【摘要】：背景:腸道炎癥和NADPH氧化酶(NADPH oxidase,NOX)介導(dǎo)的氧化應(yīng)激參與多種病理狀態(tài)下腸黏膜屏障破壞。新近研究表明肝纖維化大鼠存在腸黏膜屏障受損,但具體機(jī)制尚不明確。我們前期研究發(fā)現(xiàn)熊果酸(ursolic acid,UA)能夠抑制NOX介導(dǎo)的氧化應(yīng)激改善肝纖維化,另有研究表明UA通過抗炎和抗氧化作用緩解實(shí)驗(yàn)性結(jié)腸炎。在四氯化碳(CCl4)誘導(dǎo)的肝纖維化大鼠中,UA能否通過抑制腸道炎癥和NOX介導(dǎo)的氧化應(yīng)激保護(hù)腸黏膜屏障,值得進(jìn)一步研究。目的:探討UA對(duì)CCl4誘導(dǎo)的肝纖維化大鼠腸黏膜屏障的影響及其可能機(jī)制。方法:將雄性SD大鼠隨機(jī)分為3組:空白對(duì)照組、肝纖維化組和UA治療組。取肝組織行HE和天狼星紅染色以觀察肝臟病理變化;取回腸組織行HE染色以觀察回腸病理變化,Western blot檢測(cè)回腸緊密連接蛋白claudin 1和occludin表達(dá),免疫組化測(cè)定回腸腸上皮細(xì)胞caspase 3蛋白表達(dá),ELISA法檢測(cè)血清脂多糖(lipopolysaccharides,LPS)、降鈣素原(procalcitonin,PCT)和C反應(yīng)蛋白(c-reactive protein,CRP)含量;熒光定量PCR和ELISA法分別檢測(cè)回腸腫瘤壞死因子α(tumor necrosis factor alpha,TNF-α)m RNA和蛋白表達(dá);硫代巴比妥酸(thibabituric acid,TBA)法和比色法分別檢測(cè)回腸丙二醛(malondialdehyde,MDA)含量和總抗氧化能力(total antioxidant capacity,T-AOC),Western blot檢測(cè)回腸NOX相關(guān)亞基(P67phox和NOX2)蛋白表達(dá)。結(jié)果:1.熊果酸對(duì)肝纖維化大鼠肝臟組織病理改變的影響空白對(duì)照組大鼠肝小葉結(jié)構(gòu)清晰,匯管區(qū)及中央靜脈周圍無膠原沉積;肝纖維化組大鼠肝小葉結(jié)構(gòu)紊亂,肝細(xì)胞脂肪變性及壞死明顯,肝纖維化評(píng)分(2.67±0.36)明顯高于空白對(duì)照組(0.43±0.29)(P0.01);UA治療組大鼠肝小葉結(jié)構(gòu)、肝細(xì)胞脂肪變性及壞死較肝纖維化組明顯改善,肝纖維化評(píng)分(1.70±0.37)較肝纖維化組明顯下降(P0.01)。2.熊果酸對(duì)肝纖維化大鼠腸黏膜屏障的影響2.1熊果酸對(duì)肝纖維化大鼠回腸組織病理改變的影響空白對(duì)照組大鼠回腸黏膜結(jié)構(gòu)清晰,腸絨毛排列規(guī)整,固有層無充血、水腫等;肝纖維化組大鼠回腸黏膜結(jié)構(gòu)紊亂,腸黏膜損傷評(píng)分(2.51±0.34)明顯高于空白對(duì)照組(0.28±0.21)(P0.01);UA治療組大鼠回腸黏膜結(jié)構(gòu)較肝纖維化組改善,腸黏膜損傷評(píng)分(2.04±0.33)低于肝纖維化組(P0.05)。2.2熊果酸對(duì)肝纖維化大鼠回腸緊密連接蛋白claudin1和occludin表達(dá)的影響肝纖維化組回腸緊密連接蛋白claudin 1和occludin表達(dá)較空白對(duì)照組明顯減少(P值均0.01);與肝纖維化組相比,UA治療組回腸緊密連接蛋白claudin1和occludin表達(dá)增加(P值均0.05)。2.3熊果酸對(duì)肝纖維化大鼠回腸腸上皮細(xì)胞凋亡蛋白caspase 3表達(dá)的影響肝纖維化組回腸腸上皮細(xì)胞凋亡蛋白caspase 3表達(dá)較空白對(duì)照組明顯增加(P0.01);與肝纖維化組相比,UA治療組回腸腸上皮細(xì)胞凋亡蛋白caspase 3表達(dá)減少(P0.05)。2.4熊果酸對(duì)肝纖維化大鼠血清LPS、PCT和CRP含量的影響肝纖維化組血清LPS和PCT含量較空白對(duì)照組明顯增加(P值均0.01),而CRP含量無明顯改變(P0.05);與肝纖維化組相比,UA治療組血清LPS和PCT含量減少(分別為P0.01和P0.05),CRP含量無明顯變化(P0.05)。3.熊果酸對(duì)肝纖維化大鼠腸道炎癥的影響肝纖維化組回腸TNF-αm RNA和蛋白表達(dá)較空白對(duì)照組增加(P值均0.01);與肝纖維化組相比,UA治療組回腸TNF-αm RNA和蛋白表達(dá)減少(分別為P0.05和P0.01)。4.熊果酸對(duì)肝纖維化大鼠腸道氧化應(yīng)激的影響4.1熊果酸對(duì)肝纖維化大鼠回腸MDA含量和T-AOC的影響肝纖維化組回腸MDA含量較空白對(duì)照組明顯增加(P0.01),而T-AOC無明顯改變(P0.05);與肝纖維化組相比,UA治療組回腸MDA含量明顯減少(P0.01),T-AOC無明顯變化(P0.05)。4.2熊果酸對(duì)肝纖維化大鼠回腸NOX亞基P67phox和NOX2蛋白表達(dá)的影響肝纖維化組回腸NOX亞基P67phox和NOX2蛋白表達(dá)較空白對(duì)照組增加(分別為P0.01和P0.05);與肝纖維化組相比,UA治療組回腸NOX亞基P67phox和NOX2蛋白表達(dá)減少(分別為P0.01和P0.05)。結(jié)論:1.腸道炎癥和NADPH氧化酶介導(dǎo)的氧化應(yīng)激參與肝纖維化大鼠腸黏膜屏障破壞。2.熊果酸具有保護(hù)肝纖維化大鼠腸黏膜屏障作用,機(jī)制可能與抑制腸道炎癥和NADPH氧化酶介導(dǎo)的氧化應(yīng)激相關(guān)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2

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