Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats.

The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8 mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of α-smooth muscle actin (α-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-ß1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-κB p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis.

[Effects of Artesunate on hepatic fibrosis and its mechanism].

OBJECTIVE: To investigate the effects of Artesunate(Art) on the LX-2 cell. METHODS: The cultured hepatic stellate cells were divided into control group and Art-treated groups with 250,350,450 µmol/L. The rate of cellular proliferation was detected by MIT assay, the content of ceramide (Cer)was determined by HPLC method, the content of hydroxyproline (Hyp) was determined by enzyme digestion method, the expressions of PPAR-γ, p53 and Caspase 3 were detected by Western blot. RESULTS: Compared with control group, IX-2 treated with Art were inhibited in a concentration-dependent manner(P < 0.01). Art could significantly increase the content of cerarnide in LX-2 ( P <0.01), and the content of Hyp was significantly decreased (P <0.05, P <0.01). The expressions of PPAR-γ, p53 and Caspase 3 were increased compared with that of control group(P < 0.01). CONCLUSION: Artesunate could inhibit the proliferation and induce apoptosis of hepatic stellate cells through upregulating ceramide.

Artesunate ameliorates hepatic fibrosis induced by bovine serum albumin in rats through regulating matrix metalloproteinases.

The effect of Artesunate on anti-hepatic fibrosis was discovered by our team for the first time. In order to investigate the effect of Artesunate on hepatic fibrosis induced by Bovine serum albumin (BSA) in rats and understand the initiatory mechanism of its effect, several experiments were conducted in this assay. HE staining and Masson׳s Trichrome staining were employed in observation of morphological changes. The content of hydroxyproline in the hepatic tissue was determined by using an acid hydrolyzation method. In addition, the expression of Matrix metalloproteinase-13 (MMP-13) and type I collagen were tested by western blotting respectively. The expression of Matrix metalloproteinase-2(MMP-2), Matrix metalloproteinase-9 (MMP-9) were determined by Gelatin Zymography Assay. Also, we use immunohistochemical studies to measure the expression of α-SMA. The final results indicated that Artesunate could dramatically attenuate the extent of hepatic fibrosis showed by histopathological sections of hepatic tissues, significantly decrease the content of hydroxyproline and efficiently inhibit the protein expression of MMP-2, MMP-9, α-SMA and type I collagen. Artesunate could as well promote the expression of MMP-13 at the same time. In conclusion, the results not only suggested that Artesunate could ameliorate hepatic fibrosis, but also suggested the anti-fibrogenic mechanisms of Artesunate might be associated with inhibiting the activation of HSCs, decreasing the expression of MMP-2, MMP-9 and increasing the expression of MMP-13.These results would bring new insights for the treatment for hepatic fibrosis.

Effects of Fufang Biejia Ruangan pills on hepatic fibrosis in vivo and in vitro.

AIM: To explore the protective effect and the relevant mechanisms of Fufang Biejia Ruangan Pills (FFBJRGP) on hepatic fibrosis in vivo and in vitro. METHODS: Hepatic fibrosis was induced by carbon tetrachloride composite factors. Adult Wistar rats were randomly divided into four groups: normal control group; hepatic fibrosis model group; FFBJRGP-treated group at a daily dose of 0.55 g/kg; and colchicine-treated group at a daily dose of 0.1 g/kg. The effects of FFBJRGP on liver function, serum levels of hyaluronic acid (HA), type IV collagen (CIV), type III procollagen (PC III), laminin (LN), histopathology, and expression of transforming growth factor (TGF-ß1) and Smad3 in hepatic fibrosis were evaluated in vivo. The effects of FFBJRGP on survival rate, hydroxyproline content and cell cycle distribution were further detected in vitro. RESULTS: Compared with the hepatic fibrosis model group, rats treated with FFBJRGP showed a reduction in hepatic collagen deposition and improvement in hepatic lesions. Compared with those of the model group, the activities of alanine aminotransferase (62.0 ± 23.7 U/L) and aspartate aminotransferase (98.8 ± 40.0 U/L) in the FFBJRGP-treated group were decreased (50.02 ± 3.7 U/L and 57.2 ± 30.0 U/L, respectively, P < 0.01). Compared with those in the model group, the levels of PCIII (35.73 ± 17.90 µg/mL), HA (563.82 ± 335.54 ng/mL), LN (89.57 ± 7.59 ng/mL) and CIV (29.20 ± 6.17 ng/mL) were decreased to 30.18 ± 9.41, 456.18 ± 410.83, 85.46 ± 7.51 and 28.02 ± 9.45 ng/mL, respectively. Reverse-transcriptase polymerase chain reaction and Western blotting also revealed that expression of TGF-ß1 and Smad3 were down-regulated in vivo. Cell proliferation was inhibited, the level of hydroxyproline was decreased compared with the control group (P < 0.01), and the cell cycle was redistributed when exposed to FFBJRGP in vitro. CONCLUSION: FFBJRGP inhibits hepatic fibrosis in vivo and in vitro, which is probably associated with downregulation of fibrogenic signal transduction of the TGF-ß-Smad pathway.

[Impact of artesunate on the expression and secretion of transforming growth factor-b1 of primary rat hepatic stellate cells].

OBJECTIVE: To investigate the impact of the Artemisia annua plant-derived drug, artesunate, on proliferation of primary rat hepatic stellate cells (HSCs), and to analyze the underlying molecular mechanisms of its anti-fibrogenic effects involving the inhibition of transforming growth factor-beta 1 (TGF-b1) expression and secretion in liver. METHOD: Isolated, cultured, and activated primary rat HSCs were divided into sixteen groups, including one untreated control group and fifteen artesunate-treated experimental groups with 125, 150, 175, 200 or 225 mumol/L for 24, 48 or 72 hours. The rate of cellular proliferation was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. TGF-b1 mRNA expression was evaluated by reverse transcription-polymerase chain reaction and protein expression was evaluated by Western blotting. Enzyme-linked immunosorbent assay was used to evaluate secreted levels of TGF-b1 protein. RESULTS: Artesunate significantly inhibited proliferation of cultured HSCs in a dose- and time-dependent manner (all, P less than 0.01). After 24 hours of exposure, the inhibition ratios of the various artesunate concentrations were: 6.06%+/-1.44% (125 mumol/L), 21.47%+/-5.57% (150 mumol/L), 42.00%+/-7.36% (175 mumol/L), 67.12%+/-4.55% (200 mumol/L), and 79.83%+/-3.67% (225 mumol/L). Artesunate significantly inhibited the TGF-b1 mRNA expression in HSCs, and the higher the drug concentration, the higher the degree of inhibition (all, P less than 0.01). In addition, artesunate significantly inhibited the expression of intracellular and secreted TGF-b1 protein (all, P less than 0.01). In response to artesunate (mumol/L concentrations), the TGF-b1 levels were (164.24+/-6.88) pg/ml (0µmol/L), (102.68+/-4.45) pg/ml (150µmol/L), (86.54+/-5.56) pg/ml (175µmol/L), and (56.55+/-5.66) pg/ml (200µmol/L). CONCLUSION: Artesunate exerts anti-fibrogenic effects on HSCs in vitro, possibly by reducing the expression, translation and secretion of TGF-b1.

[Therapeutic effect of Haobieyangyinruanjianfang on the mouse liver fibrosis caused by schistosomiasis].

OBJECTIVE: To explore therapeutic effect of Haobieyangyinruanjianfang (HBYYRJ) on mouse liver fibrosis by schistosomiasis. METHODS: Mice except for normal control were infected with Japanese schistosome cercarias, after 12 weeks, infected mice were divided into 7 groups: low HBYYRJ group, middle HBYYRJ group, high HBYYRJ group, Fufangbiejiaruangan tablet (FFBJRG) group, colchicine group, 3 months infection group and 6 months infection group. Hepatic fibrosis was found in 3 months infection group. Liver hydroxyproline (Hyp) was determined, matrix metalloproteinase-2 and 9 (MMP-2, MMP-9) were detected with gelatin zymography, serum hyaluronic acid (HA) and precollagen III (PC-III) were detected using RIA. RESULTS: HBYYRJ obviously reduced hepatic fibrosis (probability value less than 0.01). Collagen and HA in 3 months infection group and 6 months infection group were higher than that in normal group (probability value less than 0.01), collagen in high and middle HBYYRJ groups and HA in middle and low HBYYRJ groups were lower than that in 6 months infection group (P less than 0.01, probability value less than 0.05). The expression of MMP-9 and MMP-2 in 3 months infection group and 6 months infection group was higher than that in normal group (probability value less than 0.01), The expression of MMP-9 in three HBYYRJ groups and the expression of MMP-2 in high HBYYRJ group were lower than that in 6 months infection group (probability value less than 0.05). CONCLUSION: HBYYRJ can reduce liver fibrosis caused by schistosomiasis.

Effects of Haobie Yangyin Ruanjian decoction on hepatic fibrosis induced by carbon tetrachloride in rats.

AIM: To explore the anti-fibrotic effect of Haobie Yangyin Ruanjian decoction (HYRD) on CCl(4)-induced hepatic fibrosis in rats and its modulation on the transforming growth factor (TGF) beta-Smad signaling pathway. METHODS: Fifty-six healthy Wistar rats were randomly divided into five groups: normal control group (n = 6), CCl(4)-induced hepatic fibrosis group (n = 14) and three treatment groups (the treated rats received HYRD via oral administration at daily dosages of 8.2, 2.5 and 0.82 g/kg, respectively) of HYRD (n = 12, respectively). Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride solution (CCl(4) dissolved in peanut oil, 4:6, V/V) with 0.5 mL/100 g body weight for the first time, and then 0.3 mL/100 g body weight twice a week for 8 wk. In the former 2 wk, rats were raised by feedstuff I (80% corn meal, 20% lard, 0.5% cholesterol). After 2 wk, they were raised by feedstuff II (corn meal and 0.5% cholesterol). Except for the control group, 30% alcohol solution was given orally to each rat every other day from the beginning, 1 mL for each rat. Liver function parameters and hepatic hydroxyproline content were detected by chromatometry. Serum levels of hyaluronic acid (HA), type IV collagen (CIV), type III precollagen (PCIII) and laminin (LN) were assayed with radioimmunoassay. Deposition of collagen was observed with hematoxylin-eosin staining and collagen staining. Gene expression of TGFbeta1 and Smad3 were detected with real-time reverse transcriptase-polymerase chain reaction and Western blotting, respectively. RESULTS: The serum levels of alanine transaminase and aspartate transaminase were increased in the model group compared with the control group (P < 0.01), and they were decreased in the three treatment groups compared with the model group. The serum levels of total protein and albumin were decreased in the model group and increased in the three treatment groups. The hepatic hydroxyproline content and serum levels of PCIII, HA, LN and CIV were markedly increased in the model group compared with the control group, and decreased in the treatment groups. The gene expression of TGFbeta1 and Smad3 was enhanced in the model group compared with the control group, and HYRD could down regulate their expression. CONCLUSION: HYRD can inhibit hepatic fibrosis induced by CCl(4) in rats, which is probably associated with its down-regulation on fibrogenic signal transduction of TGFbeta-Smad pathway.

[A fluorospectrophotometric determination of nitrite in blood].

AIM: To establish a fluorospectrophotometric assay for the measurement of nitrite in blood. METHODS: Interference from hemoglobin and other blood ingredients was removed through sulfuric acid and phosphotungstic acid pretreatment. Fluorescence of 1-[H]-naphthotriazole from the reaction of 2,3-diaminonaphthalene with nitrite was determined with fluorospectrophotometry. RESULTS: The following conditions were proper: Serum or plasma was treated with sulfuric acid and phosphotungstic acid pretreatment for two times, 2,3-diaminonaphthalene of 0.63 mmol x (L(-1)) was used, reaction solution pH and final pH were about 1.60 and 1.70 respectively, solution containing 2,3-diaminonaphthalene and supernatant after pretreatment was water-bathed at 20 degrees C for 15 minutes. The lower limit of detection was 24.27 nmol x L(-1). Nitrite determined in peripheral blood of healthy people was (10.91 +/- 2.38) micromol x L(-1), and its 95% distribution range was (6.24-15.57) micromol x L(-1). CONCLUSION: It's a relatively sensitive, specific, simple method. It's of some value to the study of nitric oxide.

[Changes in pro-inflammatory cytokines and media and peptide hormones during multiple organ dysfunction syndrome following acute abdominal diseases].

OBJECTIVE: To inquire into effects of cytokines and other inflammatory media, and peptide hormones during multiple organ dysfunction syndrome (MODS) subsequent to acute abdominal diseases. METHODS: In 19 patients with MODS due to acute abdominal diseases, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), thromboxane B(2) (TXB(2)), 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), endotoxin, gene-related peptide(CGRP), endothelin-1 (ET-1) and substance P (SP) in plasma, and lipid peroxide (LPO) and nitric oxide (NO) in serum were determined dynamically. RESULTS: Both TNF-alpha and IL-6 at increased significantly in MODS patients; IL-6 on day 0 in patients without treatment of endoscopic retrograde bile duct drainage (ERBD) were higher than that in patients with correspondent treatment, IL-6 in severe acute cholangitis patients was higher than that in patients with acute necrotic pancreatitis, it approached 24,000 ng/L during toxic shock. TNF-alpha and IL-6 during early stage of MODS were higher than that during systemic inflammatory response syndrome (SIRS) respectively. Endotoxin and LPO levels in MODS patients increased significantly. The levels of NO in emergency patients with MODS was elevated, but lowered in patients with acute necrotic pancreatitis, hepatocarcinoma, advanced age's patients with long time fever due to hepatic abscess. TXB(2) and 6-keto-PGF(1alpha) during early stage rose significantly, both decreased after treatment. ET-1 and CGRP during early stage increased significantly, SP peaked on day 0. CONCLUSION: The level of IL-6 persistently higher than 300 ng/L suggests the diagnosis of MODS. The levels of IL-6 and TNF-alpha could be taken as an indication of the degree of SIRS. NO maybe either increased or decreased, ET-1, CGRP, TXB(2), 6-keto-PGF(1alpha), endotoxin, and LPO are found to be increased MODS.