灵芝多糖对HepG2 细胞诱导的肝癌小鼠肠道菌群及其菌群代谢功能的调节作用

∗基金项目:北京市自然科学基金资助项目(编号:20198485)作者单位:101100北京市通州区120紧急救援中心(于雷,孙超);包头医学院第二附属医院普通外科(张曼旭)
第一作者:于雷,男,41岁,大学本科,主治医师㊂E-mail: mailto:ylajj@ ㊃实验性肝炎㊃
灵芝多糖对HepG2细胞诱导的肝癌小鼠肠道菌群
及其菌群代谢功能的调节作用∗
于雷,孙超,张曼旭
㊀㊀ʌ摘要ɔ㊀目的㊀研究灵芝多糖对HepG2细胞诱导的肝癌小鼠肠道菌群及其菌群代谢功能的调节作用㊂方法㊀采用HepG2细胞构建肝癌小鼠模型,将21只造模成功的肝癌小鼠随机分为模型组㊁灵芝多糖(GLP)和益生菌处理组,每组7只,另选择正常小鼠7只作为对照组,给予药物处理组动物GLP或益生菌灌胃,另两组采用生理盐水灌胃,连续干预2w㊂采用镜检法和变性梯度凝胶电泳法(DGGE)检测小鼠肠道菌群结构㊁多样性及其脂肪酸代谢产物水平㊂结果㊀肝癌小鼠肠道双歧杆菌㊁乳酸杆菌㊁大肠埃希菌和肠球菌数量分别为(2.3ʃ0.5)ˑ1010CFU/g㊁(12.2ʃ2.2)ˑ109CFU/g㊁(3.9ʃ1.5)ˑ107CFU/g和(5.0ʃ1.2)ˑ1010CFU/g,显著高于对照组[分别为(0.94ʃ0.18)ˑ1010CFU/g㊁(3.49ʃ0.66)ˑ109CFU/g㊁
(1.12ʃ0.11)ˑ107CFU/g和(0.57ʃ0.06)ˑ1010CFU/g,P<0.05],GLP干预组小鼠肠道上述菌群分别为(1.1ʃ0.1)ˑ1010
CFU/g㊁(6.5ʃ1.1)ˑ109CFU/g㊁(1.8ʃ0.2)ˑ107CFU/g和(1.5ʃ0.1)ˑ1010CFU/g,均显著低于模型组(P<0.05);GLP处理组小鼠肠道菌群丰富度㊁多样性指数和均匀度分别为(7.2ʃ1.1)S㊁(4.8ʃ1.1)H和(1.0ʃ0.2)E,显著高于模型组ʌ分别为(5.7ʃ1.2)S㊁(3.4ʃ0.5)H㊁(0.7ʃ0.1)E,P<0.05ɔ;GLP干预组小鼠肠道菌群乙酸㊁丙酸㊁正丁酸和D-乳酸水平分别为
(37.9ʃ3.3)mmol/L㊁(3.9ʃ0.2)mmol/L㊁(2.3ʃ0.3)mmol/L和(0.2ʃ0.0)g/L,与模型组ʌ分别为(26.8ʃ3.2)mmol/L㊁
(1.8ʃ0.4)mmol/L㊁(1.8ʃ0.2)mmol/L和(0.5ʃ0.1)g/L比,差异显著(P<0.05)㊂结论㊀肝癌小鼠肠道菌群出现了失调,
而应用GLP可能帮助改善机体肠道微生态紊乱状态,其意义还有待探讨㊂
㊀㊀ʌ关键词ɔ㊀肝细胞癌;灵芝多糖;肠道菌群;小鼠
㊀㊀DOI:10.3969/j.issn.1672-5069.2021.04.006
㊀㊀Regulation of intestinal floras and their metabolism functions by ganoderma lucidum plysaceharide in mice with HepG2 cell-induced implanted cancer㊀Yu Lei,Sun Chao,Zhang Manxu.120Emergency Rescue Center,Tongzhou101100, Beijing,China
㊀㊀ʌAbstractɔ㊀Objective㊀The aim of this experimental study was to implore the regulation of intestinal floras and their metabolism functions by ganoderma lucidum plysaceharide(GLP)in mice with HepG2cell-induced implanted cancer.Methods㊀The subcutaneous cancer in mice was established by HepG2cell implantation,and21mice with successful implanted cancer were randomly divided into model,GLP-intervened and probiotics-intervened groups,with7in each.7normal mice were selected as control.The GLP and probiotics were administered intragastrically,while normal saline were given by gavage in model and control for2weeks.The diversity of intestinal floras,and fatty acid metabolites were detected by microscopy and denaturing gradient gel electrophoresis(DGGE).Results㊀The number of Bifidobacterium,Lactobacillus,Escherichia Coli and Enterococcus in model group were(2.3ʃ0.5)ˑ1010CFU/g,(12.2ʃ2.2)ˑ109CFU/g,(3.9ʃ1.5)ˑ107CFU/g and(5.0ʃ1.2)ˑ1010CFU/g,all significantly higher than[(0.94ʃ0.18)ˑ1010CFU/g,(3.49ʃ0.66)ˑ109CFU/g,(1.12ʃ0.11)ˑ107CFU/g and(0.57ʃ0.06)ˑ1010CFU/g,respectively,P<0.05]in the control,ylajj@ while they were(1.1ʃ0.1)ˑ1010CFU/g,(6.5ʃ1.1)ˑ109 CFU/g,(1.8ʃ0.2)ˑ107CFU/g and(1.5ʃ0.1)ˑ1010CFU/g in㊀GLP-intervened group(P<0.05);the abundance,the diversity index and uniformity of intestinal floras in GLP-intervened group were(7.2ʃ1.1)S,(4.8ʃ1.1)H and(1.0ʃ0.2)E,all significantly higher than[(5.7ʃ1.2)S,(3.4ʃ0.5)H and(0.7ʃ0.1)E,respectively,P<0.05]in the model;the intestinal flora acetic acid,propionic acid and n-butyric acid in GLP-intervened group were(37.9ʃ3.3)mmol/L,(3.9ʃ0.2)mmol/L and
(2.3ʃ0.3)mmol/L,all significantly higher than[(26.8ʃ3.2)
mmol/L,(1.8ʃ0.4)mmol/L and(1.8ʃ0.2)mmol/L,
respectively,P<0.05],while the D-lactic acid was(0.2ʃ
0.0)g/L,much lower than(0.5ʃ0.1)g/L in the model(P<
0.05).Conclusion㊀The imbalance of intestinal floras is
closely related to the carcinogenesis in mice,and the GLP
administration might correct the intestinal microecological disorders,which warrants further investigation.㊀㊀ʌKey wordsɔ㊀Hepatoma;Ganoderma lucidum plysaceharide;Intestinal flora;Mice
㊀㊀人类各种疾病,包括各种代谢性疾病㊁肠道疾病㊁癌症和自身免疫性疾病等均与肠道微生物失调紧密相关,其中肝脏疾病的关联性尤为显著[1]㊂很多学者[2,3]研究发现肝细胞癌㊁肝硬化和非酒精性脂肪性肝病等患者体内存在肠道菌种及其代谢的紊乱状态㊂肝癌作为临床上高发性恶性肿瘤之一,不仅严重影响人类的生存质量和生命健康,同时对社会和家庭经济带来沉重的负担[4]㊂近几年来,很多中医药研究机构试图尝试研发辅助药物以帮助治疗肝癌患者,例如灵芝三萜和加味柴苓汤等[5,6]㊂灵芝多糖(ganoderma lucidum plysaceharide,GLP)是从灵芝中提取的一种主要活性组分㊂药理学研究揭示GLP 在调节免疫功能㊁抗癌㊁抗氧化㊁抗衰老和抗辐射等方面起着至关重要的作用[7]㊂以往研究[8]主要围绕GLP对肝癌小鼠肠道粘膜免疫功能的影响或肝癌细胞增殖和迁移的影响等㊂本研究采用HepG2细胞构建小鼠肝癌模型,并初步探讨了GLP对其肠道菌群及其菌群代谢功能的调节作用㊂
1㊀材料与方法
1.1动物㊁细胞㊁药品和试剂㊀SPF级昆明小鼠,体质量为(17.5ʃ
2.2)g,购自武汉大学实验动物资源中心(动物合格证号为16054418)㊂饲养在24~26ħ,相对湿度为53~55%的室内,通风换气10次/h,光照12h,黑暗12h,自由获取食物和水;HepG2肝癌细胞株由中国医学科学院肿瘤细胞库提供㊂培养方法:从液氮罐中取出HepG2细胞㊁复苏,用DMEM (含10%胎牛血清)培养基,在37ħ㊁5%CO2细胞培养箱中培养24h,待细胞呈对数生长期时终止培养, 1200r/m离心10min,弃去上清液,再用DMEM(含10%胎牛血清)重悬细胞,制成2ˑ105个/mL的细胞悬液,备用;GLP由西安明泽生物科技股份有限公司提供㊂益生菌由辉凌公司提供,用前稀释至50%浓度㊂精提基因组DNA裂解液:50mmol/L Tris-HCL㊁50mmol/L EDTA㊁500mmol/L NaCl㊁4%EDTA,将pH调至8.0㊂应用上下游引物(上海生物工程公司)扩增细菌16s rRNA V3可变区㊂
1.2肝癌小鼠模型的建立㊀用注射器取上述HepG2细胞悬液200μL,注射至小鼠右腋下部位,密切观察小鼠皮下成瘤情况㊂当出现可见瘤体时,表示造模初步成功㊂然后,每周测量肿瘤结节直径㊂当达到1cm时,表示造模成功,用于后续实验㊂本次总共成功构建肝癌模型小鼠21只㊂
1.3干预与分组㊀将上述造模成功的肝癌小鼠随机分为模型组㊁GLP组和益生菌组,每组7只㊂另随机选择7只正常小鼠作为对照组㊂GLP和益生菌用量参照临床上成人的用药剂量,采取人体-小鼠体表面积比值折算后应用2倍量,灌胃;在模型组和对照组,给予等量的生理盐水,灌胃,1次/d,连续干预2w㊂
1.4小鼠肠道菌群组成结构检测㊀镜检法:采用颈椎或尾椎脱臼法处死小鼠,剥离皮下肿瘤组织,分离小肠,严格按照无菌操作取出一定量的肠道内容物,于含有玻璃珠和灭菌蒸馏水的三角瓶内,于摇床上120 r/m震荡30min,以利于各类微生物充分分散㊂采用混菌法和涂布法将上述菌液以适宜的稀释比例接种于相应培养基,置于37ħ培养箱培养24~48h㊂取出培养液,于显微镜下观察,计数肠道内容物的菌群丰度㊂以每克肠道内容物含特定类型细菌数表示,每个稀释度重复检测3次;变性梯度凝胶电泳法(denaturing gradient gel electrophoresis,DGGE):取小鼠粪便,置于干净的2mL无菌EP管中,添加一定量的镐珠,加入精提基因组DNA裂解液700μL,吹打㊁充分混匀,再将苯酚/氯仿/异戊醇以25/24/1比例加入250μL并混匀㊂在旋涡震荡仪上震荡3min, 12000r/m离心5min,取上清于新的无菌EP管中,加入10mol/L乙酸铵700μL,冰上放置5min,12000 r/m离心10min,取上清并再次用苯酚/氯仿/异戊醇抽提2次,氯仿抽提2次,再加入异丙醇并置于-20ħ冰箱30min,使DNA沉淀㊂离心并弃掉上清液,用70%乙醇洗涤沉淀,最后用双蒸水50μL溶解DNA,备用;PCR扩增16s rRNA V3可变区㊂PCR 反应体系:Taq DNA聚合酶1
2.5μL㊁10μM F0.5μL㊁10μM R0.5μL㊁DNA模板1.0μL,ddH2O补足至25μL㊂PCR反应条件采用Touch down程序:94ħ预变性5min,94ħ变性30s,65ħ退火30s,72ħ延伸70s,共20个循环,每个循环退火温度依次降低0.5ħ,温度递降范围为65~56ħ,最后以56ħ退火温度循环10次;配制浓度为8%聚丙烯酰胺凝胶,变性梯度范围为30%~58%㊂将上述DNA扩增产物上样,并置于1ˑTAE缓冲液中电泳,电泳条件:电压
220v,预电泳5min,60ħ㊁电压85v电泳16h;硝酸银染色㊁拍照,应用Quantity One软件分析;DGGE指纹图谱分析㊂应用相似性聚类分析法将DGGE胶通过扫描仪输入到电脑,再应用Molecular Analysis软件分析各细菌序列相似性,每一个条带表示特定菌种,然后通过序列比对分析和计算得出菌种丰富度㊁多样性指数和均匀度㊂
1.5小鼠肠道菌群脂肪酸代谢产物检测㊀于干预后2w收集各组小鼠粪便,采用气相色谱法检测短链脂肪酸(乙酸㊁丙酸㊁正丁酸)水平;采用Megazyme D-乳酸定量检测试剂盒测定D-乳酸含量㊂1.6统计学分析㊀应用SPSS21.0软件处理数据㊂计量资料以(xʃs)表示,采用方差分析,P<0.05表示差异具有统计学意义㊂
2㊀结果
2.1四组小鼠肠道菌群比较㊀GLP处理组和益生菌处理组小鼠肠道双歧杆菌㊁乳酸杆菌㊁大肠埃希菌和肠球菌数量显著低于模型组(P<0.05);模型组小鼠肠道双歧杆菌㊁乳酸杆菌㊁大肠埃希菌和肠球菌数量显著高于对照组(P<0.05);但GLP处理组和益生菌处理组小鼠肠道双歧杆菌㊁乳酸杆菌㊁大肠埃希菌和肠球菌数量与对照组基本一致(P>0.05,表1)㊂
表1㊀四组小鼠肠道菌群(xʃs)比较
只数双歧杆菌(ˑ1010CFU/g)乳酸杆菌(ˑ109CFU/g)大肠埃希菌(ˑ107CFU/g)肠球菌(ˑ1010CFU/g)对照组70.8ʃ0.2 4.4ʃ0.9 1.4ʃ0.10.7ʃ0.2
模型组7 2.6ʃ0.5①13.0ʃ1.7① 4.4ʃ1.0① 4.6ʃ1.0①GLP7 1.1ʃ0.1② 6.5ʃ1.1② 1.8ʃ0.2② 1.5ʃ0.1②益生菌7 2.2ʃ0.47.3ʃ1.6 2.1ʃ0.4 2.1ʃ0.3
㊀㊀与对照组比,①P<0.05;与模型组比,②P<0.05 2.2四组小鼠粪便DGGE图谱指标比较㊀GLP处理组和益生菌处理组小鼠肠道菌群丰富度㊁多样性指数和均匀度较显著高于模型组(P<0.05);模型组小鼠肠道菌群丰富度㊁多样性指数和均匀度均显著低于对照组(P<0.05,表2)㊂
2.3四组小鼠菌群代谢功能指标比较㊀GLP组㊁益生菌组小鼠肠道菌群代谢产物短链脂肪酸乙酸㊁丙酸㊁正丁酸均显著高于模型组(P<0.05),D-乳酸显著低于模型组(P<0.05);模型组乙酸㊁丙酸㊁正丁酸均显著低于对照组(P<0.05),D-乳酸显著高于对照组(P<0.05);GLP组㊁益生菌组小鼠肠道菌群代谢产物短链脂肪酸乙酸㊁丙酸㊁正丁酸㊁D-乳酸与对照组基本一致(P>0.05,表3)㊂
表2㊀四组小鼠粪便DGGE图谱指标(xʃs)比较
例数丰富度(S)多样性指数(H)均匀度(E)对照组78.0ʃ1.6 4.9ʃ1.0 1.0ʃ0.2模型组7 5.7ʃ1.2① 3.4ʃ0.5①0.7ʃ0.1①GLP组77.2ʃ1.1② 4.8ʃ1.1② 1.0ʃ0.2②
益生菌组77.1ʃ1.3② 4.7ʃ1.0②0.9ʃ0.1②㊀㊀与对照组比,①P<0.05;与模型组比,②P<0.05
表3㊀四组小鼠肠道菌群代谢产物水平(xʃs)比较
例数乙酸(mmol/L)丙酸(mmol/L)正丁酸(mmol/L)D-乳酸(g/L)对照组743.2ʃ3.7 4.3ʃ0.3 2.7ʃ0.40.1ʃ0.0模型组726.8ʃ3.2① 1.8ʃ0.4① 1.8ʃ0.2①0.5ʃ0.1①GLP组737.9ʃ3.3② 3.9ʃ0.2② 2.3ʃ0.3②0.2ʃ0.0②
益生菌组737.8ʃ3.3② 3.5ʃ0.2② 2.2ʃ0.3②0.3ʃ0.1②
㊀㊀与对照组比,①P<0.05;与模型组比,②P<0.05
3㊀讨论
肠道菌群失调指的是肠道内菌群结构发生剧烈的改变,益生菌数量急剧下降,同时伴随有害细菌的肆意滋生,使肠道代谢功能紊乱[9-11]㊂本研究结果显示,肝癌模型小鼠肠道双歧杆菌㊁乳酸杆菌㊁大肠埃希菌和肠球菌数量较对照组均显著升高,表明肝癌的形成改变了小鼠肠道菌群的种类和数量㊂在干预后,GLP组和益生菌处理组小鼠肠道上述菌群数量较模型组均显著减少,且接近对照组水平,表明
GLP干预可以降低肝癌小鼠体内双歧杆菌㊁乳酸杆
菌㊁大肠埃希菌和肠球菌数量,有利于维持肠道菌群
的平衡㊂益生菌是目前临床上广泛使用的有益活性
微生物,可用于改善肠道功能,帮助营养物质的消化
和吸收[12-16]㊂DGGE分析结果显示GLP处理组和益生菌处理组小鼠肠道菌群丰富度㊁多样性指数和
均匀度较模型组均显著升高,进一步验证了GLP参
与调控肝癌小鼠肠道的微生态失衡,其作用与益生
菌相当㊂
短链脂肪酸,诸如乙酸㊁丙酸和正丁酸,为机体
肠道菌群独有的经典重要代谢产物,这些指标可以
从侧面揭示肠道内厌氧性菌群代谢的功能状态[17]㊂本研究结果发现,GLP处理组和益生菌处理组肠道乙酸㊁丙酸㊁正丁酸水平较模型组均显著升高,且接近对照组小鼠水平,表明GLP干预有利于肝癌小鼠肠道菌群代谢功能的恢复,改善机体代谢紊乱状态,其机制与调节机体肠道菌群平衡密切相关[18]㊂D-乳酸是衡量肠道菌群增殖能力的指标,可用于指示肠道细菌增殖程度[19]㊂GLP处理组和益生菌处理组肠道D-乳酸较模型组均显著降低,表明GLP和益生菌处理均能够降低与D-乳酸代谢相关的有害细菌增殖,其机制可能与调控肠道内酸碱度㊁调控肠道粘膜免疫相关分子水平和抑制酪氨酸激酶信号转导通路有关[20]㊂
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(收稿:2020-10-29)
(本文编辑:陈从新)。