OBJECTIVES: To prepare a stable mouse model of chronic liver fibrosis induced by dietary vitamin A (VA) deficiency combined with CCl₄ injections. METHODS: A total of 126 Balb/c mice were randomized into 3 groups for feeding with a normal VA diet or a VA-deficient diet containing 500 or 200 IU/kg VA. After 4 weeks of feeding, half of the mice in each group were given intraperitoneal injections of 5% CCl₄ (10 mL/kg, twice a week) for 8 weeks. Serum retinol, ALT/AST and liver index of the mice were examined, liver tissue pathologies were observed with HE and Masson staining, and liver fibrosis score and oxidative stress level were evaluated. RESULTS: Four weeks of VA-deficient feeding, especially at 200 IU/kg, significantly lowered serum retinol level of the mice. CCl₄ injections for 8 weeks obviously increased liver index and ALT/AST and caused obvious liver fibrosis in all the mice, but liver pathologies were more severe in the 2 VA-deficient groups; severe liver necrosis with inflammatory cell infiltration was observed in 200 IU/kg VA group, where 2 mice died. After discontinuation of CCl₄, the mice with normal dietary VA showed gradual recovery of the liver index, ALT/AST, liver cord structure and liver fibrosis; the mice with VA deficiency, however, showed no significant improvements in these parameters, and the mice with 200 IU/kg VA still had serious abdominal adhesion, false lobules and massive inflammatory cell infiltration with a fibrosis stage score of 3. The oxidative damage index 8-OHdG was significantly higher in 500 IU/kg VA group than in normal VA group after CCl₄ modeling. CONCLUSIONS Feeding with diet containing 500 IU/kg VA for 4 weeks and 10 mL/kg CCl₄ injections for 8 weeks can result in stable moderate to severe liver fibrosis in mice without spontaneous reversal at 8 weeks of drug withdrawal.
Animals ; Mice ; Mice, Inbred BALB C ; Disease Models, Animal ; Carbon Tetrachloride ; Vitamin A Deficiency/complications* ; Male ; Liver Cirrhosis/etiology* ; Oxidative Stress ; Vitamin A/blood*

Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.
Mice ; Animals ; Interferon-gamma/metabolism* ; Interleukin-17/metabolism* ; Interleukin-33/metabolism* ; Cytokines/metabolism* ; Carbon Tetrachloride ; Th2 Cells ; Interleukin-4/metabolism* ; Liver Cirrhosis/pathology* ; Th1 Cells ; Th17 Cells/pathology* ; Immunity

The development of acute liver injury can result in liver cirrhosis, liver failure, and even liver cancer, yet there is currently no effective therapy for it. The purpose of this study was to investigate the protective effect and therapeutic mechanism of Lyciumbarbarum polysaccharides (LBPs) on acute liver injury induced by carbon tetrachloride (CCl₄). To create a model of acute liver injury, experimental canines received an intraperitoneal injection of 1 mL/kg of CCl₄ solution. The experimental canines in the therapy group were then fed LBPs (20 mg/kg). CCl₄-induced liver structural damage, excessive fibrosis, and reduced mitochondrial density were all improved by LBPs, according to microstructure data. By suppressing Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), promoting the production of sequestosome 1 (SQSTM1)/p62, nuclear factor erythroid 2-related factor 2 (Nrf2), and phase II detoxification genes and proteins downstream of Nrf2, and restoring the activity of anti-oxidant enzymes like catalase (CAT), LBPs can restore and increase the antioxidant capacity of liver. To lessen mitochondrial damage, LBPs can also enhance mitochondrial respiration, raise tissue adenosine triphosphate (ATP) levels, and reactivate the respiratory chain complexes I‒V. According to serum metabolomics, the therapeutic impact of LBPs on acute liver damage is accomplished mostly by controlling the pathways to lipid metabolism. 9-Hydroxyoctadecadienoic acid (9-HODE), lysophosphatidylcholine (LysoPC/LPC), and phosphatidylethanolamine (PE) may be potential indicators of acute liver injury. This study confirmed that LBPs, an effective hepatoprotective drug, may cure acute liver injury by lowering oxidative stress, repairing mitochondrial damage, and regulating metabolic pathways.
Animals ; Dogs ; Antioxidants/metabolism* ; Carbon Tetrachloride ; Chemical and Drug Induced Liver Injury/drug therapy* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Liver ; Metabolic Networks and Pathways ; Mitochondria/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Polysaccharides/pharmacology* ; Lycium/chemistry*

OBJECTIVE: To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro. METHODS: Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl₄)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed. RESULTS: High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01). CONCLUSIONS Amygdalin can dramatically alleviate liver fibrosis induced by CCl₄ in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.
Rats ; Male ; Mice ; Animals ; Transforming Growth Factor beta/metabolism* ; Amygdalin/therapeutic use* ; Endothelial Cells/metabolism* ; Olive Oil/therapeutic use* ; Rats, Wistar ; Smad Proteins/metabolism* ; Liver Cirrhosis/metabolism* ; Liver ; Transforming Growth Factor beta1/metabolism* ; Signal Transduction ; Collagen Type I/metabolism* ; Carbon Tetrachloride ; Hepatic Stellate Cells

OBJECTIVES: Hepatic fibrosis is a serious pathological consequence of chronic liver disease. Mycophenolate mofetil (MMF) is a commonly used immunosuppressant after organ transplant. However, the relationship between MMF and hepatic fibrosis remains unclear. This study aims to explore the effect of MMF on hepatic fibrosis in mice and the potential mechanism. METHODS: A total of 24 mice (male, 8-week old, C57BL/6) were randomly divided into a control group, a MMF group, a carbon tetrachloride (CCl₄) group and a CCl₄+MMF group (n=6 in each group). After the mice were sacrificed, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. The liver tissues were taken up for Masson staining and collagen I (COL1) immunohistochemistry. The levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were detected by Western blotting. Finally, the levels of mRNA for TGF-β1, α-SMA, and COL1 were detected using real-time PCR. RESULTS: Compared with the CCl₄ group, the ALT and AST levels were lower (both P<0.05), the degree of liver fibrosis was alleviated, and the deposition of COL1 in the liver was significantly decreased (P<0.01) in the CCl₄+MMF group. Compared with the CCl₄ group, the protein expression levels of TGF-β1 and α-SMA were significantly decreased (both P<0.05) and the relative expression levels of TGF-β1, α-SMA and COL1 mRNA in the liver were significantly decreased (all P<0.05) in the CCl₄+MMF. CONCLUSIONS MMF could reduce CCl₄-induced hepatic fibrosis, which might be related to the inhibition of TGF-β1. This study is expected to provide a target for the treatment of hepatic fibrosis.
Male ; Animals ; Mice ; Mice, Inbred C57BL ; Mycophenolic Acid/therapeutic use* ; Carbon Tetrachloride/toxicity* ; Transforming Growth Factor beta1/genetics* ; Liver Cirrhosis/drug therapy* ; RNA, Messenger

Landfill is one of the important sources of carbon tetrachloride (CT) pollution, and it is important to understand the degradation mechanism of CT in landfill cover for better control. In this study, a simulated landfill cover system was set up, and the biotransformation mechanism of CT and the associated micro-ecology were investigated. The results showed that three stable functional zones along the depth, i.e., aerobic zone (0-15 cm), anoxic zone (15-45 cm) and anaerobic zone (> 45 cm), were generated because of long-term biological oxidation in landfill cover. There were significant differences in redox condition and microbial community structure in each zone, which provided microbial resources and favorable conditions for CT degradation. The results of biodegradation indicated that dechlorination of CT produced chloroform (CF), dichloromethane (DCM) and Cl^- in anaerobic and anoxic zones. The highest concentration of dechlorination products occurred at 30 cm, which were degraded rapidly in aerobic zone. In addition, CT degradation rate was 13.2-103.6 μg/(m²·d), which decreased with the increase of landfill gas flux. The analysis of diversity sequencing revealed that Mesorhizobium, Thiobacillus and Intrasporangium were potential CT-degraders in aerobic, anaerobic and anoxic zone, respectively. Moreover, six species of dechlorination bacteria and eighteen species of methanotrophs were also responsible for anaerobic transformation of CT and aerobic degradation of CF and DCM, respectively. Interestingly, anaerobic dechlorination and aerobic transformation occurred simultaneously in the anoxic zone in landfill cover. Furthermore, analysis of degradation mechanism suggested that generation of stable anaerobic-anoxic-aerobic zone by regulation was very important for the harmless removal of full halogenated hydrocarbon in vadose zone, and the increase of anoxic zone scale enhanced their removal. These results provide theoretical guidance for the removal of chlorinated pollutants in landfills.
Bacteria/metabolism* ; Biodegradation, Environmental ; Carbon Tetrachloride/metabolism* ; Methane/metabolism* ; Waste Disposal Facilities

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-β1 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- β1-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-β signaling pathway, thereby preventing TGF-β1-mediated activation of hepatic stellate cells, which may be a possible mechanism by which hydronidone alleviates CCl₄-induced liver fibrosis in rats.
Animals ; Male ; Rats ; Carbon Tetrachloride/metabolism* ; Collagen Type I ; Hepatic Stellate Cells/pathology* ; Hydroxyproline/therapeutic use* ; Liver Cirrhosis ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Smad Proteins/metabolism* ; Transforming Growth Factor beta1/metabolism*

OBJECTIVE: To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis (L.) Juss. (EDC) in human hepatic stellate cells (HSCs) in vitro and in a carbon tetrachloride (CCl₄)-induced hepatic fibrosis mouse model in vivo. METHODS: For in vitro study, HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h, respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay, EdU staining, transmission electron microscopy, immunofluorescence staining, and Western blot, respectively. For in vivo experiments, mice were intraperitoneally injected with CCl₄ (2 ° L/g, adjusted to a 25% concentration in olive oil), 3 times per week for 6 weeks, to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (n=10), including control, model, positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α -smooth muscle actin (α -SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot. RESULTS: EDC reduced pathological damage associated with liver fibrosis, downregulated the expression of α -SMA and upregulated the expression of LC3B (P<0.05), both in HSCs and the CCl₄-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α -SMA protein expression levels (P<0.01). The results also found that the levels of phosphoinositide (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and p-p70S6K all decreased after EDC treatment (P<0.05). CONCLUSIONS EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.
Acetates ; Animals ; Autophagy ; Carbon Tetrachloride ; Hepatic Stellate Cells ; Liver/pathology* ; Liver Cirrhosis/pathology* ; Male ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism*

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-1β(IL-1β) 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-β(TGF-β), 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-1β 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-β, 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-β/Smad4 signaling pathway.
Mice ; Male ; Animals ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Olive Oil/therapeutic use* ; Carbon Tetrachloride/metabolism* ; Liver Cirrhosis/metabolism* ; Liver ; Superoxide Dismutase/metabolism* ; Transforming Growth Factor beta/metabolism*

This study aims to investigate the efficacy of forsythiaside A(FTA) against CCl_4-induced liver fibrosis and the mechanism. Specifically, activities of serum alanine/aspartate aminotransferase(ALT/AST) and hydroxyproline(HYP) level in liver were detected, and pathological morphology of liver was observed based on hematoxylin-eosin(HE) staining, Masson's trichrome staining, and Sirius red staining of liver. On this basis, the effect of FTA on liver fibrosis was evaluated. The mRNA expression of actin alpha 2/α-smooth muscle actin(Acta2/α-SMA), transforming growth factor β(Tgfβ), collagen Ⅰ alpha 1(Col1 a1), and collagen Ⅲ alpha 1(Col3 a1) in liver tissue and hepatic stellate cells(HSC) was determined by qPCR, and the protein expression of α-SMA in liver tissue and HSC was measured by Western blot to assess the inhibition of FTA on HSC activation. The protein expression of α-SMA, vi-mentin(Vim), vascular endothelial cadherin(Ve-cadherin), and platelet endothelial cell adhesion molecule-1(PECAM-1/CD31) was measured by Western blot to evaluate the reverse of endothelial-mesenchymal transition(EMT) by FTA. The efficacy of FTA in relieving CCl_4-induced liver fibrosis was evidenced by the alleviation of hepatocyte necrosis, liver inflammation, and hepatic collagen deposition. FTA decreased the mRNA expression of Acta2, Tgfβ, Col1 a1, and Col3 a1 and protein expression of α-SMA both in vivo and in vitro. FTA reversed the increase of α-SMA and Vim and the decrease of CD31 and Ve-cadherin in livers from mice treated with CCl_4. Therefore, FTA alleviated CCl_4-induced liver fibrosis in mice via suppressing HSC activation and reversing EMT.
Animals ; Mice ; Actins/metabolism* ; Alanine Transaminase/blood* ; Carbon Tetrachloride/metabolism* ; Collagen/metabolism* ; Hepatic Stellate Cells ; Liver/drug effects* ; Liver Cirrhosis/genetics* ; RNA, Messenger/metabolism* ; Transforming Growth Factor beta/metabolism* ; Glycosides/therapeutic use*