摘要
目的探讨微小RNA(microRNA,miR)-140缺失对小鼠非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)发展的影响及机制。方法选取60只C57BL/6J小鼠,采用随机数字表法分为对照组、模型组和miR-140抑制组,每组20只。对照组小鼠采用普通动物饲料喂养,模型组和miR-140抑制组采用高脂饮食喂养。从第4周开始,miR-140抑制组每日腹腔注射mimics inhibitor质粒,持续1周,对照组及模型组腹腔注射相应体积的生理盐水。在第4周结束时,每组选取2只小鼠验证是否造模成功,随后腹膜内注射10%水合氯醛(0.3 ml/100 g)麻醉,股动脉取血,同时取出肝脏。计算肝脏脂肪变积分、炎症坏死灶积分、气球样变积分及非酒精性脂肪性肝炎活动度积分(non-alcoholic steatohepatitis activity score,NAS)。采用酶联免疫吸附法检测小鼠肝组织天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、丙氨酸氨基转移酶(Alanine aminotransferase,ALT)、肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)及白细胞介素-6(interleukin-6,IL-6)水平。采用实时荧光定量逆转录聚合酶链式反应(real-time fluorescent quantitative reverse transcription polymerase chain reaction,qRT-PCR)检测小鼠肝组织miR-140、核因子κB(nuclear factor kappa-B,NF-κB)及Toll样受体-4(Toll-like receptor4,TLR-4)mRNA相对表达量。采用免疫组织化学法检测NF-κB和TLR4蛋白水平。结果对照组、模型组和miR-140抑制组小鼠肝组织脂肪变积分[(0.00±0.00)分vs(3.12±0.23)分vs(4.80±0.16)分]、炎症坏死灶积分[(0.52±0.21)分vs(2.95±0.24)分vs(4.13±0.28)分]、气球样变积分[(0.00±0.00)分vs(1.98±0.26)分vs(2.65±0.20)分]、NAS积分[(0.48±0.32)分vs(7.29±0.34)分vs(9.41±0.51)分]、AST[(43.24±6.89)U/L vs(83.21±10.98)U/L vs(129.36±11.14)U/L]、ALT[(60.21±12.36)U/L vs(83.21±10.98)U/L vs(135.36±9.34)U/L]、TNF-α[(145.77±6.46)μg/L vs(267.86±6.98)μg/L vs(439.45±6.98)μg/L]、IL-6[(47.13±15.95)μg/L vs(187.66±9.47)μg/L vs(334.14±12.74)μg/L]、miR-140 mRNA相对表达量(4.96±0.21 vs 1.29±0.49 vs 0.86±0.54)、NF-κB mRNA相对表达量(0.96±0.21 vs 2.29±0.49 vs 4.56±0.54)、TLR4 mRNA相对表达量(0.89±0.39 vs 3.01±0.43 vs 5.81±0.26)、NF-κB蛋白相对表达水平(2.63±0.32 vs 3.14±0.29 vs 5.15±0.31)和TLR4蛋白相对表达水平(3.96±0.31 vs 5.01±0.26 vs 6.95±0.34)差异均有统计学意义(P均<0.001)。模型组肝组织脂肪变积分、炎症坏死灶积分、气球样变积分、NAS积分、AST、ALT、TNF-α、IL-6、NF-κB mRNA相对表达量、TLR4 mRNA相对表达量、NF-κB蛋白相对表达水平和TLR4蛋白相对表达水平均显著高于对照组,miR-140 mRNA相对表达量显著低于对照组,差异均有统计学意义(P均<0.001)。miR-140抑制组肝组织脂肪变积分、炎症坏死灶积分、气球样变积分、NAS积分、AST、ALT、TNF-α、IL-6、NF-κB mRNA相对表达量、TLR4 mRNA相对表达量、NF-κB蛋白相对表达水平和TLR4蛋白相对表达水平均显著高于模型组,miR-140 mRNA相对表达量显著低于模型组,差异均有统计学意义(P均<0.001)。结论miR-140缺失可促进小鼠NAFLD的发展,其机制与miR-140抑制导致肝组织NF-κB和TLR4 mRNA相对表达量及蛋白表达水平升高有关。
Objective To investigate the effects and mechanism of microRNA-140 deletion on the development of non-alcoholic fatty liver disease(NAFLD)in mice.Methods Total of sixty C57BL/6J mice were selected and divided into control group,model group and miR-140 inhibition group according to random number table,20 mice in each group.Mice in control group were fed with ordinary animal feed,while mice in model group and miR-140 inhibition group were fed with a high-fat diet.At the beginning of the 4th week,mice in miR-140 inhibition group were intraperitoneally injected with mimics inhibitor plasmid daily for 1 week,mice in control group and model group were intraperitoneally injected with corresponding volume of saline.At the end of the 4th week,two mice in each group were sacrificed to verify whether the modeling was successful,and then 10%chloral hydrate(0.3 ml/100 g)was used to anesthetize the mice,blood from the femoral artery and the liver were collected.Liver steatohepatitis score,inflammatory necrosis score,ballooning score and non-alcoholic steatohepatitis activity score(NAS)were calculated.Enzyme-linked immunosorbent assay was used to detect the levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6).Real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-140,nuclear factor kappa-B(NF-κB)and Toll-like receptors 4(TLR-4)mRNA in liver tissues.Immunohistochemistry was used to detect the levels of NF-κB and TLR4 protein.Results Liver tissue steatosis score[(0.00±0.00)points vs(3.12±0.23)points vs(4.80±0.16)points],inflammatory necrosis score[(0.52±0.21)points vs(2.95±0.24)points vs(4.13±0.28)],balloon integration[(0.00±0.00)points vs(1.98±0.26)points vs(2.65±0.20)points],NAS integration[(0.48±0.32)points vs(7.29±0.34)points vs(9.41±0.51)points],AST[(43.24±6.89)U/L vs(83.21±10.98)U/L vs(129.36±11.14)U/L],ALT[(60.21±12.36)U/L vs(83.21±10.98)U/L vs(129.36±11.14)U/L],TNF-α[(145.77±6.46)μg/L vs(267.86±6.98)μg/L vs(439.45±6.98)μg/L],IL-6[(47.13±15.95)μg/L vs(187.66±9.47)μg/L vs(334.14±12.74)μg/L],relative expression of miR-140 mRNA(4.96±0.21 vs 1.29±0.49 vs 0.86±0.54),relative expression of NF-κB mRNA(0.96±0.21 vs 2.29±0.49 vs 4.56±0.54),relative expression of TLR4 mRNA(0.89±0.39 vs 3.01±0.43 vs 5.81±0.26),relative expression of NF-κB protein(2.63±0.32 vs 3.14±0.29 vs 5.15±0.31)and relative expression of TLR4 protein(3.96±0.31 vs 5.01±0.26 vs 6.95±0.34)of mice in control group,model group and miR-140 deletion group were statistically significant(all P<0.001).Liver tissue steatosis score,inflammatory necrosis score,balloon integration,NAS integration,AST,ALT,TNF-α,IL-6,relative expression of NF-κB mRNA,relative expression of TLR4 mRNA,relative expression of NF-κB protein and relative expression of TLR4 protein of mice in model group were significantly higher than those in control group(all P<0.001),relative expression of miR-140 mRNA of mice in model group was significantly lower than that in control group(P<0.001).Liver tissue steatosis score,inflammatory necrosis score,balloon integration,NAS integration,AST,ALT,TNF-α,IL-6,relative expression of NF-κB mRNA,relative expression of TLR4 mRNA,relative expression of NF-κB protein and relative expression of TLR4 protein of mice in miR-140 deletion group were significantly higher than those in model group(all P<0.001),relative expression of miR-140 mRNA of mice in miR-140 inhibition group was significantly lower than that in model group(P<0.001).Conclusions The deletion of microRNA-140 can promote the development of NAFLD in mice,and the mechanism maybe related to the increase of expression of NF-κB,TLR4 mRNA and protein in liver tissue.
作者
寇小妮
解新科
郝明霞
宋春荣
吴维
Kou Xiaoni;Xie Xinke;Hao Mingxia;Song Chunrong;Wu Wei(Department of Liver Diseases,Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,Xianyang 712000,Shaanxi Province,China)
出处
《中国肝脏病杂志(电子版)》
CAS
2020年第3期34-40,共7页
Chinese Journal of Liver Diseases:Electronic Version
基金
陕西省科技厅社会发展领域项目(2018SF295)。