熊果酸对肝纤维化大鼠肠黏膜屏障的保护作用及其机制探讨
发布时间：2018-08-13 16:01
【摘要】：背景:肠道炎症和NADPH氧化酶(NADPH oxidase,NOX)介导的氧化应激参与多种病理状态下肠黏膜屏障破坏。新近研究表明肝纤维化大鼠存在肠黏膜屏障受损,但具体机制尚不明确。我们前期研究发现熊果酸(ursolic acid,UA)能够抑制NOX介导的氧化应激改善肝纤维化,另有研究表明UA通过抗炎和抗氧化作用缓解实验性结肠炎。在四氯化碳(CCl4)诱导的肝纤维化大鼠中,UA能否通过抑制肠道炎症和NOX介导的氧化应激保护肠黏膜屏障,值得进一步研究。目的:探讨UA对CCl4诱导的肝纤维化大鼠肠黏膜屏障的影响及其可能机制。方法:将雄性SD大鼠随机分为3组:空白对照组、肝纤维化组和UA治疗组。取肝组织行HE和天狼星红染色以观察肝脏病理变化;取回肠组织行HE染色以观察回肠病理变化,Western blot检测回肠紧密连接蛋白claudin 1和occludin表达,免疫组化测定回肠肠上皮细胞caspase 3蛋白表达,ELISA法检测血清脂多糖(lipopolysaccharides,LPS)、降钙素原(procalcitonin,PCT)和C反应蛋白(c-reactive protein,CRP)含量;荧光定量PCR和ELISA法分别检测回肠肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)m RNA和蛋白表达;硫代巴比妥酸(thibabituric acid,TBA)法和比色法分别检测回肠丙二醛(malondialdehyde,MDA)含量和总抗氧化能力(total antioxidant capacity,T-AOC),Western blot检测回肠NOX相关亚基(P67phox和NOX2)蛋白表达。结果:1.熊果酸对肝纤维化大鼠肝脏组织病理改变的影响空白对照组大鼠肝小叶结构清晰,汇管区及中央静脉周围无胶原沉积;肝纤维化组大鼠肝小叶结构紊乱,肝细胞脂肪变性及坏死明显,肝纤维化评分(2.67±0.36)明显高于空白对照组(0.43±0.29)(P0.01);UA治疗组大鼠肝小叶结构、肝细胞脂肪变性及坏死较肝纤维化组明显改善,肝纤维化评分(1.70±0.37)较肝纤维化组明显下降(P0.01)。2.熊果酸对肝纤维化大鼠肠黏膜屏障的影响2.1熊果酸对肝纤维化大鼠回肠组织病理改变的影响空白对照组大鼠回肠黏膜结构清晰,肠绒毛排列规整,固有层无充血、水肿等;肝纤维化组大鼠回肠黏膜结构紊乱,肠黏膜损伤评分(2.51±0.34)明显高于空白对照组(0.28±0.21)(P0.01);UA治疗组大鼠回肠黏膜结构较肝纤维化组改善,肠黏膜损伤评分(2.04±0.33)低于肝纤维化组(P0.05)。2.2熊果酸对肝纤维化大鼠回肠紧密连接蛋白claudin1和occludin表达的影响肝纤维化组回肠紧密连接蛋白claudin 1和occludin表达较空白对照组明显减少(P值均0.01);与肝纤维化组相比,UA治疗组回肠紧密连接蛋白claudin1和occludin表达增加(P值均0.05)。2.3熊果酸对肝纤维化大鼠回肠肠上皮细胞凋亡蛋白caspase 3表达的影响肝纤维化组回肠肠上皮细胞凋亡蛋白caspase 3表达较空白对照组明显增加(P0.01);与肝纤维化组相比,UA治疗组回肠肠上皮细胞凋亡蛋白caspase 3表达减少(P0.05)。2.4熊果酸对肝纤维化大鼠血清LPS、PCT和CRP含量的影响肝纤维化组血清LPS和PCT含量较空白对照组明显增加(P值均0.01),而CRP含量无明显改变(P0.05);与肝纤维化组相比,UA治疗组血清LPS和PCT含量减少(分别为P0.01和P0.05),CRP含量无明显变化(P0.05)。3.熊果酸对肝纤维化大鼠肠道炎症的影响肝纤维化组回肠TNF-αm RNA和蛋白表达较空白对照组增加(P值均0.01);与肝纤维化组相比,UA治疗组回肠TNF-αm RNA和蛋白表达减少(分别为P0.05和P0.01)。4.熊果酸对肝纤维化大鼠肠道氧化应激的影响4.1熊果酸对肝纤维化大鼠回肠MDA含量和T-AOC的影响肝纤维化组回肠MDA含量较空白对照组明显增加(P0.01),而T-AOC无明显改变(P0.05);与肝纤维化组相比,UA治疗组回肠MDA含量明显减少(P0.01),T-AOC无明显变化(P0.05)。4.2熊果酸对肝纤维化大鼠回肠NOX亚基P67phox和NOX2蛋白表达的影响肝纤维化组回肠NOX亚基P67phox和NOX2蛋白表达较空白对照组增加(分别为P0.01和P0.05);与肝纤维化组相比,UA治疗组回肠NOX亚基P67phox和NOX2蛋白表达减少(分别为P0.01和P0.05)。结论:1.肠道炎症和NADPH氧化酶介导的氧化应激参与肝纤维化大鼠肠黏膜屏障破坏。2.熊果酸具有保护肝纤维化大鼠肠黏膜屏障作用,机制可能与抑制肠道炎症和NADPH氧化酶介导的氧化应激相关。
[Abstract]:BACKGROUND: Intestinal inflammation and NADPH oxidase (NOX)-mediated oxidative stress are involved in the destruction of intestinal mucosal barrier in a variety of pathological conditions. Recent studies have shown that intestinal mucosal barrier is damaged in rats with hepatic fibrosis, but the specific mechanism is still unclear. Our previous study found that ursolic acid (UA) can inhibit NOX-mediated oxidation. Stress ameliorates hepatic fibrosis, and other studies have shown that UA relieves experimental colitis through anti-inflammatory and anti-oxidative effects. Whether UA protects intestinal mucosal barrier by inhibiting intestinal inflammation and NOX-mediated oxidative stress in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4) deserves further study. Methods: Male SD rats were randomly divided into three groups: blank control group, hepatic fibrosis group and UA treatment group. Hepatic tissues were stained with HE and Sirius red to observe the pathological changes of liver, ileal tissues were stained with HE to observe the pathological changes of ileum, and ileal tight junction protein C was detected by Western blot. The expression of laudin-1 and occludin, the expression of caspase-3 protein in ileal epithelial cells were detected by immunohistochemistry, the levels of lipopolysaccharides (LPS), procalcitonin (PCT) and C-reactive protein (CRP) in serum were detected by ELISA, and the levels of tumor necrosis factor-alpha (TNF-alpha) were detected by fluorescence quantitative PCR and ELISA, respectively. Factor alpha, TNF-a) m RNA and protein expression, malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) in ileum were detected by thibabituric acid (TBA) and colorimetry respectively, and NOX-related subunits (P67phox and NOX2) protein expression in ileum were detected by Western blot. The pathological changes of liver tissue in rats with hepatic fibrosis showed that the structure of hepatic lobules in blank control group was clear, and there was no collagen deposition around portal area and central vein. The hepatic lobule structure, hepatocyte steatosis and necrosis of the treatment group were significantly improved compared with the hepatic fibrosis group, and the liver fibrosis score was significantly decreased (P 0.01). 2. Effect of ursolic acid on intestinal mucosal barrier of hepatic fibrosis rats 2.1 Effect of ursolic acid on ileal pathological changes of hepatic fibrosis rats in blank control group The structure of ileal mucosa was clear, the villi were arranged regularly, the lamina propria was not congested, edema, etc. The score of ileal mucosa structure disorder and intestinal mucosa injury in hepatic fibrosis group was significantly higher than that in blank control group (0.28 + 0.21) (P 0.01). The structure of ileal mucosa in UA group was improved and the score of intestinal mucosa injury was (2.04 + 0.3). 3) The expression of claudin 1 and occludin in the ileum of rats with hepatic fibrosis was lower than that of the control group (P 0.05). The expression of claudin 1 and occludin in the ileum of rats with hepatic fibrosis was significantly lower than that of the control group (P 0.01). Effects of ursolic acid on expression of Caspase-3 in ileal epithelial cells of hepatic fibrosis rats The expression of Caspase-3 in ileal epithelial cells of hepatic fibrosis group was significantly higher than that of control group (P 0.01). Effects of ursolic acid on serum LPS, PCT and CRP levels in liver fibrosis rats were significantly higher than those in blank control group (P 0.01), but CRP levels did not change significantly (P 0.05); Compared with liver fibrosis group, serum LPS and PCT levels in UA treatment group were significantly lower (P 0.01 and P 0.05), CR levels were significantly higher (P 0.01 and P 0.05). Ursolic acid had no significant effect on intestinal inflammation in hepatic fibrosis rats (P 0.05). 3. The expression of TNF-alpha m RNA and protein in ileum of hepatic fibrosis rats was higher than that of blank control group (P 0.01); compared with hepatic fibrosis group, the expression of TNF-alpha m RNA and protein in ileum of UA treatment group was lower (P 0.05 and P 0.01, respectively). Effects of intestinal oxidative stress 4.1 Ursolic acid on MDA content and T-AOC content in ileum of rats with hepatic fibrosis were significantly increased in hepatic fibrosis group than in blank control group (P The expression of NOX subunit P67phox and NOX2 protein in ileum of rats with hepatic fibrosis was increased in hepatic fibrosis group compared with blank control group (P 0.01 and P 0.05 respectively); compared with hepatic fibrosis group, the expression of NOX subunit P67phox and NOX2 protein in ileum of UA treatment group decreased (P 0.01 and P 0.05 respectively). Ursolic acid can protect the intestinal mucosal barrier of hepatic fibrosis rats. The mechanism may be related to the inhibition of intestinal inflammation and NADPH oxidase-mediated oxidative stress.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
本文编号：2181478
[Abstract]:BACKGROUND: Intestinal inflammation and NADPH oxidase (NOX)-mediated oxidative stress are involved in the destruction of intestinal mucosal barrier in a variety of pathological conditions. Recent studies have shown that intestinal mucosal barrier is damaged in rats with hepatic fibrosis, but the specific mechanism is still unclear. Our previous study found that ursolic acid (UA) can inhibit NOX-mediated oxidation. Stress ameliorates hepatic fibrosis, and other studies have shown that UA relieves experimental colitis through anti-inflammatory and anti-oxidative effects. Whether UA protects intestinal mucosal barrier by inhibiting intestinal inflammation and NOX-mediated oxidative stress in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4) deserves further study. Methods: Male SD rats were randomly divided into three groups: blank control group, hepatic fibrosis group and UA treatment group. Hepatic tissues were stained with HE and Sirius red to observe the pathological changes of liver, ileal tissues were stained with HE to observe the pathological changes of ileum, and ileal tight junction protein C was detected by Western blot. The expression of laudin-1 and occludin, the expression of caspase-3 protein in ileal epithelial cells were detected by immunohistochemistry, the levels of lipopolysaccharides (LPS), procalcitonin (PCT) and C-reactive protein (CRP) in serum were detected by ELISA, and the levels of tumor necrosis factor-alpha (TNF-alpha) were detected by fluorescence quantitative PCR and ELISA, respectively. Factor alpha, TNF-a) m RNA and protein expression, malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) in ileum were detected by thibabituric acid (TBA) and colorimetry respectively, and NOX-related subunits (P67phox and NOX2) protein expression in ileum were detected by Western blot. The pathological changes of liver tissue in rats with hepatic fibrosis showed that the structure of hepatic lobules in blank control group was clear, and there was no collagen deposition around portal area and central vein. The hepatic lobule structure, hepatocyte steatosis and necrosis of the treatment group were significantly improved compared with the hepatic fibrosis group, and the liver fibrosis score was significantly decreased (P 0.01). 2. Effect of ursolic acid on intestinal mucosal barrier of hepatic fibrosis rats 2.1 Effect of ursolic acid on ileal pathological changes of hepatic fibrosis rats in blank control group The structure of ileal mucosa was clear, the villi were arranged regularly, the lamina propria was not congested, edema, etc. The score of ileal mucosa structure disorder and intestinal mucosa injury in hepatic fibrosis group was significantly higher than that in blank control group (0.28 + 0.21) (P 0.01). The structure of ileal mucosa in UA group was improved and the score of intestinal mucosa injury was (2.04 + 0.3). 3) The expression of claudin 1 and occludin in the ileum of rats with hepatic fibrosis was lower than that of the control group (P 0.05). The expression of claudin 1 and occludin in the ileum of rats with hepatic fibrosis was significantly lower than that of the control group (P 0.01). Effects of ursolic acid on expression of Caspase-3 in ileal epithelial cells of hepatic fibrosis rats The expression of Caspase-3 in ileal epithelial cells of hepatic fibrosis group was significantly higher than that of control group (P 0.01). Effects of ursolic acid on serum LPS, PCT and CRP levels in liver fibrosis rats were significantly higher than those in blank control group (P 0.01), but CRP levels did not change significantly (P 0.05); Compared with liver fibrosis group, serum LPS and PCT levels in UA treatment group were significantly lower (P 0.01 and P 0.05), CR levels were significantly higher (P 0.01 and P 0.05). Ursolic acid had no significant effect on intestinal inflammation in hepatic fibrosis rats (P 0.05). 3. The expression of TNF-alpha m RNA and protein in ileum of hepatic fibrosis rats was higher than that of blank control group (P 0.01); compared with hepatic fibrosis group, the expression of TNF-alpha m RNA and protein in ileum of UA treatment group was lower (P 0.05 and P 0.01, respectively). Effects of intestinal oxidative stress 4.1 Ursolic acid on MDA content and T-AOC content in ileum of rats with hepatic fibrosis were significantly increased in hepatic fibrosis group than in blank control group (P The expression of NOX subunit P67phox and NOX2 protein in ileum of rats with hepatic fibrosis was increased in hepatic fibrosis group compared with blank control group (P 0.01 and P 0.05 respectively); compared with hepatic fibrosis group, the expression of NOX subunit P67phox and NOX2 protein in ileum of UA treatment group decreased (P 0.01 and P 0.05 respectively). Ursolic acid can protect the intestinal mucosal barrier of hepatic fibrosis rats. The mechanism may be related to the inhibition of intestinal inflammation and NADPH oxidase-mediated oxidative stress.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
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