Ginkgolide B inhibits cell proliferation and promotes cell apoptosis of MH7A human fibroblast-like synoviocytes through PI3K/AKT pathway
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摘要:
探讨银杏内酯B(ginkgolide B,GB)对MH7A人成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)的增殖抑制作用及其潜在机制。采用20 μg/L肿瘤坏死因子-α(tumor necrosis factor-a,TNF-α)刺激MH7A构建关节炎细胞模型。经不同浓度GB作用于MH7A细胞后,CCK-8法检测细胞活力;Transwell实验检测细胞侵袭力;流式细胞术检测细胞凋亡率和细胞周期;实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)和蛋白免疫印迹分别检测基因转录和蛋白表达量。与对照组相比,GB对细胞活力的抑制作用呈现出一定的浓度和时间依赖性;GB显著抑制细胞侵袭力、增加细胞凋亡率和G0/G1期比例;GB显著上调细胞Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)和p21 mRNA和下降Bcl-2、髓系白血病1(myeloid cell leukemia 1,Mcl-1)、蛋白激酶B(protein kinase B,PKB;又称AKT)、磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)、Cyclin D1和细胞周期调节蛋白激酶4(cyclin-dependent kinase 4,CDK4)mRNA转录水平;同时,GB显著上调Bax、p21和Cleaved-caspase 3蛋白和下调Bcl-2、Mcl-1、p-AKT、p-PI3K、Cyclin D1和CDK4蛋白表达量,且伴有p-PI3K/PI3K、p-AKT/AKT和Bcl-2/Bax比值的降低。综上,GB通过抑制PI3K/AKT信号通路,阻滞MH7A细胞G1期向S期转化、抑制细胞活力和侵袭力,并诱导MH7A人成纤维样滑膜细胞凋亡。
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关键词:
- 银杏内酯B /
- 磷脂酰肌醇3-激酶/蛋白激酶B /
- 类风湿性关节炎 /
- MH7A细胞 /
- 凋亡
Abstract:To explore the inhibitory effect of ginkgolide B (GB) on MH7A human fibroblast-like synoviocytes (FLS) and its potential mechanism. Firstly, 20 μg/L tumor necrosis factor-α (TNF-α) was pretreated with MH7A to establish a cell model of arthritis. After incubation of MH7A cells with various concentrations of GB, CCK-8 assay, Transwell assay, and flow cytometry (FCM) were separately used to detect cell viability, cell invasion, and cell apoptosis rate and cell cycle; Real-time quantitative PCR and Western blot assay were performed to detect the apoptosis- and cycle-related gene transcriptions and protein expressions, respectively. The results showed that compared with the control group, GB dose- and time-dependently suppressed cell viability to a greater extent; GB significantly reduced cell invasive ability and increased cell apoptosis rate and proportion of G0/G1 phase in MH7A cells, along with increased transcription levels of Bcl-2-associated X protein (Bax) and p21 mRNA and decreased transcription levels of Bcl-2, myeloid cell leukemia 1(Mcl-1), protein kinase B (PKB; AKT), IP3K, Cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA; GB remarkably increased expression levels of Bax, p21, and cleaved-Caspase 3 protein and decreased expression levels of Bcl-2, Mcl-1, p-AKT, p-PI3K, Cyclin D1, and CDK4 protein, with decreased ratios of p-PI3K/PI3K, p-AKT/AKT, and Bcl-2/Bax. In conclusion, GB blocks the G1-to-S cell cycle transition, suppresses cell viability and cell invasion and induces cell apoptosis of MH7A human RA-FLS via suppressing the PI3K/AKT signaling pathway.
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Keywords:
- ginkgolide B /
- PI3K/AKT /
- rheumatoid arthritis /
- MH7A cell /
- cell apoptosis
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类风湿性关节炎(rheumatoid arthritis,RA)是一种由免疫系统异常激活和免疫耐受引起的自身免疫性疾病,其特征是过度滑膜增生和进行性关节破坏[1]。流行病学调查显示:RA几乎发生于全世界任何地区或各个民族,以女性多见,主要与遗传、雌激素、生殖和神经内分泌等宿主危险因素以及吸烟、空气暴露、外源性感染等环境风险因素有关[2];RA在全球的发病率为0.5%~1.0%,在中国大陆为0.42%[3]。成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)是人类RA滑膜组织中增生性滑膜的主要细胞类型,在高水平肿瘤坏死因子-α(tumor necrosis factor,TNF-α)等炎症因子的持续刺激下,表现出肿瘤样异常增殖、侵袭、凋亡障碍以及滑膜组织炎性增生和关节破坏等病理特点[4],由此可见FLS功能改变在RA进展过程中起到关键作用。因此,通过有效抑制RA-FLS异常过度增殖和侵袭以及促进其凋亡可能对减轻软骨破坏和改善RA临床症状有着重要作用。
MH7A是由RA患者的FLS经SV40T抗原基因转染而来的永生化细胞株,延续了人RA-FLS的特点,目前已成为RA病理机制和体外药物筛选的主要研究载体之一[5]。了解中草药有效成分在FLS凋亡中的诱导途径,不仅有助于阐明中药治疗RA的分子机制,而且有利于从天然草药中寻找新的候选治疗药物[6]。银杏内酯B(ginkgolide B,GB)是从银杏叶内提取的萜内酯类单体成分,具有抗炎、抗氧化活性和抗血栓形成的作用[7]。本实验通过研究GB对TNF-α诱导的MH7A细胞活力、侵袭力和凋亡的影响,初步探讨基于磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/AKT)通路的相关蛋白,为临床抗RA的新药开发利用提供理论依据。
1. 材 料
1.1 试 剂
高糖DMEM培养基(美国Sigma-Aldrich公司);CCK-8试剂盒(日本同仁化学研究所);Matrigel胶(BD美国Biosciences公司);凋亡检测试剂盒(美国BD公司);TRIzol试剂盒[宝日医生物技术(北京)有限公司];SYBR®Green荧光定量检测试剂盒和GB(美国Sigma公司);放射免疫沉淀法(radioimmunoprecipitation assay,RIPA)细胞裂解液(武汉博士德生物工程有限公司);BCA试剂盒(广州伟伯科技有限公司);Bax、Bcl-2、Mcl-1、cleaved-Caspase 3、CyclinD1、CDK4、p21和p-AKT抗体(美国Abcam公司);PI3K和p-PI3K抗体(美国Cell Signaling Technology公司);AKT和GAPDH抗体(美国Proteintech公司);辣根过氧化物酶标记的羊抗兔IgG(美国Jackson Immuno Research公司);其他试剂均为市售分析纯。
细胞株来源于上海冠导生物科技有限公司。
1.2 仪 器
Multiskan FC酶标仪(美国ThermoFisher公司);IX53倒置显微镜(日本Olympus公司);FACS Calibur流式细胞分析仪(美国BD公司)。
2. 方 法
2.1 细胞培养
MH7A细胞培养参照文献[8−9]。MH7A细胞置于含10% FBS、青霉素(100 U/mL)-链霉素(100 μg/mL)的DMEM培养基中,置37 ℃、5% CO2培养箱培养。隔天加入含20 μg/L TNF-α的DMEM培养基中继续培养,选取TNF-α刺激下MH7A对数生长期的细胞进行传代和后续试验。
2.2 CCK-8法检测细胞活力
离心收集经0.25%胰酶消化处理的MH7A细胞(每升5×107个),将每孔单细胞悬液100 μL接种于96孔板,置37 ℃、5% CO2细胞培养箱培养。待细胞贴壁生长24 h后,将原培养液更换为20 μg/L TNF-α的DMEM培养基和不同质量浓度的GB(0、10、20、40和80 μg/mL),分别继续培养24、48和72 h。弃去含药培养液,每孔加入CCK-8应用液10 μL,37 ℃避光反应,1 h后应用Multiskan FC酶标仪测量450 nm处各孔的吸收度A,用各实验组A与对照组A的比值表示细胞活力。
2.3 Transwell实验检测细胞侵袭力
将MH7A细胞(约每孔1.0×104个)接种到涂有Matrigel胶(1∶5稀释)的Transwell小室(直径6.5 mm,孔径8.0 μm)的上室,置于终浓度为20 μg/L TNF-α的DMEM培养基中,分别加入质量浓度为0、10、20和40 μg/mL GB;下室中加入含有20%胎牛血清的DMEM培养基。在37 ℃、5% CO2条件下培养,24 h后用磷酸盐缓冲液(phosphate buffered saline,PBS)洗涤小室3次,并用棉签擦拭小室内表面的细胞,用4%多聚甲醛固定迁移至小室外膜下的细胞20 min,然后用0.1%结晶紫染色10 min。再次用PBS洗涤细胞后,采用Image J软件(2.6版)随机拍摄倒置显微镜下5个视野中各组细胞图像并进行计数。
2.4 流式细胞术(flow cytometry, FCM)检测细胞凋亡率
离心收集经0.25%胰酶消化处理的MH7A细胞(每升5×107个),将每孔单细胞悬液2 mL接种于6孔板,置37 ℃、5% CO2细胞培养箱,待细胞贴壁生长24 h后,更换原培养液为含20 μg/L TNF-α以及含质量浓度0、10、20和40 μg/mL GB的DMEM培养基,继续培养48 h。胰酶消化后收集细胞,预冷PBS洗涤细胞2次,加结合缓冲液(1×)500 μL悬浮细胞,并依次加入膜联蛋白V(annexin V)-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)5 μL和碘化丙啶(propidium iodide,PI)5 μL,轻轻混匀,置4 ℃暗处孵育15 min,采用FCM检测细胞凋亡率,并用BD Accuri C6 软件分析实验数据。
2.5 FCM检测细胞周期
同“2.4”项下方法预处理MH7A细胞48 h,胰酶消化细胞,PBS洗涤细胞2次;经预冷的70%乙醇固定并低温下孵育3 h,离心洗涤后,悬浮于50 μg/mL PI染液中,室温下避光振摇,调整细胞浓度为每毫升1×106个,30 min内上FCM检测细胞周期分布。
2.6 实时荧光定量聚合酶链式反应(RT-qPCR)检测mRNA转录
同“2.4”预处理MH7A细胞48 h,消化收集细胞,采用TRIzol试剂盒提取总核糖核酸(ribonucleic acid,RNA)和测定其浓度、反转录试剂盒逆转录成互补脱氧核糖核酸(complementary DNA,cDNA),以GAPDH为内参,按照SYBR®Green荧光定量检测试剂盒的操作流程进行PCR定量测定目的基因mRNA表达。引物序列由上海生物工程有限公司合成,Bcl-2正义链为5′-GGTGGGGTCATGTGTGTGG-3′,反义链为5′-CGGTTCAGGTACTCAGTCATCC-3′;Bax正义链为5′-CCCGAGAGGTCTTTTTCCGAG-3′,反义链为5′-CCAGCCCATGATGGTTCTGAT-3′;CyclinD1正义链为5′-GCTGCGAAGTGGAAACCATC-3′,反义链为5′-CCTCCTTCTGCACACATTTGAA-3′;AKT正义链为5'-TCCTCCTCAAGAATGATGGCA-3',反义链为5'-GTGCGTTCGATGACAGTGGT-3';PI3K正义链为5'-AAACAGAGCCAAAGGGAAGG-3',反义链为5'-ATACC AGCCACAAAGGCTTC-3';Mcl-1正义链为5'-AGACGATGTGAAATCG-3',反义链为5'-TAACTAGCCAGTCCCG-3';CDK4正义链为5'-GAGGCGACTGGAGGCTTTT-3',反义链为5'-GGATGTGCACAGACGTCC-3';p21正义链为5'-GCCCA GTGGACAGCGAGCAG-3',反义链为5'-GCCGGCGTTTGGAGTGGTAG-3';GAPDH正义链为5′-GCACCGTCAAGGCTGAGAAC-3′,反义链为5′-TGGTGA AGACGCCAGTGGA-3′。PCR扩增程序如下:95 ℃变性30 s,95 ℃变性5 s,60 ℃退火30 s,65 ℃延长1 min,35个循环,每个样品设3个复孔。最后用2-ΔΔCt方法计算目的基因mRNA相对表达量。
2.7 Western blot检测蛋白表达
同“2.4”项下方法预处理MH7A细胞48 h,消化收集细胞,加入RIPA细胞裂解液后置于冰上裂解30 min,以提取各组总蛋白。经
1000 r/min离心5 min,采用BCA试剂盒测定上清液蛋白含量。蛋白变性后十二烷基硫酸钠聚丙烯酰胺凝胶电泳,转膜,5%脱脂牛奶封闭,1 h后用含Tween-20的Tris 盐缓冲液(tris-borate-sodium Tween-20,TBST)漂洗3次(×15 min),分别加入1∶1000 稀释的Bax、Bcl-2、Mcl-1、cleaved-Caspase 3、CyclinD1、CDK4、p21、AKT、p-AKT、PI3K、p-PI3K和GAPDH一抗,置4 °C摇床孵育过夜。TBST漂洗后加入1∶5000 稀释的辣根过氧化物酶标记的羊抗兔IgG二抗,常温孵育1 h。TBST再次漂洗后加入电化学发光工作液显色,应用Image J 1.44软件分析各蛋白条带灰度值。2.8 统计学分析
计量资料以$ \bar{x } \pm s $表示,组间比较采用t检验或方差分析;计数资料以率(%)表示,组间比较采用χ2检验;用SPSS 26.0统计学软件进行数据分析,以P < 0.05为差异有统计学意义。
3. 结 果
3.1 对MH7A细胞活力和细胞侵袭力的影响
与对照组相比,同一干预时间点,各组MH7A细胞活力伴随着GB浓度的增大呈现出下降趋势;同一质量浓度GB(< 80 μg/mL)作用MH7A细胞不同时间,细胞活力伴随着作用时间延长呈现出下降趋势,差异均有统计学意义(P < 0.05)。当GB达80 μg/mL时,48 和72 h的细胞存活率均小于50%,但两组间差异无统计学意义(P > 0.05)(图1)。鉴于上述CCK-8检测结果,选择10,20和40 μg/mL GB为后续实验研究。
Figure 1. Effects of ginkgolide B (GB) on MH7A cell viability and cell invasion ($ \bar{x } \pm s $,n=6)A: Cell viability determined by CCK-8 assay after incubation of MH7A cells with 0 (control group),10,20,40, and 80 μg/mL GB for 24,48 and 72 h; B: Representative image of cell invasion detected by Transwell assay after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 24 h; C: Bar graph of cell invasion ability in MH7A cells *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P<0.05, ##P < 0.01 vs 10 μg/mL GB group经不同质量浓度GB作用MH7A细胞24 h,Transwell试验结果显示:与对照组相比,TNF-α刺激下的MH7A细胞侵袭能力伴随着GB浓度的升高而显著降低(P < 0.05)。且20和40 μg/mL GB组细胞侵袭力均较10 μg/mL GB组明显下降(P < 0.05)。
3.2 对MH7A细胞凋亡率的影响
经不同浓度GB作用MH7A细胞48 h,各组细胞凋亡率均较对照组细胞增高,并且细胞凋亡率伴随着GB浓度的增大呈现出增高趋势(P < 0.05)(图2)。
Figure 2. GB on cell apoptosis ($\bar{x } \pm s $, n=3)A: Representative flow cytometry analysis of cell apoptosis after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Bar graph of cell apoptosis in MH7A cells *P < 0.05, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group3.3 对MH7A细胞周期的影响
与对照组相比,不同浓度GB作用MH7A细胞48 h后,各组G0/G1期细胞比例呈上升趋势(P < 0.05),S期细胞比例呈下降趋势(P < 0.05),但G2/M期细胞比例呈下降趋势,但各组间差异无统计学意义(P > 0.05)(图3),提示GB浓度依赖性地阻滞MH7A细胞于G0/G1期。
Figure 3. Effect of GB on cell cycle ($ \bar{x } \pm s $, n=3)A: Representative FCM analysis of cell cycle after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 48 h; B: Bar graph of cell cycle distribution in MH7A cells *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group3.4 对MH7A细胞mRNA转录水平的影响
与对照组相比,不同浓度GB作用MH7A细胞48 h,各组Bax 和p21 mRNA转录水平均显示出升高趋势,而Bcl-2、Cyclin D1、CDK4、AKT、PI3K 和Mcl-1 mRNA转录水平均呈现出下降趋势,且随着GB质量浓度的增大,作用效果越明显(P < 0.05)(图4)。
Figure 4. Effect of GB on the transcription levels of mRNA ($ \bar{x } \pm s $, n=3)qRT-PCR analysis of levels of transcription mRNA after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h *P < 0.05, **P < 0.01, *** P < 0.001 vs control group; # P < 0.05, ##P < 0.01 vs 10 μg/mL GB group; &P < 0.05 vs 20 μg/mL GB group3.5 对MH7A细胞凋亡相关蛋白表达水平的影响
与对照组相比,不同浓度GB作用MH7A细胞48 h,各组Bax和cleaved-Caspase 3蛋白表达量均呈现出上升趋势,而Bcl-2、Mcl-1、p-AKT和p-PI3K蛋白表达量均呈现出下降趋势(图5-A,5-B),并伴有p-PI3K/PI3K、p-AKT/AKT(图5-C)和Bcl-2/Bax比值降低(图5-D),且随着GB质量浓度的增加,作用效果越明显(P < 0.05)。
Figure 5. Effect of GB on the expression levels of apoptosis-related protein ($ \bar{x } \pm s $, n=3)A: Representative image of Western blot assay after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Relative expression levels of apoptosis-related proteins; C: Ratio of phosphorylated to non-phosphorylated proteins; D: Ratio of Bcl-2 to Bax protein *P < 0.05, **P < 0.01, *** P < 0.001 vs control group; # P<0.05, ##P<0.01 vs 10 μg/mL GB group; &P<0.05 vs 20 μg/mL GB group3.6 对MH7A细胞中周期相关蛋白表达水平的影响
与对照组相比,不同浓度GB作用MH7A细胞48 h,各组p21蛋白表达量呈现出上升趋势,而CyclinD1和CDK4蛋白表达量均呈现出下降趋势,且伴随着GB质量浓度的增加,作用效果越明显(P < 0.05)(图6-A,6-B)。
Figure 6. Effect of GB on the expression levels of cell cycle-related protein ($ \bar{x } \pm s $, n=3)MH7A cells were collected and the expression levels of protein were quantitatively determined by Western blot assay after incubation with 0 (control group),10,20, and 40 μg/mL GB for 48 h *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group; &P<0.05 vs 20 μg/mL GB group4. 讨 论
目前,临床上针对RA的治疗目标主要是控制疾病症状、减缓疾病进一步损害[10]。中药银杏叶的二萜类化合物GB具有明显的抗炎和抗肿瘤等药理作用[11−12]。GB通过下调TLR4-NF-κB通路抑制小胶质BV2细胞活化及炎症反应[13]。最近研究表明,GB还能够通过抑制JAK2/STAT3信号通路发挥其抗肿瘤作用[12]。本研究中GB干预高浓度TNF-α持续刺激下的MH7A细胞,其肿瘤样异常增殖和侵袭能力均呈现出明显下降。Xie等[14]证实了GB具有减轻类风湿关节炎模型中小鼠关节软骨和骨质的破坏等作用,其机制与Wnt5a/JNK/NF-κB轴调控的RA-FLSs凋亡和炎性细胞因子释放有关。上述数据表明GB有助于抑制RA滑膜组织中FLS异常增殖和侵袭,缓解RA的症状和疾病进展。
RA发病机制复杂,涉及细胞内多种信号分子的调节,其中P13K/AKT信号通路为重要的信号转导通路之一[6,15]。活化的AKT激活或抑制PI3K/AKT信号通路的下游靶点,在细胞增殖、侵袭转移、周期和凋亡相关靶点的调控中发挥着重要作用[16]。在各种促进细胞炎性增殖的反应中,首先可通过调节G1期检查点分子和活化AKT信号传导来促进G1期到S期转变,从而加速人非小细胞肺癌的生长[17]。值得注意的是,1×10−9~1×10−5 mol/L GB浓度依赖性抑制牛主动脉平滑肌细胞增殖,其与细胞周期G1期转换到S期阻滞有关[18]。本研究中GB通过下调Cyclin D1和CDK4以及上调p21基因转录和蛋白表达,阻滞MH7A细胞G1期向S期转化,从而影响DNA复制和抑制其增殖。
细胞凋亡是细胞的一种基本生物学现象,在多细胞生物去除不需要的或异常细胞中起着关键性作用[19]。研究发现,PI3K/AKT通路中AKT活化可激活其下游靶点Bax,增强Bcl-2因子的转录,发挥抗凋亡作用[20−21]。Caspase家族中的caspase 3是细胞凋亡最重要的调控蛋白,参与细胞凋亡启动和执行过程[22]。GB通过活性氧生成、c-Jun N末端激酶激活、线粒体膜电位丧失和caspase-3激活诱导小鼠胚胎干细胞(ESC-B5)凋亡[23]。本研究中GB剂量依赖性地上调Bax基因转录和蛋白表达量、下调MH7A细胞中Bcl-2、Mcl-1、AKT和PI3K基因转录以及p-PI3K、p-AKT、Bcl-2和Mcl-1蛋白表达量,致使p-PI3K/PI3K、p-AKT/AKT和Bcl-2/Bax比值下降。其中Bcl-2/Bax比值下降可导致线粒体外孔膜形成,释放细胞色素C入胞质,激活下游的cleaved-Caspase 3表达量增加,并进一步促进MH7A细胞凋亡。
综上所述,GB通过抑制PI3K/AKT信号通路,阻滞MH7A细胞G1期向S期转化、抑制细胞活力和侵袭力,并诱导人成纤维样滑膜MH7A细胞凋亡。这可能是GB抗RA的信号分子调节机制之一,但该作用机制还有待于进一步体内研究。
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Figure 1. Effects of ginkgolide B (GB) on MH7A cell viability and cell invasion ($ \bar{x } \pm s $,n=6)
A: Cell viability determined by CCK-8 assay after incubation of MH7A cells with 0 (control group),10,20,40, and 80 μg/mL GB for 24,48 and 72 h; B: Representative image of cell invasion detected by Transwell assay after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 24 h; C: Bar graph of cell invasion ability in MH7A cells *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P<0.05, ##P < 0.01 vs 10 μg/mL GB group
Figure 2. GB on cell apoptosis ($\bar{x } \pm s $, n=3)
A: Representative flow cytometry analysis of cell apoptosis after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Bar graph of cell apoptosis in MH7A cells *P < 0.05, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group
Figure 3. Effect of GB on cell cycle ($ \bar{x } \pm s $, n=3)
A: Representative FCM analysis of cell cycle after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 48 h; B: Bar graph of cell cycle distribution in MH7A cells *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group
Figure 4. Effect of GB on the transcription levels of mRNA ($ \bar{x } \pm s $, n=3)
qRT-PCR analysis of levels of transcription mRNA after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h *P < 0.05, **P < 0.01, *** P < 0.001 vs control group; # P < 0.05, ##P < 0.01 vs 10 μg/mL GB group; &P < 0.05 vs 20 μg/mL GB group
Figure 5. Effect of GB on the expression levels of apoptosis-related protein ($ \bar{x } \pm s $, n=3)
A: Representative image of Western blot assay after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Relative expression levels of apoptosis-related proteins; C: Ratio of phosphorylated to non-phosphorylated proteins; D: Ratio of Bcl-2 to Bax protein *P < 0.05, **P < 0.01, *** P < 0.001 vs control group; # P<0.05, ##P<0.01 vs 10 μg/mL GB group; &P<0.05 vs 20 μg/mL GB group
Figure 6. Effect of GB on the expression levels of cell cycle-related protein ($ \bar{x } \pm s $, n=3)
MH7A cells were collected and the expression levels of protein were quantitatively determined by Western blot assay after incubation with 0 (control group),10,20, and 40 μg/mL GB for 48 h *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs 10 μg/mL GB group; &P<0.05 vs 20 μg/mL GB group
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