrow transplantation is not yet resolved. Irradiation of the lung induces pulmonary pneumonitis and fibrosis. Loss of Nrf2 promotes TGF-?/Smad signaling that induces ATF3 suppression of Nrf2-mediated target gene expression. This, in turn, results in elevated reactive oxygen species (ROS) and isolevuglandin adduction of protein that impairs collagen degradation, and may contribute to radiation-induced chronic cell injury. Loss of Nrf2 impairs ?Np63 stem/progenitor cell mobilization after irradiation, while promoting alveolar type 2 cell epithelial-mesenchymal transitions into myofibroblasts. These studies identify Nrf2 as an important factor in the radiation response of normal tissue.
Project description:Tissue stem cells are maintained in quiescence under physiological conditions but proliferate and differentiate to replenish mature cells under stressed conditions. The KEAP1-NRF2 system plays an essential role in stress response and cytoprotection against redox disturbance. To clarify the role of the KEAP1-NRF2 system in tissue stem cells, we focused on hematopoiesis in this study and used Keap1-deficient mice to examine the effects of persistent activation of NRF2 on long-term hematopoietic stem cells (LT-HSCs). We found that persistent activation of NRF2 due to Keap1 deficiency did not change the number of LT-HSCs but reduced their quiescence in steady-state hematopoiesis. During hematopoietic regeneration after bone marrow (BM) transplantation, persistent activation of NRF2 reduced the BM reconstitution capacity of LT-HSCs, suggesting that NRF2 reduces the quiescence of LT-HSCs and promotes their differentiation, leading to eventual exhaustion. Transient activation of NRF2 by an electrophilic reagent also promotes the entry of LT-HSCs into the cell cycle. Taken together, our findings show that NRF2 drives the cell cycle entry and differentiation of LT-HSCs at the expense of their quiescence and maintenance, an effect that appears to be beneficial for prompt recovery from blood loss. We propose that the appropriate control of NRF2 activity by KEAP1 is essential for maintaining HSCs and guarantees their stress-induced regenerative response.
Project description:Hepatic stellate cells (HSCs) are the primary source of matrix components in liver disease such as fibrosis. Phosphatidylinositol 3-kinase (PI3K) signaling in HSCs has been shown to induce fibrogenesis. In this study, we evaluated the anti-fibrotic activity of a novel imidazopyridine analogue (HS-173) in human HSCs as well as mouse liver fibrosis. HS-173 strongly suppressed the growth and proliferation of HSCs and induced the arrest at the G2/M phase and apoptosis in HSCs. Furthermore, it reduced the expression of extracellular matrix components such as collagen type I, which was confirmed by an in vivo study. We also observed that HS-173 blocked the PI3K/Akt signaling pathway in vitro and in vivo. Taken together, HS-173 suppressed fibrotic responses such as cell proliferation and collagen synthesis by blocking PI3K/Akt signaling. Therefore, we suggest that this compound may be an effective therapeutic agent for ameliorating liver fibrosis through the inhibition of PI3K signaling.
Project description:Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.
Project description:Hepatic stellate cells (HSC) orchestrate the deposition of extracellular matrix (ECM) and are the primary effector of liver fibrosis. Several factors, including TGF-?1, PDGF and oxidative stress, have been shown to trigger HSC activation. However, the involvement of cellular defence mechanisms, such as the activation of antioxidant response by Nrf2/Keap1 in the modulation of HSC activation is not known. The aim of this work was to elucidate the role of Nrf2 pathway in HSC trans-differentiation involved in the development of fibrosis. To this end, we repressed Nrf2 and Keap1 expression in HSC with specific siRNAs. We then assessed activation markers, as well as proliferation and migration, in both primary and immortalised human HSCs exposed to Smad inhibitors (SB-431542 hydrate and SB-525334), TGF-?1 and/or PDGF. Our results indicate that knocking down Nrf2 induces HSC activation, as shown by an increase in ?SMA-positive cells and by gene expression induction of ECM components (collagens and fibronectin). HSC with reduced Nrf2-levels also showed an increase in migration and a decrease in proliferation. We could also demonstrate that the activation of Nrf2-deficient HSC involves the TGF-?1/Smad pathway, as the activation was successfully inhibited with the two tested Smad inhibitors. Moreover, TGF-?1 elicited a stronger induction of HSC activation markers in Nrf2 deficient cells than in wild type cells. Thus, our data suggest that Nrf2 limits HSCs activation, through the inhibition of the TGF-?1/Smad pathway in HSCs.
Project description:Milk thistle contains silybin, which is a potential iron chelator. We aimed to determine whether silybin reduced iron absorption in patients with hereditary haemochromatosis. In this crossover study, on three separate occasions, 10 patients who were homozygous for the C282Y mutation in the HFE gene (and fully treated) consumed a vegetarian meal containing 13.9?mg iron with: 200?ml water; 200?ml water and 140?mg silybin (Legalon Forte); or 200?ml tea. Blood was drawn once before, then 0.5, 1, 2, 3 and 4?h after the meal. Consumption of silybin with a meal resulted in a reduction in the postprandial increase in serum iron (AUC±s.e.) compared with water (silybin 1726.6±346.8 versus water 2988.8±167; P<0.05) and tea (silybin 1726.6±346.8 versus tea 2099.3±223.3; P<0.05). In conclusion, silybin has the potential to reduce iron absorption, and this deserves further investigation, as silybin could be an adjunct in the treatment of haemochromatosis.