枸杞多糖对肝纤维化小鼠肠道菌群及血液中相关炎性因子的影响

Effects of lycium barbarum polysaccharides on intestinal flora and related inflammatory factors in blood of liver fibrosis mice

目的分析枸杞多糖(lycium barbarum polysaccharides,LBP)对肝纤维化小鼠肠道内指定菌群、血液中炎性因子、肝组织内纤维化相关因子含量的影响,探讨枸杞多糖对肝组织纤维化的保护机制。方法采取结扎小鼠胆总管使肝组织发生纤维化的方法,建立20只肝纤维化小鼠模型,将其均等分为模型组和LBP治疗组,另选10只健康小鼠为对照组。LBP治疗组:自术后第3周开始,每天灌胃30μg/g的LBP溶液5 mL;对照组和模型组:每天同时间灌胃三蒸水5 mL。3组均持续治疗2周。qRT-PCR技术检测肠道内指定菌群的变化;酶联免疫吸附试验(enzyme-linked immunosor-bent assay,ELISA)检测血液里炎性因子:白介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)、白介素-10(interleukin-10,IL-10)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的含量;蛋白印迹分析肝组织内纤维化相关因子:Ⅰ型胶原、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及转化生长因子-β1(transforming growth factor-β1,TGF-β1)的相对值。结果与对照组相比,模型组肝内出现坏死区域,面积不一且数量多,结缔组织含量及胆小管数量增多;肠道内容物中大肠杆菌(179.27±30.93)和产气荚膜梭菌(234.93±40.17)均增加,双歧杆菌(15.86±4.98)和乳酸杆菌(28.12±7.30)均减少(均P<0.01);血液里炎性因子:IL-1β[(46.30±4.15)pg/mL]、IL-6[(19.18±2.04)pg/mL]及TNF-α[(85.28±7.49)ng/mL]含量升高,而IL-10[(28.68±2.58)ng/mL]含量降低(均P<0.05);纤维化相关因子:Ⅰ型胶原(1.209±0.053)、α-SMA(0.416±0.012)及TGF-β1(0.675±0.018)的相对值上升(均P<0.05)。与模型组相比,LBP治疗组肝内坏死区域面积缩小且数量减少,结缔组织含量及胆小管数量减少;肠道内容物中大肠杆菌(121.56±20.25)和产气荚膜梭菌(117.83±24.50)均下降,双歧杆菌(60.08±13.60)和乳酸杆菌(92.50±19.42)均增多(均P<0.01);血液里炎性因子:IL-1β[(19.33±1.68)pg/mL]、IL-6[(11.79±1.70)pg/mL]及TNF-α[(50.24±4.90)ng/m L]含量降低,而IL-10[(58.61±5.17)ng/mL]含量升高(均P<0.05);纤维化相关因子:Ⅰ型胶原(0.760±0.020)、α-SMA(0.176±0.008)及TGF-β1(0.420±0.016)的相对值下降(均P<0.01)。结论枸杞多糖能通过调节小鼠肠道内容物中指定菌群及血液里炎性因子含量来降低肝组织纤维化病变程度。

Objective To analyze the effects of lycium barbarum polysaccharides(LBP)on the contents of designated flora in intestinal tract,inflammatory factors in blood and fibrosis related factors in liver tissue of liver fibrosis mice,explore the protective mechanism of LBP on liver fibrosis.Methods 20 mouse models of hepatic fibrosis were established by ligating their common bile duct,and they were equally divided into model group and LBP treatment group.Another 10 healthy mice were selected as the control group.The mice in LBP treatment group were given intragastric administration 5 mL solution with 30μg/g LBP every day from the third week after operation.The mice in control group and model group were given 5 mL of tridistilled water by gavage at the same time every day.Three groups were treated for two weeks.The qRT-PCR was used to detect the changes of designated flora in intestinal tract.The enzyme-linked immunosorbent assay(ELISA)was used to detect the contnts of inflammatory factors in blood,including interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor-α(TNF-α).The Western blot analysis was used to analyze the relative value of fibrosis related factors in liver tissue,including type I collagen,α-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1).Results Compare with the control group,different sizes and quantities necrotic areas appeared in the liver of the model group,and the content of connective tissue and the number of bile ducts increased,Escherichia coli(179.27±30.93)and Clostridium perfringens(234.93±40.17)in intestinal contents increased,Bifidobacterium(15.86±4.98)and Lactobacillus(28.12±7.30)decreased(all P<0.01),the contents of blood inflammatory factor IL-1β[(46.30±4.15)pg/mL],IL-6[(19.18±2.04)pg/mL]and TNF-α[(85.28±7.49)ng/mL]increased,while content of IL-10[(28.68±2.58)ng/mL]decreased(all P<0.01),the relative value of fibrosis related factors type I collagen(1.209±0.053),α-SMA(0.416±0.012)and TGF-β1(0.675±0.018)increased(all P<0.05).Compare with the model group,the area and quantity of intrahepatic necrosis in LBP treatment group decreased,the content of connective tissue and the number of bile ducts decreased,Escherichia coli(121.56±20.25)and Clostridium perfringens(117.83±24.50)in intestinal contents decreased,Bifidobacterium(60.08±13.60)and Lactobacillus(92.50±19.42)increased(all P<0.01),the contents of blood inflammatory factor IL-1β[(19.33±1.68)pg/mL],IL-6[(11.79±1.70)pg/mL]and TNF-α[(50.24±4.90)ng/mL]decreased,while content of IL-10[(58.61±5.17)ng/mL]increased(all P<0.01),the relative value of fibrosis related factors type I collagen(0.760±0.020),α-SMA(0.176±0.008)and TGF-β1(0.420±0.016)decreased(all P<0.05).Conclusion The lycium barbarum polysaccharides can reduce the degree of liver fibrosis by regulating the designated flora in the intestinal tract and the content of inflammatory factors in the blood of mice.