大黄素诱导急性胰腺炎胰腺细胞凋亡机制的实验研究

目的 从凋亡信号传导的角度探讨中药大黄素治疗大鼠急性胰腺炎的分子生物学机制。方法 将44只雄性Wistar大鼠随机分为正常组，非治疗组，大黄素组，以腹腔注射雨蛙肽物方法诱导大鼠急性胰腺炎模型。并于大黄素治疗后6，24，48，72，96小时处死大鼠。应用HE染色比较胰腺组织病理学改变，应用Tunel法检测胰腺细胞凋亡指数，应用RT-PCR技术检测治疗前后凋亡调控基因Bak和BaxmRNA表达。结果 大黄素干预治疗急性胰腺炎后96小时淀粉酶值显低于未治疗组，胰腺细胞凋亡指数显高于未治疗组，凋亡调控基因BakmRNA的表达与未治疗组之间无显性差异。而BaxmRNA的表达显高于未治疗组。结论 大黄素治疗实验性急性胰腺炎的机制可能与干预凋亡调控基因有关。诱导凋亡调控基因Bax表达增强可能是干预凋亡信号传导的重要机制，而与Bak基因表达无关。     

大黄素抗肝纤维化作用的实验研究

目的 研究大黄素抗肝纤维化作用。方法 采用40％四氯化碳给大鼠皮下注射制备肝纤维化模型并以小、中和大剂量大黄素（20mg/kg、40mg/kg和80mg/kg体重）干预,测定肝功能、血清透明质酸、层粘连蛋白及肝组织胶原蛋白,并通过光镜观察肝组织病理变化,免疫组织化学法检测肝组织α－肌蛋白表达。结果 大黄素组较模型组：（1）肝功能明显改善：谷氨酸转氨酶及碱性磷酸酶显著降低,总蛋白及白蛋白显著升高;（2）血清透明质酸及层粘连蛋白显著降低;（3）肝组织胶原蛋白含量明显减少;（4）肝组织纤维化程度明显改善;（5）肝组织α－肌动蛋白表达减少。结论 大黄素具有抗肝纤维化作用。     

Effects of emodin and baicalein on rats with severe acute pancreatitis

AIM: To investigate the therapeutic effects of emodin in combination with baicalein on severe acute pancreatitis (SAP) rats and to explore the mechanism of SAP. METHODS: A total of 112 SAP rats induced by retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct, randomly assigned to a untreated group and three treated groups emodin group, combined emodin and baicalein group, and sandostatin group. Meanwhile, another 28 other rats were selected as sham operation (SO) group. There were 28 rats in each group, 8 rats were in 3 and 6 h groups respectively, and 12 rats in 12 h group. At each time-points, survival rates,ascites volumes, pathological lesion scores of pancreas tissues,serum amylase, tumor necrosis factor-α and IL-6 levels were determined as the indexes of therapeutic effects. RESULTS: The survival rate at 12 h was significantly higher in three treated groups than in untreated group.The ascites volume at 12 h was remarkably less in combined and sandostatin groups than in emodin group,but there was no difference between combined group and sandostatin group (P>0.05). Serum amylase levels at all time-points were significantly lower in three treated groups than in untreated group. However, they had no difference among treated groups (P>0.05).Serum TNF-α were lower in three treated groups than in untreated group at all time points. Among the three treated groups, at 6 h, the TNF-α levels of combination and sandostatin groups were lower than those of emodin group. These was no difference between combined and sandostantin. Serum IL-6 concentration at 3 h were lower in combined and sandostatin groups than in untreated group, but at 6 and 12 h they were lower in all treated groups than in untreated group and the combined and sandostatin groups and in emodin group, no difference was found between combined and sandostatin groups at all time-points (P>0.05). The pathological scores of pancreas at all time points were significantly lower in three treated groups than in the untreated group, and at 6, 12 h, the scores of combined and sandostatin groups were lower than in emodin group. There was no difference between combined and sandostatin groups (P>0.05). CONCLUSION: Combination of emodin with baicalein has significant therapeutic effects on SAP rats.     

A sustained prostacyclin analog,ONO-1301, attenuates pancreatic fibrosis in experimental chronic pancreatitis induced by dibutyltin dichloride in rats

BackgroundONO-1301, a novel sustained-release prostacyclin agonist, has an anti-fibrotic effect on the lungs, heart, and kidneys that is partly associated with the induction of hepatocyte growth factor (HGF). This study examined the anti-fibrotic effect of ONO-1301 on chronic pancreatitis (CP) progression.MethodsCP was induced in rats in vivo by dibutyltin dichloride (DBTC). Seven days after DBTC injection (day 7), a slow-release form of ONO-1301 (10 mg/kg; ONO-1301–treated group) or vehicle (DBTC-treated group) was injected. On days 14 and 28, we evaluated the histopathological CP score and mRNA expressions of HGF, cytokines, and collagen in the pancreas by real-time RT-PCR. In vitro, monocytes and pancreatic stellate cells (PSCs) were isolated from normal rat spleen and pancreas, respectively. The cytokine and collagen expressions of monocytes and PSCs were detected by real-time RT-PCR, and PSCs proliferation was examined by BrdU assay.ResultsHistopathological CP scores in vivo improved in the ONO-1301–treated group compared to the DBTC-treated group, particularly inflammatory cell infiltration on day 14 and interstitial fibrosis on day 28. HGF mRNA increased significantly after ONO-1301 administration, whereas IL-1β, TNF-α, TGF-β, MCP-1, and collagen mRNA decreased significantly. Cytokine expression in monocytes was suppressed in vitro not only by HGF, but also ONO-1301 alone. However, neither ONO-1301 nor HGF affected the proliferation, or cytokine or collagen expression of PSCs.ConclusionsONO-1301 suppresses pancreatic fibrosis in the DBTC-induced CP model by inhibiting monocyte activity not only with induction of HGF but also by ONO-1301 itself.     

Animal models for the study of hepatic fibrosis

Animal models are being used for several decades to study fibrogenesis and to evaluate the anti-fibrotic potential of therapies and strategies. Although immensely valuable for our understanding of pathophysiological processes, they remain models and none of them reproduces a human disease. Each model (meaning stimulus, design, strain and species) displays specific characteristics in the nature of the pathogenesis, the topography and the evolution of fibrosis. We review here the most used as well as some newly described but potentially interesting models including models for studying biliary, immune, alcohol-induced, NASH-associated and viral fibrosis and provide insight on underlying disease processes and practical details. We attempted to delineate the benefits, advantages, limitations and drawbacks of those models. We also report the new opportunities provided by genetically engineered mice for tracking and manipulating cells that participate to fibrosis. Finally, we emphasize the importance of adapting study design to the question addressed.     

干扰素α干预对大鼠胰腺纤维化的影响

目的 观察干扰素α(INF-α)对DDC诱导的胰腺纤维化大鼠模型纤维增生程度、胰腺星状细胞活化标志物α-平滑肌肌动蛋白(α-SMA)和细胞外基质成分Ⅲ型胶原蛋白表达的影响.方法 Wistar大鼠40只随机数字法分成对照组、纤维化组和干扰素组.纤维化组和干扰素组每周2次腹腔内注射DDC,干扰素组在造模同时每天皮下注射INF-α10万U.6周末取材,光镜下观察胰腺病理学变化.免疫组化法测定胰腺组织中α-SMA、Ⅲ型胶原蛋白的表达.结果 第4周起纤维化组大鼠体重增长缓慢甚至下降,干扰素组体重仍缓慢增长,5周后两组差异显著[(309.8±19.7)g与(277.3±19.9)g,P＜0.05].纤维化组胰腺组织纤维化表现明显,纤维化分值、Masson染色值、α-SMA和Ⅲ型胶原蛋白相对表达量分别为2.679±0.899、218.713±36.102、148.971±30.686和88.142±42.581;干扰素组纤维化减轻,上述指标分别为1.952±0.219、114.732±24.912、77.237±9.275和59.952±25.498,均较纤维化组显著降低(P＜0.05).结论 给予INF-α能显著减轻纤维化程度和α-SMA、Ⅲ型胶原表达,对DDC诱导的大鼠胰腺纤维化有一定的预防作用.     

胰蛋白酶在猪慢性胰腺炎胰腺组织中的分布及免疫组化阳性标准

目的:建立慢性胰腺炎(CP)胰蛋白酶免疫组化阳性判断标准,探讨胰蛋白酶在CP中的分布特征及与CP严重程度的关系.方法:取正常猪4例及不同程度CP猪14例的胰腺组织,用链菌素亲生物素-过氧化酶连接法(streptavidin peroxidase conjugated method,SP)免疫组化法检测胰蛋白酶在实验性CP胰腺组织中的分布特征并初步建立阳性判断标准,分析胰蛋白酶的阳性标准与CP严重程度的相关性.结果:胰蛋白酶在CP胰腺组织主要表现为腺泡细胞质内、间质及胰管内棕色或棕黄色染色,染色程度在轻度CP以强阳性( )为主,中度CP以中等阳性( )为主,重度CP以弱阳性(±)为主,3组染色程度之间差异有统计学意义(P<0.05).胰蛋白酶强阳性染色与慢性胰腺炎严重程度具有相关性(r=0.742).结论:胰蛋白酶原异常活化可见于腺泡细胞质内、间质及胰管内,建立了胰蛋白酶阳性判断标准.随着慢性胰腺炎实质纤维化,胰蛋白酶阳性率降低.     

重症胰腺炎二十碳烯酸的异常代谢与大黄素,施他宁的作用

目的：探讨重症胰腺炎（AHNP）胰组织出血、坏死与二十碳烯酸类的异常代谢关系。方法：以牛黄胆酸钠诱发大鼠AHNP模型，测定前列腺素E2（PGE2）、6-酮-前列腺素F1a（6－keto－PGE1a）、血栓烷B2（TXB2）等变化，并以大黄素、施他宁药物干预，以了解AHNP时二十碳烯酸类的异常代谢和上述药物的作用。结果：重症胰腺炎时血浆TXB2显著增高．发病6小时达假手术组的4．5倍，而6－keto－PGF1a或PGE2的测定值则呈降低趋势．应用大黄素或施他宁后，TXB2测定值显著降低，施他宁组TXB2测定值较之于大黄素组降低更为显著；6－keto－PGE1a和PGE2则呈上升趋势．给药两组12小时生存率高于非治疗组；给药两组胰腺细胞坏死等病理损害减轻．结论：大黄素和施他宁对AHNP时TXB2等异常代谢有明显调整作用，与此相关的改善微循环和细胞保护机制可能是两药治疗AHNP的重要药理基础；联合应用大黄素与施他宁可能会有协同作用．     

Pathophysiology of chronic pancreatitis induced by dibutyltin dichloride joint ethanol in mice

AIM: To search for a new chronic pancreatitis model in mice suitable for investigating the pathophysiological processes leading to pancreatic fibrosis.METHODS: The mice were randomly divided into 2 groups(n = 50), control group and model group. The mice in model group were given ethanol(10%) in drinking water after injection of dibutyltin dichloride(DBTC)(8 mg/kg BW) in tail vein. The mice in control group were injected with only solvent into tail vein( 60 % ethanol, 20% glycerine and 20% normal saline) and drank common water. At days 1, 7, 14, 28, and 56 after application of DBTC or solvent, 10 mice in one group were killed at each time point respectively. Blood was obtained by inferior vena cava puncture. The activity of amylase, concentration of bilirubin and hyaluronic acid in serum were assayed. The pancreas was taken to observe the pancreatic morphology by HE staining, and to characterize the pancreatic fibrosis by Masson staining. The expression of F4/80, CD3 and fibronectin(FN) were assayed by immuno-histochemistry or Immunofluorescence technique. Collagen typeⅠ(COL1A1) in pancreas were detected by Western blot. The expression of matrix metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinases-1(TIMP-1) m RNA in the pancreas was assessed by real time PCR.RESULTS: DBTC induced an acute edematous pancreatitis within 1 d. The dilated acini, scattered acinar cell necrosis, and inflammatory cells were found at day 7. Extensive infiltration with inflammatory cells following deposition of connective tissue was observed at day 14. At day 28, level of pancreatic fibrosis was aggravated. The pancreatic tissue was replaced by an extended interstitial fibrosis at the end of 2 mo. There was significant difference in the level of amylase, bilirubin and hyaluronic acid in serum between control group and model group(P 0.05). The level of COL1A1 and FN in pancreas increased. The expression of MMP-1 m RNA in pancreas decreased, but TIMP-1 m RNA increased at model group.CONCLUSION: DBTC joint Ethanol drinking can induce chronic pancreatitis in accordance with the pathophysiological modification of human. DBTC joint Ethanol-induced pancreatitis in mice is an effective and handy experimental method. The model is suitable to study the mechanism of pancreatic fibrosis in chronic pancreatitis.     

Quantitative evaluation of pancreatic tumor fibrosis using shear wave elastography

Background & aimsThere is no established non-invasive method for diagnosis of pancreatic fibrosis. Shear wave elastography (SW-EG) may be a candidate for this purpose. The aims of this study were to assess the reproducibility of SW-EG in the normal imaging pancreas (Phase 1) and to evaluate the diagnostic performance of SW-EG for pancreatic fibrosis classified histologically (Phase 2).MethodsPhase 1: This included 127 cases that underwent SW-EG of the normal imaging pancreas. SW-EG was measured at least five times in the pancreatic parenchyma and the median of repeated measurements was defined as the pancreatic elastic modulus (PEM). Phase 2: This included 53 cases that underwent SW-EG of the pancreatic parenchyma preoperatively and in which pancreas parenchyma were evaluated histologically. Histological fibrosis was graded in 4 stages: normal, mild, moderate, and severe.ResultsPhase 1: Median PEM in the head, body, and tail of the pancreas were 3.23, 3.17, and 2.91 kPa, respectively, with no significant difference among regions (P = 0.554). The intraclass correlation coefficient showed good reproducibility (ρ = 0.71) after 5 measurements. Phase 2: There was a significant positive correlation between PEM and the histological pancreatic fibrosis stage (r_s = 0.63, P < 0.001). Areas under the receiver operating characteristic curve for the accuracy of SW-EG for diagnosis of pancreatic fibrosis were 0.85 (≥mild), 0.84 (≥moderate), and 0.87 (severe).ConclusionSW-EG can be used to determine the stage of pancreatic fibrosis non-invasively with high accuracy and reproducibility.     

大黄素对实验性淤胆型肝炎的治疗作用及其机制

目的 探讨大黄素对淤胆型肝炎的治疗作用及其机制.方法 S D大鼠分为5组:大黄素组,熊去氧胆酸组、地塞米松组、模型组、正常对照组,除正常对照组外,其余4组均给予α-异硫氰酸萘酯50 mg/kg一次灌胃大鼠建立淤胆型肝炎动物模型,并给予相应的药物干预,造模后24、48、72 h分别处死大鼠.免疫组织化学法检测肝组织中核因子κB表达.实时荧光定量PCR检测肝组织中早期生长反应因子1、中性粒细胞趋化因子1,巨噬细胞炎症蛋白-2 mRNA表达,Westernblot检测肝组织中细胞间黏附分子1蛋白表达.双抗体夹心酶联免疫吸附法检测肝组织肿瘤坏死因子α、白细胞介素6含量,硫代巴比妥酸比色法、黄嘌呤氧化酶法及比色法分别检测肝组织中丙二醛、超氧化物歧化酶、髓过氧化物酶含量.结果 (1)造模后24、48,72 h,大黄素组血清总胆红素分别为(32.8±3.7)umol/L、(61.0±16.4)μmol/L和(10.8±4.5) μmol/L,直接胆红素分别为(26.0±3.1)lamol/L、(49.4±18.2)μmol/L和(8.0±3.0)μmol/L;ALT分别为(313.7±49.8)U/L、(664.3±96.5)U/L和(200.3±60.3)U/L,均明显低于模型组,P值均<0.01,差异有统计学意义.(2)造模后24、48 h,大黄素组的核因子κ B p65核表达阳性细胞百分率分别为24.1%和9.8%,模型组分别为48.3%和26.5%,P值均<0.01,差异有统计学意义.(3)造模后24、48 h,大黄素组的细胞因子诱导的中性粒细胞趋化因子1、巨噬细胞炎症蛋白-2mRNA表达、细胞间黏附分子1蛋白表达、肿瘤坏死因子α、白细胞介素6含量均较模型组显著降低(P值均<0.01).结论 大黄素可显著改善实验性淤胆型肝炎大鼠的肝功能,其作用机制可能与抑制NF-κB信号通路有关.     

DDC递增法诱导大鼠胰腺纤维化动物模型的研究

目的建立DDC剂量递增腹腔内注射诱发大鼠胰腺纤维化动物模型方法,并与常规恒量DDC腹腔内注射方法比较。方法70只Wistar大鼠随机分为4组:生理盐水注射组、低剂量DDC恒量注射组(600 mg/kg/次。每周2次,共8次)、高剂量DDC恒量注射组(1 000 mg/kg/次,每周2次,共8 次)及DDC剂量递增注射组(首次400 mg/kg/次,以10%浓度连续递增,每周2次,共8次)。每周于注射后2d h、48 h、1周、2周、3周、4周6个时间点分别随机处死3只大鼠,并对胰、肝、肾组织进行病理学诊断与胶原纤维测定。实验中观察动物并发症(鼻出血,尿失禁,震颤)的发生率及死亡率。结果DDC递增组大鼠并发症较少,但成模时间及纤维化程度与恒量注射组无显著性差异,纤维化程度增加过程较为渐进,提示更为接近胰腺纤维化的发展过程。结论DDC注射递增法可以建立大鼠胰腺纤维化动物模型, 此模型较常规恒定DDC腹腔注射法更为优越。     

Quantitative measurement of fibrosis in pancreatic tissue Evaluation of a colorimetric method

A colorimetric method has been used to quantify the collagen contained in 23 specimens of pancreatic tissue (11 controls and 12 chronic pancreatitis). The method takes advantage of the selective capacity of Sirius red to stain collagen protein and of rapid green to stain noncollagen protein. The results obtained by this method were compared with those of standard morphometry to determine tissue fibrosis. With the morphometric method, the values of the control group were 6.6 ± 4.0% (fiber area/total area), and those of chronic pancreatitis 66.0 ± 19.0% (difference 59.4, 95% confidence interval for difference: 47.2–71.6,P< 0.001). The values obtained with the colorimetric method were 89.1 ± 11.6 μg collagen/mg total protein in the control group, and 132.7 ± 25.3 μg collagen/mg total protein in the chronic pancreatitis group (difference 43.6, 95% confidence interval for difference: 26.3–61.0,P< 0.001). A highly significant correlation (r = 0.847;p< 0.001) was observed between the amount of collagen measured colorimetrically and the degree of fibrosis determined morphometrically. These results demonstrate that the colorimetric method is a reproducible, simple, and rapid technique to quantitate fibrosis in histological preparations of pancreatic tissue.     

Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCU-induced hepatic fibrosis

AIM: To explore the anti-fibrotic effect of Oxymatrine on CCU-induced liver fibrosis in rats and its modulation on the TGFbeta-Smad signaling pathway.METHODS: One hundred healthy male SD rats were randomly divided into three groups: normal group (/7 = 20), treatment group of Oxymatrine (n = 40) and CCU-induced fibrosis group {n = 40). Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride (CCU soluted in liquid paraffin with the concentration of 300 g/L, the dosage of injection was 3 mL/kg, twice per week for 8 wk). The treated rats received Oxymatrine via celiac injection at a dosage of 10 mg/kg twice a week at the same time. The deposition of collagen was observed with H&E and Masson staining. The concentration of serum TGF-pi was assayed with ELISA. The gene expression of Smads and CBP (CREB binding protein) was detected with in situ hybridization (ISH) and immunohistochemistry (IH), respectively. All the experimental figures were scanned and analyzed with special figure-analysis software.RESULTS: A significant reduction of collagen deposition and rearrangement of the parenchyma was noted in the liver tissue of Oxymatrine-treated rats. The semi-quantitative histological scores (2.43 ± 0.47 um2 vs 3.76 ± 0.68, P < 0.05) and average area of collagen in those rats were significantly decreased when compared with hepatic cirrhosis model rats (94.41 ± 37.26 urn2 vs 290.86 ± 89.37 um2, P < 0.05). The gene expression of Smad 3 mRNA was considerably decreased in the treated animals. The A value of Smad 3 mRNA was lower in the treated rats than the model rats (0.034 ± 0.090 us 0.167 ± 0.092, P < 0.05). Contrarily, the A value of Smad 7 mRNA was increased considerably in the treated animals (0.175 ± 0.065 vs 0.074 ± 0.012, P < 0.05). There was an obvious decrease in the expression of CBP mRNA in treated rats as illuminated by a reduction of its A value when compared with model rats (0.065 ± 0.049 us 0.235 ± 0.025, P < 0.001).CONCLUSION: Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats. Oxymatrine could promote the expression of Smad 7 and inhibit the expression of Smad 3 and CBP in CCU-induced hepatic fibrosis in SD rats, could modulate the fibrogenic signal transduction of TGFp-Smad pathway.     

胰管内支架治疗慢性胰腺炎

目的 探讨胰管内支架引流术治疗慢性胰腺炎的临床疗效。方法 对１４例临床及影像学检查确诊的慢性胰腺炎伴胰管狭窄患者在内镜下进行了胰管内支架引流术,并对术后腹痛缓解率、胃纳、脂肪泻、体重变化及并发症发生率作了近期及远期了随访观察。结果 １４例患者均在内镜下内支架一次性置入成功,支架规格为５～１０Ｆ,术后随访２８～５２０ｄ,平均２１０ｄ,１４例患者术后近期（〈３个月）腹痛缓解率为９２．９％（１３／１４）     

Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCl4-induced hepatic fibrosis

AIM： To explore the anti-fibrotic effect of Oxymatrine on CCl4-induced liver fibrosis in rats and its modulation on the TGFbeta-Smad signaling pathway. METHODS： One hundred healthy male SD rats were randomly divided into three groups： normal group （n = 20）, treatment group of Oxymatrine （n = 40） and CCh-induced fibrosis group （n = 40）. Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride （CCh soluted in liquid paraffin with the concentration of 300 g/L, the dosage of injection was 3 mL/kg, twice per week for 8 wk）. The treated rats received Oxymatrine via celiac injection at a dosage of 10 mg/kg twice a week at the same time. The deposition of collagen was observed with H＆E and Masson staining. The concentration of serum TGF-β1 was assayed with ELISA. The gene expression of Smads and CBP （CREB binding protein） was detected with in situ hybridization （ISH） and immunohistochemistry （IH）, respectively. All the experimental figures were scanned and analyzed with special figure-analysis software. RESULTS： A significant reduction of collagen deposition and rearrangement of the parenchyma was noted in the liver tissue of Oxymatrine-treated rats. The semi- quantitative histological scores （2.43 ± 0.47 μm^2 vs 3.76 ±0.68, P 〈 0.05） and average area of collagen/in those rats were significantly decreased when compared with hepatic cirrhosis model rats （94.41 ± 37.26 μm^2 vs 290.86 ± 89.37 μm^2, P 〈 0.05）. The gene expression of Smad 3 mRNA was considerably decreased in the treated animals. The A value of Smad 3 mRNA was lower in the treated rats than the model rats （0.034 ± 0.090 vs 0.167 ± 0.092, P 〈 0.05）. Contrarily, the A value of Smad 7 mRNA was increased considerably in the treated animals （0.175 ± 0.065 vs 0.074 vs 0.012, P 〈 0.05）. There was an obvious decrease in the expression of CBP mRNA in treated rats as illuminated by a reduction of its A value when compared with model rats （0.065±0.049 vs 0.235 ?     

大黄素对小鼠急性肝损伤过程中Toll样受体4表达的影响及其机制

通过实时PCR及蛋白质印迹法测定肝组织中Toll样受体(TLR)4 mRNA及蛋白质表达,并检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,研究大黄素对脂多糖诱导小鼠急性肝损伤的作用及其机制。结果显示大黄素处理各组肝组织中TLR4 mRNA及蛋白质表达水平、血清中ALT和AST水平均明显低于模型组,大黄素能减轻脂多糖诱导的肝组织病理学损伤。提示大黄素可能通过抑制TLR4的表达而对脂多糖诱导的急性肝损伤起保护作用。