[Adeno-associated virus-mediated hepatocyte-specific NDUFA13 overexpression protects against CCl4-induced liver fibrosis in mice by inhibiting hepatic NLRP3 activation].

OBJECTIVE: To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms. METHODS: BALB/C mice (7 to 8 weeks old) were divided into normal group, CCl4 group, CCl4+AAV-NC group and CCl4+AAV-NDU13 group (n=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4 twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7-10 days prior to CCl4 injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1ß, and α-SMA and collagen Ⅲ was analyzed with immunofluorescence assay. RESULTS: HE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl4 injection (P < 0.001). NDUFA13 expression markedly decreased in CCl4-treated mice (P < 0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression (P < 0.001). In CCl4+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes (P < 0.001), significantly decreased TNF-α and IL-1ß secretion (P < 0.001), and inhibited hepatic stellate cell activation (P < 0.05) and collagen formation in the liver (P < 0.001). CONCLUSION: Mitochondrial NDUFA13 overexpression in hepatocytes protects against CCl4- induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.

Metabolomics reveals that chronic restraint stress alleviates carbon tetrachloride-induced hepatic fibrosis through the INSR/PI3K/AKT/AMPK pathway.

Hepatic fibrosis (HF) could be developed into liver cirrhosis or even hepatocellular carcinoma. Stress has an important role in the occurrence and development of various considerable diseases. However, the effect of a certain degree stress on HF is still controversial. In our study, stress was simulated with regular chronic restraint stress (CRS) and HF model was induced with CCl4 in mice. We found that CRS was able to attenuate CCl4-induced liver injury and fibrosis in mice. Surprisingly, behavioral analysis showed that the mice in the HF group exhibited depression-like behavior. Further, the metabolomic analysis revealed that 119 metabolites and 20 metabolic pathways were altered in mice liver, especially the betaine metabolism pathway. Combined with the results of Ingenuity Pathway Analysis (IPA), the key proteins INSR, PI3K, AKT, and p-AMPK were identified and verified, and the results showed that CRS could upregulate the protein levels and mRNA expression of INSR, PI3K, AKT, and p-AMPK in liver tissues of HF mice. It suggested that CRS alleviated CCl4-induced liver fibrosis in mice through upregulation of the INSR/PI3K/AKT/AMPK pathway. Proper stress might be a potential therapeutic strategy for the treatment of chronic liver disease, which provided new insights into the treatment of HF. KEY MESSAGES: Chronic restraint stress mitigated CCl4-induced liver injury and hepatic fibrosis. CCl4-induced liver fibrosis could cause depression-like behavior. Chronic restraint stress altered metabolomic profiles in hepatic fibrosis mice, especially the betaine metabolism pathway. Chronic restraint stress increased betaine levels in liver tissue. Chronic restraint stress regulated the INSR/PI3K/AKT/AMPK signaling pathway in hepatic fibrosis mice.

Aloin Attenuates Oxidative Stress, Inflammation, and CCl4-Induced Liver Fibrosis in Mice: Possible Role of TGF-ß/Smad Signaling.

Liver fibrosis refers to the excessive buildup of extracellular matrix (ECM) components in liver tissue. It is considered a pathological response to liver damage for which there is no effective treatment. Aloin, an anthraquinone compound isolated from the aloe plant, has shown good pharmacological effects in the treatment of gastric cancer, ulcerative colitis, myocardial hypertrophy, traumatic brain injury, and other diseases; however, its specific impact on liver fibrosis remains unclear. To address this gap, we conducted a study to explore the mechanisms underlying the potential antifibrotic effect of aloin. We constructed a mouse liver fibrosis model using carbon tetrachloride (CCl4) dissolved in olive oil as a modeling drug. Additionally, a cellular model was developed by using transforming growth factor ß1 (TGF-ß1) as a stimulus applied to hepatic stellate cells. After aloin intervention, serum alanine aminotransferase, hepatic hydroxyproline, and serum aspartate aminotransferase were reduced in mice after aloin intervention compared to CCl4-mediated liver injury without aloin intervention. Aloin relieved the oxidative stress caused by CCl4 via reducing hepatic malondialdehyde in liver tissue and increasing the level of superoxide dismutase. Aloin treatment decreased interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and increased the expression of IL-10, which inhibited the inflammatory response in liver injury. In addition, aloin inhibited the activation of hepatic stellate cells and reduced the level of α-smooth muscle actin (α-SMA) and collagen type I. In cell and animal experiments, aloin attenuated liver fibrosis, acting through the TGF-ß/Smad2/3 signaling pathway, and mitigated CCl4- and TGF-ß1-induced inflammation. Thus, the findings of this study provided theoretical data support and a new possible treatment strategy for liver fibrosis.

Empagliflozin suppresses hedgehog pathway, alleviates ER stress, and ameliorates hepatic fibrosis in rats.

Worldwide mortality from hepatic fibrosis remains high, due to hepatocellular carcinoma and end stage liver failure. The progressive nature of hepatic fibrosis from inflammation to cicatrized tissues warrants subtle intervention with pharmacological agents that hold potential. Empagliflozin (Empa), a novel hypoglycemic drug with antioxidant and anti-inflammatory properties, has lately been proposed to have additional antifibrotic activities. In the current study, we examined the antifibrotic effect of the Empa through modulating the activity of hepatic stellate cells by hedgehog (Hh) pathway. We also assessed the markers of inflammatory response and endoplasmic reticulum (ER) stress. Male Albino rats were treated with either CCl4 (0.4 mg/kg twice/week) and/or Empa (10 mg/kg/day) for eight weeks. In this study, CCl4 rats had active Hh signaling as indicated by overexpression of Patched 1, Smoothened and Glioblastoma-2. CCl4 induced ER stress as CHOP expression was upregulated and ERAD was downregulated. CCl4-induced inflammatory response was demonstrated through increased levels of TNF-α, IL-6 and mRNA levels of IL-17 while undetectable expression of IL-10. Conversely, Empa elicited immunosuppression, suppressed the expression of Hh markers, and reversed markers of ER stress. In conclusion, Empa suppressed CCl4-induced Hh signaling and proinflammatory response, meanwhile embraced ER stress in the hepatic tissues, altogether provided hepatoprotection.

Kaempferol attenuates carbon tetrachloride (CCl4)-induced hepatic fibrosis by promoting ASIC1a degradation and suppression of the ASIC1a-mediated ERS.

BACKGROUND: Kaempferol is a flavonoid derived from the herb, Kaempferia galanga L., in addition to exhibiting a wide range of pharmacological properties, kaempferol is also an anti-inflammatory, anti-lipid metabolizing, and anti-oxidative stress agent. The underlying molecular mechanisms of its effects on vascular endothelial growth factor (VEGF) secretion and activation of hepatic stellate cells (HSCs) are yet unknown. Activated HSCs induces VEGF release and extracellular matrix (ECM) accumulation which are important factors in hepatic fibrosis. PURPOSE: Our aim is to explore how kaempferol may affect hepatic fibrosis and the mechanisms behind its effects. METHODS: The in vivo model was Sprague-Dawley rats induced with carbon tetrachloride (CCl4). Histological staining was used to observe histological features of the liver. The levels of (alanine aminotransferase) ALT and (aspartate aminotransferase) AST were detected by the corresponding kits. Platelet-derived growth factor (PDGF) was used to stimulate the HSC-T6 rat hepatic stellate cells. The mechanisms underlying this process were investigated using a variety of molecular approaches, including immunofluorescence, RT-qPCR, and western blotting. Moreover, intracellular Ca2+ were observed by laser confocal microscope. RESULTS: It was found that kaempferol significantly reduced the expression of ASIC1a, VEGF, α-SMA and Collagen-I proteins in a model of CCl4-induced hepatic fibrosis in rats. In HSC-T6, kaempferol inhibits activation of HSCs by decreasing expression of ASIC1a, eIF2α, p-eIF2α and ATF-4. Laser confocal fluorescence showed that kaempferol inhibited Ca2+ influx and reduced Ca2+ concentration around the endoplasmic reticulum. Molecular docking and cellular thermal shift assay (CETSA) results further indicated that kaempferol interacted with ASIC1a. We found that kaempferol may promote the degradation of ASIC1a and inhibited ASIC1a- mediated upregulation of ERS. CONCLUSION: The data from our in vivo experiments demonstrate that kaempferol effectively attenuates hepatic fibrosis. In vitro studies we further propose a novel mechanism of kaempferol against hepatic fibrosis which can interact with ASIC1a and promote ASIC1a degradation while inhibiting the activation and VEGF release of HSCs by suppressing the ASIC1a-eIF2α-ATF-4 signaling pathway.

Protective effect of water extracts of Veronicastrum latifolium (Hemsl.) Yamazaki on carbon tetrachloride-induced liver fibrosis in mice and its effect on intestinal flora.

Liver fibrosis refers to a reversible event of repair and reconstruction following injury due to various etiologies, and its continuous development will lead to cirrhosis and liver cancer. Abnormal alterations in intestinal microbiota can hasten the development of hepatic fibrosis and damage. Veronicastrum latifolium (Hemsl.) Yamazaki (VLY) is a classic drug applied extensively for managing acute and chronic hepatitis, liver cirrhosis and ascites in ethnic minority areas of Guizhou Province, China, which possesses broad-spectrum pharmacological activities. In view of the crucial role of intestinal microbiota in the development of liver fibrosis, the present study attempted to investigate the effects of VLY aqueous extract on ameliorating CCl4-elicited liver fibrosis in mice and on intestinal microbiota and to explore its possible mechanism. Phytochemical analysis showed that VLY water extract contained a variety of components, particularly rich in organic acids and their derivatives, flavonoids, phenolic acids, nucleotides and their derivatives, carbohydrates and other compounds. VLY water extract remarkably alleviated CCl4-induced liver damage and fibrosis in mice, improved liver histology, and improved liver function abnormalities. VLY water extract also inhibited the activation of hepatic stellate cells and invasion of intrahepatic inflammatory cells. Additionally, sequencing the 16 s rDNA gene revealed that VLY water extract changed the intestinal microbiota composition in liver fibrotic mice. It elevated the Firmicutes/Bacteroidota ratio and enriched the relative Lactobacillus richness, which is capable of mitigating fibrosis and inflammation in impaired liver. In summary, through modulation of inflammation and intestinal microbiota, VLY water extract can reduce the CCl4-elicited liver fibrosis.

Farnesoid X receptor activation is required for the anti-inflammatory and anti-oxidative stress effects of Alisol B 23-acetate in carbon tetrachloride-induced liver fibrosis in mice.

Previous studies have shown that Alisol B 23-acetate (23ABA) had potent liver-protection effects, however, its roles and potential mechanisms in carbon tetrachloride (CCl4)-induced liver fibrosis remain to be determined. The present study aimed to investigate the effects of 23ABA on CCl4-induced liver fibrosis and tried to elucidate the underlying mechanisms by focusing on regulating of farnesoid X receptor (FXR). In vivo study found that 23ABA alleviated the CCl4-induced liver injury, and showed no obvious systemic toxicity on mice. 23ABA inhibited the collagen production, decreased sera levels of hyaluronic acid (HA) and procollagen type III (PC-III), lowered mRNA expression of α-smooth muscle actin (α-SMA), fibronectin, collagen I and collagen III in livers of mice. 23ABA inhibited the mRNA expressions and the sera levels of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α), as well as decreased the expression of cyclooxygenase 2 (COX-2) in fibrotic livers of mice. Besides, 23ABA decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased glutathione (GSH) level, enhanced activities of superoxide dismutase (SOD) and glutathione reductase (GR) as well as increased mRNA expression of nuclear factor-E2-related factor 2 (Nrf2), glutamate-cysteine ligase, catalytic subunit (GCLC) and glutamate-cysteine ligase, modifier subunit (GCLM). Further study showed that the anti-liver injury and anti-fibrotic effects of 23ABA were abrogated by FXR antagonist guggulsterone (GS) in vivo. In addition, the inhibition effects of 23ABA on liver inflammation and oxidative stress were also weakened by treatment with GS in CCl4-induced fibrotic mice livers. In conclusion, the protective effects of 23ABA against CCl4-induced liver injury and fibrosis, due to FXR-mediated regulation of liver inflammation and oxidative stress.

Axitinib attenuates the progression of liver fibrosis by restoring mitochondrial function.

Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma, which may eventually lead to liver failure and even death. No direct anti-fibrosis drugs are available at present. Axitinib is a new generation of potent multitarget tyrosine kinase receptor inhibitors, but its role in liver fibrosis remains unclear. In this study, a CCl4-induced hepatic fibrosis mouse model and a TGF-ß1-induced hepatic stellate cell model were used to explore the effect and mechanism of axitinib on hepatic fibrosis. Results confirmed that axitinib could alleviate the pathological damage of liver tissue induced by CCl4 and inhibit the production of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. It also inhibited collagen and hydroxyproline deposition and the protein expression of Col-1 and α-SMA in CCl4-induced liver fibrosis. In addition, axitinib inhibited the expression of CTGF and α-SMA in TGF-ß1-induced hepatic stellate cells. Further studies showed that axitinib inhibited mitochondrial damage and reduced oxidative stress and NLRP3 maturation. The use of rotenone and antimycin A confirmed that axitinib could restore the activity of mitochondrial complexes I and III, thereby inhibiting the maturation of NLRP3. In summary, axitinib inhibits the activation of HSCs by enhancing the activity of mitochondrial complexes I and III, thereby alleviating the progression of liver fibrosis. This study reveals the strong potential of axitinib in the treatment of liver fibrosis.

Peptides from Antarctic krill (Euphausia superba) ameliorate acute liver injury in mice induced by carbon tetrachloride via activating the Nrf2/HO-1 pathway.

This study aimed to evaluate the hepatoprotective effects of peptides from Antarctic krill (AKP) on carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in mice and the underlying molecular mechanisms. ICR mice were pretreated with AKP (500 mg kg-1, i.g.) and silybin (30 mg kg-1, i.g.) for 15 days before CCl4 (0.25 mL per kg BW, i.p.) injection. To assess hepatocellular damage and molecular indices, the serum and liver tissue were evaluated at harvest. The results showed that AKP pretreatment remarkably attenuated CCl4-induced liver injury, which was identified by the decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alleviation of hepatocyte necrosis, and inhibition of the levels of the pro-inflammatory factors TNF-α and IL-1ß compared to those for silymarin. AKP pretreatment also enhanced the redox balance by reducing the concentrations of MDA and 8-iso-PG and increasing the activities of SOD, GSH and GSH-PX in the liver of mice. In addition, AKP upregulated oxidative stress-related mRNA expressions of Nrf2, Keap1, HO-1, and NQO1 and further activated the protein expression on the Nrf2/HO-1 singling pathway. In summary, AKP might be a promising hepatoprotective nutraceutical against ALI and its underlying mechanisms are associated with activation of the Nrf2/HO-1 pathway.

Glaucocalyxin A attenuates carbon tetrachloride-induced liver fibrosis and improves the associated gut microbiota imbalance.

Liver fibrosis refers to the pathophysiological process of dysplasia on the connective tissue of the liver, caused by a variety of pathogenic factors. Glaucocalyxin A (GLA) has anticoagulation, antibacterial, anti-inflammation, antioxidant and antitumour properties. However, whether GLA ameliorates liver fibrosis or not is still unclear. In this study, a liver fibrosis model was established using male C57BL/6 mice. The mice were treated with 5 and 10 mg/kg GLA via intraperitoneal injection, respectively. The ones that were treated with 5 mg/kg OCA were used as the positive control group. The levels of liver function, liver fibrosis biomarkers and liver pathological changes were then evaluated. We also explored the effects of GLA on inflammatory response and liver cell apoptosis. In addition, we investigated the gut microbiota mechanisms of GLA on liver fibrosis. The results from this study that GLA could significantly decrease the level of liver function (AST, ALT, TBA) and liver fibrosis (HA, LN, PC-III, IV-C). On the other hand, a significant decrease in inflammation levels (IL-1ß, TNF-α) were also noted. GLA also improves CCl4-induced pathological liver injuries and collagen deposition, in addition to decreasing apoptosis levels. In addition, an increase in the ratio of Bacteroidetes and Firmicutes in liver disease was also observed. GLA also improves the gut microbiota. In conclusion, GLA attenuates CCl4-induced liver fibrosis and improves the associated gut microbiota imbalance.

Dietary oxidized frying oil activates hepatic stellate cells and accelerates the severity of carbon tetrachloride- and thioacetamide-induced liver fibrosis in mice.

Deep-frying is a common cooking practice worldwide, and after repeated heating's, the oil undergoes various chemical reactions, including hydrolysis, polymerization, lipid oxidation, and the Maillard reaction. Studies have pointed out that oxidized dietary frying oil may cause teratogenesis in mice and increase cancer and cardiovascular risks. The liver is the main organ involved in dietary nutrient catabolism, detoxification, bile production, and lipid metabolism. Nevertheless, the effects of oxidized frying oil exposure on the activation of hepatic stellate cells (HSCs) and liver fibrosis are still unclear. In this study, we showed that exposure to oxidized frying oil enhanced the sensitivity of HSCs to transforming growth factor (TGF)-ß1-induced α-smooth muscle actin (α-SMA), collagen 1a2, collagen 1a1, metalloproteinase-2, and phosphorylated smad2/3 activation. In both carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis mouse models, we showed that long-term administration of a 10% fried oil-containing diet significantly upregulated fibrogenesis genes expression and deposition of hepatic collagen. Furthermore, long-term fried oil exposure not only promoted macrophage infiltration and increased inflammatory-related gene expression, but also accumulated excess cholesterol and lipid peroxidation in the liver tissues. In conclusion, our study demonstrated that feeding a fried oil-containing diet may trigger TGF-ß1-induced HSCs activation and thereby promote liver damage and fibrosis progression through enhancing the inflammatory response and lipid peroxidation.

Alternanthera brasiliana L. extract alleviates carbon tetrachloride-induced liver injury and fibrotic changes in mice: Role of matrix metalloproteinases and TGF-ß/Smad axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera brasiliana L. is a flowering plant belonging to the family Amaranthaceae and is popularly known as "penicillin". It is used in folk medicine to treat infections, coughs, wound healing, and inflammatory diseases. AIM OF THE STUDY: We investigated the effect of Alternanthera brasiliana L. leaves hydroalcoholic extract (AB) against oxidative stress, inflammation, and fibrotic changes in an experimental model of carbon tetrachloride (CCl4)-induced liver injury and fibrosis in mice. MATERIALS AND METHODS: Thirty-six male Balb/C mice were randomized into five groups: normal control, AB control, CCl4 control, CCl4 + AB-200 mg/kg, and CCl4 + AB-400 mg/kg. In mice, liver injury was induced by intraperitoneal injection of CCl4 (20% in corn oil, 5 ml/kg body weight) thrice a week for six consecutive weeks. AB extract at two doses (200 mg/kg and 400 mg/kg body weight) was administered orally for six consecutive weeks. Liver injury-related serum markers (ALT, AST, ALP), antioxidants (GSH, GST, SOD, and vitamin C), pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-18, ultrasonographic and histological alterations, proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), nuclear factor-κB (p65) (NF-κB), nod-like receptor protein 3 (NLRP3), and TGF-ß/Smad signaling were accessed. LC-Q-TOF-MS/MS analysis of AB was performed. RESULTS: AB treatment significantly decreased the CCl4-induced rise in serum ALT, AST, and ALP activities and improved the histological alterations. Compared with the CCl4-treated group, treatment with AB significantly restored the hepatic antioxidants and reduced the pro-inflammatory cytokines in the liver. The antioxidant activity of AB may be attributed to its terpenoid constituents, which was confirmed by LC-Q-TOF-MS/MS analysis. The CCl4-induced rise in expression of MMP-2 and MMP-9 and decrease in TIMP-1 were markedly restored in the AB-treated groups. Further findings revealed a significant reduction in the protein levels of phospho-NF-κB (p65), NLRP3, TGF-ß, pSmad2/3, collagen I, and α-smooth muscle actin (α-SMA) in the AB treatment groups. CONCLUSIONS: The hepatoprotective effect of AB may be attributed to the high content of terpenoid compounds and alleviates liver injury and associated fibrotic changes through modulating MMPs, NF-κB (p65), and the TGF-ß/Smad axis.

Ginsenoside Rd Inhibited Ferroptosis to Alleviate CCl4-Induced Acute Liver Injury in Mice via cGAS/STING Pathway.

Carbon tetrachloride (CCl4)-induced lipid peroxidation associated with hepatic oxidative stress and cell death is an important mechanism of acute liver injury (ALI). Ginsenoside Rd is considered an active ingredient of ginseng. Evidence suggests that ginsenoside Rd may improve ischaemic stroke, nerve damage, cancer and other diseases involving apoptosis, inflammation, oxidative stress, mitochondrial injury and autophagy. However, the effects of ginsenoside Rd on CCl4-induced ALI and its underlying mechanisms are still unclear. In this study, 0.25% CCl4 was injected intraperitoneally in mice to establish a CCl4-induced ALI model. In the Rd treatment group, Rd (10, 20[Formula: see text]mg/kg) doses were injected intraperitoneally 1[Formula: see text]h before and 23[Formula: see text]h after CCl4 administration. Ferroptosis inducer imidazole ketone erastin (IKE) was injected intraperitoneally 4[Formula: see text]h before CCl4 administration to explore the mechanism. The blood and liver were collected 24[Formula: see text]h after CCl4 administration to investigate the effect and mechanism of ginsenoside Rd on CCl4-induced ALI. Our results showed that ginsenoside Rd inhibited CCl4-induced ALI in mice. Ginsenoside Rd also downregulated CCl4-induced serum and liver iron, 4-hydroxynonenal, and 8-hydroxy-2 deoxyguanosine levels. Furthermore, it upregulated glutathione and glutathione peroxidase 4 levels. In addition, ginsenoside Rd downregulated the expression of cGAS and STING. Subsequently, the ferroptosis inducer imidazole ketone erastin significantly reversed the hepatoprotective effect and influence of ginsenoside Rd with regard to the indicators mentioned above. Our study confirmed that ginsenoside Rd ameliorated CCl4-induced ALI in mice, which was related to the reduction of ferroptosis. Simultaneously, the ginsenoside Rd-mediated inhibition of the cGAS/STING pathway contributed to its antiferroptosis effect. In conclusion, our results suggested that ginsenoside Rd inhibited ferroptosis via the cGAS/STING pathway, thereby protecting mice from CCl4-induced ALI. These results suggested ginsenoside Rd may be used as a potential intervention treatment against CCl4-induced ALI.

Experimental VX2 Rabbit Liver Tumor Model in Carbon Tetrachloride-Induced Cirrhosis of the Liver.

Liver cirrhosis is a major underlying factor in the development of hepatocellular carcinoma. Currently, there is an unmet need for midsize experimental vertebrate models that would offer reproducible implantable liver tumors in a cirrhotic liver background. This study establishes a protocol for a syngeneic rabbit model of VX2 liver cancer with underlying liver cirrhosis induced using carbon tetrachloride (CCl4). Male New Zealand white rabbits (n = 3) received CCl4 by intragastric administration once weekly. Concentrations started at 5% v/v CCl4 dissolved in olive oil. CCl4 dosing was progressively increased every week by 2.5% v/v increments for the duration of treatment (16 weeks total). VX2 tumors were then orthotopically implanted into the left hepatic lobe and allowed to grow for 3 weeks. Cross-sectional imaging confirmed the presence of hepatic tumors. Gross and histopathological evaluations showed reproducible tumor growth in the presence of liver cirrhosis in all animals.

The effects of taraxasterol on liver fibrosis revealed by RNA sequencing.

Effective treatment of liver fibrosis remains a challenging medical problem. Taraxasterol (TAR) has anti-inflammatory, anti-tumor and hepatoprotective effects. Studies have shown that TAR has good biological activity against liver injury induced by various factors. However, the anti-fibrotic effect of TAR and its mechanism are never clarified. The purpose of this study was to investigate the effects of TAR in liver fibrosis and to reveal its possible mechanism by RNA sequencing. Our results suggested that TAR attenuated CCl4-induced hepatocyte necrosis, inflammatory infiltration and ECM deposition. TAR inhibited the levels of ALT, AST, ALP, γ-GT, LN, HA, PC III and IV-C in serum and TNF-α, IL-6, IL-1ß and MDA in liver. In addition, TAR increased the activities of SOD and GSH-Px in liver. RNA sequencing analysis of liver tissues revealed that CCl4 and TAR significantly altered 4,155 genes and 2,675 genes, respectively. TAR reversed changes in ECM-related genes. More specifically, TAR mediated the expression of genes related to the activation of the Hippo pathway, while inhibiting the expression of genes related to the activation of HIF-1α, TGF-ß/Smad, and Wnt pathways. In the validation experiments, the qRT-PCR results showed that the expression levels of Yap1, Tead3, Hif1α, Vegfa, Tgfß1, Want3a, and Ctnnb1 mRNA were consistent with the RNA sequencing results. The Western blot results showed that TAR inhibited the levels of TGF-ß1 and p-Smad2. In addition, the results in vitro were consistent with those in vivo. Therefore, we concluded that TAR improved CCl4-induced liver fibrosis by regulating Hippo, HIF-1α, TGF-ß/Smad and Wnt pathways.

Carthami flos extract against carbon tetrachloride-induced liver fibrosis via alleviating angiogenesis in mice.

BACKGROUND: Angiogenesis is a pathological phenomenon contribute to the development of chronic liver diseases, and anti-angiogenic therapy is an effective strategy to alleviate liver fibrosis. Carthami flos, a medicinal and edible herb, has the effects of improving blood circulation and regulating angiogenesis. However, the anti-angiogenic effect of Carthami flos in liver fibrosis remains unknown. METHODS: We investigated the protective effect and therapeutic mechanism of Carthami flos extract (CFE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The liver injury and collagen deposition were observed and evaluated by conducting HE, Masson, and Sirius red staining, testing the serum biochemical indexes (ALT, AST, ALP, γ-GT), and measuring the contents of HYP and four indexes of liver fiber (Col-IV, LN, HA, PC-III). Simultaneously, the expressions of α-SMA and Collagen-I were detected to determine the activation of hepatic stellate cells (HSCs). Subsequently, we measured the expressions of angiogenesis-related proteins such as PDGFRB, ERK1/2, p-ERK1/2, MEK, p-MEK, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, and the mRNA levels of PDGFRB and VEGFA. Additionally, immunofluorescence staining and RT-qPCR assays were carried out to ascertain the expressions of continuous endothelial markers CD31, CD34 and vWF, and scanning electron microscope analysis was performed to observe the number of sinusoidal endothelial fenestrations. RESULTS: Herein, we found that CFE could significantly reduce liver injury and collagen deposition, like the same effect of colchicine. CFE significantly alleviated CCl4-induced liver injury and fibrosis, mainly manifested by reducing the levels of ALT, AST, ALP and γ-GT and decreasing the contents of HYP, Col-IV, LN, HA and PC-III. Additionally, CCl4 promoted the activation of HSCs by increasing the expressions of α-SMA and Collagen-I, while CFE could rectify the condition. Moreover, CFE treatment prevented the CCl4-induced the up-regulation of PDGFRB, p-MEK, p-ERK1/2, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, suggesting that CFE might provide the protection against abnormal angiogenesis. In the meantime, the gradual disappearance of sinusoidal capillarization after CFE treatment was supported by the decreased the contents of CD31, CD34 and vWF, as well as the increased number of sinusoidal endothelial fenestrae. CONCLUSION: In this study, the reduction of collagen deposition, the obstruction of HSCs activation, the inactivation of angiogenic signaling pathways and the weakening of hepatic sinusoidal capillarization jointly confirmed that CFE might be promising to resist angiogenesis in liver fibrosis via the PDGFRB/ERK/HIF-1α and VEGFA/AKT/eNOS signaling pathways. Nevertheless, as a potential therapeutic drug, the deeper mechanism of Carthami flos still needs to be further elucidated.

[Therapeutic effect of Jingfang Granules on CCl_4-induced liver fibrosis in mice and its mechanism].

To investigate the therapeutic effect of Jingfang Granules on carbon tetrachloride(CCl_4)-induced liver fibrosis in mice and its mechanism. Forty-nine 8-week-old male C57 BL/6 J mice were randomly divided into a blank group, a CCl_4 group, a silybin group(positive control, 100 mg·kg~(-1))+CCl_4, a Jingfang high-dose(16 g·kg~(-1)) group, a Jingfang high-dose(16 g·kg~(-1))+CCl_4 group, a Jingfang medium-dose(8 g·kg~(-1))+CCl_4 group, and a Jingfang low-dose(4 g·kg~(-1))+CCl_4 group, with 7 mice in each group. The mice in the blank group and Jingfang high-dose group were intraperitoneally injected olive oil solution, and mice in other groups were intraperitoneally injected with 10% CCl_4 olive oil solution(5 mL·kg~(-1)) to induce liver fibrosis, twice a week with an interval of 3 d, for 8 weeks. At the same time, except for the blank group and CCl_4 group, which were given deionized water, the mice in other groups were given the corresponding dose of drugs by gavage once daily for 8 weeks with the gavage volume of 10 mL·kg~(-1). All mice were fasted and freely drank for 12 h after the last administration, and then the eyeballs were removed for blood collection. The liver and spleen were collected, and the organ index was calculated. The levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bile acid(TBA), and triglyceride(TG) in the serum of mice were detected by an automated analyzer. Tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1ß(IL-1ß) levels were detected by enzyme-linked immunosorbent assay(ELISA). Kits were used to detect the contents of superoxide dismutase(SOD), malondialdehyde(MDA), and glutathione(GSH) in the liver tissue. Pathological changes in the liver tissue were observed by hematoxylin-eosin(HE), Masson, and Sirius red staining. Western blot was used to detect protein expressions of transforming growth factor-ß(TGF-ß), α-smooth muscle actin(α-SMA) and Smad4 in the liver tissue. The results indicated that Jingfang Granules significantly reduced the organ index, levels of ALT, AST, TBA,TG, TNF-α, IL-6, and IL-1ß in the serum, and the content of MDA in the liver tissue of mice with CCl_4-induced liver fibrosis. Jingfang Granules also significantly increased the content of SOD and GSH in the liver tissue. Meanwhile, Jingfang Granules down-regulated the protein levels of TGF-ß, α-SMA, and Smad4. Furthermore, Jingfang Granules had no significant effect on the liver tissue morphology and the above indexes in the normal mice. In conclusion, Jingfang Granules has obvious therapeutic effect on CCl_4-induced liver fibrosis, and its mechanism may be related to reducing the expression of pro-inflammatory factors, anti-oxidation, and regulating TGF-ß/Smad4 signaling pathway.

[Hydroxynitone suppresses hepatic stellate cell activation by inhibiting TGF-ß1 phosphorylation to alleviate CCl4-induced liver fibrosis in rats].

OBJECTIVE: To investigate the effect of hydronidone on CCl4-induced liver fibrosis in rats and explore the possible mechanism. METHODS: Sixty-six male SD rats were randomized into 5 groups, including a control group (n=10), a liver fibrosis model group (n=20), 2 hydronidone dose groups (100 and 250 mg/kg; n=12), and a pirfenidone (250 mg/kg) treatment group (n= 12). Rat models of liver fibrosis were established by subcutaneous injection of CCl4 in all but the control group. Hydronidone and pirfenidone were given daily at the indicated doses by intragastric administration for 6 weeks. After the treatments, serum samples were collected from the rats for detecting liver function parameters, and hydroxyproline content in the liver tissue was determined. Inflammation and fibrosis in the liver tissue were observed using HE staining and Sirius Red staining. In the cell experiment, human hepatic stellate cell line LX-2 was stimulated with TGF-ß1 and treated with hydronidone or pirfenidone, and the expression levels of α-SMA, collagen type I and phosphorylated Smad3, phosphorylated p38, phosphorylated ERK1/2 and phosphorylated Akt were detected with Western blotting. RESULTS: In the rat models of liver fibrosis, treatment with hydronidone obviously improved the liver functions, reduced the content of hydroxyproline in the liver tissue, and significantly alleviated liver fibrosis (P < 0.05). In LX-2 cells, hydronidone dose-dependently decreased the expression levels of α-SMA and collagen type I. In TGF- ß1-stimulated cells, the phosphorylation levels of Smad3, P38, ERK, and Akt increased progressively with the extension of the treatment time, but this effect was significantly attenuated by treatment with hydronidone (P < 0.05). CONCLUSION: Hydronidone can inhibit the phosphorylation of the proteins in the TGF-ß signaling pathway, thereby preventing TGF-ß1-mediated activation of hepatic stellate cells, which may be a possible mechanism by which hydronidone alleviates CCl4-induced liver fibrosis in rats.

Coprinus comatus polysaccharides ameliorated carbon tetrachloride-induced liver fibrosis through modulating inflammation and apoptosis.

Liver fibrosis is a serious global public health problem, owing to a lack of effective treatment. Coprinus comatus polysaccharides (CP), isolated from C. comatus, possess multiple biological activities. In our work, water-soluble polysaccharides (CPa) from CP were obtained by column chromatography. We attempted to investigate the anti-liver fibrosis ability of CPa and the underlying mechanisms of its activity against liver fibrosis in vivo and in vitro, as well as its structure. In vivo results showed that CPa reduced the release of inflammatory factors and apoptosis by modulating the TLR4/MyD88/NF-κB, Bcl-2/Bax and caspase family signaling pathways, thereby attenuating serum enzymes, ROS, α-SMA, collagen III, TGFß1, p-Smad3, and collagen volume fraction, and increasing the defense capacity of the antioxidant defense system in tetrachloride (CCl4)-induced liver fibrosis mice. The in vitro result was used to verify that, in vivo, CPa regulated the TLR4/MyD88/NF-κB, Bcl-2/Bax and caspase family signaling pathways to prevent the activation of HSCs and accelerate HSCs apoptosis in activated LX-2 cells. Thus, CPa could attenuate liver fibrosis by mediating inflammation and apoptosis. Meantime, the structural analysis showed that CPa is a polysaccharide with α- and ß-configurations including Fuc, Man, Gal and Glc with a Mw of 524 kDa. These findings indicate that CPa could be developed into functional foods and drugs against liver fibrosis.

Igf2bp2 knockdown improves CCl4-induced liver fibrosis and TGF-ß-activated mouse hepatic stellate cells by regulating Tgfbr1.

Progressive liver fibrosis is a dynamic process characterized by the net accumulation of extracellular matrix (ECM), which could eventually develop into cirrhosis, leading to malignant transformation. In this study, insulin-like growth factor 2 mRNA binding protein 2 (Igf2bp2) was found to be up-regulated in carbon tetrachloride (CCl4)-induced liver fibrosis and transforming growth factor-beta 1 (TGF-ß)-activated hepatic stellate cells (HSCs). Igf2bp2 knockdown in the CCl4-induced hepatic fibrosis mice model significantly improved CCl4-induced liver damage by decreasing necrosis and fibrotic septa, reducing hydroxyproline levels, and down-regulating fibrotic markers levels. In TGF-ß-activated HSCs, Igf2bp2 knockdown partially attenuated TGF-ß-induced cellular effects by suppressing HSCs viability and DNA synthesis and reducing the ECM-associated factors such as α-SMA, COLLAGEN I, and COLLAGEN III. Integrative network and signaling analysis revealed that the Igf2bp2 could bind to Tgfbr1. Transforming growth factor-beta receptor 1 (Tgfbr1) was found to be significantly up-regulated in the fibrotic liver and activated HSCs, and positively correlated with Igf2bp2. Tgfbr1 knockdown partially eliminated TGF-ß-induced fibrotic changes and Igf2bp2 overexpression effects on TGF-ß-activated HSCs in vitro. Moreover, Igf2bp2 overexpression promoted the phosphorylation of SMAD2/SMAD3, AKT, and PI3K, whereas Tgfbr1 knockdown exhibited the opposite effect; Tgfbr1 knockdown also partially attenuated the effects of Igf2bp2 overexpression on the phosphorylation of SMAD2/SMAD3, AKT, and PI3K. In closing, Igf2bp2 and Tgfbr1 are up-regulated in CCl4-induced liver fibrosis and TGF-ß-activated mHSCs. Igf2bp2 knockdown improved CCl4-induced liver fibrosis and TGF-ß-activated HSCs by targeting Tgfbr1, possibly through the PI3K/Akt pathway.