Liver diseases are major global health problems. Ginseng extract has antioxidant, immune-modulatory and anti-inflammatory activities. This study investigated the effect of ginseng extract on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Male Wistar rats were divided into four groups: control group, ginseng group, CCl4 group and CCl4 + ginseng group. Liver injury was induced by the intraperitoneal (I.P) injection of 3 ml/kg CCl4 (30% in olive oil) weekly for 8 weeks. The control group was I.P injected with olive oil. The expression of genes encoding transforming growth factor beta (TGF-β), type I TGF-β receptor (TβR-1), type II TGF-β receptor (TβR-II), mothers against decapentaplegic homolog 2
 (Smad2), Smad3, Smad4, matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor matrix metalloproteinase-1 (TIMP-1), Collagen 1a2 (Col1a2), Collagen 3a1 (Col3a1), interleukin-8 (IL-8) and interleukin -10 (IL-10) were measured by real-time PCR. Treatment with ginseng extract decreased hepatic fat deposition and lowered hepatic reticular fiber accumulation compared with the CCl4 group. The CCl4 group showed a significant increase in hepatotoxicity biomarkers and up-regulation of the expression of genes encoding TGF-β, TβR-I, TβR-II, MMP2, MMP9, Smad-2,-3, -4, and IL-8 compared with the control group. However, CCl4 administration resulted in the significant down-regulation of IL-10 mRNA expression compared with the control group. Interestingly, ginseng extract supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl4. ginseng extract had an anti‐fibrosis effect via the regulation of the TGF‐β1/Smad signaling pathway in the CCl4‐induced liver fibrosis model. The major target was the inhibition of the expression of TGF‐β1, Smad2, and Smad3.

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Effect of ginseng extract on the TGF-β1 signaling pathway in CCl 4 -induced liver fibrosis in rats

Liver fibrosis is considered to be a result of chronic liver pathological changes, and hepatic stellate cells (HSCs) play an important role during this process. Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities. This study focused on the effects of evodiamine on carbon tetrachloride (CCl4)-induced liver fibrosis in rats and HSCs in vitro via the TGF-β1/Smad signaling pathway. A liver fibrosis rat model was established by the intraperitoneal injection of CCl4 (3 ml/kg, 30% in olive oil). Evodiamine (15 and 25 mg/kg) was administered orally for 8 weeks. HSCs were treated with different evodiamine concentrations. The results indicated that evodiamine could improve the histopathological abnormalities in liver tissues and decrease the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hydroxyproline, and total bilirubin (TBIL). Concentrations of IL-6, tumor necrosis factor-α (TNF-α), collagen-I (COL-I), and collagen-III (COL-III) were reduced by evodiamine. Western blotting and real-time PCR showed that protein expression of transforming growth factor-β (TGF-β1), p-Smad 2/3 (phosphorylation of Smad 2/3), and smooth muscle alpha-actin (α-SMA) as well as mRNA expression of TGF-β1 and α-SMA in liver tissues were downregulated by evodiamine. The cell proliferation, production of hydroxyproline, and the protein expression of TGF-β1, p-Smad 2/3, and α-SMA in HSCs were dose-dependently reduced by evodiamine. Collectively, evodiamine had an antifibrosis effect in CCl4-induced liver fibrosis, and reduced HSCs proliferation and collagen metabolism in vitro. The major mechanism was downregulation of relative expression of TGF-β1, p-Smad 2/3, and α-SMA.

Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway

Cistanche tubulosa is a traditional Chinese herbal medicine that is widely used for regulating immunity. Phenyl ethanol glycosides (CPhGs) from this plant are the primarily efficacious materials. This aim of this study was to evaluate the preventive and therapeutic effects of CPhGs on BSA-induced hepatic fibrosis in rats and related molecular mechanisms involving hepatic stellate cells. Biejiarangan (BJRG), another traditional Chinese herbal medicine, was used as a positive control. In in vivo experiments, 75 SD rats were randomly divided into 6 groups: normal (distilled water-treated), model (BSA-treated), positive drug (BSA-treated + BJRG 600 mg/kg/day), and BSA-treated + CPhGs (125, 250, and 500 mg/kg/day) groups. The liver and spleen indices, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (IV-C), hydroxyproline (Hyp), and transforming growth factor β
 1 (TGF-β
 1) were measured in rat livers. Histopathological grades for liver fibrosis were assessed for each group using H&E and Masson’s trichrome staining. The expression of TGF-β
 1, collagen I (Col-I) and collagen III (Col-III) were determined by an immunohistochemical staining method. These effects were further evaluated in vitro by determining expression levels of NF-κB p65 and Col-I by quantitative real-time PCR analyses. Col-I protein expression was also examined by western blotting. All dose groups (125, 250, and 500 mg/kg/day) of CPhGs significantly reduced the liver and spleen index, decreased ALT, AST, HA, LN, PCIII, IV-C serum levels, TGF-β
 1 content (P < 0.01, P < 0.01, and P < 0.01), and Hyp content. CPhGs also markedly alleviated the swelling of liver cells and effectively prevented hepatocyte necrosis and inflammatory cell infiltration. Immunohistochemical results showed that CPhGs significantly reduced the expression of TGF-β
 1 (P < 0.01, P < 0.01, and P < 0.01), Col- I, and Col-III. The in vitro effects of CPhGs (100, 75, 50, and 25 ug/ml) on HSC-T6 showed that CPhGs significantly reduced mRNA expression of NF-κB p65 and Col-I, and CPhGs also downregulated Col-I protein expression. CPhGs have a significant anti-hepatic fibrosis effect, and may be used as hepatoprotective agents for treatment of hepatic fibrosis.

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Preventive effects of phenylethanol glycosides from Cistanche tubulosa on bovine serum albumin-induced hepatic fibrosis in rats

Background Body weight gain is a social issue all over the world. When body weight increased, hepatic fat accumulation also increased and it causes fatty liver disease. Therefore, developing a new treatment method and elucidating its mechanism is necessary. L-citrulline (L-Cit) is a free amino acid found mainly in watermelon. No reports regarding its effects on the improvement of hepatic steatosis and fibrogenesis are currently available. The aim of this study was to clarify the effect and the mechanism of L-Cit on inhibition of body weight gain and hepatic fat accumulation in high-fat and high-cholesterol fed SHRSP5/Dmcr rats. Methods L-Cit or water (controls) was administered to six-week-old male SHRSP5/Dmcr rats by gavage for nine weeks. We recorded the level of body weight and food intake while performing the administration and sacrificed rats. After that, the blood and lipid metabolism-related organs and tissues were collected and analyzed. Results L-Cit treatment reduced body weight gain and hepatic TC and TG levels, and serum levels of AST and ALT. L-Cit enhanced AMPK, LKB1, PKA, and hormone-sensitive lipase (HSL) protein phosphorylation levels in the epididymal fat. L-Cit treatment improved steatosis as revealed by HE staining of liver tissues and enhanced AMPK and LKB1 phosphorylation levels. Moreover, activation of Sirt1 was higher, while the liver fatty acid synthase (FAS) level was lower. Azan staining of liver sections revealed a reduction in fibrogenesis following L-Cit treatment. Further, the liver levels of TGF-β, Smad2/3, and α-SMA, fibrogenesis-related proteins and genes, were lower in the L-Cit-treated group. Conclusions From the results of analysis of the epididymal fat and the liver, L-Cit inhibits body weight gain and hepatic fat accumulation by activating lipid metabolism and promoting fatty acid β-oxidation in SHRSP5/Dmcr rats.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/fsn3.2439

L-citrulline inhibits body weight gain and hepatic fat accumulation by improving lipid metabolism in a rat nonalcoholic fatty liver disease model

The aqueous extracts of three different Artemisia species (Artemisia capillaris, Artemisia iwayomogi, & Artemisia annua) were evaluated for their antioxidant potential and antiproliferation, antifibrotic effect using activated rat HSC-T6 cells. The antioxidant activities of these plant extracts were assessed by different in vitro methods, such as free radical scavenging assays by DPPH, Superoxide, Hydroxyl, Nitric oxide and H2O2, and Ferric reducing antioxidant power (FRAP), total antioxidant abilities, and Iron chelating activity. The total phenolic and flavonoid contents were examined as well. Antifibrotic effects were investigated for three Artemisia species by measuring the hydroxyproline content and analyzing the gene expression of the two main fibrogenic cytokines of TGF-β and α-SMA in activated HSC-T6 cells. Among the tested Artemisia species, Artemisia capillaries is highest in TPC, TFC, TAC and FRAP while compared to others with 900.91 ± 22.81 (μg/g of GAE), 2918.83 ± 109.37 (μg/g of QE), 43.21 ± 0.28 (μg/g of GAE) and 3880.24 ± 340.37 (μg/g of FeSO4) respectively. The aqueous extracts of all Artemisia species reduced the proliferation of activated HSC – T6 cells. Regarding antifibrotic effect, the hydroxyproline content was significantly reduced in a concentration-dependent manner while treated with extracts. Further the activated HSCs drastically upregulated the fibrogenic cytokines (TGF-β, α-SMA), while the addition of three Artemisia extracts (50 μg/mL) significantly reduced these alterations. However, Artemisia capillaris and Artemisia iwayomogi enormously reduced both TGF-β and α-SMA expressions.

In vitro assessment of three different artemisia species for their antioxidant and anti-fibrotic activity

Objective: To investigate the effect of gypenosides on TGF‐β1/Smad pathway in liver fibrosis induced by carbon tetrachloride (CCl4) in rats. Method: Liver fibrosis in rats was induced by CCl4 subcutaneous injection over nine weeks, using 3 mg/kg of 100% CCl4 for the first injection and then 2 ml/kg of 50% CCl4‐olive oil solution, twice per week. At the beginning of the seventh week, the rats stimulated by CCl4 were divided into model group (n=12), gypenosides group (n=11) and colchicine group (n=11). The rats in the gypenosides and colchicine groups were administered with gypenosides (200 mg/kg.d) and colchicine (0.1mg/kg.d), respectively. The rest were administered with sterile water. At the end of the ninth week, hepatic hydroxyproline (Hyp) was tested and the histological changes in liver tissue were observed, as well as hepatic α‐smooth muscle actin (α‐SMA), transforming growth factor‐β1 (TGF‐β1), transforming growth factor‐β1 receptor (TβR‐I), transforming growth factor‐β2 receptor(TβR‐II), Smad2, phosphorylated Smad2 (p‐Smad2), Smad3 and phosphorylated Smad3 (p‐Smad3). Results: At the end of the experiment, two rats had died in the model group but none in the gypenosides and colchicine groups. With the stimulation of CCl4, hepatic Hyp content increased significantly in the model group but clearly decreased in the gypenosides and colchicine groups. Histological detection revealed serious steatosis, inflammation and fibrosis as well as collagen deposition in the liver tissue of the model group, but these were alleviated in the gypenosides and colchicine groups. The protein expressions of hepatic α‐SMA, TGF‐β1, TβR‐I, TβR‐II, Smad2, p‐Smad2 and p‐Smad3 were all up‐regulated in the model group. In the gypenosides‐treated group, the protein expressions of hepatic α‐SMA, TGF‐β1, Smad2, p‐ Smad2 and p‐Smad3 were all down‐regulated. In the colchicine‐treated group, hepatic α‐SMA, TGF‐β1, Smad2 and p‐Smad3 were also down‐regulated. The protein expressions of hepatic TβR‐I and TβR‐II were not inhibited significantly in the gypenosides‐ or colchicine‐ treated groups compared to the model group. 1 Lin Liu, Xue-mei Li, Liang Chen, Qin Feng, Lili Xu, Yi-yang Hu, Jing-hua Peng: The Effect of Gypenosides on TGF-s 1 /Smad Pathway in Liver Fibrosis Induced by Carbon Tetrachloride in Rats www.intechopen.com ARTICLE Int. j. integr. med., 2013, Vol. 1, 23:2013 Conclusion: Gypenosides alleviated liver fibrosis induced by CCl4 in rats, which is probably related to their inhibitory effects on hepatic stellate cell activation and the protein expression of TGF‐β1, Smad2, p‐Smad2 and p‐Smad3.

Yi-yang Hu

Papers

International Journal of Integrative Medicine