摘要
为揭示柑橘果胶寡糖H1(POSH1)对胆固醇代谢的调控机制,采用巨噬细胞模型,基于胆固醇代谢外排、摄取与合成机制对POSH1调控胆固醇代谢进行了研究。结果显示:POSH1和POSH1的肠道菌群发酵产物(P24)均通过显著上调肝X受体α-ATP结合盒转运体ABCA1/ABCG1通路中关键基因的蛋白表达量来促进巨噬细胞胆固醇外排。POSH1对巨噬细胞胆固醇摄取相关基因的蛋白表达无显著影响,但P24却可以通过显著抑制A类清道夫受体和分化抗原簇36的蛋白表达来抑制巨噬细胞对胆固醇的摄取。POSH1和P24均可通过显著抑制胆固醇合成基因3-羟基-3甲基-戊二酰辅酶A还原酶蛋白的表达来抑制巨噬细胞胆固醇的合成。研究结果表明,POSH1通过促进胆固醇排出、抑制胆固醇摄取与合成调控胆固醇代谢,这是POSH1调控胆固醇代谢的机制之一,且该机制与肠道菌群发酵POSH1有关。
高胆固醇血症作为心血管疾病的高风险因子,胆固醇外排、摄取与合成在其病理发展过程中起到了重要作用。与正常人相比,家族性高胆固醇血症患者体内负责胆固醇外排的ATP结合盒转运体A1基因(ABCA1)和ATP结合盒转运体G1基因(ABCG1)表达被抑制,由此导致的胆固醇外排功能受损是先天性
巨噬细胞胆固醇代谢平衡稳态与高胆固醇血症的病理发展过程密切相
笔者所在团队前期研究发现,柑橘果胶寡糖H1(POSH1)作为一种可以被肠道菌群水解利用的水溶性膳食纤维,其对高脂膳食小鼠胆固醇水平具有明显的改善效果,且POSH1发挥调控胆固醇代谢作用与胆固醇排出和合成密切相
1)试验原料。本研究所用原料为实验室自制的柑橘果胶寡糖级分H1(以下均简称为POSH1),由过氧化氢高温水解柑橘皮果胶所得,方法见文献[
2)主要试剂。Gibco RPMI 1640培养基、Gibco胎牛血清、High capacity cDNA反转录试剂盒、SYBR Green Master mix、LPS、蛋白Marker和蛋白酶抑制剂均购于Thermo Fisher Scientific公司。TRIzol Reagent购于美国Invitrogen公司。Pam3CSK4购于Bio-techne公司。PMA购于Sigma公司。引物合成由Integrated DNA Technologies公司合成。Pierc
3)仪器设备。101-2AB电热鼓风干燥箱,天津泰斯特仪器有限公司;BBS-SDC洁净工作台,济南鑫贝西生物技术有限公司;ABI StepOnePlus实时荧光定量PCR仪,美国应用生物系统公司;Thermofisher Veriti 96 梯度PCR仪,美国应用生物系统公司;NanoDrop微量分光光度计,Thermo Fisher Scientific公司;Anaerobox IV厌氧培养箱,美国GENE SCIENCE公司;DYY-6C电泳槽,北京六一仪器厂;TGL-16高速离心机,苏州雨泽仪器有限公司;SMR16.1酶标仪,优尔生生命科学装备有限公司;AX-Ⅱ暗匣,广东粤华医疗器械厂有限公司。
液体LB培养基配方:10 g胰蛋白胨、5 g酵母提取物、5 g氯化钠,1 mol/L NaOH调pH至7.4,去离子水定容至1 L,高压灭菌备用。POSH1培养基:每100 mL液体LB培养基加入5 g POSH1。
POSH1体外培养:粪便接种液是将10人粪便经0.01 mol/L灭菌PBS缓冲液稀释为0.1 g/mL的粪便悬浮液,由瑞典卡罗林斯卡医学院Clinical Microbiology系的Christian G. Giske教授实验室提供。将该接种液按1%(V/V)接种到POSH1培养基中,37.0 °C厌氧培养24 h,对照组为不加POSH1的LB液体培养基。24 h后,4 °C、4 000 r/min离心10 min取上清液收集,该上清液即为肠道菌群发酵产物。对照组的肠道菌群发酵产物记为N24,POSH1组的肠道菌群发酵产物记为P24。上清液过0.22 μm滤膜后,-80 °C储存待用。
取出-80 ℃冰箱冻存的细胞,将其于37 ℃恒温水浴锅融化。随后,转移至15 mL无菌离心管离心;再加入8 mL含10% FBS、1% 10 U/mL青霉素和10 mg/mL链霉素混合液的RPMI 1640培养基,1 000 r/min离心5 min;离心结束后小心吸出上清,取1 mL培养液至离心管中,混匀吹打3次;将有细胞的1 mL培养液加至75 c
细胞传代:将离心机转速调至1 000 r/min,15 mL离心管离心THP-1细胞5 min。小心弃去培养基,再加入3~5 mL培养基吹打混匀,并于无菌条件下将其转移至75 c
细胞培养条件:37 ℃恒温,5% CO2。
待THP-1细胞数目维持在1×1
向分化好的细胞中加入RPMI 1640培养基(含10% FBS、1% 10 U/mL青霉素和10 mg/mL链霉素混合液),分别给予POSH1和P24,共15组。
1)POSH1效果研究。分别为阴性对照组(NC)、POSH1(5 mg/mL)、Pam3CSK4 (Pam,20 ng/mL)、LPS(10 ng/mL)、POSH1+Pam3CSK4(PP)和POSH1+LPS(PL),共6组。其中,Pam3CSK4和LPS(分别为TLR2和TLR4激动剂)组为阳性对照组。POSH1预处理THP-1巨噬细胞1 h,其余组各加等体积灭菌PBS缓冲液;1 h后各组相应加入LPS或Pam3CSK4处理6 h(共计7 h)后,收集细胞用于PCR检测。
2)肠道菌群发酵产物效果研究。分别为阴性对照组(NC)、P24(1%,V/V)、N24(1%,V/V)、Pam3CSK4(Pam,20 ng/mL)、LPS(10 ng/mL)、P24+Pam3CSK4(P24P)、P24+LPS(P24L)、N24+Pam3CSK4(N24P)和N24+LPS(N24L),共9组。其中,Pam3CSK4和LPS组(分别为TLR2和TLR4激动剂)为阳性对照组。P24或者N24预处理THP-1巨噬细胞1 h,其余组各加等体积灭菌PBS缓冲液;1 h后再加入LPS或Pam3CSK4处理6 h(共计7 h)后,收集细胞用于后续试验检测。
1)细胞中总蛋白提取和变性。RIPA工作液使用时先加入各种抑制剂,1 mL RIPA工作液=790 μL RIPA+100 μL蛋白酶抑制剂母液(10×)+100 μL磷酸化蛋白酶抑制剂母液(10×)+10 μL 100 mmol/L PMSF母液。RIPA工作液配好后,放在冰上预冷。用TBS缓冲液润洗贴壁细胞2~3次,最后1次尽量吸干残留液。加入适当体积的细胞总蛋白提取试剂(使用前数分钟内加入蛋白酶抑制剂)于培养板/瓶内裂解3~5 min。期间反复震动培养板/瓶,使试剂与细胞充分接触。用细胞刮刀将细胞及试剂刮下,收集到1.5 mL离心管中。冰浴30 min,期间用移液器反复吹打,确保细胞完全裂解。4 ℃、12 000 r/min离心5 min,收集上清,即为总蛋白溶液。每个样品总蛋白溶液加入5×loading buffer,涡旋混匀,98 ℃加热10 min,-80 ℃保存。
2)SDS-PAGE电泳。根据蛋白的相对分子质量大小,配制10%的分离胶和5%的浓缩胶,为防止胶凝固应在加入TEMED后立即灌胶;将干净的玻璃板固定好,然后加入预先配制好的分离胶,并加水液封除去气泡。待胶凝固后,用滤纸吸干玻璃板中水分,然后加入浓缩胶并插入梳子,待胶再次凝固后轻轻拔出梳子;处理好的各组蛋白样品进行电泳。浓缩胶电压为80 V,分离胶电压为130 V,待条带到达玻璃板底部时结束电泳。
3)转膜。PVDF膜于甲醇中活化1 min,将夹子的黑色面放入装有转移液的玻璃平皿中,从下到上依次放入1层海绵垫、3层滤纸、分离胶、PVDF膜、3层滤纸,最后盖上1层海绵垫,排掉气泡,夹紧夹子,加入预冷的转膜液,设定电流为250 mA,于冰浴中转膜1 h。
4)封闭。将上一步转好的PVDF膜放入适量5%脱脂奶粉中,于脱色摇床上振荡封闭1 h。
5)孵育一抗。一抗按照试剂与耗材表中的比例用一抗稀释液稀释,将封闭后的PVDF膜装入杂交袋中4 °C孵育过夜。用TBST洗5次,每次5 min。
6)孵育二抗。二抗按照试剂与耗材表中的比例用5%脱脂奶粉稀释。将膜二抗中孵育1 h。用TBST洗5次,每次5 min。
7)显影。滴加新鲜配制的ECL混合溶液到膜的蛋白面侧,发光检测;根据不同的光强度调整曝光条件,显影、定影。将胶片进行扫描存档,AlphaEaseFC软件处理系统分析目标带的光密度值。
ABCA1和ABCG1是介导机体胆固醇外排的基因,且二者的表达受LXRα受体的调控。POSH1对巨噬细胞胆固醇外排相关基因的表达影响如

图1 POSH1对巨噬细胞胆固醇外排基因ABCA1和ABCG1表达水平的影响
Fig.1 Effects of POSH1 on the expression of macrophage cholesterol efflux gene ABCA1 and ABCG1
NC、POSH1、Pam、PP、LPS和PL分别代表阴性对照组、POSH1、Pam、POSH1+Pam、LPS和POSH1+LPS组;每组数据用平均值±标准误差表示,*,P< 0.05、**,P< 0.01、***,P< 0.001,****,P< 0.000 1。Macrophages were determined for protein expression of ABCA1 and ABCG1. The cells were treated either PBS,POSH1,Pam,LPS,POSH1+Pam (PP),and POSH1+LPS (PL). Values are presented as the means ± SEM. *,P< 0.05;**,P< 0.01;***,P< 0.001;****,P< 0.000 1。下同 The same as below.
P24对巨噬细胞胆固醇外排相关基因的表达影响如

图2 P24对巨噬细胞胆固醇外排基因ABCA1和ABCG1表达水平的影响
Fig.2 Effects of P24 on the expression of macrophage cholesterol efflux gene ABCA1 and ABCG1
NC、P24、N24、Pam、P24P、N24P、LPS、P24L和N24L分别代表阴性对照组、POSH1的肠道菌群发酵产物、对照组的肠道菌群发酵产物、Pam、POSH1的肠道菌群发酵产物+Pam、对照组的肠道菌群发酵产物+Pam、LPS、POSH1的肠道菌群发酵产物+LPS和对照组的肠道菌群发酵产物+LPS。Macrophages were determined for protein expression of ABCA1 and ABCG1. The cells were treated either PBS,P24,N24,Pam,LPS,or P24+Pam (P24P),or P24+LPS (P24L),or N24+Pam (N24P),and N24+LPS (N24L).
POSH1和P24对LXRα的表达变化如

图3 P24对巨噬细胞胆固醇外排基因LXRα表达水平的影响
Fig.3 Effects of P24 on the expression of macrophage cholesterol efflux gene LXRα
清道夫受体是CD36和SR-A导致巨噬细胞胆固醇蓄积并大量摄取修饰膜蛋白入胞的关键基因,POSH1对CD36和SR-A在巨噬细胞蛋白的表达量影响如

图4 POSH1对胆固醇摄取基因CD36和SR-A表达水平的影响
Fig.4 Effects of POSH1 on the expression of cholesterol uptake gene CD36 and SR-A
P24对清道夫受体CD36和SR-A基因的表达的影响如

图5 P24对胆固醇摄取基因CD36和SR-A表达水平的影响
Fig.5 Effects of P24 on the expression of cholesterol uptake gene CD36 and SR-A
POSH1和P24对胆固醇合成基因HMGCR的表达量影响结果如

图6 POSH1和P24对胆固醇合成基因HMGCR表达水平的影响
Fig.6 Effects of POSH1 and P24 on the expression of cholesterol synthesis gene HMGCR
本研究对POSH1和P24对巨噬细胞胆固醇外排、摄取与合成的影响进行了考察。ABCA1和ABCG1是巨噬细胞胆固醇外排的关键调控因
CD36和SR-A是参与巨噬细胞胆固醇摄取的关键基
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