Interleukin-1ß upregulates matrix metalloproteinase-13 gene expression via c-Jun N-terminal kinase and p38 MAPK pathways in rat hepatic stellate cells.

Matrix metalloproteinase-13 (MMP-13) is crucial in the cleavage and remodeling of the extracellular matrix (ECM), and its expression levels are decreased following the induction of liver fibrosis. The aim of the present study was to investigate the role of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in interleukin (IL)-1ß-mediated MMP-13 gene expression in rat hepatic stellate cells (HSCs). In the present study, we demonstrated that IL-1ß is capable of activating JNK and p38 in a time-dependent manner and the inhibition of the JNK pathway is able to increase MMP-13 mRNA expression; however, the inhibition of the p38 MAPK pathway is capable of inhibiting MMP-13 gene expression. These data demonstrate that IL-1ß is able to promote MMP-13 mRNA expression in rat HSCs and the JNK and p38 MAPK pathways were involved in this process. In summary, IL-1ß-induced MMP-13 mRNA expression is possibly mediated by cytoplasmic JNK and p38 MAPK pathways, and they play a distinct role in this process. Thus, the JNK and p38 MAPK pathway co-operatively mediate MMP-13 mRNA expression in rat HSCs.

Role of c-Jun N-terminal kinase and p38/activation protein-1 in interleukin-1ß-mediated type I collagen synthesis in rat hepatic stellate cells.

Interleukin-1 (IL-1) may play a role in maintaining hepatic stellate cell (HSC) in activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathway that is stimulated by IL-1 in HSC remains to be fully elucidated. The aims of this study were to investigate the role of c-Jun N-terminal kinase (JNK) and p38/activation protein (AP-1) in IL-1ß-mediated type I collagen synthesis in rat HSCs. Here, we show that IL-1ß could activate JNK and p38 in a time-dependent manner, and that inhibition of the JNK pathway could increase collagen synthesis; however, inhibition of the p38 pathway could inhibit collagen synthesis. Furthermore, IL-1ß activated AP-1 in a time-dependent manner in rat HSCs. These data demonstrate that L-1ß could promote the synthesis of type I collagen in rat HSCs, and the JNK and p38/AP-1 pathways were involved in this process. In summary, IL-1ß-induced collagen synthesis is possibly mediated by cytoplasmic JNK and p38/AP-1 pathways. Therefore, drugs that block the p38/AP-1 pathway may inhibit liver extracellular matrix synthesis and suppress liver fibrosis.

Differential role of YiGanKang decoction in IL-1ß induction of IL-1RI and AP-1 in rat hepatic stellate cell.

BACKGROUND/AIM: YiGanKang, a combination of Chinese herbs, has anti-fibrosis effects in chronic liver diseases. The present study tries to demonstrate differential role of YiGanKang Decoction in interleukin-1ß (IL-1ß) induction of the type I receptor (IL-1RI) and the activator protein 1 (AP-1) in rat hepatic stellate cell (HSC). METHODS: Flow cytometry was used to detect the IL-1RI expression. Electrophoretic mobility shift assays was used to detect AP-1 activity. RESULTS: IL-1RI expression after IL-1ß treatment for 0, 2, 10, 60, and 120 min was 227.08 (13.15), 268.43 (8.93), 442.97 (7.00), 367.66 (14.70), and 261.58 (15.08), respectively. The differences between IL-1RI expression after treatment for 10 and 60 min were significantly higher than the corresponding values in the control (P<0.01, P<0.01, respectively); After pretreatment with YiGanKang Decoction, IL-1RI expression induced by IL-1ß was not decrease obviously; IL-1ß could activate AP-1 in rat HSCs (P<0.01). Meanwhile YiGanKang Decoction could inhibit activity of AP-1 induced by IL-1ß (P<0.01), and the inhibition rate was 42.71%. CONCLUSION: YiGanKang Decoction could not decrease IL-1RI expression, but it could inhibit activity of AP-1 in rat HSCs induced by IL-1ß.

Suppressive effects of YiGanKang, a combination of Chinese herbs, on collagen synthesis in hepatic stellate cell.

BACKGROUND/AIM: Clinical practice and animal research demonstrated that YiGanKang, a combination of Chinese herbs, has anti-fibrosis effects in chronic liver diseases. However, the mechanism is not clear. The present study is to investigate the inhibiting mechanism of YiGanKang on collagen type I synthesis induced by Interleukin-1ß(IL-1 ß) in rat hepatic stellate cells (HSCs). METHODS: Cultured rat HSCs were divided into 4 groups, control, IL-1ß treated group, IL-1ß+YiGanKang group and IL-1ß+SB203580 (the p38 mitogen-activated protein kinase inhibitor) treated group. The expression of p38 mitogen-activated protein kinase (p38 MAPK), was evaluated by Western blot, collagen type I synthesis was examined by (3)H-Pro incorporation. RESULTS: Type I collagen synthesis in HSCs increased significantly under the stimulation of IL-1ß for 24h, YiGanKang could inhibit p38 expression and type I collagen synthesis, SB203580, a p38 inhibitor, can significantly reduce type I collagen synthesis. CONCLUSION: IL-1ß could stimulate the synthesis of type I collagen in rat HSCs, p38 mediate signal pathway between IL-1ß and was type I collagen production. YiGanKang inhibits HSCs collagen synthesis induced by IL-1ß via p38 inhibition.

[The role of apoptosis and the related genes in non-alcoholic steatohepatitis].

OBJECTIVE: To study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH). METHODS: An experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit. RESULTS: In MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05). CONCLUSIONS: In our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.

Diabetes mellitus with hepatic infarction: a case report with literature review.

Hepatic infarction rarely occurs due to the double supply of arterial and portal inflow. A 53-year-old man with diabetes mellitus developed multiple hepatic infarctions after an episode of fever and diarrhea. The infarction was documented by pathology after partial liver resection. Several causes of hepatic infarction may present in this patient: dehydration and hypotension caused by fever and diarrhea, type 2 diabetes and administration of glibenclamide, diabetic ketoacidosis and widespread atherosclerosis. We suggest that diabetic patient with elevated liver enzyme should be considered the possibility of hepatic infarction.

Comparison of protocatechuic aldchyde in Radix Salvia miltiorrhiza and corresponding pharmacological sera from normal and fibrotic rats by high performance liquid chromatography.

AIM: To observe the effect of protocatechuic aldchyde on the proliferation of hepatic stellate cells (HSCs). METHODS: Liver fibrosis was induced in rats by carbon tetrachloride (CCl4). Then normal and fibrotic drug sera were extracted from rats. The effects of protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza on HSC growth were determined by CCK-8. The protocatechuic aldchyde was separated by high performance liquid chromatography (HPLC) in a Alltima C18 column (250 mm mult 4.6 mm, 5 microm) with a mobile phase of acetonitrile-4% glacial acetic acid solution (gradient elution) at the wavelength of 281 nm. RESULTS: Protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza were found to have inhibitory effects on proliferation of rat HSCs. Raw Radix Salvia miltiorrhiza had a stronger inhibitory effect than the drug sera. The fibrotic drug sera showed a higher suppressive effect than the normal drug sera (P < 0.05). Protocatechuic aldchyde was found in crude materials of both Radix Salvia miltiorrhiza and its corresponding drug sera. The average recovery (n = 6) was 110.5% for raw Salvia miltiorrhiza Bge, 102% for normal drug sera and 105.2% for fibrotic drug sera. The relative standard devitation (RSD) was 0.37%, 1.96% and 1.51%, respectively (n = 6). The contents of protocatechuic aldchyde were 0.22%, 0.15% and 0.19%, respectively (n = 6) (P < 0.05). The RSD was 0.33%, 0.75% and 1.24% (n = 6) for raw material of Radix Salvia miltiorrhiza, normal drug sera and fibrotic drug sera, respectively. The samples were stable for 6 d. CONCLUSION: Protocatechuic aldchyde can inhibit the growth of HSCs. HPLC is suitable for the determination of virtual bioactive components of Chinese herbal medicines in vitro.

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells.

AIM: To study the relationship between interleukin-1beta (IL-1beta) up-regulating tissue inhibitor of matrix metalloproteinase-1 (TIMMP-1) mRNA expression and phosphorylation of both c-jun N-terminal kinase (JNK) and p38 in rat hepatic stellate cells (HSC). METHODS: RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1beta-induced JNK and p38 activities in rat HSC. RESULTS: TIMMP-1 mRNA expression (1.191+/-0.079) was much higher after treatment with IL-1beta (10 ng/mL) for 24 h than in control group (0.545+/-0.091) (P<0.01). IL-1beta activated JNK and p38 in a time-dependent manner. After stimulation with IL-1beta for 0, 5, 15, 30, 60 and 120 min,the JNK activity was 0.982+/-0.299,1.501+/-0.720, 2.133+/-0.882, 3.360+/-0.452, 2.181+/-0.789, and 1.385+/-0.368, respectively. There was a significant difference in JNK activity at 15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) in comparison to that at 0 min. The p38 activity was 1.061+/-0.310,2.050+/-0.863, 2.380+/-0.573, 2.973+/-0.953, 2.421+/-0.793, and 1.755+/-0.433 at the 6 time points (0, 5, 15, 30, 60 and 120 min) respectively. There was a significant difference in p38 activity at 5 min (P<0.05),15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) compared to that at 0 min.TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 (10 micromol/L, 1.022+/-0.113; 20 micromol/L, 0.869+/-0.070; 40 micromol/L, 0.666+/-0.123). Their decreases were all significant (P<0.05, P<0.01, P<0.01) in comparison to control group (without SP600125 treatment, 1.163+/-0.107). In the other 3 groups pretreated with different concentrations of SB203580 (10 micromol/L, 1.507+/-0.099; 20 micromol/L, 1.698+/-0.107; 40 micromol/L,1.857+/-0.054), the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group (without SB203580 treatment, 1.027+/-0.061) with a significant statistical significance (P<0.01). CONCLUSION: IL-1beta has a direct action on hepatic fibrosis by up-regulating TIMMP-1 mRNA expression in rat HSC. JNK and p38 mitogen-activated protein kinases (MAPKs) are involved in IL-1beta-induced TIMMP-1 gene expression,and play a distinct role in this process, indicating that p38 and JNK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.

Effects of pharmacological serum from normal and liver fibrotic rats on HSCs.

AIM: To make drug sera of Salvia miltiorrhiza and Yigankang, both of which are Chinese herbs that activate bleeding and eliminate stasis, in normal rats and those with liver fibrosis, respectively. To investigate and compare the effects of the two different drug sera on the proliferation and activation of hepatic stellate cells (HSCs). METHODS: Some rats were induced with liver fibrosis: 40% carbon tetrachloride (CCl4) subcutaneous injection, twice a week for 9 wk. Salvia miltiorrhiza, Yigankang, colchicines and normal saline were administered into the stomachs of normal rats and those with liver fibrosis. Drug sera were extracted 5 d later. HSCs in vitro were cultivated in different drug sera for 24 h. The rates of proliferation and expression of alpha-smooth muscle actin (alpha-SMA) were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and immunocytochemistry stain, respectively. RESULTS: The drug sera from normal and liver fibrotic rats could be used to cultivate HSCs and to observe the effects of the corresponding components of herbs on HSCs. Salvia miltiorrhiza and Yigankang had better inhibitory effects on HSCs than colchicines (MTT: normal drug serum: Salvia miltiorrhiza 0.42+/-0.08, Yigankang 0.32+/-0.10 vs colchicines 0.45+/-0.12 pathological drug serum: Salvia miltiorrhiza 0.33+/-0.02, Yigankang 0.26+/-0.01 vs colchicines 0.41+/-0.09. P<0.05). The drug sera of Salvia miltiorrhiza, Yigankang from liver fibrotic rats had a stronger inhibitory effect than the same ones from normal rats (MTT: Salvia miltiorrhiza: normal drug serum 0.42+/-0.08 vs pathological drug serum 0.33+/-0.02. Yigankang: normal drug serum 0.32+/-0.10 vs pathological drug serum 0.26+/-0.01. P<0.05). CONCLUSION: Salvia miltiorrhiza and Yigankang could inhibit the expression of alpha-SMA and the proliferation of HSCs. The drug sera from normal and liver fibrotic rats had different effects on HSCs, probably due to different metabolic processes, effective components and different quantities of drug contents in drug sera from rats with different states of liver.

[Effects of Radix Salviae Miltiorrhizae on Ca2+ in hepatic stellate cells].

OBJECTIVE: To investigate the effects of Radix Salviae Miltiorrhizae (RSM) on intracellular free calcium in hepatic stellate cells (HSCs). METHODS: After the model of hepatic fibrosis was established in SD rats, RSM [20 ml/(kg x d)] was given via gastrogavage to the rats of treatment groups while the same volume of 0.9% NaCl was given to the rats of control groups twice a day for 6 consecutive days. Then the blood sample was drawn from the inferior vena cava, and the serum was extracted for pharmacological studies. After 24 h incubation with 10% drug serum, HSCs were loaded with Fluo-3/AM, a Ca2+ marker, and were observed with laser scanning confocal microscopy (LSCM). RESULTS: By comparison with controls, both RSM pharmacological serums decreased [Ca2+]i in HSCs significantly in the condition of using Ang II or not (P<0.05). CONCLUSION: RSM decreased [Ca2+]i in activated HSCs, which may be one of important ways to block liver fibrosis.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats.

AIM: To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCl4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCl4-induced liver fibrosis by Yi-gan-kang. At wk 6, 10, 14 and 20 (baseline for CCl4 or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for alpha-SMA, type I collagen and in situ hybridization of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCl4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in microm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, alpha-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCl4- and pig serum-induced rat models.

Effects of killing Helicobacter pylori quadruple therapy on peptic ulcer: a randomized double-blind clinical trial.

AIM: To study the therapeutic efficacy of a Chinese and Western integrated regimen, killing Helicobacter pylori quadruple therapy on H pylori-associated peptic ulcers (PU). METHODS: With prospective and double-blind controlled method, seventy-five active PU patients with H pylori infection were randomized to receive one of the following three regimens: (1) new triple therapy (group A: lansoprazole 30 mg qd, plus clarithromycin 250 mg bid, plus amoxycillin 500 mg tid, each for 10 d); (2) killing Hp quadruple therapy(group B: the three above drugs plus killing H pylori capsule 6 capsules bid for 4 wk) and (3) placebo(group C: gastropine 3 tablets bid for 4 wk). H pylori eradication and ulcer healing quality were evaluated under an endoscope 4 wk after treatment. The patients were followed up for 5 years. RESULTS: Both the healing rate of PU and H pylori eradication rate in group B were significantly higher than those in group C (100% and 96.4% vs 20% and 0%, respectively, P<0.005), but there was no significant difference compared to those in group A (88% and 92%, P>0.05). The healing quality of ulcer in group B was superior to that in groups C and A (P<0.05). The recurrence rate of PU in group B (4%) was lower than that in group A (10%) and group C (100%, P<0.01). CONCLUSION: Killing Helicobacter pylori quadruple therapy can not only promote the eradication of H pylori and healing quality of ulcer but also reduce recurrence rate of ulcer.

Current research of hepatic cirrhosis in China.

Hepatic cirrhosis is a common disease that poses a serious threat to public health, and is characterized by chronic, progressive and diffuse hepatic lesions preceded by hepatic fibrosis regardless of the exact etiologies. In recent years, considerable achievements have been made in China in research of the etiopathogenesis, diagnosis and especially the treatment of hepatic fibrosis, resulting in much improved prognosis of hepatic fibrosis and cirrhosis. In this paper, the authors review the current status of research in hepatic fibrosis, cirrhosis and their major complications.

[Effects of aldosterone and spironolactone on the proliferation and collagen synthesis of hepatic stellate cells in rats].

OBJECTIVE: To investigate the effects of aldosterone and spironolactone on the proliferation and collagen synthesis of hepatic stellate cells. METHODS: Rat HSC were incubated with aldosterone or spironolactone, an aldosterone receptor antagonist at different concentrations. The proliferation of HSC was measured by (3)H-thymidine incorporation, and the collagen synthesis by (3)H-Proline. The changes of HSC cycle were analyzed by FCM. RESULTS: At dose of 10(-4) mol/L, ALD increased the incorporation of (3)H-TdR and (3)H-Pro (P < 0.05), but didn't at the lower doses. Spironolactone significantly inhibited HSC proliferation and collagen synthesis. The effective concentration was among 10(-4) mol/L - 10(-6) mol/L, and in dose-dependent manner. The percentage of G(1) phase of the cell cycle was significantly increased, and the cell proliferation suppression of spironolactone is associated with cell arrest in G(1) phase. Co-stimulation with aldosterone and spironolactone did not have significant effects on HSC proliferation and collagen synthesis compared with the control group. CONCLUSION: The proliferation and collagen synthesis of HSC might be enhanced by ALD at higher concentration, but was inhibited by spironolactone.

The expression of hTERT mRNA and cellular immunity in gastric cancer and precancerosis.

AIM: To observe the expression of Human telomerase reverse transcriptase (hTERT) in gastric carcinomas and precancerosis lesions, to evaluate the immune state of such patients, and to then study the clinical significance of hTERT and immune state for the diagnosis, treatment and prognosis of gastric cancer. METHODS: In situ hybridization was used to detect the expression of hTERT mRNA in 116 endoscopic of gastric mucosa. Analyzed tissue samples were as follows: 30 cases of chronic superficial gastritis (CSG), 44 of precancerosis lesions (including 27 of chronic atrophic gastritis, 8 of adenomatous polyp and 9 of gastric ulcer) and 42 of gastric cancer (GC). In addition, the T lymphocyte subsets (CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+)) and natural killer cells (NK) in peripheral blood were determined by flow cytometric analysis (FCM) in 30 cases of CSG, 27 of precancerosis (chronic atrophic gastritis, CAG), and 42 of GC. The data were compared with those of normal control (NC). RESULTS: The detected positive rate of hTERT varied as follows: 86 % (36/42) in GC, 36 % (16/44) in precancerosis lesions and 0 % (0/30) in CSG. The expression of hTERT mRNA was not associated with patient gender, tumor location, macroscopic type, lymph node metastasis, or degree of differentiation. It was found that the CD3(+), CD4(+) of the CSG group were lower than that of NC (P<0.05). Meanwhile, the T lymphocyte subsets (CD3(+), CD4(+), CD4(+)/CD8(+) ratio) and NK cells of CAG were remarkably lower than that of NC and CSG groups (P<0.05-0.01). Values of T cells and NK cells of the GC group were significantly abnormal when compared with the CAG group (P<0.05-0.01). Furthermore, with tumor progression, the function of T cells was weakened gradually. CONCLUSION: The expression of telomerase may be a crucial step in gastric carcinogenesis and increased hTERT mRNA may serve as a novel marker for diagnosis of GC. The immune state of patients with GC and precancerosis was somewhat depressed, which indicates the importance of cellular immunological assays in cancer patients.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells.

AIM: To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P<0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P<0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P<0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P<0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P<0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

[Effects of ET-1 on isolated perfused rat liver and vascular rings at two stages of cirrhosis].

OBJECTIVE: To investigate the effects of ET-1 on isolated perfused rat liver and vascular rings at early and late stages of cirrhosis. METHODS: Liver cirrhosis was induced by an intraperitoneal injection of 50% CCl(4) (0.3 ml/100 g, twice a week). In the 9th and 14th weekend after injecting CCl(4), the isolated perfused liver and vascular rings were performed to evaluate effects of four concentrations of ET-1 on early and late stages of cirrhosis. RESULTS: The Ppv of L-HC group at baseline was higher than that of E-HC group, both were higher than that of the controls. However, there showed no differences on Phv in these groups. With the concentration of ET-1 increasing, PVP was elevated accordingly in E-HC and L-HC group. L-HC group showed higher PVP compared with E-HC group, both were higher than the controls. While in isolated vascular rings, with the deteriorating of cirrhosis, the cumulative response curves showed right-shift. 0.1 nmol/L ET-1 showed mild relaxation on vascular rings in L-HC group. CONCLUSION: ET-1 can increase the PVP, especially with the deterioration of cirrhosis, there showed higher reaction compared with normal controls. The vascular rings showed low response on the contrary. So ET-1 plays an important role in the pathogenesis of portal hypertension. In view of its different roles on liver and vascular rings at early and late stages, administration of different selective antagonist of ET receptor at different stages of cirrhosis should be well considered.

Clinical and experimental study of effect of Raondix Salviae Militiorrhiza and other blood-activating and stasis-eliminating Chinese herbs on hemodynamics of portal hypertension.

AIM:To study the effects of Radix Salviae Militiorrhiza (RSM), other blood-activating and stasis-eliminating Chinese herbs on hemodynamics of portal hypertension.METHODS:Portal pressure of cirrhotic dogs after chronic common bile duct ligation was measured directly; portal blood flow in patients with liver cirrhosis were detected by ultrasound Doppler.RESULTS:After administration of RSM and Radix Angelicae Sinensis (RAS) by intravenous infusion in cirrhosis dogs, the portal venous pressure (Ppv), wedge hepatic venous pressure (WHVP), hepatic venous pressure gradient (HVPG), were significantly decreased (P < 0.05-0.01), but the mean arterial pressure (MAP), and the heart rate (HR) remained unchanged. When nifedipine was used, Ppv, WHVP, MAP and HR were significantly decreased (P < 0.05), and the MVPG unchanged (P > 0.05). After administration of RSM, RSM+nifedipine and RSM+Hirudin+Nifedpin for 10-12 weeks, the diameter of portal vein (Dpv), spleen vein (Dsv), the portal venous flow (Qpv) and splenic venous flow (Qsv) in patients with hepatic cirrhosis were significantly lowered (P < 0.05-0.01), and the effect of RAS was weaker.CONCLUSIONS: The efficacy of decreasing Ppv by Chinese herbs RSM, RAS, etc. as compared with nifedipine, demonstrated that the Chinese herbs were slower in action than that of nifedipine, but more long-lasting and without side effects. Hence, long-term administration of Chinese herbs, would be more beneficial.