Potential therapeutic effects of a traditional Chinese formulation, BJ-JN, on liver fibrosis induced by carbon tetrachloride in rats

Aim of the study

to evaluate the anti-fibrotic effects of BJ-JN (a traditional Chinese formulation) in CCl₄-induced liver fibrosis in rats.

Materials and methods

BJ-JN (0.5, 1.0, 2.0 g/kg) was administrated via gavage once a day starting from the fifth weeks after the CCl₄ treatment for subsequent 9 weeks. Evaluated with liver and spleen index, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), nitric oxide (NO), hepatic malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, as well as with histopathologic changes of liver. The proliferation and collagen synthesis of primary hepatic stellate cells (HSCs) from normal, model and BJ-JN (2.0 g/kg) treatment rats were examined with ³H-TdR and ³H-Pro uptake assay, respectively.

Results

BJ-JN (0.5, 1.0, 2.0 g/kg) effectively reduced the elevated levels of liver and spleen index, serum ALT, AST, NO, HA, and hepatic MDA contents, enhance the reduced hepatic SOD activity in CCl₄-treated rats. The histopathological analysis suggested that BJ-JN obviously alleviated the degree of liver fibrosis induced by CCl₄. The proliferation and collagen synthesis of HSC isolated from BJ-JN (2.0 g/kg) treatment rats were remarkably inhibited.

Conclusions

Those results suggest BJ-JN has a protective and therapeutic effect on liver fibrosis induced by CCl₄, which might be associated with its anti-oxidative activity, inhibitory activity on HSC proliferation and collagen synthesis.     

Pharmacological evaluation of Ipomoea asarifolia (Desr.) against carbon tetrachloride-induced hepatotoxicity in rats

Ethnopharmacological relevance

Ipomoeaasarifolia (Desr.) Roem. and Schult. is used traditionally in some parts of Africa for the treatment of a variety of diseases. This study attempts to validate its hepatoprotective activity by evaluating the prophylactic and curative properties of the methanolic extract of Ipomoea asarifolia (IA) leaves.

Materials and Methods

Liver damage was induced by administering 0.5 ml/kg of an equal mixture of carbon tetrachloride (CCl₄) in olive oil intraperitoneally on alternate days, for 5 days and the plant extract was given orally daily, for 7 days at doses of 100, 200 and 400 mg/kg.

Results

Pre-treatment with the extract significantly (P<0.05) decreased CCl₄-induced elevation in serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase, triglycerides, bilirubin and cholesterol, better than the standard drug silymarin at 100 mg/kg. In the curative study, IA significantly (P<0.05) reversed CCl₄-induced liver damage, comparable to silymarin. Hepatoprotective potential was further supported by decrease in pentobarbitone sleeping time and improved hepatic tissue histopathology.

Conclusion

These results indicate that I. asarifolia leaves have potent hepatoprotective activity against CCl₄-induced hepatic damage in rats.     

Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model

Ethnopharmacological relevance

Artemisia capillaris and Artemisia iwayomogi, both members of the Compositae family, have been indiscriminately used for various liver disorders as traditional hepatotherapeutic medicines in Korea for many years.

Aim of the study

In this study, the anti-hepatofibrotic effects of Artemisia capillaris and Artemisia iwayomogi were comparatively analyzed using a carbon tetrachloride (CCl₄)-induced liver fibrosis rat model.

Materials and methods

Hepatic fibrosis was induced via a 10-week course of intraperitoneal CCl₄ injections (50% dissolved in olive oil, 2 mL/kg, twice per week). Water extract of Artemisia capillaris (AC) or Artemisia iwayomogi (AI) was orally administered six times per week from the 5th to the 10th week.

Results

AI (50 mg/kg) significantly attenuated the CCl₄-induced excessive release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum (p < 0.05), and hydroxyproline and malondialdehyde (MDA) contents in liver tissue (p < 0.05). Further, AI markedly ameliorated the depletion of total antioxidant capacity (TAC), glutathione (GSH), and superoxide dismutase (SOD) in liver tissue (p < 0.01). Unexpectedly, AC did not exert any effects on the above parameters. Histopathological and immunohistochemical analyses revealed that AI drastically reduced inflammation, necrosis, fatty infiltration, collagen accumulation, and activation of hepatic satellite cells in liver tissue. These changes were not observed with AC treatment. Several critical genes of fibrosis-related cytokines including transforming growth factor beta (TGF-β), platelet-derived growth factor beta (PDGF-β), and alpha smooth muscle actin (α-SMA) were more prominently downregulated by AI compared to AC treatment.

Conclusion

Our results show that AI exerts greater hepatoprotective and anti-fibrotic effects as compared with AC via enhancing antioxidant capacity and downregulating fibrogentic cytokines.     

Action mechanism of Yi Guan Jian Decoction on CCl4 induced cirrhosis in rats

Aim of study

To investigate action mechanism of Yi Guan Jian Decoction on cirrhosis induced by CCl₄ in rats.

Material and methods

CCl₄ (3 mL/kg) for the first time and then olive oil CCl₄ solution 50% (2 mL/kg) was administered hypodermically to rats twice each week for 12 weeks. At the end of 8th week, rats were randomly divided into CCl₄ control group (n = 10), Yi Guan Jian Decoction group (n = 9) and Xiao Chai Hu Decoction group (n = 9). Yi Guan Jian Decoction and Xiao Chai Hu Decoction were oral administrated per day respectively for 4 weeks, concomitantly continued CCl₄ administration. At 12th weekend, the rats were sacrificed for sampling and detection of liver function, histological changes of liver tissue, liver tissue hydroxyproline content and expression of α-SMA, CD68, MMP-13, TIMP-1, TIMP-2, Caspase-12, HGFα, MMP-2, MMP-9 and hepatocyte apoptotic index.

Results and conclusions

(1) Compared with that of normal rats, expression of α-SMA, CD68 and TIMP-1 in liver tissue of 8 week model group rats increases significantly (P < 0.01), moreover further increased in the 12 week of model group. However, MMP-13, HGFα, TIMP-2 content decreases gradually and the statistical difference is seen between each time point (P < 0.01). Activity of MMP-2, MMP-9, content of Caspase-12 and hepatocyte apoptotic index increased gradually at 4th, 8th, 12th week. (2) Compared to that of the same time point model group, activity of MMP-9 and contents of MMP-13, TIMP-2 and HGFα in Yi Guan Jian Decoction group improves significantly (P < 0.01), and activity of MMP-2 and contents of α-SMA, TIMP-1, Caspase-12 and hepatocyte apoptotic index decreases significantly (P < 0.01). This work suggests that Yi Guan Jian Decoction exerts significant therapeutic effect on CCl₄-induced cirrhosis in rats, through mechanism of inhibiting hepatocytes apoptosis and hepatic stellate cells activation, and regulating the function of Kupffer cell.

Ethnopharmacological relevance

This study investigates the mechanism of Yi Guan Jian against cirrhosis from aspect of heptocytes apoptosis and hepatic stellate cells activation. It suggest that although of unknown bioactive ingredients, mechanism of traditional Chinese medicine recipe against cirrhosis can be disclosed and of profound significance.     

Herb medicine Gan-fu-kang attenuates liver injury in a rat fibrotic model

Aim of the study

To verify therapeutic effects of Gan-fu-kang (GFK), a traditional Chinese medicine compound, in a rat model and to investigate the underlying mechanisms.

Materials and methods

Liver fibrosis was established by 12 weeks of carbon tetrachloride (CCl₄) treatment (0.5 mg/kg, twice per week) followed by 8 weeks of “recovery” in rats. Rats randomly received GFK (31.25, 312.5 and 3125 mg/kg/day, p.o.) or vehicle from weeks 9 to 20, and were sacrificed at the end of week 20 for histological, biochemical, and molecular biological examinations. In a separate set of experiments, rats received 12 weeks of CCl₄ treatment, concomitant with GFK (312.5 mg/kg/day, p.o.) during the same period in some subjects, but were then sacrificed immediately. An additional group of rats receiving no CCl₄ treatment served as normal controls.

Results and conclusions

(1) CCl₄ treatment resulted in severe liver damage and fibrosis. (2) In the main block of the 20-week study, GFK attenuated liver damage and fibrosis. (3) In the 12-week study, GFK produced prevention effect against hepatic injury. (4) GFK suppressed the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), type I collagen, platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor-β chains (PDGFRβ) and mitogen-activated protein kinases (MAPKs)/active protein-1 (AP-1) signal pathways. Taken together, these results indicated that GFK could attenuate liver injuries in both settings. Our findings also suggest that the AP-1 pathway is the likely molecular substrate for the observed GFK effects.     

Antioxidative and hepatoprotective effect of CGX, an herbal medicine, against toxic acute injury in mice

Aim

CGX, a modified traditional Chinese herbal drug whose name means “liver cleaning,” is used to treat various liver disorders. This study investigated the protective effects of CGX and its mechanisms.

Material and methods

After pretreating ICR mice twice daily with CGX (po, 50 or 100 mg/kg) or distilled water for three consecutive days, acute liver injury was induced by a single injection of CCl₄ (ip, 10 mL/kg of 0.2% in olive oil) (n = 8 per group).

Results

Pretreatment with CGX significantly attenuated the elevation in biochemical parameters, such as alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) in serum, and the malondialdehyde concentrations in liver tissue. Pretreatment with CGX significantly restored the reduction of catalase activity and glutathione (GSH) content, but not superoxide dismutase (SOD) activity, and it inhibited the CCl₄-induced high expression of iNOS and TNF-α in hepatic tissue.

Conclusion

This study showed that CGX has hepatoprotective effects against free radical-induced acute injury via primarily antioxidative properties.     

A Chinese herbal medicine, Gexia-Zhuyu Tang (GZT), prevents dimethylnitrosamine-induced liver fibrosis through inhibition of hepatic stellate cells proliferation

Ethnopharmacological evidence

Gexia-Zhuyu Tang (GZT), also called Gexiazhuyu decoction (GXZYD), is a traditional Chinese herbal medicine for chronic liver diseases such as cirrhosis and liver fibrosis.

Aim of the study

In this study, we have investigated the affects of GZT on a rat model of dimethylnitrosamine (DMN)-induced liver fibrosis.

Materials and methods

In this study, the protective effects of GZT on DMN-induced liver fibrosis were measured using a rat model. Following 5 weeks of DMN-treatment (8 mg/kg, i.p., given 3 consecutive days each week), oral administration of GZT at 1.8 g/kg daily via oral gavage for 2 weeks beginning at week 13.

Results

Both body and liver weights were significantly decreased. The reductions in body and liver weights corresponded with increasing liver damage severity. Furthermore, GZT-treatment remarkably decreased the levels of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase), and the mRNA expression levels of collagen alpha-1(I) and alpha-smooth muscle actin (alpha-SMA) in DMN-induced hepatic fibrosis. In addition, hepatic stellate cells (HSCs) play a major role in various types of liver fibrosis through initial myofibroblast transformation. The proliferation of HSCs was inhibited by GZT. Treatment with GZT also induced HSC apoptosis in a dose- and time-dependent manner. GZT treatment induced HSC apoptosis by facilitating Ca²⁺ release from the mitochondria within 6 h. Subsequently, caspases 3 and 12 were elevated by 72 h after treatment.

Conclusions

Our studies indicate that GZT exhibited both hepatoprotective and antifibrogenic effects in DMN-induced hepatic injury. These findings suggest that GZT may be useful in preventing the development of hepatic fibrosis.     

The hepatoprotective and antifibrotic effects of Saururus chinensis against carbon tetrachloride induced hepatic fibrosis in rats

Ethnopharmacological relevance

Saururus chinensis (Lour.) Baill (Saururaceae) has been used in Chinese folk medicine for treatment of various diseases, such as edema, jaundice, gonorrhea, antipyretic, diuretic, and antiinflammatory agents.

Aim of the study

Our aim was to evaluate the hepatoprotective and antifibrotic effects of Saururus chinensis extract (SC-E) in carbon tetrachloride (CCl₄) induced liver fibrosis rats.

Materials and methods

The SC-E (70 mg/kg) was administrated via gavage once a day starting from the onset of CCl₄ treatment (14 weeks) for subsequent 8 weeks. Evaluated with liver index, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), hepatic malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity, total cholesterol (TC), triglyceride (TG), total lipoprotein (TP), albumin (ALB), hydroxyproline (HYP), total antioxidant capacity (T-AOC), laminin (LN), type III collagen terminal peptide (PC-IIINP), and type IV collagen (IV-C), as well as with histopathologic changes of liver.

Results

SC-E effectively reduced the elevated levels of liver index, serum ALT, AST, HA, and hepatic MDA contents, enhance the reduced hepatic SOD activity in CCl₄-treated rats. The histopathological analysis suggested that SC-E obviously alleviated the degree of liver fibrosis induced by CCl₄.

Conclusions

Those results suggest SC-E has protective and therapeutic effects on liver fibrosis induced by CCl₄.     

Fuzheng Huayu inhibits carbon tetrachloride-induced liver fibrosis in mice through activating hepatic NK cells

Aim of the study

Fuzheng Huayu (FZHY) is a Chinese compound herbal preparation which consists of six Chinese herbs. This study examines the preventative effects of FZHY on liver fibrosis induced by carbon tetrachloride (CCl₄) and explores its possible mechanisms of action.

Materials and methods

Liver fibrosis was induced in male C57BL/6N mice by injecting a 10% CCl₄ solution intraperitoneal twice a week for six weeks. After 6 weeks of treatment, serum ALT and AST assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of alpha-smooth muscle actin (SMA) were measured by quantitative real-time PCR and western blot. Hepatic natural killer (NK) cells were isolated from liver and evaluated by FACS.

Results

Upon pathological examination, the FZHY-treated mice showed significantly reduced liver damage. The expression of α-SMA increased markedly upon treatment with CCl₄ and the increase was reversed by FZHY treatment. FZHY treatment also enhanced the activation of hepatic NK cells and the production of interferon-gamma (IFN-γ). The protective effects of FZHY were reversed in the mice that were depleted of NK cells by anti-ASGM-1 Ab treatment.

Conclusions

FZHY can efficiently inhibit CCl₄-induced liver fibrosis. Furthermore, the depletion of NK cells attenuates the protective effects of FZHY. We conclude that FZHY could be an effective drug for liver fibrosis, and its mechanism of action involves the activation of hepatic NK cells.     

Fraxinus rhynchophylla ethanol extract attenuates carbon tetrachloride-induced liver fibrosis in rats via down-regulating the expressions of uPA,MMP-2, MMP-9 and TIMP-1

Aim of the study

To investigate the effect of Fraxinus rhynchophylla ethanol extract (FR_EtOH) on liver fibrosis induced by carbon tetrachloride (CCl₄) in rats.

Materials and methods

Rat hepatic fibrosis was induced by oral administration of CCl₄. Sixty SD rats were divided randomly into 6 groups: control, CCl₄ group, silymarin group and three FR_EtOH-treated groups. Except for the rats in control group, all rats were administered orally with CCl₄ (20%, 0.2 mL/100 g body weight) twice a week for 8 weeks. Rats in FR_EtOH groups were treated daily with FR_EtOH (0.1, 0.5 and 1.0 g/kg, p.o.) throughout the whole experimental period. Liver function parameters (such as activities of serum GOT and GPT levels), activities of liver anti-oxidant enzymes (such as catalase, SOD, GPx) and expressions of uPA, tPA, MMP-2, MMP-9 and TIMP-1, -2, -3, -4 in the liver fibrosis pathway were detected.

Results

The results showed that FR_EtOH (0.1, 0.5 and 1.0 g/kg BW) significantly reduced the elevated activities of sGOT and sGPT caused by CCl₄. FR_EtOH (0.1 and 0.5 g/kg BW) and significantly increased the activities of GSH-Px. The histopathological study showed that FR_EtOH (0.1 and 0.5 g/kg BW) reduced the incidence of liver lesions, including hepatic cells cloudy swelling, lymphocytes infiltration, cytoplasm vacuolization hepatic necrosis and fibrous connective tissue proliferated induced by CCl₄ in rats. In our study it was showed that CCl₄-treated group significantly increased the protein levels of uPA, MMP-2, MMP-9 and TIMP-1. FR_EtOH (0.1 and 0.5 g/kg BW) could inhibit the protein levels of uPA, MMP-2, MMP-9 and TIMP-1. Finally, the amount of esculetin in the FR_EtOH was 33.54 mg/g extract.

Conclusions

Oral administration of FR_EtOH significantly reduces CCl₄-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular fibrosis by its free radical scavenging ability. FR_EtOH down-regulated the expressions of uPA, MMP-2 and MMP-9 in CCl₄-induced liver fibrosis in rats.     

Therapeutic efficacy of Traditional Chinese Medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats

Background/aims

Hepatic fibrosis is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. TCM 319 recipe is a Chinese Medicine formula which consists of six Chinese herbs. In this study, we investigated the anti-fibrotic efficacy and mechanisms of TCM 319 recipe.

Methods

Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). 34 male adult SD rats were allocated into five groups (group 1—concomitant CCl₄ and TCM 319 recipe for 8 weeks; group 2—CCl₄ for 4 weeks and then CCl₄ and TCM 319 recipe for 4 weeks; group 3—CCl₄ alone for 8 weeks; group 4—TCM 319 recipe only for 8 weeks; group 5—untreated controls). After 8 weeks of treatment, serum ALT assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of platelet derived growth factor (PDGF-B), PDGF-Rβ, and transforming growth factor-beta 1 (TGF-β1) were measured by quantitative RT-PCR and western blot.

Results

TCM 319 recipe reduced liver injury and attenuated hepatic fibrosis in group 1 compared with that in group 3. TCM 319 recipe suppressed the mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1). In addition, treatment with TCM 319 recipe significantly down-regulated mRNA expression of PDGF-B and PDGF-Rβ, and it also suppressed protein expression of PDGF-Rβ and TGF-β1.

Conclusions

TCM 319 recipe extracts could attenuate hepatic fibrosis induced by CCl₄ in rats. The anti-fibrotic effect of TCM 319 recipe is associated with the down-regulation of mRNA expression of TIMP-1, PDGF-B and PDGF-Rβ, and with the suppression of protein expression of PDGF-Rβ and TGF-β1.     

Inhibitory effects of salvianolic acid B on CCl4-induced hepatic fibrosis through regulating NF-κB/IκBα signaling

Ethnopharmacological relevance

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl₄-induced hepatic fibrosis.

Materials and methods

Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10 mg/kg body weight (L group), or high SA-B of 20 mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting.

Results

SA-B was shown to reduce CCl₄-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10–20 mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group.

Conclusions

This study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.     

Polyprenols from Taxus chinensis var. mairei prevent the development of CCl4-induced liver fibrosis in rats

Ethnopharmacological relevance

The aim of this study was to investigate the anti-fibrotic effects and the possible underlying mechanisms of taxus polyprenols (TPs) isolated from the needles of Taxus chinensis var. mairei.

Materials and methods

The animals were randomly divided into normal control with vehicles only (olive oil), rat model given CCl₄ only, CCl₄+low TPs (48 mg/kg), CCl₄+medium TPs (120 mg/kg), CCl₄+high TPs (300 mg/kg), and CCl₄+Polyene phosphatidylcholine (PP, 120 mg/kg). The rat model of liver fibrosis was induced by subcutaneous injection of 40% (v/v) of CCl₄ diluted in olive oil (3 mL/kg body weight) twice per week for 8 weeks. Liver histopathological study was performed. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and albumin (ALB) of the serum were determined for evaluating the liver function. In order to reveal the possible mechanisms of the anti-fibrotic effects, oxidative stress level, hepatic collagen metabolism, and hepatic stellate cells (HSCs) activation were investigated. Furthermore, the mRNA expression of the fibrotic-related factors was measured by the quantitative real-time RT-PCR.

Results

TPs successfully attenuated liver injury induced by CCl₄ shown by histopathological sections of livers and improved liver function as indicated by decreased ALT, AST and ALP levels and increased ALB levels in serum of the rats. TPs significantly increased the hepatic Cu/Zn SOD and GSH-Px activities along with GSH content while a remarkable decrease in MDA content. Both immunohistochemical staining and mRNA expression levels of α-SMA indicated a profound suppression of HSCs activation. Furthermore, it significantly inhibited the mRNA expression of the pro-fibrotic cytokines Col α1(I), Col α1(Ш), MMP-2, TIMP-1, TIMP-2, PDGF-β, TGF-β1, CTGF and TNF-α and restored the hepatoprotective factor HGF.

Conclusion

These results suggest that the protective effects of TPs in chronic CCl₄-induced liver fibrosis might be related with the reduction of oxidative damage, the inhibition of HSCs activation, the down-regulation of pro-fibrogenic stimuli and the protection of hepatocytes.     

Antifibrotic activity of Taraxacum officinale root in carbon tetrachloride-induced liver damage in mice

Aim of the study

Dandelion (Taraxacum officinale) has been traditionally used in the treatment of various liver disorders. The present study was aimed to assess the efficacy of dandelion root water–ethanol extract (DWE) in carbon tetrachloride (CCl₄)-induced hepatic fibrosis.

Materials and Methods

The mice were treated with CCl₄ dissolved in olive oil (20%, v/v, 2 ml/kg) intraperitoneally (i.p.), twice a week for 4 weeks. DWE was administered i.p. once daily for next 10 days, in doses of 200 and 600 mg/kg of body weight. The degree of hepatic fibrosis was determined by hydroxyproline content and Mallory trichrome staining. Oxidative stress was determined by measuring hepatic superoxide dismutase (Cu/Zn SOD) activity. The expression and specific tissue distribution of glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA), and metallothionein (MT) I/II in the liver were determined by immunohistochemistry.

Results

Hepatic Cu/Zn SOD activity has been decreased in intoxicated mice and normalized in DWE treated groups. MT I/II immunopositivity was strongly reduced in the CCl₄ group. DWE treatment successfully decreased hepatic fibrinous deposits, restored histological architecture, and modulate the expression of GFAP and α-SMA. Concomitantly, MT I/II expression increased in the DWE treated groups.

Conclusions

Our results suggest the therapeutic effect of DWE on CCl₄-induced liver fibrosis by the inactivation of hepatic stellate cells and the enhancement of hepatic regenerative capabilities. The present results provide scientific evidence to substantiate the traditional use of Taraxacum officinale root in hepatic disorders.     

Effect and mechanism of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) on hepatic fibrosis induced by carbon tetrachloride in rats

Ethnopharmacological relevance

Methyl helicterate is a triterpenoid isolated from Helicteres angustifolia (Sterculiaceae), one of the valuable traditional Chinese herbs. Antifibrotic activities of H. angustifolia have been extensively proved.

Aim of the study

The purpose of this study was to investigate the effect of methyl helicterate (MH) on liver fibrosis in rats induced by carbon tetrachloride (CCl₄) and to explore its underlying mechanism.

Materials and methods

Hepatic fibrosis was induced in male Sprague–Dawley (SD) rats by intragastric administration with 2 ml/kg CCl₄ (mixed 1:1 in peanut oil) twice a week for 12 weeks. To evaluate the effect of MH (16.72, 33.45, 66.90 mg/kg) on hepatic fibrosis, liver function, histological study and hepatic fibrosis evaluation were performed. Liver function was assessed by determining the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb) and total protein (TP). The biomarkers such as hydroxyproline (Hyp), hyaluronic acid (HA), type III precollagen (PCIII) and laminin (LN) were examined for the evaluation of hepatic fibrosis. The underlying mechanism was investigated by measuring oxidative stress level and detecting the expression of TGF-β1 mRNA and Smad3 protein.

Results

MH (33.45, 66.90 mg/kg) treatment significantly inhibited the loss of body weight and the increase of liver index in rats induced by CCl₄. MH also improved the liver function as indicated by decreasing serum enzymatic activities of ALT, AST, TP and Alb (P<0.05). Histological results indicated that MH alleviated liver damage and reduced the formation of fibrous septa. Moreover, MH significantly decreased liver Hyp, HA, LN and PCIII (P<0.05). Research on mechanism showed that MH could markedly reduce liver malondialdehyde (MDA) concentration, increase activities of liver superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and inhibit the expression of TGF-β1 mRNA and Smad3 protein (P<0.05).

Conclusions

Our findings indicated that MH can inhibit CCl₄-induced hepatic fibrosis, which may be ascribed to its radical scavenging action, antioxidant activity, and modulation of TGF-β-Smad3 signaling pathway.     

Xuefuzhuyu decoction inhibition of angiogenesis attenuates liver fibrosis induced by CCl4 in mice

Ethnopharmacological relevance

Xuefuzhuyu decoction (XFZY) is a well-known traditional Chinese herbal formulation composed of 11 herbs. It is an effective treatment for cardiovascular and chronic liver diseases. The aim of the study is to investigate the role of XFZY on angiogensis in hepatic fibrogenesis, and identify the possible mechanism.

Material and methods

Liver fibrosis was induced by intraperitoneal injection of Carbon tetrachloride (CCl₄) in C57BL/6 mice for 6 weeks. From week 4 to week 6, the CCl₄-injected mice were randomly divided into three groups, followed by oral administration of Sorafenib, XFZY and water for 3 weeks. Biochemical parameters, hydroxyproline (Hyp) content and histological changes of the liver were determined. The expressions of alpha smooth muscle actin (α-SMA), collagen I, CD31 and vascular endothelial grow factor (VEGF) were assessed by immunohistochemistry and western blot. The protein expressions of VEGFR-2, hypoxia inducing factor (HIF)-1α, asymmetric dimethylarginine (ADMA) and dimethylarginine hydrolase (DDAH) 1 were determined by western blot. The mRNA levels of α-SMA, VEGF and HIF-1α were measured by RT-PCR.

Results

Both Sorafenib and XFZY improved biochemical parameters of the liver fibrosis mice. A significant reduction in Hyp content was found in the XFZY-treated mice as well as the Sorafenib-treated mice. Changes in histopathology showed that Sorafenib and XFZY decreased inflammatory and fibrotic stages of the liver in fibrosis mice. Compared to CCl₄ model group, Sorafenib and XFZY decreased α-SMA, collagen I, CD31, VEGF, VEGFR-2, HIF-1α and ADMA, and increased the expression of DDAH1.

Conclusion

XFZY inhibits liver fibrosis not only through inhibiting collagen deposition but also through an antiangiogenic effect on the fibrotic liver. Moreover, the antiangiogenic mechanism of XFZY involves alleviating hypoxia and protecting liver sinusoidal endothelial cell function.     

Isolation of gentiopicroside from Gentianae Radix and its pharmacokinetics on liver ischemia/reperfusion rats

Ethnopharmacological relevance

Gentiopicroside (GPS) is a secoiridoid glucoside isolated from the ethanol extract of Gentianae Radix with a content of 13%, which has been used for centuries in Chinese as a digestive aid.

Aim of the study

This study investigates the pharmacokinetics of GPS and its metabolic pathway for the liver ischemia/reperfusion (I/R) in rats.

Materials and methods

The experimental animals were anesthetized intraperitoneally (i.p.) with a mixture of urethane (1.0 g/kg) and α-chloralose (0.1 g/kg). A midline laparatomy was performed and the liver hilum was gently exposed. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with silk thread for 30 min. Ischemia was followed by a sudden reperfusion after removing the occluding threads. After 60 min reperfusion, the rats received a single intravenous 5 mg/kg dose of GPS.

Results

The area under concentration curve (AUC) was significantly increased; however, the clearance (Cl) was significantly decreased in the liver I/R rats. Furthermore, after pretreated with SKF-525A (50 mg/kg, i.p.), a cytochrome P450 (CYP) inhibitor, AUC, elimination half-life (t_1/2) and the mean residence time (MRT) of GPS in rat blood were significantly increased, suggesting that CYP was involved in the metabolism of GPS. For the group without liver I/R, GPS was administered at doses of 5 mg/kg and 100 mg/kg intravenously and orally, respectively. The pharmacokinetic results indicated that the AUC was 565 ± 95.1 and 1163 ± 273 min μg/mL and the t_1/2 of GPS was 71 ± 9 and 106 ± 17 min after intravenous and oral administration, respectively. The oral bioavailability of GPS was 10.3 ± 2.4% in the rats.

Conclusions

The status of I/R might prolong the disposition of GPS, and the plasma concentration of GPS in the liver I/R injury rats was significantly increased. The increased body exposure of GPS in the treatment of liver I/R may result from the decreased metabolism of GPS mediated by CYP in the liver.     

Protective effect of Actiniopteris radiata (Sw.) Link. against CCl4 induced oxidative stress in albino rats

Ethnopharmacological relevance

Actiniopteris radiata is a herb with great medicinal value and is evaluated for hepatoprotective activity. To investigate the protective effect of ethanolic extract of Actiniopteris radiata (EEAR) on CCl₄ induced oxidative stress in male Wistar albino rats.

Materials and methods

EEAR were administered for 8 consecutive weeks to rats. Group I – control; Group II – toxin control (30% CCl₄); Group III and Group IV received EEAR (250 and 500 mg/kg respectively). Antioxidant status in liver were estimated by determining the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx); as well as by determining the levels of lipid peroxidation (LPO) and reduced glutathione (GSH). In addition, isoenzyme pattern and mRNA expression of the antioxidants were studied. Partial characterization of EEAR was performed by Liquid chromatography–mass spectrometry (LC–MS).

Results

CCl₄ induced oxidative stress as evidenced from increase in LPO along with reduction of SOD, CAT, GPx and GSH. Treatment with EEAR (250 and 500 mg/kg) mitigated the CCl₄ induced oxidative stress. An analysis of the isozyme pattern of these antioxidant enzymes revealed variations in SOD2, CAT, GPx2 and GPx3 in CCl₄ treated rats, which were normalized after EEAR treatment. Furthermore, expression of genes for the antioxidant enzymes, were down-regulated by CCl₄ treatment, which were reversed by EEAR. The results of partial characterization of EEAR by LC–MS revealed the presence of rutin and other 7 unknown phenolic derivatives.

Conclusions

These findings suggest the protective effect of EEAR against CCl₄ induced oxidative stress might be attributed to the presence of flavonoids and phenolic compounds.     

Hepatoprotective effects of Coptidis rhizoma aqueous extract on carbon tetrachloride-induced acute liver hepatotoxicity in rats

Aim of the study

Coptidis rhizoma (CR, Chinese name is Huanglian) has been used in treating infectious and inflammatory diseases for two thousand years in Traditional Chinese Medicine (TCM). Its related pharmacological basis for the therapeutics has been studied intensively, but CR can also be used for vomiting of “dampness-heat type or acid regurgitation” due to “liver-fire attacking stomach” in TCM, whose symptoms seem to link the hepatic and biliary disorders, yet details in the therapies of liver diseases and underlying mechanism(s) remain unclear. To clarify this ethnopharmacological relevance, hepatoprotective effect of Coptidis rhizoma aqueous extract (CRAE) and its possible mechanism were studied in rats intoxicated with carbon tetrachloride (CCl₄) in the present study.

Materials and methods

Sprague–Dawley (SD) rats aged 7 weeks old were intraperitoneally injected with CCl₄ at a dose of 1.0 ml/kg as a 50% olive oil solution. The rats were orally given the CRAE at doses of 400, 600, 800 mg/kg and 120 mg/kg berberine body weight (BW) after 6 h of CCl₄ treatment. At 24 h after CCl₄ injection, samples of blood and liver were collected and then biochemical parameters and histological studies were carried out.

Results

The results showed that CRAE and berberine inhibited significantly the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and increased the activity of superoxide dismutase (SOD). Observation on the hepatoprotective effect of berberine was consistent to that of CRAE.

Conclusion

The study is the first time to demonstrate that CRAE has hepatoprotective effect on acute liver injuries induced by CCl₄, and the results suggest that the effect of CRAE against CCl₄-induced liver damage is related to antioxidant property.     

Aegiceras corniculatum extract suppresses initial and late phases of inflammation in rat paw and attenuates the production of eicosanoids in rat neutrophils and human platelets

Aim of the study

The present study is designed to explore the anti-inflammatory potential of Aegiceras corniculatum Linn. Blanco stems extracts and their mechanism of action against various pro-inflammatory mediators and to validate its traditional use against inflammatory diseases.

Materials and methods

Rat paw edema and peritonitis models were employed for in vivo studies. For in vitro studies human platelets and rat neutrophils were stimulated with Ca²⁺-ionophore A23187 leading to the production of various pro-inflammatory metabolites, i.e., 12-HTT, 12-HETE and LTB₄ and 5-HETE which were quantified by HPLC.

Results

The highly polar methanol extract (100 mg/kg) caused ∼90% reduction in the carrageenan- and prostaglandin E2-induced paw edema in rats. It also caused the inhibition of cycloxygenase-1 metabolite, 12-HHT (IC₅₀ 41.1 ± 1.5 μg/ml) with a concomitant rise in 12-lipoxygenase metabolite, 12-HETE in A23187 stimulated human platelets. Conversely, the non-polar hexane extract attenuated (IC₅₀ 0.36 ± 0.12 μg/ml) 12-HETE formation with a parallel rise in 12-HHT, thereby displaying a selectivity towards 12-lipoxygenase. Non-polar hexane extract also antagonized the production of 5-lipoxygenase metabolites, i.e., leukotriene B₄ and 5-HETE in the rat neutrophils. Furthermore, ethyl acetate extract inhibited both COX and 5-LOX with a marked decline in the production of 12-HHT (IC₅₀ 0.08 ± 0.002 μg/ml) and LTB₄ (IC₅₀ 0.86 ± 0.03 μg/ml), respectively. The anti-inflammatory effect of hexane and ethyl acetate extracts was also reflected by the diminution of carrageenan-induced cell infiltration in rat peritoneum. Additionally, plant extracts caused ∼60% suppression in dextran-induced paw edema implying that they also ameliorate histamine and serotonin release.

Conclusion

Hexane, ethyl acetate and methanol extracts derived from Aegiceras corniculatum possess significant anti-inflammatory activity via multiple mechanisms and validate their traditional use against inflammation-related diseases.