复方鳖甲软肝片及其联合婴儿双歧杆菌抗肝纤维化的实验研究

发布时间：2018-08-29 09:21

【摘要】：目的:探讨复方鳖甲软肝片及其和婴儿双歧杆菌联用治疗实验性肝纤维化的作用及其机制。方法:采用三种不同的肝纤维化动物模型研究复方鳖甲软肝片及婴儿双歧杆菌治疗实验性肝纤维化的作用,全文共分四个部分:(1)复方鳖甲软肝片对四氯化碳(CCl4)诱导的大鼠化学损伤性肝纤维化的防治作用。采用CCl4制备大鼠肝纤维化模型。将雄性SD大鼠随机分为:正常组,模型组,复方鳖甲软肝片0.6 g/kg、1.2g/kg和2.4g/kg组,秋水仙碱0.5mg/kg组。除正常组外,各组大鼠给予纯CCl4溶液皮下注射,之后每4天皮下注射40%CCl4橄榄油溶液建立肝纤维化动物模型;正常组腹腔注射等量生理盐水。治疗组在给予CCl4同时灌胃给予复方鳖甲软肝片0.6 g/kg、1.2g/kg和2.4g/kg组,秋水仙碱0.5mg/kg;正常组和模型组等量饮用水灌胃。连续给药8周后,测定大鼠肝脏重量(liver weight,LW)、肝脏质量指数(liver weight index,LWI)、肝功能,血清透明质酸(hyaluronic acid,HA)和III型前胶原肽(precollagen pepride III,PCIII),肝脏羟脯氨酸(hydroxyproline,Hyp)含量,病理切片HE染色和苦味酸天狼猩红染色观察肝脏组织病变情况和胶原纤维含量,测定肝脏组织血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)、抗氧化物和氧化应激产物、基质金属蛋白酶(matrix metalloproteinase,MMP)和基质金属蛋白酶抑制因子(tissue inhibitors of metaslloproteinases,TIMP),蛋白免疫印迹法观察药物对肝脏TGF-β1/smad信号通路的影响。(2)复方鳖甲软肝片对大鼠酒精性肝纤维化的防治作用。建立酒精诱导大鼠肝纤维化模型。雄性SD大鼠随机分为:正常对照组,模型对照组,复方鳖甲软肝片0.6g/kg组,复方鳖甲软肝片1.2g/kg组,复方鳖甲软肝片2.4g/kg组,秋水仙碱0.5mg/kg组。除正常对照组给予蒸馏水灌胃外,各组大鼠每天上午给予白酒灌胃,每天下午各组大鼠给予相应的药物溶液或蒸馏水,给药体积15ml/kg,连续10周后,测定大鼠肝脏重量,肝脏质量指数、肝功能,血清透明质酸和III型前胶原肽,病理切片HE染色观察肝脏组织病理结构,测定肝脏组织抗氧化物、氧化应激产物和I/III型胶(Collagen I/III)原蛋白浓度,RT-PCR法检测肝脏CYP2E1m RNA相对表达量,蛋白免疫印迹法检测肝脏转化生长因子-β1(transforming growth factor,TGF-β1)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达量。(3)复方鳖甲软肝片和婴儿双歧杆菌连用联用对刀豆蛋白诱导的小鼠免疫损伤性肝纤维化的防治作用。用刀豆蛋白诱导免疫性小鼠肝纤维化模型,将雄性Balb/c小鼠随机分为8组:正常对照组,模型对照组,复方鳖甲软肝片0.9 g/kg组,复方鳖甲软肝片1.8g/kg组,复方鳖甲软肝片3.6g/kg组,复方鳖甲软肝片1.8g/kg和婴儿双歧杆菌0.5g/kg联用组,婴儿双歧杆菌0.5g/kg组,秋水仙碱0.75mg/kg组。正常对照组静脉注射给予注射用生理盐水,其余各组小鼠按12.5mg/kg剂量静脉注射给予刀豆蛋白溶液,每周一次,造模同时给药,正常对照组和模型对照组给予蒸馏水灌胃,其余各组给予相应的药物溶液,每日1次,持续7周后,检测小鼠肝脏重量,肝脏质量指数、肝功能,血清透明质酸和III型前胶原肽蛋白浓度,肝脏组织TNF-α、IL-6、IL-10、MMP-1、MMP-2、MMR-9、Collagen I/III的蛋白浓度,TGF-β1 m RNA和蛋白表达量。(4)肝纤维化大鼠的肝脏彩色多普勒超声图像及实时组织弹性成像的研究。用肝脏彩色超声多普勒图像及实时组织弹性成像技术观察复方鳖甲软肝片对CCl4和酒精诱导的肝纤维化大鼠肝脏生理结构的影响。结果:(1)复方鳖甲软肝片能显著降低CCl4致肝纤维化大鼠肝脏重量,肝脏质量指数,改善肝功能,缓解肝脏病变;减少血清HA、PCIII,肝脏Hyp,丙二醛(malondialdehyde,MDA)、MMP-2、MMP-9、TIMP-1、TGF-β1、p-samd2/3和Collagen I/III表达,提高肝脏谷胱甘肽过氧化物酶(glutathion peroxidase,GSH-Px),超氧化物岐化酶(superoxide dismutase,SOD)活力,上调MMP-1和smad7表达。(2)复方鳖甲软肝片能显著降低酒精致肝纤维化大鼠的肝脏重量,肝脏质量指数,改善肝功能,缓解肝脏病变;减少血清HA、PCIII,肝脏Hyp、MDA、Collagen I/III、TGF-β1和α-SMA含量,下调肝脏CYP2E1m RNA表达,提高肝脏谷GSH-Px,SOD活力。(3)复方鳖甲软肝片能显著降低刀豆蛋白致免疫性肝纤维化小鼠的肝脏重量,肝脏质量指数,改善肝功能,降低肝脏MMP-2、MMP-9、TIMP-1、TGF-β1和Collagen I/III表达,升高肝脏MMP-1含量,但其对免疫诱导所引起的炎症因子表达无明显作用。婴儿双歧杆菌能有效的降低免疫性肝纤维化小鼠肝脏组织TNF-α,IL-6浓度,升高IL-10浓度。复方鳖甲软肝片与婴儿双歧杆菌联用对于免疫所致肝纤维化治疗效果优于单用。(4)治疗组的大鼠的肝脏弹性蓝色区域明显小于模型组。结论:(1)复方鳖甲软肝片具有良好的抗肝纤维化作用,能够显著改善肝功能,抑制ECM成分过度合成。其作用机制与抗氧化应激,调节肝脏MMPs和TIMPs表达,干预TGF-β1/smad信号通路有关,其抗纤维化作用机制与婴儿双岐杆菌不同。(2)婴儿双歧杆菌可能通过免疫调节作用发挥抗纤维化作用,作用机制与复方鳖甲软肝片不同。两者联用对于肝纤维化治疗具有良好的协同作用。(3)超声实时组织弹性成像技术有助于肝纤维化的实时监测。
[Abstract]:Objective: To investigate the effect and mechanism of Biejia Ruangan Tablet and its combination with Bifidobacterium infantis on experimental hepatic fibrosis. Methods: Three different animal models of hepatic fibrosis were used to study the effect of Biejia Ruangan Tablet and Bifidobacterium infantis on experimental hepatic fibrosis. CCl4-induced hepatic fibrosis in rats was induced by CCl4. Rats were randomly divided into normal group, model group, compound Biejiaruangan tablets 0.6 g/kg, 1.2 g/kg and 2.4 g/kg groups, colchicine 0.5 mg/kg group. Except normal group, rats in each group were given pure CCl4 solution. Liver fibrosis animal model was established by subcutaneous injection of 40% CCl4 olive oil every 4 days; normal group was injected with normal saline intraperitoneally. The treatment group was given CCl4 and compound Biejia Ruanggan tablets 0.6 g/kg, 1.2 g/kg and 2.4 g/kg, colchicine 0.5 mg/kg. The normal group and model group were given the same amount of drinking water for 8 weeks. After that, liver weight (LW), liver weight index (LWI), liver function, serum hyaluronic acid (HA), precollagen pepride III (PCIII), liver hydroxyproline (Hyp), pathological section HE staining and picric acid Sirius red staining were used to observe the liver. Tissue pathological changes and collagen fibers content were measured. Angiotensin II (Ang II), antioxidants and oxidative stress products, matrix metalloproteinase (MMP) and tissue inhibitors of metaslloproteinases (TIMP) in liver tissues were detected by Western blot. (2) Preventive and therapeutic effects of compound Biejia Ruangan tablets on alcoholic liver fibrosis in rats. Establishment of alcohol-induced liver fibrosis model in rats. Male SD rats were randomly divided into normal control group, model control group, compound Biejia Ruangan tablets 0.6g/kg group, compound Biejia Ruangan tablets 1.2g/kg group, and compound Biejia Ruangan tablets 1.2g/kg group. Liver tablet 2.4g/kg group, colchicine 0.5mg/kg group. Except for the normal control group, rats in each group were given distilled water by gastric lavage every morning. Rats in each group were given corresponding drug solution or distilled water every afternoon, and the volume of administration was 15ml/kg. After 10 weeks, liver weight, liver mass index, liver function and serum hyaluronic acid were measured. Hepatic histopathological structure was observed by HE staining, antioxidants, oxidative stress products and Collagen I/III proprotein concentrations were measured, the relative expression of CYP2E1m RNA in liver was detected by RT-PCR, and transforming growth factor-beta 1 (transforming growth factor) was detected by Western blot. TGF-beta 1 and alpha-smooth muscle actin (alpha-SMA) expression. (3) Preventive and therapeutic effects of Bifidobacterium infantis and Bifidobacterium compound Biejiaruangan tablets on liver fibrosis induced by concanavalin in mice with immune injury. The normal control group, the model control group, the compound Biejia Ruangan tablet 0.9 g/kg group, the compound Biejia Ruangan tablet 1.8 g/kg group, the compound Biejia Ruangan tablet 3.6 g/kg group, the compound Biejia Ruangan tablet 1.8 g/kg and the infant Bifidobacterium 0.5 g/kg group, the infant Bifidobacterium 0.5 g/kg group, the colchicine 0.75 mg/kg group were injected intravenously into the normal control group. The mice in the other groups were given concanavalin solution by intravenous injection at a dose of 12.5mg/kg once a week. The mice in the normal control group and model control group were given distilled water by gastric lavage. The mice in the other groups were given corresponding drug solution once a day for 7 weeks. The liver weight, liver mass index, liver function and serum transparency were measured. Concentrations of plasmic acid and type III procollagen peptide protein, liver tissue TNF-a, IL-6, IL-10, MMP-1, MMP-2, MMR-9, Collagen I/III protein, TGF-beta 1 m RNA and protein expression. (4) Color Doppler ultrasonography and real-time tissue elastography of liver fibrosis in rats. Results: (1) Compound Biejia Ruangan tablets could significantly reduce the liver weight, liver mass index, improve liver function and alleviate liver pathological changes in CCl4-induced liver fibrosis rats, reduce serum HA, PCIII, liver Hyp and malondialdehyde (malondi). Aldehyde, MDA, MMP-2, MMP-9, TIMP-1, TGF-beta 1, p-samd2/3 and Collagen I/III expression, increase liver glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity, and up-regulate the expression of MMP-1 and Smad7 in alcoholic-induced liver fibrosis rats. (2) Compound Biejia Ruangan Tablet can significantly reduce the liver fibrosis. Weight, liver mass index, improve liver function, alleviate liver disease; reduce serum HA, PCIII, liver Hyp, MDA, Collagen I/III, TGF-beta 1 and alpha-SMA content, down-regulate liver CYP2E1m RNA expression, increase liver gluten GSH-Px, SOD activity. (3) Compound Biejia Ruanggan Tablet can significantly reduce the liver weight of mice with immune liver fibrosis induced by concanavalin. BMI, MMP-2, MMP-9, TIMP-1, TGF-beta 1 and Collagen I/III expression in liver were decreased and MMP-1 content in liver was increased, but the expression of inflammatory factors induced by immune induction was not significantly affected. Biejia Ruangan Tablet combined with Bifidobacterium infantis is superior to single therapy in the treatment of immune-induced liver fibrosis. (4) The elastic blue area of the liver in the treatment group was significantly smaller than that in the model group. The mechanism is related to antioxidant stress, regulating the expression of MMPs and TIMPs in the liver, interfering with TGF-beta 1/smad signaling pathway, and its anti-fibrosis mechanism is different from that of Bifidobacterium infantis. (2) Bifidobacterium infantis may play an anti-fibrosis role through immune regulation, and the mechanism is different from that of Biejia Ruangan Tablet. (3) Ultrasound real-time tissue elastography is helpful for real-time monitoring of liver fibrosis.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R725.7

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