Nrf2抗氧化通路在CCl4所致大鼠肝毒性中的保护作用

目的 研究Nrf2氧化损伤通路在CCl₄所致大鼠急性肝损伤中的保护作用。方法 将20只雄性Wistar大鼠随机分为溶剂对照组和CCl₄ 组,每组10只,另选10只雄性Wistar大鼠,通过载体进行转基因大鼠雄原核显微注射,获得了目的基因Nrf2-tk整合与特异表达的转基因大鼠,作为CCl₄+Nrf2整合组。溶剂对照组静脉给予1%聚山梨酯-80,共4 d,CCl₄组和Nrf2-tk整合组静脉给予1%聚山梨酯-80,共4 d,第4天给予1%聚山梨酯-80 30 min后,静脉给予7.5 mg/kg CCl₄,24 h 后处死大鼠。测定血清中AST、ALT和LDH的水平,分别测定肝脏组织中MDA、GSH、GSSG的含量,并计算GSH/GSSG 比值。留取肝脏组织,常规石蜡包埋切片,HE染色,光学显微镜下观察肝脏组织的病理变化。结果 和溶剂对照组相比,CCl₄组大鼠的血清AST,ALT 和LDH 的水平明显升高(P < 0.05),Nrf2-tk转基因组大鼠的AST,ALT 和 LDH 的水平亦有轻度升高,但差异无统计学意义(P > 0.05)。肝脏的MDA 含量以及 GSH/GSSG 比值显示Nrf2-tk整合组可以有效降低CCl₄造成的脂质过氧化损伤和谷胱甘肽的消耗,肝脏病理观察结果显示和CCl₄组相比,Nrf2-tk整合组明显减轻了CCl₄造成的损伤。结论 Nrf2抗氧化损伤通路在CCl₄所致大鼠急性肝损伤中的起着重要的保护作用。     

肝细胞生长因子对高氧暴露早产鼠肺泡Ⅱ型上皮细胞增殖与凋亡的影响

目的:观察肝细胞生长因子(hepatocyte growth factor,HGF)对高氧暴露体外培养的早产鼠肺泡Ⅱ型上皮细胞(Type Ⅱ alveolar epithelial cells,AEC Ⅱ)增殖、凋亡及功能的影响,探讨HGF对高氧肺损伤保护作用的机制.方法:原代培养早产鼠AEC Ⅱ,纯化后随机分为4组:空气组(Air)、高氧组(HO)、空气 HGF组(Air HGF)、高氧 HGF组(HO HGF).采用流式细胞术及免疫印迹(Western blot)法观察AEC Ⅱ的增殖和凋亡情况,RT-PCR分析各组肺泡表面活性物质蛋白(surfactant associated protein,SP)SPA,SPB,SPC mRNA表达.结果:(1)各组细胞周期比例及细胞凋亡显示,与Air组比,HO组Sub G1(代表凋亡细胞比例),G0期/G1期细胞比例明显增高(P＜0.01),G2期/M期细胞比例明显降低(P＜0.01);Air HGF组S期细胞比例明显增高(P＜0.01).与HO组比,HO HGF组G0期/G1期细胞比例显著降低(P＜0.01),S期和G2期/M期细胞比例明显增高(P＜0.01).(2)各组增殖细胞核抗原表达显示,HO组显著低于Air组(P＜0.01),Air HGF组明显高于Air组(P＜0.05);HO HGF明显高于HO组(P＜0.05).(3)各组SPA,SPB,SPC mRNA表达显示,HO组强度较Air组弱(P＜0.01),而HO HGF组较HO组明显增加(P＜0.05).结论:高氧导致早产鼠AEC Ⅱ增殖抑制,凋亡增加,SPA,SPB,SPC mRNA表达降低,HGF可部分抑制这种变化,提示HGF对高氧肺损伤起一定的保护作用.     

注射用丹酚酸A防治肝纤维化的实验研究

目的探讨注射用丹酚酸A抗肝纤维化的作用,为丹酚酸A的临床应用提供理论依据。方法采用CCl4体外诱导肝细胞损伤,观察丹酚酸A对肝细胞活性及其细胞培养上清液ALT、AST、LDH水平和细胞裂解液中SOD活性和MDA含量的变化;另采用皮下注射CCl4诱导大鼠肝纤维化模型,观察丹酚酸A对肝纤维化大鼠血清LN、HA、SOD和MDA含量的影响以及肝脏组织病理改变情况。结果与模型对照组比,丹酚酸A高、低剂量组和Vit E组的细胞存活率显著提高(P0.01),丹酚酸A高剂量组ALT、AST和LDH活性显著降低(P0.01),丹酚酸A高剂量组和Vit E组SOD活性明显升高(P0.05),MDA含量显著降低(P0.05);体内试验发现,与模型对照组比,丹酚酸A高剂量组纤维化大鼠的血清LN和HA水平显著降低(P0.05),高、低剂量组SOD活性显著升高(P0.05,P0.01),MDA含量显著降低(P0.01,P0.05),并能改善肝脏病理形态。结论注射用丹酚酸A可通过抗脂质过氧化作用,起到保护肝细胞,减轻肝纤维化的作用。     

麝香保心丸对肝硬化大鼠心肌组织即早基因与结缔组织生长因子的影响

目的 研究麝香保心丸对肝硬化致心肌重构的抑制作用及其机制。方法 以sc体积分数40% CCl₄橄榄油溶液3 mL/kg,每周2次,共8周的方法制备大鼠肝纤维化模型,第2周末随机取5只大鼠肝组织作HE染色,观察肝纤维化程度,造模大鼠分别ig麝香保心丸22.5、45 mg/kg,共给药6周,第8周末分别取各组大鼠肝组织、左室心肌组织行组织病理学及透射电镜观察,观察各组大鼠肝脏、心肌组织病理及心肌组织超微结构改变;以RT-PCR和免疫组化染色分析心肌组织中c-fos和c-jun mRNA以及结缔组织生长因子（CTGF）、I型和III型胶原的表达变化。结果 造模大鼠均存在不同程度的肝纤维化及心肌损伤。与模型组比较,低剂量麝香保心丸组大鼠肝纤维化程度无明显差异（P＞0.05）,但心肌损伤程度减轻,心肌组织中c-fos mRNA、c-jun mRNA、CTGF、I型和III型胶原表达明显降低（P＜0.05、0.01）。高剂量麝香保心丸组大鼠肝纤维化及心肌损伤程度较低剂量组减轻（P＜0.05）,心肌组织中c-fos mRNA、c-jun mRNA、CTGF、I型和III型胶原表达均低于低剂量组（P＜0.05、0.01）。结论 肝硬化大鼠心肌组织心肌即早基因活化、诱导CTGF过度表达而致心肌重构,麝香保心丸可通过抑制心肌即早基因表达,降低心肌CTGF水平而发挥心肌保护作用,其效应呈剂量依赖性。     

Hepcidin-orchestrated Hemogram and Iron Homeostatic Patterns in Two Models of Subchronic Hepatic injury

Objective This study was designed to evaluate hematological disorders and the orchestrating roles of hepcidin and IL-6 in rat models of thioacetamide(TAA) and carbon tetrachloride(CCl_4) hepatotoxicity. Methods Rats were intraperitoneally injected with TAA(10 mg/100 g rat weight dissolved in isosaline) or CCl_4(100 μL/100 g rat weight diluted as 1:4 in corn oil) twice weekly for eight consecutive weeks to induce subchronic liver fibrosis. Blood and tissue samples were collected and analyzed. Results CCl_4 but not TAA significantly decreased the RBCs, Hb, PCV, and MCV values with minimal alterations in other erythrocytic indices. Both hepatotoxins showed leukocytosis, granulocytosis, and thrombocytopenia. By the end of the experiment, the erythropoietin level increased in the CCl_4 model. The serum iron, UIBC, TIBC, transferrin saturation%, and serum transferrin concentration values significantly decreased, whereas that of ferritin increased in the CCl_4 model. TAA increased the iron parameters toward iron overload. RT-PCR analysis revealed increased expression of hepatic hepcidin and IL-6 m RNAs in the CCl_4 model and suppressed hepcidin expression without significant effect on IL-6 in the TAA model. Conclusion These data suggest differences driven by hepcidin and IL-6 expression between CCl_4 and TAA liver fibrosis models and are of clinical importance for diagnosis and therapeutics of liver diseases.     

Effect of Dahuang Zhechong pills on long non-coding RNA growth arrest specific 5 in rat models of hepatic fibrosis

OBJECTIVE

To investigate the involvement of growth arrest specific 5 (GAS5), a long non-coding RNA, in the anti-hepatic fibrosis process induced by Dahuang Zhechong pill (DHZCP) in rats.

复方五仁醇胶囊抗氧化及抑制肝细胞凋亡的血清药理学研究

目的:研究复方五仁醇胶囊保肝作用的机理。方法:大鼠灌胃给予复方五仁醇胶囊制备含药血清,CCl4诱导原代培养大鼠肝细胞损伤模型,测定含药血清作用后细胞培养液中丙二醛(MDA)含量和超氧化物岐化酶(SOD)活性,Annexin Ⅴ-FITC/PI双染色流式细胞仪定量检测细胞凋亡率。结果:与模型对照组比较,含药血清组MDA含量显著降低(P<0.01)、SOD活力显著增强(P<0.01),细胞早期凋亡率、晚期凋亡及坏死率显著降低(P<0.01或P<0.05)。结论:复方五仁醇胶囊具有抗氧化和抑制肝细胞凋亡作用。     

移植静脉平滑肌细胞凋亡及相关基因表达与增殖关系的动态研究

目的 探讨移植静脉狭窄发生机制。方法 建立１００只Ｗｉｓｔａｒ大鼠自体颈静脉移植与肾下腹主动脉模型。采用透射电镜、原位ＤＮＡ片断末端标记（ＴＵＮＥＬ）和免疫组织化学法、检测移植静脉血管平滑肌细胞（ＶＳＭＣｓ）凋亡及相关基因ｂａｌ－２、ｂａｘ蛋白和增殖细胞核抗原（ＰＣＮＡ）的表达。结果 术后１￣８周,移植静脉ＴＵＮＥＬ及ＰＣＮＡ阳性ＶＳＭＣｓ多于对照静脉（Ｐ〈０．０１）;术后１￣２周为ＴＵＮＥＬ及Ｐ     

Effects of anti-CXCR4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

Objective:To investigate the expression of CXCR, on HL-60 cell line and the proliferation, apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5. an anti-CXCR4 monoclonal antibody, in eradicating the minimal residual disease. Methods:The activity of SDF-1 was inhibited by 10μg/ml 12G5. After treatment with 12G5. the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohis-tochemical technique. Results :(1) There was middling degree expression of CXCR4 on HL-60 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR4 decreased gradually. (2) After treatment for 24 h, the adhesion rates in the experiment group and the control were (39. 4±7. 9)% and (51. 4±5. 9)%, respectively. (3)After treatment for 24 h, the percentage of HL-60 cells in G0/G1 phase were (55. 21±4. 9)%, and that in S phase and G2/M phase were (30. 40±4. 1)% and (14. 39±5.2)%, respectively, with the corresponding proportions being (44. 67±2. 2) % , (45. 30±3. 7)% . and (10. 03±2. 6)% in the control. (4) The percentage of apoptotic HL-60 cells was (8. 95±1. 7)% in the experiment group, compared to (3. 97±2. 4)% in the control. (5)The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. (6)The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion :12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G0/G1 phase and promote apoptosis. It may be helpful by inhibiting the bioactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.     

非酒精性脂肪肝大鼠肝细胞凋亡与线粒体膜通透性转换孔开放的关系

目的通过研究肝细胞凋亡及线粒体膜通透性转换孔（MPTP）开放在大鼠非酒精性脂肪性肝病（NAFLD）形成中的作用,
探讨NAFLD的发病机理。方法30只雄性SD大鼠随机分为正常对照组和高脂饲料4、8、12周组;流式细胞仪检测肝细胞凋亡;
紫外分光光度计检测MPTP开放程度;免疫组织化学法检查Bcl-2、Bax在肝组织中表达情况;Western Blot检测肝脏Bax表达及
变化情况。结果与正常对照组比较,高脂4~12周组肝细胞凋亡指数增加;紫外分光光度计检测显示高脂组随着造模时间延
长,MPTP开放增加;免疫组化显示Bcl-2在4、8、12周高脂组表达,但组间比较阳性细胞数增加不明显;Bax在高脂组随造模时
间延长阳性细胞数增加;Western Blot 进一步证实Bax 在高脂组随造模时间延长蛋白表达量增加;并且随着脂肪肝的进展,
Bcl-2/Bax 比率进行性下降。结论NAFLD大鼠模型中存在肝细胞凋亡,线粒体损伤与肝细胞凋亡密切相关,MPTP开放是
NAFLD大鼠重要线粒体损伤机制,而Bax表达量增加、Bcl-2/Bax比率异常是MPTP开放的分子基础。
     

《金匮要略》大黄甘遂汤对小鼠实验性肝纤维化治疗作用的实验研究

[目的]观察研究《金匮要略》大黄甘遂汤对四氯化碳(CCl₄)导致的小鼠肝纤维化的治疗作用及机制。[方法]昆明种小鼠42只随机分为3组,正常组、模型组、治疗组。除正常组外,其余两组依高连相法改进后造模,观察53d后各组小鼠肝脏病理、脾脏质量及体质量变化。[结果]光镜下每高倍镜视野贮脂细胞密度,造模组明显高于其他两组(P<0.01)。脾脏质量各组间无显著性差异(P>0.05)。小鼠体质量,正常组明显高于其他两组(P<0.01)。[结论]大黄甘遂汤对CCl₄导致的小鼠肝纤维化有明显的治疗作用,其机制可能与抑制了贮脂细胞的激活和转化,减少了成纤维细胞的生成有关。     

激活剂蛋白-1诱骗性寡核苷酸对大鼠颈总动脉损伤后新生内膜的影响

目的探讨激活剂蛋白1(AP1)诱骗性寡核苷酸对大鼠颈总动脉损伤后新生内膜的影响及其作用机制。方法36只Wistar大鼠随机分为3组,每组12只,均建立颈总动脉损伤模型,单纯损伤组不予任何处理;Lipofectin转染试剂组局部释放Lipofectin;治疗组局部释放AP1诱骗性寡核苷酸,3组均于于术后30min、3h、7d处死13取材,观察比较内膜增生程度,免疫组化方法检测增殖细胞核抗原(PCNA)和c fos。结果单纯损伤组和Lipofectin转染试剂组7d时可见内膜明显增生,且有较多平滑肌细胞表达PCNA,c fos30min表达达高峰。而治疗组内膜增生程度减轻,PCNA、c fos的表达率明显下降,差异有显著意义(P<0.05或P<0.01)。结论局部转染AP1诱骗性寡核苷酸可明显抑制动脉损伤后的内膜增生,下调PCNA、c fos的表达。     

Curcumin down-regulates PCNA, cyclin D1 and Bcl-XL expression in human keratinocyte cell lines

Objective：To evaluate the effects of curcumin on regulating the proliferation,cell cycle distribution,apoptosis and relevant mechanisms in keratinocyte cell lines.Methods：The human immortalized human keratinocyte lines（HaCaT cells） were treated with different doses of curcumin.The effects of curcumin on cell viability were measured by MTT assay,and the cell cycle distribution and apoptosis determined by flow cytometry.The mRNA expression changes of proliferating cell nuclear antigen（PCNA）,cyclin D1 and Bcl-xL were from real-time PCR analysis and the protein levels were detected by Western blotting.Results：Data obtained in the study showed that curcumin could cause significantly inhibitory effect on proliferation in HaCaT cells in a time- and dose-dependent manner.Cell arrest at G1/S phase and significant apoptosis were observed after being treated with curcumin for 24 h.In association with these,the expression of PCNA,cyclin D1 and Bcl-xL were decreased both at mRNA and protein levels for the same treatment.Conclusion：Curcumin can inhibit proliferation,induce cell arrest at G1/S phase and cause apoptosis in HaCaT cells.The decreased expression of PCNA,cyclin D1 and Bcl-xL induced by curcumin contributes to the above effects in vitro.     

大鼠肝脏癌前病变肝细胞凋亡及Fas/FasL的表达

目的探讨肝脏癌前病变过程中肝细胞凋亡的变化及Fas和FasL的表达,以期为肝脏疾病防治提供实验依据。方法将大鼠随机分为对照组和模型组,模型组采用腹腔注射二乙基亚硝胺(DEN)制备大鼠肝脏癌前病变模型,生化检查血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、γ-谷氨酰转肽酶(γ-GT)、α-L-岩藻糖苷酶(AFU)、甲胎蛋白(AFP)和癌胚抗原(CEA)。光镜下观察肝脏组织病理学改变;使用DNA电泳法检测肝细胞的凋亡情况;应用免疫组织化学法检测Fas和FasL的表达与分布。结果对照组大鼠光镜下肝组织结构正常,模型组大鼠肝细胞肿胀,肝小叶内可见灶性坏死伴炎性细胞浸润,同时逐渐出现纤维组织增生及肝细胞再生。模型组1～8周和9～12周大鼠血清ALT、AST、γ-GT和AFU活性均高于对照组,差别有统计学意义(P<0.01);模型组9～12周大鼠血清ALT、AST、γ-GT和AFU活性高于1～8周,差别有统计学意义(P<0.05),2组AFP和CEA均正常。对照组无细胞凋亡,模型组大鼠肝脏细胞呈明显的DNA梯状条带。对照组大鼠肝组织中未见Fas和FasL表达,模型组Fas和FasL主要表达在肝脏细胞胞浆。结论大鼠肝脏癌前病变时,肝细胞凋亡增加可能与凋亡相关基因Fas和FasL表达增加有关。肝脏癌前病变大鼠肝细胞凋亡增加,可能是肝损伤加重的原因。     

Comparative study on the protective effects of Yinchenhao Decoction (茵陈蒿汤) against liver injury induced by α-naphthylisothiocyanate and carbon tetrachloride

Objective:To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine.Methods:The liver injury in the model rat was induced byα-naphthylisothiocyanate(ANIT) and carbon tetrachloride(CCl_4) respectively,and the effects of Yinchenhao Decoction(茵陈蒿汤,YCHD),a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic,on the two liver injury models were evalu...     

Malotilate completely inhibits CCl4-induced liver cirrhosis in rats: biochemical and morphological analysis.

Malotilate, diisopropyl 1,3-dithiol-2-ylidenemalonate, is a relatively recently synthesized hepatotrophic chemical substance. Its inhibitory effect on rat liver cirrhosis induced by carbon tetrachloride (CCl4) was biochemically and morphologically investigated for 10 weeks, since this chemical had been reported to suppress liver damage caused by CCl4 or in vitro collagenogenesis of human fibroblasts. Concomitant administration of malotilate with CCl4 completely suppressed liver cell necrosis and markedly inhibited fatty change of hepatocytes in the first three weeks of the experiment. During the six to ten weeks of the experimental period, liver cirrhosis was perfectly inhibited by malotilate. Previously established liver cirrhosis, however, could not be normalized by malotilate treatment. Precise mechanism of the inhibitory effect of malotilate on liver cirrhosis is not elucidated, but this substance is clearly effective for preventing liver cell damage and/or liver cirrhosis caused by CCl4.     

Inhibitory effect of PPARγ agonist on the proliferation of human pterygium fibroblasts

The effects of DK2,a peroxisome proliferator-activated receptor γ agonist,on cultured human pterygium fibroblasts (HPFs) in virto were studied.The HPFs were incubated with 0-200 μmol/L DK2 for 12-72 h.The MTT method was used to assay the bio-activity of DK2 at different doses and time.The cytotoxic effect of DK2 was measured by LDH release assay.The cell cycle distribution and apoptosis were flow cytometrically detected.The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by real-time PCR (RT-PCR) and Western blotting.The results showed that administration of 1-75 μmol/L DK2 for 12-72 h could significantly inhibit HPF proliferation in a dose-and time-dependent manner.DK2-treated cells did not release significant amount of LDH as compared with rosiglitazone-treated cells.After treatment with DK2 at concentrations of 15,25 μmol/L for 24 h,the number of HPFs in G 0 /G 1 phase was significantly increased while that in S phase was significantly decreased (P<0.05),leading to arrest at G 0 /G 1 phase.The apoptosis rates of HPF cells in drug-treated groups were significantly higher than the rate of control group (P<0.05).At the dosage range between 15-25 μmol/L,DK2 could inhibit the expression of PCNA mRNA and protein in HPFs in a dose-dependent fashion (P<0.05).It was concluded that PPARγ agonist can significantly inhibit HPF proliferation,resulting in the arrest at G 0 /G 1 phase,induce the apoptosis of HPFs,and suppress the synthesis of PCNA,in dose-and time-dependent manners.     

人肝细胞生长因子基因表达对CCl4损伤的人肝细胞及大鼠肝星状细胞株的影响及其机制

目的：探讨人肝细胞生长因子(hHGF) 基因的表达对CCl4损伤的人肝细胞及大鼠肝星状细胞株（CFSC-2G）生物学效应的影响及其对CCl4损伤的人正常肝细胞株（HL-7702）的保护作用,进一步探讨凋亡产生的机制。方法：将获得的稳定转染HL-7702细胞克隆,分为4组,Ⅰ组为转染PCI-neo-hHGF实验组,Ⅱ组为转染PCI-neo空载体对照组,Ⅲ组为仅加入脂质体的对照组,Ⅳ组为空白对照组。采用MTT法测定细胞增殖活性。采用Western blotting测定转染PCI-neo-hHGF的HL-7702细胞和CFSC-2G细胞中hHGF、caspase-3、caspase-8、caspase-9蛋白质的表达。结果：HL-7702细胞转染PCI-neo-HGF并培养48 h后,细胞的增殖活性明显高于PCI-neo空载体对照组、脂质体转染对照组和空白对照组HL-7702细胞（P<0.01）,而且随着转染PCI-neo-HGF剂量增加,细胞的增殖活性有一定的增长趋势,但各组间比较差异无显著性（P>0.05）;PCI-neo-HGF转染的HL-7702细胞caspase-3蛋白质表达量较CCl4损伤的HL-7702细胞和PCI-neo转染的HL-7702细胞明显降低,未检测到caspase-8和caspase-9蛋白质的表达;PCI-neo-HGF转染的CFSC-2G细胞caspase-3蛋白质表达量较CCl4诱导的CFSC-2G增加,caspase-9蛋白质表达也呈阳性。结论：PCI-neo-HGF可促进体外培养的HL-7702细胞的生长增殖,能够抑制CCl4损伤的HL-7702细胞的凋亡,促进大鼠CFSC-2G细胞的凋亡,其作用机制可能与上调caspase-3和caspase-9蛋白质表达有关。