Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats

AIM： To investigate the suppressive effect of saikosaponin-d （SSd） on hepatic fibrosis in rats induced by CCh injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-κB （NF-κB）, tumor necrosis factor-alpha （TNF-α） and interleukins-6 （IL-6）. METHODS： Hepatic fibrosis models were induced by subcutaneous injection of CCh at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses （1.0, 1.5 and 2.0 mg/kg） once daily for 6 wk in combination with CCh, while the control group received olive oil instead of CCh. At the end of the experiment, rats were anesthetized and killed （except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in lowdose group）. Hernatoxylin and eosin （HE） staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase （ALT）, triglyeride （TG）, albumin （ALB）, globulin （GLB）, hyaluronic acid （HA） and larninin （LN） in serum and the content of hydroxyproline （HYP） in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-α and IL-6 in liver homogenate was evaluated by enzymelinked immunosorbent assay （ELISA） and the levels of NF-κBp65 and I-κBa in liver tissue were analyzed by Western blotting. RESULTS： Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-α and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SScl markedly reduced all the above parameters compared with the model group, especially in the medium gr     

复方861治疗慢性乙型肝炎肝纤维化与早期肝硬化的临床研究

目的观察复方861对慢性乙型肝炎肝纤维化、早期肝硬化患者的抗肝纤维化效果.方法采用随机、双盲、安慰剂对照的方法,以治疗前后肝穿病理组织学为评价指标,对6家医院的慢性乙型肝炎肝纤维化患者136例,按照随机编码分别服用复方861胶囊或安慰剂胶囊共24周,观察治疗前后患者症状、体征、肝功能、肝纤维化指标[IV胶原(C Ⅳ)、层黏连蛋白(L N)、Ⅲ型前胶原N端肽(P ⅢP)、透明质酸(HA)]、基质金属蛋白酶1、2、9(MMP1、2、9)及金属蛋白酶组织抑制因子(TIMP1、2)水平以及肝病理组织学的变化.结果 5 2例治疗组、5 0例安慰剂组的患者完成治疗前后肝穿刺.治疗组患者治疗前、后血清丙氨酸氨基转移酶(ALT)分别为(68.2±68.6)U/L和(45.9±26.1)U/L、天冬氨酸氨基转移酶(AST)分别为(60.4 ± 62.6)U/L和(46.7 ± 39.0)U/L,安慰剂组患者治疗前、后血清ALT分别为(65.3±48.3)U/L和(85.4±115.5)U/L,AST分别为(60.4±44.6)U/L和(77.6±89.6)U/L,两组比较差异均有显著性,t=2.315和t=2.168,P＜0.05.治疗组血清HA、PⅢP、CⅣ、LN水平均较治疗前有所下降,但与安慰剂组相比,差异无显著性.治疗组治疗前、后血清TIMP1分别为(172.0±79.6)ng/m1和(133.5 ± 66.8)ng/ml,MMP9分别为(116.1±88.2)ng/ml和(80.4±79.0)ng/ml,较治疗前均明显下降,f=2.723和t=2.433,P＜0.05.复方861治疗前、后血清TIMP1/MMP1比值分别为4 8.3±96.3和19.9 ± 28.0,较治疗前下降,而对照组则较治疗前升高,治疗前后差值相比,两组差异有显著性,t=2.248,P＜0.05.治疗组治疗前、后肝组织炎症计分分别为14.0±6.0和10.2±6.1、纤维化计分分别为11.9±6.5和8.2=4.5,病理图像分析胶原相对含量分别为18.9%±9.5%和14.9%± 8.4%,t值为3.354～2.202,P值均＜0.05;S2期逆转率为38.9%,S 3期为53.3%,S4期为78.6%,总逆转率52.0%;安慰剂组分别为14.3%、25.0%、41.7%、20.0%,两组差异有显著性,x2值为9.766～4.478,P值均＜0.05.复方861治疗组未见明显不良反应.结论复方861治疗慢性乙型肝炎肝纤维化、早期肝硬化是可以逆转的.     

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice

AIM: To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide (TAA). METHODS: Twenty-four male C57Bl/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline (2 mL/kg), leptin (1 mg/kg), TAA (200 mg/kg), TAA (200 mg/kg) plus leptin (1 mg/kg) respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminot-ransferase (ALT) and aspartate aminotransferase (AST) in serum and superoxide dismutase (SOD), malondialdehyde (MDA) in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin (HE) staining and picric acid-Sirius red dyeing were performed. The level of α1(I) procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS: Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group (205.67±27.69 U/L vs50.67±10.46 U/L, 177.50±23.65 U/L vs 76.33±12.27 U/L, 2.60±0.18 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P<0.01) and in TAA plus leptin group (256.17±22.50 U/L vs 50.67±10.46 U/L, 234.17±27.37 U/L vs 76.33±12.27 U/L, 2.97±0.19 nmol/mg pro vs 1.91±0.14 nmol/mg pro,P<0.01). The level of SOD in livers decreased (51.80±8.36 U/mg pro vs 81.52±11.40 U/mg pro, 35.78±6.11 U/mg pro vs 81.52± 11.40 U/mg pro, P<0.01) and the level of α1(I) procollagen mRNA in liver tissues also increased (0.28±0.04 vs 0.11± 0.02, 0.54±0.07 vs 0.11±0.02, P<0.01). But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and α1(I) procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased (256.17±22.50 U/L vs 205.67± 27.69 U/L, P<0.05; 234.17±27.37 U/L vs 177.50±23.65 U/L, P<0.05; 2.97±0.19 nmol/mg pro vs 2.60±0.18 nmol/mg pro,P<0.05; 0.54±0.07 vs 0.28±0.04, P<0.01; 3.17 vs 2.00, P<0.05), and the level of SOD in liver decreased (35.78±6.11 U/mg pro vs 51.80±8.36 U/mg pro, P<0.05). There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION: Leptin can exacerbate the degree of TAA-induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.     

黄芪汤组分抑制胆汁淤积性肝纤维化大鼠胆管上皮细胞增殖及其转分化的效应

目的 探讨胆管上皮细胞增殖及其转分化在肝纤维化形成过程中的作用及黄芪汤组分抑制肝纤维化的效应和机制.方法 30只SD雄性大鼠采用胆总管结扎制备胆汁淤积性肝纤维化模型,假手术对照组仅对胆总管作分离,不结扎.胆管结扎术后l周随机分为模型对照组和药物干预组(经灌胃给予黄芪汤组分4周),5周后麻醉下处死大鼠,获取肝组织与血清标本.检测肝功能、肝组织学及羟脯氨酸(Hyp)含量;激光共聚焦显微镜观察肝组织角蛋白-7(CK7)与α-平滑肌肌动蛋白(α-SMA)共定位,Western blot检测CK7、α-SMA蛋白表达.计量资料比较采用单因素方差分析中的LSD法或非参数检验中的H检验进行统计学分析,计数资料采用Radit检验,CK7与α-SMA蛋白表达间关系采用直线回归与相关分析.结果 与假手术对照组比较,模型对照组大鼠病死率33.3%,腹水出现率90%,肝功能显著异常,肝组织肝细胞显著减少,胆管上皮细胞及纤维组织大量增生,肝组织Hyp含量、CK7及α-SMA蛋白表达显著增加(P值均<0.01),并有大量的CK7及α-SMA共定位阳性细胞表达.与模型对照组比较,药物干预组大鼠病死率、腹水出现率及脾脏质量均显著降低(P值均<0.05),肝功能显著改善,肝组织肝细胞减少及胆管上皮细胞与纤维增生程度轻,CK7与α-SMA共定位阳性细胞显著减少.药物干预组与模型对照组大鼠肝Hyp含量分别为(1026.8±132.47)μg/g和(887.4±95.56)μg/g,CK7表达量分别为0.812±0.298和0.318±0.143,α-SMA蛋白表达量分别为0.787±0.277和0.341±0.119,差异均有统计学意义(JD值均<0.01).结论 黄芪汤组分可有效抑制胆管结扎大鼠胆管上皮细胞增生及胆管上皮细胞向α-SMA阳性的肌成纤维细胞的转分化.     

Serum concentration of the aminoterminal procollagen type III peptide in the rat reflects early formation of connective tissue in experimental liver cirrhosis

A specific and sensitive radioimmunoassay for the rat aminoterminal procollagen type III peptide (PIIIP) was developed which allowed easy and sequential measurement of this peptide in the serum of individual animals. PIIIP in sera of 1-week-old rats was high (60 +/- 15.4 ng, 1 SD) falling to 15.7 +/- 4.3 ng/ml (1 SD) at 7 weeks and 6.7 +/- 2.6 ng/ml (1 SD) at 12 weeks of age. Adult animals (above 6 months of age) showed serum PIIIP levels in the narrow range of 2.5 +/- 2.33 ng/ml (2.5 SD). CCl4-induced liver damage in adult rats produced an elevated serum PIIIP (median 9.1; range 2.6-45.2 ng/ml) already after 2 weeks, rising to a mean of 33.8 ng/ml (range 22.0-47 ng/ml) after 6 weeks of continued CCl4-intoxication. In the same animals at 6 weeks, hepatic hydroxyproline was almost 5 times higher in the CCl4-group (mean 493.2; range 343.1-582.3 micrograms/100 mg dry weight) when compared with controls (109 +/- 14 micrograms/100 mg dry weight, 1 SD). These results are in complete analogy to those reported for PIIIP in sera of growing children, healthy human adults and patients with fibrogenic liver disease. Elevated serum PIIIP in rats with experimental liver fibrosis predicts the deposition of excess hepatic collagen. This novel serum test allows, for the first time, to assess altered PIIIP metabolism and hepatic fibrogenesis in individual animals as early as 2 weeks after the start of the experiment. It also reflects growth-related changes of type III collagen metabolism.     

Significance of serum procollagen-III-peptide in reflecting the therapeutic effects of calcium-channel blockers on hepatic fibrosis

Changes in serum procollagen-III-peptide (PIIIP) and intravenous tryptophan tolerance test (ITT) were studied in 121 patients with liver cirrhosis during a follow-up period of up to 18 months. The patients received either nifedipine (31 cases, 60 mg/day), verapamil (28 cases, 120 mg/day), cinnarizine (29 cases, 150 mg/day) or tetrandrine (33 cases, 150 mg/day). The significant changes were found in ITT in any of the four drugs administrated for over three months. Serum PIIIP concentration decreased significantly in patients under tetrandrine therapy for 18 months (12.06 +/- 3.91 ng/ml vs 16.57 +/- 5.69 ng/ml before treatment). These data suggest that tetrandrine therapy may have favourable effects on hepatic fibrosis and improvement of liver function in liver cirrhosis.     

In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

AIM: To investigate the effects of Danshaohuaxian (DSHX), a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group, non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO, daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT), degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively. RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs 316.17±78.48, 300.86±72.73, P<0.05) and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01), and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01) all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0.01), the COL Ⅰ and Ⅲ expression decreased (COL I: 1.07±0.96 vs 4.18±2.26, 3.22±1.44, P<0.01; COL Ⅲ: 1.09±0.58 vs 3.04±0.62, 2.23±0.58, P<0.01), the HSCs apoptotic index of HSCs (7.81±0.47 vs 1.63±0.25, 1.78±0.4, P<0.05) and the ratio of G0-G1 phase cells increased (94.30±1.33 vs 62.27±17.96, 50.53±2.25, P<0.05). The ratios of S-phase cells (3.11±1.27 vs 9.83±1.81, 11.87±1.9, P<0.05) and G2-M phase cells (2.58±0.73 vs 23.26±10.95, 13.60±1.15, P<0.01) declined. CONCLUSION: DSHX capsule shows certain therapeutic effects on hepatic fibrosis in rats and inhibits abnormal deposition of COL I and III in rat livers by promoting the apoptosis of HSCs and preventing their proliferation.     

Preventive effect of Qianggan-Rongxian Decoction on rat liver fibrosis

AIM： To study the preventive effects of Qianggan-Rongxian Decoction on liver fibrosis induced by dimethylnitrosamine （DMN） in rats.
METHODS： Male Wistar rats were randomly divided into hepatic fibrosis model group, control group and 3 treatment groups （12 rats in each group）. Except for the normal control group, all the rats received 1% DMN （10 μL/kg body weight, i.p）, 3 times a week for 4 wk. The rats in the 3 treatment groups including a high-dose DMN group （10 mL/kg）, a medium-dose DMN group （7 mL/kg）, and a low-dose DMN group （4 mL/kg） were daily gavaged with Qianggan-Rongxian Decoction, and the rats in the model and normal control groups were given saline vehicle. Enzyme-linked immunosorbent assay （ELISA） was used to determine the changes in serum hyaluronic acid （HA）, laminin （LN）, and type IV collagen levels. Serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels were measured using routine laboratory methods. Pathologic changes, particularly fibrosis, were examined by hematoxylin and eosin （HE） and Sirius red staining. Hepatic stellate cells （HSC） were examined by transmission electron microscopy.
RESULTS： Compared with the model control group, the serum levels of HA, LN, type Ⅳ collagen, ALT and AST were decreased markedly in the other groups after treatment with Qianggan-Rongxian Decoction, especially in the medium-dose DMN group （P 〈 0.05）.Moreover, the area-density percentage of collagen fibrosis was lower in the Qianggan-Rongxian Decoction treatment groups than in the model group, and a more significant drop was observed in the medium-dose DMN group （P 〈 0.05）.
CONCLUSION： Qianggan-Rongxian Decoction can inhibit hepatic fibrosis due to chronic liver injury, delay the development of cirrhosis, and notably ameliorate liver function. It may be used as a safe and effective thera-peutic drug for patients with fibrosis.     

Rosiglitazone prevents murine hepatic fibrosis induced by Schistosoma japonicum

AIM： To evaluate the effect of rosiglitazone in a murine model of liver fibrosis induced by Schistosoma japonicum infection. METHODS： A total of 50 mice were randomly and averagely divided into groups A, B, C, D and E. The mice in group A served as normal controls, while those in the other four groups were infected with Schistosoma japonicum to induce the model of liver fibrosis. Besides, the mice in groups C, D and E were treated with praziquantel, rosiglitazone and praziquantel plus rosiglitazone, respectively. NF-κB binding activity and expression of PPARγ-mRNA were determined by Western blot assay and real-time quantitative PCR. Radioimmunonassay technique was used to detect the serum content changes of TNF-α and IL-6. Histological specimens were stained with HE. Expression of TGF-β1, a-smooth muscle actin and type Ⅰand type Ⅲ collagen was detected by immunohistochemistry and multimedia color pathographic analysis system.
RESULTS： Inflammation and fibrosis in the rosiglitazone plus praziquantel treatment group （group E） were lightest among the mice infected with Schistosoma （P 〈 0.05）. To further explore the mechanism of rosiglitazone action, we found that rosiglitazone can significantly increase the expression of PPARγ [E： -18.212 ± （-3.909） vs B： -27.315 ± （-6.348） and C： -25.647 ± （-5.694）, P 〈 0.05],reduce the NF-κB binding activity （E： 88.89 ± 19.34 vs B： 141.11 ± 15.37, C： 112.89 ± 20.17 and D： 108.89 ± 20.47, P 〈 0.05）, and lower the serum level of TNF-α （E： 1.613 ± 0.420 ng/mL vs B： 2.892 ± 0.587 ng/mL, C： 2.346 ± 0.371 ng/mL and D： 2.160 ± 0.395 ng/mL, P 〈 0.05） and IL-6 （E： 0.106 ± 0.021 ng/mL vs B： 0.140 ± 0.031 ng/mL and C： 0.137 ± 0.027 ng/mL, P 〈 0.05） in mice with liver fibrosis. Rosiglitazone can also substantially reduce the hepatic expression of TGF-β1, α-SMA type Ⅰand type Ⅲ collagen in mice with liver fibrosis. CONCLUSION： The activation of PPARγ by its ligand can retard liver fi     

姜黄素治疗肝纤维化及其作用机制的初步研究

目的观察姜黄素治疗肝纤维化的效果,初步探讨其作用机制。方法四氯化碳腹腔注射制作大鼠肝纤维化模型,以丹参治疗作为阳性对照,检测血清ALT、AST、HA、LN、Ⅲ型前胶原（PCⅢ）、一氧化氮（NO）含量;检测肝组织超氧化物歧化酶（SOD）、羟脯氨酸（Hyp）、丙二醛（MDA）含量;肝组织行HE和Masson胶原染色,光镜下观察病理学改变,并按肝纤维化半定量计分系统进行评分。结果与模型组比较,姜黄素治疗组能明显降低肝纤维化时异常升高的ALT、AST、NO、HA、LN、PCⅢ、MDA、Hyp,模型对照组分别为（693.75±117.57）U/L、（892.50±105.69）U/L、（70.95±10.23）μmol/L、（468.22±93.45）mg/L、（346.44±75.08）mg/L、（279.82±54.00）μg/L、（402.25±39.16）nmol/g、（752.50±77.62）μg/g,姜黄素（每100g体重40mg）治疗组分别为（218.50±48.89）U/L、（376.60±79.13）U/L、（47.96±6.53）μmol/L、（289.96±60.43）mg/L、（107.35±27.24）mg/L、（148.95±28.63）μg/L、（236.10±30.54）nmol/g、（478.40±75.74）μg/g,P值均〈0.05;提升肝纤维化时异常降低的肝组织SOD水平,姜黄素（每100g体重40 mg）治疗组和对照组分别为（90.39±21.23）U/mg、（46.52±20.01）U/mg,P〈0.05;明显改善四氯化碳所致大鼠肝纤维化的病理学改变,肝纤维化评分明显降低（P〈0.05）,接近正常对照组,且该作用随着姜黄素剂量增大而加强。结论姜黄素具有治疗大鼠肝纤维化作用;抗脂质过氧化损伤、直接影响胶原代谢可能是其重要的作用机制。     

Usefulness of serum hepatic fibrosis markers in the diagnosis of nonalcoholic steatohepatitis (NASH)

BACKGROUND/AIMS: In the present study, we examined the usefulness of serum hepatic fibrosis markers for the diagnosis of nonalcoholic steatohepatitis (NASH). METHODOLOGY: The subjects were 16 patients with NASH and 9 patients with fatty liver (FL). All were negative for serum HBsAg, HCVAb, antibodies related with autoimmune diseases, alcohol intake, and drug abuse. We measured the biochemical markers for liver function, hepatic fibrosis markers such as type III procollagen N-peptide (PIIIP), type IV collagen (TyIV), hyaluronic acid (HA) and leptin, and compared these data with histological findings of biopsy specimens. In addition, we examined the diagnostic efficiency of fibrosis markers and leptin for NASH using receiver operating characteristic (ROC) curve. Body mass index (BMI), fasting blood sugar, triglyceride, and degree of fat droplets, inflammation, iron deposition and fibrosis were significantly higher in the NASH group compared with the FL group. RESULTS: The diagnostic efficiencies of NASH% (cut-off value) were 68% (100ng/mL) for TyIV, 68% (10ng/mL) for HA, 64% (0.62U/mL) for PIIIP and 56% (8pg/mL) for leptin. CONCLUSIONS: From these results, it is suggested that the serum hepatic fibrosis markers such as TyIV, in addition to liver biopsy, may be useful for the diagnosis of NASH.     

Effect of Chinese medicine Qinggan Huoxuefang on inducing HSC apoptosis in alcoholic liver fibrosis rats

AIM: To investigate the effect of Qinggan Huoxuefang (QGHXF) on improvement of liver function and pathology in rats, and to analyze the mechanism. METHODS: Wistar rats were divided into three groups at random: normal control group (12), micro-amount carbon tetrachloride group (CCl4)(12) and model group A (60). The model group A was ingested with the mixture (500 mL/L alcohol, 8 mL/kg per day; corn oil, 2 mL/kg per day; pyrazole, 24 mg/kg per day) once a day and intraperitoneal injections of 0.25 mL/kg of a 250 mL/L solution of CCl4 in olive oil twice a week for 12 wk. The CCl4 group received intraperitoneal injections only. At the end of 8 wk the model group A (60) was divided into 5 subgroups: model group, Xiaochaihu Chongji (XCH) group, QGHXF high dose group, moderate dose group and low dose group, and were given the drugs respectively. At the end of 12 wk, all the rats were killed and blood samples collected, as well as liver tissue. Blood samples were used for evaluation of alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (γ-GT). Liver specimens were obtained for routine HE, apoptosis gene array and flow cytometry analysis. RESULTS: A liver fibrosis animal model was successfully established. Fibrosis was obviously reduced in QGHXF high dose group, and no fibrosis formed in CCl4 group. Compared with model group the QGHXF group and XCH group could obviously decrease the level of ALT, AST, ALP, and GGT (P<0.05). QGHXF high dose group was better than XCH group in ALT (615±190 vs 867±115), and AST(1972±366 vs 2777±608). Moreover, QGHXF could reduce liver inflammation, fibrosis-induced hepatic stellate cell (HSC) apoptosis and regulate apoptosis gene expression. The HSC apoptosis rates of QGHXF groups were 22.4±3.13, 13.79±2.26 and 10.07±1.14, higher than model group, 6.58±1.04 (P< 0.05). Compared to model group, 39 genes were up-regulated, 11 solely expressed and 17 down-regulated in high dose group. CONCLUSION: QGHXF can improve liver fibrosis and induce HSC apoptosis.     

四氯化碳诱导大鼠肝纤维化脂质过氧化相关蛋白表达的动态变化及一贯煎的干预效应

目的 探讨四氯化碳(CCl4)诱导大鼠肝纤维化脂质过氧化相关蛋白表达的动态变化及一贯煎的干预效应.方法 Wistar雄性大鼠57只,其中模型组39只,正常组18只.模型大鼠腹腔注射50％的CCl4橄榄油溶液(1ml/lg),每周2次,共9周.造模3、6周后,随机抽取正常及模型大鼠各6只,处死作动态观察.其余模型大鼠随机分为模型组15只及干预组12只,模型组大鼠在8周时处死4只观察成模情况.第7周开始,继续造模的同时,干预组用一贯煎(2.682 g/kg)蒸馏水稀释灌胃,1次/d,共计3周.用药3周结束后,处死大鼠,检测肝功能、肝组织羟脯氨酸(Hyp)和丙二醛(MDA)含量、超氧化物歧化酶(SOD)与谷胱甘肽(GSH)活性,以及热休克蛋白70 (HSP70)、血红素加氧酶-1(HO-1)、转铁蛋白(Transferrin)、过氧化还原酶(Prxd)6、肝脏型脂肪酸结合蛋白(L-FABP)等的表达.计量资料采用单因素方差分析,计数资料采用Ridit分析. 结果 (1)与对照组比较,模型组大鼠6、9周时肝组织MDA含量显著升高[(4.23±0.45) nmol/mg比(2.22±0.59)nmol/mg; (6.29±1.23) nmol/mg比(2.22±0.59) nmol/mg,F值分别为60.13、66.99,P值均＜ 0.05];SOD活性显著降低[(196.94±39.20) U/mg比(264.50±30.44)U/mg,F=11.12,P＜ 0.05; (152.21±51.65) U/mg比(264.50±30.44) U/mg,F=23.11,P＜0.01];GSH含量显著降低[(48.47±7.27) nmol/mg比(60.74±9.04) nmol/mg,F=6.71,P＜0.05;(37.89±9.01) nmol/mg比(60.74±9.04)nmol/mg,F=24.06,P＜0.01];与9周模型组比较,干预组MDA显著降低[(4.25±0.86) nmol/mg比(6.29±1.23) nmol/mg,F=19.52,P＜ 0.01],SOD显著升高[(198.35±46.48) U/mg比(152.21±51.65) U/mg,F=4.65,P＜0.05],GSH显著升高[(53.73±7.54) nmol/mg比(37.89±9.01) nmol/mg,F=19.23,P＜0.01];(2)与正常组比较,9周时模型组大鼠HSP70蛋白表达量升高(1.21±0.06比0.58±0.07,F=166.87,P＜0.0l),HO-1蛋白表达量也升高(1.11±0.06比0.58±0.06,F=123.96,P＜ 0.01),Prdx6蛋白表达量降低(0.04±0.05比1.49±0.05,F=1215.85,P＜0.01),L-FABP表达量降低(0.24±0.02比1.44±0.14,F=219.05,P＜0.01),Transferrin蛋白表达量降低(0.67±0.03比1.67±0.04,F=301.35,P＜0.01).9周时,干预组HSP70和HO-1蛋白表达量分别为0.82±0.04、0.90±0.04,与9周时模型组比较,F值分别为92.31、26.89,P值均＜0.01,差异有统计学意义;9周时,干预组Prdx6、L-FABP和Transferrin蛋白表达分别为0.88±0.11、1.36±0.13、1.04±0.12,与9周时模型组比较,F值分别为150.17、237.19、27.53,P值均＜0.01,差异有统计学意义. 结论 一贯煎具有促进机体抗氧化物质生成、减轻脂质过氧化损伤的作用.     

Effect of WeiJia on carbon tetrachloride induced chronic liver injury

AIM:To study the effect of WeiJia on chronic liver injuryusing carbon tetrachloride(CCl_4)induced liver injuryanimal model.METHODS:Wistar rats weighing 180-220g were ran-domly divided into three groups:normal control group(Group A),CCl_4 induced liver injury control group(GroupB)and CCl_4 induction with WeiJia treatment group(GroupC).Each group consisted of 14 rats.Liver damage andfibrosis was induced by subcutaneous injection with 40?l_4 in olive oil at 3 mL/kg body weight twice a week foreight weeks for Groups B and C rats whereas olive oilwas used for Group A rats.Starting from the third week,Group C rats also received daily intraperitoneal injectionof WeiJia at a dose of 1.25 μg/kg body weight.Animalswere sacrificed at the fifth week(4 male,3 female),andeighth week(4 male,3 female)respectively.Degree offibrosis were measured and serological markers for liverfibrosis and function including hyaluronic acid(HA),typeIV collagen(CIV),γ-glutamyl transferase(γ-GT),alanineaminotransferase(ALT)and aspartate aminotransferase(AST)were determined.Alpha smooth muscle actin (α-SMA)and proliferating cell nuclear antigen(PCNA)immunohistochemistry were also performed.RESULTS:CCl_4 induction led to the damage of liver anddevelopment of fibrosis in Group B and Group C ratswhen compared to Group A rats.The treatment of WeiJiain Group C rats could reduce the fibrosis condition sig-nificantly compared to Group B rats.The effect could beobserved after three weeks of treatment and was moreobvious after eight weeks of treatment.Serum HA,CIV,ALT,AST and γ-GT levels after eight weeks of treatmentfor Group C rats were 58±22 μg/L(P<0.01),57±21 μg/L(P<0.01),47±10 U/L(P<0.01),139±13 U/L(P<0.05)and 52±21 U/L(P>0.05)respectively,similar to normalcontrol group(Group A),but significantly different fromCCl_4 induced liver injury control group(Group B).An in-crease in PCNA and decrease in α-SMA expression levelwas also observed.CONCLUSION:WeiJia could improve liver function andreduce liver fibrosis which might be through the inhibi-tion of stellate cell activity.     

丹参素治疗肝纤维化及其作用机制研究

目的 研究丹参素对大鼠免疫性肝纤维化的治疗作用及其怍用机制。 方法 用猪血清制作大鼠肝纤维化动物模型,自第8周起给予丹参素组大鼠腹腔注射丹参素300 mg·kg-1·d-1,同时给予模型对照组和正常对照组相同体积的双蒸水腹腔注射。第12周动物实验结束时,除留取一只模型组大鼠用原位循环灌流法提取肝星状细胞(HSC)外,其余大鼠全部处死提取血清检测透明质酸(HA)以及取肝脏标本切片做HE、VG染色。以MTT法观察不同浓度丹参素对HSC的增殖抑制作用,用流式细胞仪分析丹参素作用后HSC所处周期。 结果 丹参素干预组HA为(231.4±41.1)ng/ml,模型组(398.7±54.5)ng/ml,F=154.796,P<0.05。HE及VG染色示丹参素干预组肝纤维化程度明显轻于模型组;细胞试验表明50、100、200 mg/L丹参素对HSC作用后的MTT值(A值)分别为1.60×102±8.17×10-4、1.10×10-2±1.41×10-3和6.75×10-3±3.30×10-3,细胞对照组A值为7.18×10-2±1.71×10-3,F=1154.221,P<0.05。 结论丹参素对大鼠免疫性肝纤维化有明显的防治怍用。其主要机制为丹参素对HSC明显的增殖抑制作用。     

Effects of transmitters and interleukin-10 on rat hepatic fibrosis induced by CCl4

AIM: To study the effects of transmitters ET, AgⅡ, PGI2,CGRP and GG on experimental rat hepatic fbrosis and the antifibrogenic effects of IL-10.METHODS: One hundred SD rats were randomly divided into 3 groups: control group (N): intraperitoneal injection with saline 2 ml.kg^-1 twice a week; the fibrogenesis group (C): intraperitoneal injection with 50 % CC14 2 ml.kg^-1 twice a week; IL-10 treated group (E): besides same dosage of CC14 given, intraperitoneal injection with IL-10 4 ug.kg^-1 from the third week. In the fifth, the seventh and the ninth week,rats in three groups were selected randomly to collect plasma and liver tissues. The levels of ET, AgⅡ, PGI2, CGRP and GG were assayed by radioimmunoassay (RIA). The liver fibrosis was observed with silver staining.RESULTS: The hepatic fibrosis was developed with the increase of the injection frequency of CC14. The ET, AgⅡ, PGI2, CGRP and GG levels in serum of group N were 71.84&#177;60.2 ng.L^-1,76.21&#177;33.3 ng.L^-1, 313.03&#177;101.71 ng.L^-1, 61.97&#177;21.4 ng.L^-1 and 33.62&#177;14.37 ng.L^-1, respectively; the levels of them in serum of group C were 523.30&#177;129.3 ng.L^-1, 127.24&#177;50.0 ng.L^-1,648.91&#177;357.29 ng.L^-1, 127.15&#177;62.0 ng&#183;L^-1 and 85.26&#177;51.83ng.L^-1, respectively; the levels of them in serum of group E were 452.52&#177;99.5 ng.L^-1, 90.60&#177;44.7 ng.L^-1, 475.57&#177;179.70ng.L^-1, 102.2&#177;29.7 ng.L^-1 and 38.05&#177;19.94 ng.L^-1, respectively.The histological examination showed that the degrees of the rats liver fibrosis in group E were lower than those in group C.CONCLUSION: The transmitters ET, AgⅡ, PGI2, CGRP and GG play a significant role in the rat hepatic fibrosis induced by CCl4, IL-10 has the antagonistic action on these transmitters and can relieve the degree of the liver fibrosis.     

Animal experiment and clinical study of effect of gamma-interferon on hepatic fibrosis

AIM To evaluate the antifibrotic effect ofdifferent doses of recombinant human Gamma-Interferon (IFN-γ) in two rat models of hepaticfibrosis, and to observe its effect on moderatechronic hepatitis B virus fibrosis.METHODS Hepatic fibrosis was successfullyinduced in 150 and 196 rats by subcutaneousinjection of carbon tetrachloride (CCl_4) andintraperitoneal injection of dimethylnitrosamine(DMN), respectively. Each of the two modelgroups was divided into: ① fibrotic modelgroup; ② colchicine treatment group (0.1 mg/kg/day, gastrogavage for 8 weeks); ③ high-dose IFN-γ group (15 MU/kg per day, i.m. for 8weeks); ④ medium-dose IFN-γ group (5 MU/kgdaily, i.m. for 8 weeks); and ⑤ low-dose IFN-γgroup (1.67 MU/kg daily, i.m. for 8 weeks).Another group of 10 rats without any treatmentwas used as normal controls. At the end of theexperiment, semi-quantitative histopathologicalscores of inflammation and fibrosis, liversmooth muscle actin (α-SMA) expression level,liver hydroxyl proline content and serumhyaluronic acid levels were compared. And 47medium chronic hepatitis B viral fibrosispatients were studied. They were given IFN-γtreatment, 100MU/day i,m. for the first threemonths and 100MU qod i.m. for the next sixmonths. Semi-quantitative pathological scoresof inflammation and fibrosis and serum hepaticfibrosis indices were compared within the 9months.RESULTS In animal experiment, thepathological fibrosis scores and liver hydroxylproline content were found to be significantly lower in rats treated with different doses of IFN-γ as compared with rats in fibrotic model groupinduced by either CCl_4 or DMN, in a dose-dependent manner. For CCl_4-induced model,pathological fibrosis scores in high, medium andlow doses IFN-γ groups were 5. 10±2.88, 7.70±3.53 and 8.00±3.30, respectively, but the scorewas 14.60±7.82 in fibrotic model group.Hydroxyl proline contents were 2.83±1.18, 3.59±1.22 and 4.80±1.62, in the three IFN-γgroups, and 10.01±3.23 in fibrotic model group.The difference was statistically significant(P<0.01). Similar results were found in DMN-induced model. Pathological fibrosis scoreswere 6.30±0.48, 8.10±2.72 and 8.30±2.58, inhigh, medium and low doses IFN-γ groups, and12.60±3.57 in fibrotic model group. Hydroxylproline contents were 2.72±0.58, 3.14±0.71and 3.62±1.02, in the three IFN-γ groups, and12.79±1.54 in fibrotic model group. Thedifference was statistically significant(P<0.01). Serum hepatic fibrosis indicesdecreased significantly in the 47 patients afterIFN-γ treatment (HA: 433.38±373.00 vs 281.57±220.48; LN: 161.22±41.02 vs 146.35±44.67;PCⅢ: 192.59±89.95 vs 156.98±49.22; C-Ⅳ:156.30±44.01 vs 139.14±34.47) and thedifferences between the four indices weresignificant (P<0.05). Thirty-three patientsreceived two liver biopsies, one before and oneafter IFN-γ treatment. In thirty of 33 patientsIFN-γ had better effects according to semi-quantitative pathological scores (8.40±5.83 vs5.30±4.05, P<0.05).CONCLUSION All the three doses of IFN-γ areeffective in treating rat liver fibrosis induced byeither CCl_4 or DMN, the higher the dose, thebetter the effect. And IFN-γ is effective forpatients with moderate chronic hepatitis B viralfibrosis.     

Effects of Guiyuanfang and autologous transplantation of bone marrow stem cells on rats with liver fibrosis

AIM: To investigate the therapeutic effects of Guiyuanfang and bone marrow stem cells (BMSCs) on rats with liver fibrosis. METHODS: Liver fibrosis model was induced by carbon tetrachloride, ethanol, high lipid and assessed biochemically and histologically. Liver function and hydroxyproline contents of liver tissue were determined. Serum hyaluronic acid (HA) level and procollagen Ⅲ level were performed by radioimmunoassay. The VG staining was used to evaluate the collagen deposit in the liver. Immunohistochemical SABC methods were used to detect transplanted BMSCs and expression of urokinase plasminogen activator (uPA). RESULTS: Serum transaminase level and liver fibrosis in rats were markedly reduced by Guiyuanfang and BMSCs. HA level and procollagen Ⅲlevel were also reduced obviously, compared to model rats (HA: 47.18±10.97 ng/mL, 48.96±14.79 ng/mL; PCⅢ: 22.48±5.46 ng/mL, 26.90±3.35 ng/mL; P<0.05). Hydroxyproline contents of liver tissue in both BMSCs group and Guiyuanfang group were far lower than that of model group (1 227.2±43.1 μg/g liver tissue, 1390.8±156.3 μg/g liver tissue; P<0.01). After treatment fibrosis scores were also reduced. Both Guiyuanfang and BMSCs could increase the expression of uPA. The transplanted BMSCs could engraft, survive, and proliferate in the liver. CONCLUSION: Guiyuanfang protects against liver fibrosis. Transplanted BMSCs may engraft, survive, and proliferate in the fibrosis livers indefinitely. Guiyuanfang may synergize with BMSCs to improve recovery from liver fibrosis.     

A Study on the dynamic alterations of serum HA in rats with carbontetrachloride-induced liver fibrosis

AIM To study the clinical significance of alterations of serum hyaluronic acid in rats with carbontetrachioride-induced liver fibrosis.METHODS Rat liver fibrosis model was induced by carbon tetrachloride (CC14). The rats were divided intofive groups; group 1 (control): 0 week with no CCl4-inducing; group 2, 3, 4 and 5: 3, 6, 9 and 12 weeksafter CCl4-induction respectively. Serum HA level was analysed among various liver fibrosis groups andcontrol, and then compared the HA findings with the hepatic histopathology.RESULTS During rat liver fibrosis, serum HA levels of the liver fibrosis groups (group 2: 7.98ng/mL;group 3: 20.10 ng/mL; group 4:229.73 ng/mL; group 5:324,74 ng/mL) were significantly higher thanthat of control group (group 1:0.21 ng/mL) (P<0.01), in which group 4 and group 5 are much higher1094 times (229.73ng/mL/0.21 ng/mL) and 1546 times (324.74 ng/mL/0.21 ng/mL) than group 1respectively. When compared with each other, the serum HA levels are 38 times (7.98ng/mL/0.21 ng/mL; P<0.01, group 2 vs group 1); 2.5 times (20.10ng/mL/7.98 ng/mL; P<0.01, group 3 vsgroup 2); 11.4 times (229.73 ng/mL/20.10 ng/mL; P<0.01, group 4 vs group 3); 1.4 times (324.74 ng/mL/229.73 ng/mL; P<0.01, group 5 vs group 4) respectively.CONCLUSION The results demonstrated that the dynamic alterations of serum HA play an important rolein the early clinical diagnosis and staging of liver cirrhosis.