【摘要】：目的:探討復方鱉甲軟肝片及其和嬰兒雙歧桿菌聯(lián)用治療實驗性肝纖維化的作用及其機制。方法:采用三種不同的肝纖維化動物模型研究復方鱉甲軟肝片及嬰兒雙歧桿菌治療實驗性肝纖維化的作用,全文共分四個部分:(1)復方鱉甲軟肝片對四氯化碳(CCl4)誘導的大鼠化學損傷性肝纖維化的防治作用。采用CCl4制備大鼠肝纖維化模型。將雄性SD大鼠隨機分為:正常組,模型組,復方鱉甲軟肝片0.6 g/kg、1.2g/kg和2.4g/kg組,秋水仙堿0.5mg/kg組。除正常組外,各組大鼠給予純CCl4溶液皮下注射,之后每4天皮下注射40%CCl4橄欖油溶液建立肝纖維化動物模型;正常組腹腔注射等量生理鹽水。治療組在給予CCl4同時灌胃給予復方鱉甲軟肝片0.6 g/kg、1.2g/kg和2.4g/kg組,秋水仙堿0.5mg/kg;正常組和模型組等量飲用水灌胃。連續(xù)給藥8周后,測定大鼠肝臟重量(liver weight,LW)、肝臟質(zhì)量指數(shù)(liver weight index,LWI)、肝功能,血清透明質(zhì)酸(hyaluronic acid,HA)和III型前膠原肽(precollagen pepride III,PCIII),肝臟羥脯氨酸(hydroxyproline,Hyp)含量,病理切片HE染色和苦味酸天狼猩紅染色觀察肝臟組織病變情況和膠原纖維含量,測定肝臟組織血管緊張素Ⅱ(angiotensinⅡ,AngⅡ)、抗氧化物和氧化應激產(chǎn)物、基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)和基質(zhì)金屬蛋白酶抑制因子(tissue inhibitors of metaslloproteinases,TIMP),蛋白免疫印跡法觀察藥物對肝臟TGF-β1/smad信號通路的影響。(2)復方鱉甲軟肝片對大鼠酒精性肝纖維化的防治作用。建立酒精誘導大鼠肝纖維化模型。雄性SD大鼠隨機分為:正常對照組,模型對照組,復方鱉甲軟肝片0.6g/kg組,復方鱉甲軟肝片1.2g/kg組,復方鱉甲軟肝片2.4g/kg組,秋水仙堿0.5mg/kg組。除正常對照組給予蒸餾水灌胃外,各組大鼠每天上午給予白酒灌胃,每天下午各組大鼠給予相應的藥物溶液或蒸餾水,給藥體積15ml/kg,連續(xù)10周后,測定大鼠肝臟重量,肝臟質(zhì)量指數(shù)、肝功能,血清透明質(zhì)酸和III型前膠原肽,病理切片HE染色觀察肝臟組織病理結(jié)構(gòu),測定肝臟組織抗氧化物、氧化應激產(chǎn)物和I/III型膠(Collagen I/III)原蛋白濃度,RT-PCR法檢測肝臟CYP2E1m RNA相對表達量,蛋白免疫印跡法檢測肝臟轉(zhuǎn)化生長因子-β1(transforming growth factor,TGF-β1)和α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)表達量。(3)復方鱉甲軟肝片和嬰兒雙歧桿菌連用聯(lián)用對刀豆蛋白誘導的小鼠免疫損傷性肝纖維化的防治作用。用刀豆蛋白誘導免疫性小鼠肝纖維化模型,將雄性Balb/c小鼠隨機分為8組:正常對照組,模型對照組,復方鱉甲軟肝片0.9 g/kg組,復方鱉甲軟肝片1.8g/kg組,復方鱉甲軟肝片3.6g/kg組,復方鱉甲軟肝片1.8g/kg和嬰兒雙歧桿菌0.5g/kg聯(lián)用組,嬰兒雙歧桿菌0.5g/kg組,秋水仙堿0.75mg/kg組。正常對照組靜脈注射給予注射用生理鹽水,其余各組小鼠按12.5mg/kg劑量靜脈注射給予刀豆蛋白溶液,每周一次,造模同時給藥,正常對照組和模型對照組給予蒸餾水灌胃,其余各組給予相應的藥物溶液,每日1次,持續(xù)7周后,檢測小鼠肝臟重量,肝臟質(zhì)量指數(shù)、肝功能,血清透明質(zhì)酸和III型前膠原肽蛋白濃度,肝臟組織TNF-α、IL-6、IL-10、MMP-1、MMP-2、MMR-9、Collagen I/III的蛋白濃度,TGF-β1 m RNA和蛋白表達量。(4)肝纖維化大鼠的肝臟彩色多普勒超聲圖像及實時組織彈性成像的研究。用肝臟彩色超聲多普勒圖像及實時組織彈性成像技術(shù)觀察復方鱉甲軟肝片對CCl4和酒精誘導的肝纖維化大鼠肝臟生理結(jié)構(gòu)的影響。結(jié)果:(1)復方鱉甲軟肝片能顯著降低CCl4致肝纖維化大鼠肝臟重量,肝臟質(zhì)量指數(shù),改善肝功能,緩解肝臟病變;減少血清HA、PCIII,肝臟Hyp,丙二醛(malondialdehyde,MDA)、MMP-2、MMP-9、TIMP-1、TGF-β1、p-samd2/3和Collagen I/III表達,提高肝臟谷胱甘肽過氧化物酶(glutathion peroxidase,GSH-Px),超氧化物岐化酶(superoxide dismutase,SOD)活力,上調(diào)MMP-1和smad7表達。(2)復方鱉甲軟肝片能顯著降低酒精致肝纖維化大鼠的肝臟重量,肝臟質(zhì)量指數(shù),改善肝功能,緩解肝臟病變;減少血清HA、PCIII,肝臟Hyp、MDA、Collagen I/III、TGF-β1和α-SMA含量,下調(diào)肝臟CYP2E1m RNA表達,提高肝臟谷GSH-Px,SOD活力。(3)復方鱉甲軟肝片能顯著降低刀豆蛋白致免疫性肝纖維化小鼠的肝臟重量,肝臟質(zhì)量指數(shù),改善肝功能,降低肝臟MMP-2、MMP-9、TIMP-1、TGF-β1和Collagen I/III表達,升高肝臟MMP-1含量,但其對免疫誘導所引起的炎癥因子表達無明顯作用。嬰兒雙歧桿菌能有效的降低免疫性肝纖維化小鼠肝臟組織TNF-α,IL-6濃度,升高IL-10濃度。復方鱉甲軟肝片與嬰兒雙歧桿菌聯(lián)用對于免疫所致肝纖維化治療效果優(yōu)于單用。(4)治療組的大鼠的肝臟彈性藍色區(qū)域明顯小于模型組。結(jié)論:(1)復方鱉甲軟肝片具有良好的抗肝纖維化作用,能夠顯著改善肝功能,抑制ECM成分過度合成。其作用機制與抗氧化應激,調(diào)節(jié)肝臟MMPs和TIMPs表達,干預TGF-β1/smad信號通路有關(guān),其抗纖維化作用機制與嬰兒雙岐桿菌不同。(2)嬰兒雙歧桿菌可能通過免疫調(diào)節(jié)作用發(fā)揮抗纖維化作用,作用機制與復方鱉甲軟肝片不同。兩者聯(lián)用對于肝纖維化治療具有良好的協(xié)同作用。(3)超聲實時組織彈性成像技術(shù)有助于肝纖維化的實時監(jiān)測。
[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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R725.7

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