Volatile aromatic components and pyrolytic products in ethanol extracts of Cyclocarya paliurus by GC-MS
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摘要:
为研究青钱柳浸膏作为添加剂应用于卷烟的可行性，采用气相-离子迁移谱（GC-IMS）分析挥发性香气成分，通过单热重-气相色谱-质谱联用技术（TGA-GC/MS）模拟燃烧过程，分析青钱柳浸膏在氮气环境中的裂解产物，并对产物可能的裂解机制进行合理推测。结果表明：醛类、醇类、酮类为青钱柳浸膏的主要挥发性香气成分，占总香气成分的62.28%。在不同温度条件下，青钱柳浸膏裂解产物差异较大，在200 ℃、360 ℃、440 ℃共鉴定出79种化合物，其中24种致香成分对卷烟风格具有重要作用，包括醛、酮、醇、酚、呋喃、芳香族化合物和其他一些天然香气物质，其中含量较多的酚类化合物主要是由含有类似愈创木酚单元和丁香酚单元结构的化合物通过侧链的断裂、脱甲基化、脱甲氧基化、脱水等形成，呋喃及呋喃衍生物主要由糖类或糖苷类化合物通过断裂糖苷键和脱水形成。
Abstract:In order to investigate the feasibility of the extract from Cyclocarya paliurus as an additive in cigarettes, the volatile aromatic components were analyzed by gas chromatography-ion mobility spectrometry (GC-IMS), and C.paliurus extract was pyrolyzed to simulate cigarette smoking by TGA-GC/MS. The cracking products of C.paliurus were analyzed in a nitrogen environment, and the possible cracking mechanism of the products was reasonably speculated. The results showed that aldehydes, alcohols, and ketones were the primary volatile aroma components of the C.paliurus extract, comprising 62.28% of the total aroma components. The cracking products of C.paliurus extract varied greatly under different temperature conditions. A total of 79 compounds were identified at 200,360, and 440 ℃, among which 24 aromatic components were clearly identified as having significant effects on cigarette style, including aldehydes, ketones, alcohols, phenols, furans, benzene series, and other natural aromatic substances. Among them, phenols containing a high concentration are mainly formed by compounds containing the structure of guaiacol unit and eugenol unit by side chain cleavage, demethylation, demethoxylation, dehydration, etc. Furan and its derivatives are mainly formed by glycosides or glycoside compounds by breaking glucoside bonds and dehydration.
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肝癌复发率高、转移性强，预后差， 是全球最常见的恶性肿瘤之一[1−2]。因此，对其发生发展机制的研究及后续药物的开发迫在眉睫。肿瘤免疫微环境（tumour immune microenvironment, TIME）是由巨噬细胞、淋巴细胞、树突状细胞等免疫细胞共同构成[3]。肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）是TIME中占比最多的免疫细胞[4]。TAMs浸润的数量与肝癌的恶性程度、不良预后等密切相关[5]。TAMs可通过代谢重编程等方式影响肿瘤恶性进展[6]。因此，对TAMs进行靶向调控或改造，可为肝癌的治疗提供新的策略。
脂质代谢作为肝癌细胞代谢重编程的主要标志之一，对脂质代谢进行调控可成为一种临床肝癌治疗的候选方案[7]。例如，脂肪酸合酶（fatty acid synthase, FASN）的小分子抑制剂（TVB-2640）目前已进入肝细胞癌Ⅱ期临床试验[8]。此外，TAMs在肿瘤细胞脂质合成中也发挥着重要作用，可能通过分泌促进脂质合成途径的细胞因子和生长因子，如白细胞介素-6（interleukin-6, IL-6）、肿瘤坏死因子-α（tumor necrosis factor α, TNF-α）、TGF-β，激活肿瘤细胞内的脂质合成途径或抑制脂质代谢途径，影响乙酰辅酶 A 羧化酶（acetyl-CoA carboxylase alpha, ACC1）等关键酶的表达和活性，从而促进脂质的合成和积累[9−11]，进而为肿瘤细胞的恶性增殖提供物质基础[12]。然而，关于TAMs是否能够介导肝癌细胞的脂质代谢重编程鲜有报道。因此，深入研究TAMs如何介导肝癌细胞脂质积累的机制将有助于揭示肝癌发展的新机制，为肝癌肿瘤治疗提供更多的靶点和策略。
为此，本研究首先建立了TAMs上清诱导Hepa1-6肝癌细胞的脂滴形成的共培养体系，在此基础上，探究了TAMs与Hepa1-6互作的关键因子。进一步探究了IL-10与ACC1在肝癌细胞恶性生物学中的作用。通过对肝癌细胞脂滴形成的作用机制进行初步研究，以期为肝癌的免疫治疗提供理论依据。
1. 材 料
1.1 试 剂
高糖DMEM培养基（上海源培生物科技有限公司）；牛血清（以色列Biological Industries公司）；cell counting kit-8（CCK-8）试剂盒、乳酸脱氢酶细胞毒性检测试剂盒、油红O（上海碧云天生物科技有限公司）；细胞凋亡检测试剂盒（南京诺唯赞生物科技有限公司）；脂滴绿色荧光检测试剂盒BODIPY 493/503（美国Thermo Fisher公司）；乙酰辅酶A羧化酶-α抑制剂TOFA（美国APExBIO公司）；脂滴荧光染料ND640（美国MedChemExpress公司）；乙酰辅酶A羧化酶Rabbit pAb（成都正能生物技术有限公司）；GAPDH-HRP conjugated（美国Bioworld Technology公司）；HRP标记的山羊抗兔IgG（H+L）（上海碧云天生物科技有限公司）；羧基荧光素琥珀酰亚胺酯荧光染料（CFSE，上海翌圣生物科技股份有限公司）；碘化丙啶荧光染料（PI，上海翌圣生物科技股份有限公司） ；甘油三酯（TG）测定试剂盒（南京建成生物工程研究所）； 乳酸脱氢酶（lactate dehydrogenase，LDH）细胞毒性试剂盒（上海碧云天生物技术股份有限公司）；细胞凋亡检测试剂盒（南京诺唯赞生物科技股份有限公司）。
1.2 细胞株与质粒
小鼠肝癌细胞株Hepa1-6、小鼠永生化骨髓源性巨噬细胞iBMDM（上海中科院细胞所）；ACC1-siRNA（苏州吉玛基因股份有限公司）；ACC1 CRISPR-cas9质粒实验室自己合成，并测序验证（上海生工生物工程有限公司）。
1.3 仪 器
超净工作台、CO2细胞培养箱（美国Thermo Fisher公司）；高速冷冻离心机（德国Eppendorf公司）；酶标仪（杭州奥盛仪器有限公司）；转膜仪、实时荧光定量PCR仪（美国Bio-Rad公司）；Novo Cyte流式细胞仪（美国艾森生物公司）；数码倒置相差荧光显微镜（日本Nikon公司）。
2. 方 法
2.1 细胞培养
用含10% FBS的DMEM培养基培养Hepa1-6与iBMDM细胞。将细胞放入37 ℃、5% CO2的细胞培养箱中培养。
2.2 细胞模型
以1∶1的比例分别在培养板和Transwell小室中接种iBMDM细胞和Hepa1-6细胞，培养3 d后得到TAMs。用TAMs上清液刺激Hepa1-6细胞，研究TAMs浸润对该细胞的影响。实验中，TOFA和ND-630终浓度分别为10 μmol/L和10 nmol/L。
2.3 脂滴检测
BODIPY：用2 mmol/L BODIPY493/503染料（1∶
1000 ）对Hepa1-6细胞染色，室温避光孵育20 min，PBS洗涤，进行流式分析。油红染色：参照油红O染色试剂盒说明书。
2.4 免疫印迹试验
细胞中加入蛋白裂解液，用BCA法进行定量，蛋白变性后，用8% SDS-PAGE凝胶进行电泳，转移至PVDF膜上，用5% BSA室温封闭1 h，Acetyl Coenzyme A Carboxylase Rabbit pAb、GAPDH-HRP conjugated过夜封闭，TBST洗脱6次共30 min。HRP标记的山羊抗兔IgG（H+L）室温封闭1 h，TBST洗脱，ECL化学发光法显影，化学发光仪拍照。
2.5 甘油三酯含量检测
使用试剂盒检测甘油三酯含量，详细实验步骤参考该说明书。
2.6 siRNA干扰
将Hepa1-6按每孔1×106个的密度接种于12孔细胞培养板中，待细胞贴壁后进行转染。
2.7 ACC1 CRISPR-cas9质粒构建
使用在线工具（https://benchling.com）设计靶向鼠源ACC1不同外显子区域的gRNA，序列如下：
Table 1. Sequence for guide RNA (gRNA) about acetyl-CoA carboxylase alpha (ACC1)gRNA Forward primer Reverse primer gRNA-1 CACCGAATGCATGCGATCTATCCGT AAACACGGATAGATCGCATGCATTC gRNA-2 CACCGAAGTGTATCTGAGCTGACGG AAACCCGTCAGCTCAGATACACTTC gRNA-3 CACCGCAAACGTGAATGCTTGACCA AAACTGGTCAAGCATTCACGTTTGC 将Lenti-CRPISPRv2载体用BsmBⅠ酶酶切后回收。连接，转化，挑选单克隆进行扩大培养，提质粒后测序验证。后续进行慢病毒包装，侵染Hepa1-6细胞。
2.8 细胞活力检测（CCK-8法）
在96孔细胞培养板中，每孔加入200个Hepa1-6细胞，总体积为100 µL；然后按1∶10的比例加入CCK-8溶液；37 ℃孵育2~4 h；用酶标仪检测样品的吸收度（450 nm）。
2.9 细胞增殖（CFSE法）
首先用PBS清洗细胞，在
1000 r/min条件下离心4~5 min后收集细胞；向细胞中按1∶500的比例加入CFSE，然后置培养箱中孵育25~30 min；用PBS洗去未结合的CFSE并取一部分细胞置4 ℃冰箱中作为阳性对照；将实验组细胞继续避光培养48 h；最后收集细胞，用流式细胞仪对其进行检测。2.10 细胞周期检测
用75%的乙醇重悬细胞，−20 ℃过夜，离心弃去废液，PBS洗涤后重悬细胞，加入RNA裂解酶，37 ℃水浴15 min；然后加入PI染液，避光静置30 min；PBS洗涤后用流式细胞仪分析。
2.11 细胞毒性检测
使用乳酸脱氢酶（lactate dehydrogenase，LDH）细胞毒性试剂盒检测 ，实验步骤参考说明书。
2.12 细胞凋亡
使用细胞凋亡检测试剂盒进行检测，实验步骤参考说明书（A211-01）。
2.13 统计学分析
用GraphPadPrism8软件统计分析。数据以算数$\bar{x}\pm s $形式表示，组间两两比较采用Student’s t-test检验进行显著性检验。3组以上比较采用One-Way ANOVA方差分析进行显著性检验。P <0.05认为是存在显著性差异。
3. 结 果
3.1 TAMs浸润诱导Hepa1-6细胞积累LDs
将鼠肝癌细胞Hepa1-6和iBMDM进行共培养（TAMs组），使用q-PCR与流式检测未共培养或共培养后的iBMDM的表型，实验发现M2相关标志物精氨酸酶-1（arginase-1, ARG1）、血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、IL-10的mRNA水平显著升高，细胞程序性死亡配体1（programmed cell death ligand 1，PD-L1）的表达也有升高趋势（P < 0. 01，图1-A、图1-B），结果表明共培养后iBMDM偏向M2型且PD-L1表达升高。
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Figure 1. tumor-associated macrophage (TAMs) model establishmentA: q-PCR for CD206, ARG1, VEGFα, IL-10, inducible nitric oxide synthase (iNOS), interleukin-12 subunit alpha (IL12A), TNFα mRNA expression levels; B: flow cytometry was used to detect the expression of PD-L1 in TAMs*P < 0. 05,**P < 0. 01,***P < 0. 001使用浓度分别为10%、20%、30%、40%的TAMs上清液（即TAMs上清液占完全培养基的比例）刺激Hepa1-6细胞。流式结果显示，随着TAMs上清液浓度的增加，Hepa1-6细胞LDs积累量逐渐增高，且TAMs上清液含量为30%和40%时并未出现显著性差异，即使用30%的TAMs上清液即可增强Hepa1-6细胞LDs的积累（P < 0. 05，图2-A、图2-B）。当用30% TAMs上清液刺激Hepa1-6细胞1，2，3 d后，LDs积累量呈现逐渐上升趋势（P <0.05，图2-C、图2-D）。因此，构建了一个TAMs浸润诱导肝癌LDs积累细胞模型，该模型用30% TAMs上清液与Hepa1-6细胞共同培养3 d，在此过程中，Hepa1-6细胞LDs积累量增加，这些LDs积累量增加的Hepa1-6细胞被标记为LDhigh Hepa1-6。油红染色实验结果表明，TAMs上清液刺激后，Hepa1-6细胞LDs增加（图2-E）。采用q-PCR检测细胞内围脂滴蛋白家族的mRNA水平表达量，结果表明脂蛋白1（Plin1）、脂蛋白4（Plin4）、脂蛋白5（Plin5）基因表达量显著性提高（P <0.05，图2-F）。以上结果表明，TAMs浸润可以诱导Hepa1-6细胞LDs的积累。
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Figure 2. LDhigh Hepa1-6 cell model constructs and TAMs infiltration induced accumulation of LDs in hepatocellular carcinoma cellsA and B: Hepa1-6 was stimulated with different concentrations of TAMs supernatants (10%, 20%, 30%, 40%), stained with BODIPY 493/503, and LDs accumulation in Hepa1-6 was detected by flow cytometry; C and D: 30% TAMs supernatants were stimulated for 1, 2 and 3 days, stained with BODIPY 493/503 and flow cytometry was used to detect the accumulation of LDs in Hepa1-6; E: Oil Red O labelled LDs; F: q-PCR for Plin 1, Plin 2, Plin 3, Plin 4, Plin5 mRNA expression levels*P < 0. 05, **P < 0. 01,***P < 0. 001 vs control group3.2 TAMs通过分泌IL-10影响肝癌细胞积累LDs
为了深入了解TAMs如何诱导Hepa1-6细胞累积LDs，使用q-PCR对IL-10、TGF-β、白细胞介素12（IL-12）和TNF-α的mRNA表达水平进行了检测。结果显示，TAMs组IL-10和TGF-β的表达水平均较高，其中IL-10的表达水平尤其显著（P <0.001，图3-A）。为进一步探讨IL-10对Hepa1-6细胞LDs积累的影响，采用不同质量浓度（1、5和10 ng/mL）的IL-10刺激Hepa1-6细胞24 h，用BODIPY493/503标记的脂滴，流式结果显示，当IL-10质量浓度为10 ng/mL时，Hepa1-6细胞的LDs含量显著增加（P <0.001，图3-B、图3-C）。以上结果表明，肿瘤相关巨噬细胞分泌的IL-10可能是促进Hepa1-6细胞积累脂滴的重要机制之一。
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Figure 3. IL-10 secretion by TAMs induces accumulation of LDs in hepatocellular carcinoma cells A: q-PCR for IL-10, TGFβ, IL-12, TNFα mRNA expression levels; B and C: Stimulation with different concentrations of IL-10, BODIPY 493/503 staining, and flow cytometric detection of LDs accumulation in Hepa1-6, MFI: Mean fluorescence intensity*P < 0. 05,**P < 0. 01,***P < 0. 001为了进一步说明IL-10在TAMs中的作用，采用IL-10封闭抗体处理LDhigh Hepa1-6，与未处理的LDhigh Hepa1-6相比，其脂滴生成明显减少（图4-A），q-PCR结果显示，IL-10封闭后的LDhigh Hepa1-6 ACC1、Plin4、Plin5表达量有降低趋势（图4-B，P <0.05）。流式结果显示，IL-10封闭后抑制了LDhigh Hepa1-6 细胞增殖能力，促进了LDhigh Hepa1-6细胞凋亡（图4-C~图4-E）。以上结果表明，TAMs可以通过分泌IL-10促进肝癌细胞LDs的积累。
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Figure 4. IL-10 deletion affects lipid droplet formation, cell proliferation and apoptosisA: Oil Red O labelled LDs; B: q-PCR for ACC1, Plin4, Plin5 mRNA expression levels; C: CFSE staining labelling to detect the effect of IL-10 deletion on the proliferation of Hepa1-6 cells; D and E: Effects of IL-10 deletion on apoptosis in Hepa1-6 cells*P < 0. 05 ,**P < 0. 01, ***P < 0. 001 vs control group3.3 肝癌细胞中ACC1高表达且与肝癌不良预后密切相关
为了进一步探究肝癌细胞积累LDs的机制, q-PCR检测了脂质合成相关基因ACC1，FASN，酰基辅酶A合成酶长链家族成员（acyl coenzyme A synthetase long chain family, ACSL），二酯酰甘油酰基转移酶1（diacylglycerol-O-acyltransferase homolog 1, DGAT1）与酯酰基转移酶2（diacylglycerol-O-acyltransferase homolog 2, DGAT2）的表达水平。结果显示，TAMs上清液刺激后, LDhigh Hepa1-6细胞中ACC1的表达显著增高（P <0.01，图5-A）。同时，Western blot结果显示LDhigh Hepa1-6细胞中ACC1的表达水平也显著提升（图5-B）。
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Figure 5. High expression of ACC1 in LDhigh Hepa1-6 cells and implications for cancerA: q-PCR for ACC1, FASN, ACSL, DGAT1, DGAT2 mRNA expression levels; B: Western blotting for ACC1 protein levels; C: ACC1 expression levels in clinical samples of hepatocellular carcinoma compared with normal tissues; D: ACC1 expression levels in different hepatocellular carcinoma stages (stage1, stage2, stage3, stage4); E: ACC1 expression levels in different hepatocellular carcinoma grades (grade1, grade2, grade3, grade4); F: Survival curves of patients with ACC1 high expression and F: Survival curves of patients with high ACC1 expression and low ACC1 expression; G: Survival curves of patients with high ACC1 expression and low ACC1 expression in different liver cancer grades (grade1, grade2, grade3, grade4) (*P < 0. 05,**P < 0. 01,***P < 0. 001)基于以上结果，本研究利用TCGA数据库对LIHC肝癌临床样本中ACC1表达水平进行分析，结果显示，与正常组织相比，肝癌组织中ACC1的表达水平显著上调，且ACC1的表达随着HCC的恶化有增高趋势（P <0.01，图5-C~图5-E）。因此推测ACC1可能调控肝癌细胞LDs的积累。
接下来，利用TCGA数据库，对ACC1高表达与HCC患者生存期之间的关系进行研究，结果显示，ACC1表达水平与HCC患者生存呈负相关性，表明ACC1高表达不利于HCC患者生存（P <0.001，图5-F-G）。
3.4 ACC1介导LDhigh Hepa1-6细胞LDs积累
为了探究ACC1是否调控Hepa1-6细胞LDs积累，Western blot结果显示，ACC1抑制剂TOFA和ND-630能抑制ACC1表达（图6-A），与其他人报道一致。分别用BODIPY493/503和油红O标记LDs，结果显示，TOFA和ND-630能显著减少LDhigh Hepa1-6细胞中LDs的积累（P < 0. 001，图6-B~图6-D）。对Hepa1-6细胞中甘油三酯（TG）含量进行检测，结果显示，TOFA和ND-630处理后，LDhigh Hepa1-6细胞中的TG含量显著减少（P <0.05，图6-E）。
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Figure 6. ACC1 mediates the accumulation of LDs in LDhigh Hepa1-6 cellsA: Western blotting to detect ACC1 protein level; B and C: BODIPY 493/503 staining and flow cytometry to detect the accumulation of LDs in TOFA and ND-630-treated Hepa1-6 cells, MFI: Mean fluorescence intensity; D: Oil Red O labelling of LDs; E: TG content of the cells; F: q-PCR to detect the expression level of ACC1 mRNA and assess the effect of ACC1-siRNA interference; G: Western blotting to detect ACC1 expression level and assess the effect of ACC1-siRNA interference; H and I: BODIPY 493/503 staining and flow cytometry to detect the effect of siRNA interference on the accumulation of LDs in Hepa1-6, MFI: Mean fluorescence intensity (*P < 0. 05,**P < 0. 01,***P < 0. 001 vs control group)利用siRNA技术设计了3条siRNA（ACC1-siRNA-563，ACC1-siRNA-1722，ACC1-siRNA-4838），进行ACC1敲减细胞模型构建，并通过q-PCR和Western blot检测了干扰效果。结果表明，3条siRNA均能降低Hepa1-6细胞中ACC1的表达，其中ACC1-siRNA-4838效果最好，ACC1敲减效率为68%（P <0.001，图6-F~图6-G）。接下来，对LDs的积累进行检测，结果显示，ACC1-siRNA-4838干扰后，LDs积累减少（P <0.05，图6-H~图6-I）。
此外，构建了慢病毒包装的ACC1 CRISPR-cas9质粒，侵染Hepa1-6细胞敲除ACC1，Western blot验证敲除效果，结果显示敲除后ACC1明显减少（标记为ACC1-KO Hepa1-6）（图7-A）。用BODIPY 493/503标记LDs，结果显示TAMs上清刺激后，ACC1-KO Hepa1-6细胞LDs显著减少（P <0.01，图7-B、图7-C）。通过检测TG含量发现，相比于LDhigh Hepa1-6细胞，TAMs上清液刺激后，ACC1-KO Hepa1-6细胞中TG含量显著减少（P <0.01，图7-D）。
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Figure 7. ACC1 knockdown inhibits the accumulation of LDs in LDhigh Hepa1-6 cellsA: Western blotting to detect the expression level of ACC1 and evaluate the effect of ACC1 knockdown; B and C: BODIPY 493/503 staining and flow cytometry to detect the effect of ACC1 knockdown on the accumulation of LDs in Hepa1-6; D: triglyceride (TG) content of the cells, MFI: Mean fluorescence intensity*P < 0. 05,**P < 0. 01,***P < 0. 001通过以上3种方式干预ACC1（抑制剂、小干扰RNA和敲除）来研究其对Hepa1-6细胞LDs积累的影响。结果表明，抑制或敲除ACC1可以显著减少脂肪积累，表明ACC1可能是调控细胞内脂肪积累的关键分子。
3.5 ACC1调控LDhigh Hepa1-6细胞增殖
为了探究ACC1介导的LDs积累是否会促进Hepa1-6细胞恶性生物学行为，首先研究了ACC1对Hepa1-6细胞毒性以及增殖的影响。TOFA和ND-630处理24 h后，Hepa1-6细胞LDH释放显著增加，表明抑制ACC1会促进Hepa1-6细胞死亡（P <0.001，图8-A）。CCK-8结果显示，使用TOFA和ND-630处理后，LDhigh Hepa1-6细胞活力显著降低，说明抑制ACC1会降低Hepa1-6细胞增殖（P <0.001，图8-B）。相比于LDhigh Hepa1-6细胞，TAMs上清刺激后，ACC1-KO Hepa1-6细胞活力降低，表明敲除ACC1会使Hepa1-6细胞增殖能力降低（P <0.001，图8-C）。
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Figure 8. ACC1 affects Hepa1-6 cell viabilityA: Detection of LDH release to assess the toxicity of TOFA and ND-630 on Hepa1-6 cells; B: CCK-8 to detect the effect of TOFA and ND-630 on the viability of Hepa1-6 cells; C: CCK-8 to detect the effect of ACC1 knockdown on the viability of Hepa1-6 cells***P < 0. 001, **P < 0. 01, *P < 0. 05为了进一步评估肿瘤细胞增殖能力，用CFSE对Hepa1-6细胞进行荧光标记。流式结果表明TOFA和ND-630会抑制LDhigh Hepa1-6细胞的增殖（图9-A）。同时，敲除ACC1的Hepa1-6细胞增殖能力显著降低（图9-B）。细胞周期检测结果显示，TOFA和ND-630处理后，S期细胞比例增加，即抑制ACC1会阻滞细胞周期在S期（图9-C~9-D）。此外，对ACC1-KO Hepa1-6细胞周期分析显示，其S期细胞比例增加，进一步验证了抑制ACC1会阻滞细胞周期在S期（图9-E~9-F）。因此，以上结果表明ACC1调控Hepa1-6细胞的增殖能力，抑制ACC1可以将细胞周期阻滞在S期，导致肿瘤细胞增殖能力降低。
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Figure 9. ACC1 regulates Hepa1-6 cell proliferationA: CFSE staining labeling to detect the effect of TOFA and ND-630 on the proliferation of Hepa1-6 cells; B: CFSE staining labeling to detect the effect of ACC1 knockdown on the proliferation of Hepa1-6 cells; C and D: Effect of TOFA and ND-630 on the cell cycle of Hepa1-6 cells; E and F: The effect of ACC1 knockdown on the cell cycle of Hepa1-6 cells3.6 ACC1影响LDhigh Hepa1-6细胞凋亡
用AnnexinV-FITC和PI对细胞进行标记，流式分析结果显示，TOFA处理后，LDhigh Hepa1-6早期凋亡无显著差异，晚期凋亡显著增强，用ND-630处理后，细胞早期凋亡和晚期凋亡都显著增强（图10-A、图10-B）。并且与LDhigh Hepa1-6细胞相比，ACC1-KO Hepa1-6细胞早期凋亡和晚期凋亡均显著增强（图10-C、图10-D）。实验结果表明，ACC1调控Hepa1-6细胞凋亡，抑制ACC1会促进肿瘤细胞凋亡。
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Figure 10. ACC1 regulates apoptosis in Hepa1-6 cellsA and B: Effects of TOFA and ND-630 on apoptosis in Hepa1-6 cells; C and D: Effects of ACC1 knockdown on apoptosis in Hepa1-6 cells*P < 0. 05,**P < 0. 01,***P < 0. 0014. 讨 论
脂质代谢作为肝癌代谢重编程的主要标志之一，在肝癌细胞中产生更多的脂滴，以此为肝癌细胞的恶性生长与转移提供能量[13−14]。因此，对肝癌中脂滴形成的机制进行探究，有利于为肝癌的潜在靶点提供理论基础。本研究建立了iBMDM上清液诱导Hepa1-6产生脂滴的共培养模型，即iBMDM与Hepa1-6共培养3 d后，得到TAMs。TAMs上清与Hepa1-6共培养3 d后便可有效诱导肿瘤细胞的脂滴生成。并在此基础上，本研究探究了脂滴形成促进肝癌细胞恶性生物学行为的机制，旨在对肝癌的免疫治疗提供新的思路。
TAMs是大多数肿瘤中浸润最多的一类免疫细胞，通过M2表型、促进血管生成、产生基质金属蛋白酶和产生抑制性受体等机制在肿瘤发生、发展和转移中发挥关键性作用。基于清除TAMs、抑制TAMs募集和对TAMs进行重编程等手段，靶向TAMs已成为肿瘤免疫治疗的新型策略[15]。Zhang等[16]发现TAMs分泌白细胞介素6（IL-6）能够作用于胶质瘤细胞，促进胶质瘤细胞中3-磷酸肌醇蛋白激酶1（PDPK1）介导的磷酸化，进而促进了胶质瘤细胞的有氧糖酵解和肿瘤生长。使用IL-6中和抗体可以抑制经TAMs促进的胶质瘤细胞的有氧糖酵解和肿瘤生长。另外研究发现，TAMs通过摄取较高的葡萄糖，以此来增强糖胺生物合成，并伴随分泌更多的组织蛋白酶B到TME中，进而促进肿瘤转移和化疗耐药性[17]。然而，关于TAMs如何调控肿瘤细胞脂质合成的报道较少。本研究发现，TAMs通过释放IL-10可促进Hepa1-6细胞的脂滴囤积，进而促进肿瘤的生长。IL-10可通过IL-10-DDIT4-mTOR通路影响细胞的糖脂代谢，但巨噬细胞分泌的IL-10如何影响肝癌细胞的脂滴形成，仍需做进一步研究。
肝癌细胞中，脂肪酸主要通过FA合成产生。ACC1是脂肪酸从头合成第一步的限速酶，是脂质生物合成的关键代谢酶[18−19]。本研究发现LDhigh Hepa1-6细胞中的ACC1表达水平显著升高。同时，TCGA数据表明，肝癌患者肿瘤组织中ACC1的表达水平也显著提高，且ACC1的高表达与HCC患者的不良预后有密切关系。在本研究中，抑制ACC1表达后，LDhigh Hepa1-6细胞中LDs积累量显著减少，表明ACC1是调控LDs积累的关键分子。
LDs在维持脂质稳态、调节细胞应激、蛋白质处理等方面发挥着至关重要的作用。本研究采用了3种模型：ACC1抑制剂TOFA、ND-630，以及ACC1 siRNA干扰和ACC1 CRISPR-cas9敲除，对ACC1进行抑制，旨在探讨其在肝癌细胞恶性行为中的影响。实验结果显示，抑制ACC1可有效减少LDs的积累，抑制LDhigh Hepa1-6细胞的增殖，并促进细胞凋亡的发生。这些结果表明，ACC1是调控LDs积累的关键因子。研究表明，ACC1在肝癌细胞中的表达水平通常较高，与肝癌的发生和发展密切相关[20]。ACC1在肝癌细胞中扮演着重要角色，一是通过合成脂肪酸满足能量需求，二是调节脂滴形成，维持肝癌细胞生存和增殖[21]。此外，在肝癌前期，由于细胞受到损伤或炎症刺激，ACC1的表达水平可能会逐渐上调[22]。在肝癌发展过程中，ACC1的表达和活性可能会进一步增加，以满足肿瘤细胞快速增殖和生长的需求[23]。然而，在肝癌晚期，肿瘤细胞可能会经历代谢异常，导致脂质代谢紊乱和脂滴形成受到抑制[24]。在这种情况下，ACC1的表达和活性可能会下降，因为肿瘤细胞对脂肪酸的需求减少。这种变化可能与肝癌细胞的代谢适应和生长状态密切相关。因此，ACC1在不同肝癌病理阶段的表达和活性变化可能反映了肝癌细胞代谢适应性的调节和肿瘤生长状态的变化。总之，ACC1在肝癌细胞中的作用机制及其潜在的临床意义值得深入研究，有望为肝癌的治疗提供新的思路和方法。
综上所述，本研究揭示了TAMs能促进肝癌细胞的脂滴形成，且TAMs释放的IL-10可能会通过调控ACC1的表达在此过程中发挥作用。肝癌细胞通过ACC1介导的脂质从头合成在其增殖和凋亡等恶性生物学中扮演了关键作用。本研究确认，ACC1是肝癌细胞脂质代谢和恶性行为调控的关键因子，其抑制不仅可以减少TAMs促进的肝癌细胞脂滴积累，还可以抑制肝癌细胞的增殖并诱导凋亡，显示了其作为治疗靶点的潜力。
本刊编委徐寒梅教授团队在Signal Transduction and Targeted Therapy上对核酸药物的最新研究及
未来发展进行总结与展望近日，中国药科大学江苏省合成多肽药物发现与评价工程研究中心徐寒梅教授团队在Signal Transduction and Targeted Therapy（IF=40.8）杂志发表题为“Nucleic acid drugs: recent progress and future perspectives”的综述文章。本文以孙小艺、Sarra Setrerrahmane、李臣诚、胡加亮为主要作者，徐寒梅教授为通信作者。中国药科大学为该论文第一通信单位。 全文链接：https://doi.org/10.1038/s41392-024-02035-4 -
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Figure 1. GC-IMS topographic three-dimensional plot of C.paliurus extract qql-jg:Qingqianliu-jingao
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Figure 2. GC-IMS topographic plot of C. paliurus extract (top viewport)RIP:Reactive ion peak
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Figure 3. GC-IMS fingerprint of volatile components of C. paliurus extract Each row represents the signal peak of one sample while each column represents the same volatile component in different samples. Colors represent the content of a volatile compound, and the brighter the color is, the higher the content
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Figure 4. Thermogravimetry(TG) and derivative thermogravimetry(DTG) curves of C.paliurus extract
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Figure 5. TGA-GC/MS of C.paliurus extract at 200 ℃(A), 360 ℃(B), 440 ℃(C),respectively
Table 1 Qualitative results of volatile compounds in C. paliurus extract
Type Compound CAS Formula RI RT/s DT/s Relative content/% Acids Acetic acid-M C64197 C2H4O2 1506.9 1184.727 1.058 3.14 Acids Acetic acid-D C64197 C2H4O2 1515.8 1201.702 1.165 1.26 Alcohols Propylene glycol C57556 C3H8O2 1713.8 1579.779 1.113 0.69 Alcohols Linalool C78706 C10H18O 1543.1 1253.848 1.207 0.82 Alcohols (Z)-2-Penten-1-ol C1576950 C5H10O 1330.8 848.484 0.944 8.55 Alcohols 3-Methyl-1-butanol C123513 C5H12O 1218.3 664.611 1.238 0.60 Alcohols 1-Penten-3-ol C616251 C5H10O 1167.8 579.743 0.949 6.19 Alcohols 1- Butanol-M C71363 C4H10O 1152.7 551.423 1.182 0.83 Alcohols 1- Butanol-D C71363 C4H10O 1152.2 550.596 1.387 0.38 Alcohols 2-Methyl-1-propanol C78831 C4H10O 1102.4 457.152 1.372 2.53 Alcohols 1-Propanethiol C107039 C3H8S 861.7 251.243 1.165 0.96 Aldehydes (2E,4E)-2,4-Heptadienal C4313035 C7H10O 1500.5 1172.396 1.213 1.25 Aldehydes (2E,4E)-2,4-Hexadienal-M C142836 C6H8O 1443.2 1063.027 1.129 2.56 Aldehydes (2E,4E)-2,4-Hexadienal-D C142836 C6H8O 1444.9 1066.292 1.452 6.41 Aldehydes (E)-2-Hexenal-M C6728263 C6H10O 1231.5 684.312 1.184 1.85 Aldehydes (E)-2-Hexenal-D C6728263 C6H10O 1230.5 682.852 1.514 0.31 Aldehydes 3-Methyl-2-butenal-M C107868 C5H8O 1212.5 655.855 1.093 1.00 Aldehydes 3-Methyl-2-butenal-D C107868 C5H8O 1214.4 658.774 1.362 1.07 Aldehydes (E)-2-Pentenal-M C1576870 C5H8O 1124.0 497.672 1.110 7.42 Aldehydes (E)-2-Pentenal-D C1576870 C5H8O 1123.1 496.018 1.361 1.20 Aldehydes 1-Hexanal-M C66251 C6H12O 1101.0 454.671 1.271 1.23 Aldehydes 1-Hexanal-D C66251 C6H12O 1102.4 457.152 1.560 0.28 Aldehydes 1-Pentanal C110623 C5H10O 969.8 317.399 1.185 1.03 Aldehydes 2-Methyl butanal C96173 C5H10O 930.6 293.417 1.399 0.81 Aldehydes Butanal C123728 C4H8O 850.9 244.628 1.114 0.73 Aldehydes Acrolein C107028 C3H4O 872.5 257.859 1.055 1.22 Esters Ethyl 2-hydroxypropanoate C97643 C5H10O3 1352.6 889.996 1.150 0.52 Esters Ethyl 2-oxopropanoate C617356 C5H8O3 1253.0 716.417 1.149 0.77 Esters Ethyl acetate C141786 C4H8O2 903.6 276.879 1.334 0.43 Ketones 2-Methyl-2-hepten-6-one C110930 C8H14O 1342.5 870.827 1.179 0.42 Ketones 1-Hydroxy-2-propanone C116096 C3H6O2 1307.9 804.705 1.230 0.73 Ketones Cyclohexanone-M C108941 C6H10O 1294.5 779.167 1.155 1.87 Ketones Cyclohexanone-D C108941 C6H10O 1292.9 776.248 1.456 0.26 Ketones 2-Hexanone C591786 C6H12O 1062.7 408.362 1.198 1.39 Ketones 2,3-Pentadione C600146 C5H8O2 1043.1 386.035 1.307 2.13 Ketones 1-Penten-3-one C1629589 C5H8O 1006.0 343.861 1.302 0.82 Ketones 2-Butanone C78933 C4H8O 918.5 285.975 1.243 1.82 Others 2,6-Dimethylpyrazine C108509 C6H8N2 1320.5 828.783 1.134 0.40 Others 2-Methylpyrazine-M C109080 C5H6N2 1308.3 805.434 1.069 1.70 Others 2-Methylpyrazine-D C109080 C5H6N2 1307.9 804.705 1.398 6.75 Others β-Pinene C127913 C10H16 1123.5 496.845 1.223 0.62 Others 2-Propenenitrile C107131 C3H3N 992.8 331.457 1.088 6.50 RI:Retention index; RT:Retention time;DT:Drift time 
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Table 2 Pyrolysis products of C.paliurus extract at 200 ℃,360 ℃,and 440 ℃
RT/s Compound CAS Match R.Match Relative content/% 200 ℃ 360 ℃ 440 ℃ 2.528 Acetic acid 64-19-7 898 941 7.222 ND ND 2.557 2-Methyl-furan 534-22-5 735 810 ND 9.426 ND 2.863 2,4-Hexadiene 5194 -51-4886 886 6.653 ND ND 3.152 2-Propanone, 1-hydroxy- 116-09-6 844 905 6.395 ND ND 3.546 2,3-Pentanedione 600-14-6 848 848 1.792 ND ND 3.704 Furan, 2,5-dimethyl- 625-86-5 821 838 ND 2.405 ND 4.739 Toluene 108-88-3 866 883 ND 2.317 3.991 5.192 Cyclopentanone 120-92-3 849 859 ND 0.393 ND 5.381 Heptane,2,4-dimethyl 2213 -23-2865 865 ND ND 1.058 5.504 3(2H)-Furanone, dihydro-2-methyl- 3188 -00-9800 800 0.854 ND ND 5.986 2-Cyclopenten-1-one 930-30-3 787 912 ND 2.244 ND 6.028 Furfural 98-01-1 878 889 23.993 ND ND 6.422 Furfuryl alcohol 98-00-0 907 907 10.180 ND ND 6.575 Ethylbenzene 100-41-4 863 919 ND ND 0.977 6.704 2-Propanone, 1-(acetyloxy)- 592-20-1 847 855 0.990 ND ND 6.763 p-Xylene 106-42-3 935 937 ND 3.699 4.945 6.963 4-Cyclopentene-1,3-dione 930-60-9 886 886 2.101 ND ND 7.192 o-Xylene 95-47-6 870 906 ND ND 1.436 7.357 Nonane 111-84-2 854 854 ND ND 1.117 7.404 2-Cyclopenten-1-one, 2-methyl- 1120 -73-6802 849 ND 0.871 ND 7.498 Ethanone, 1- (2-furanyl)- 1192 -62-7843 843 ND 2.015 ND 7.792 p-Benzoquinone 106-51-4 821 839 ND 5.066 1.061 8.375 Benzene, propyl- 103-65-1 907 907 ND ND 0.202 8.504 2-Furancarboxaldehyde, 5-methyl- 620-02-0 916 921 4.019 ND ND 8.51 Benzene, 1-ethyl-2-methyl- 611-14-3 914 930 ND ND 1.368 8.569 Benzene, 1-ethyl-4-methyl- 622-96-8 877 885 ND ND 1.326 8.663 Benzene,1,2,4-trimethyl- 95-63-6 912 912 ND ND 0.764 8.769 Phenol 108-95-2 953 973 ND 10.862 4.216 9.192 3,3,5-Trimethyl-1,5-heptadiene 74630 -29-8807 838 ND 0.804 ND 9.604 Mesitylene 108-67-8 847 920 ND 2.104 2.331 9.68 Benzene, 1-methyl-3-(1-methylethyl)- 535-77-3 800 811 ND 2.122 0.515 9.774 D-Limonen 5989 -27-5916 924 ND 2.818 ND 9.816 Benzyl alcohol 100-51-6 800 800 2.343 1.990 ND 10.022 Benzene, 1-ethynyl-4-methyl- 766-97-2 863 894 ND ND 0.142 10.092 Phenol,2-methyl- 95-48-7 928 928 ND 0.669 2.042 10.227 Benzene, 1,4-diethyl- 105-05-5 839 859 ND ND 0.921 10.38 Acetophenone 98-86-2 854 854 ND 0.367 ND 10.445 p-Cresol 106-44-5 917 919 ND 3.687 3.714 10.586 2 5-Furandicarboxaldehyde 823-82-6 918 937 3.093 ND ND 10.598 1-Ethyl-2,4-dimethylbenzene 874-41-9 872 872 ND ND 0.580 10.698 Mequinol 150-76-5 850 923 ND 1.648 ND 10.798 Benzene, 1-methyl-4-(1-methylethenyl)- 1195 -32-0832 895 ND 0.815 ND 10.939 Undecane 1120 -21-4900 900 ND ND 0.705 11.033 Phenol, 2,5-dimethyl- 95-87-4 800 875 ND ND 0.706 11.104 Benzofuran, 2-methyl- 4265 -25-2881 881 ND ND 0.294 11.292 Benzene, 1,2,4,5-tetramethyl- 95-93-2 916 916 ND ND 0.735 11.457 Phenol, 2-ethyl- 90-00-6 808 866 ND ND 0.370 11.651 Phenol, 2,4-dimethyl- 105-07-9 843 928 ND ND 2.307 11.927 Phenol, 4-ethyl- 123-07-9 901 964 ND 1.406 ND 12.492 α-Terpineol 98-55-2 866 877 3.324 1.254 12.715 Benzofuran, 2,3-dihydro- 496-16-2 800 872 ND 1.552 ND 12.81 Benzofuran,4,7-dimethyl 28715 -26-6808 808 ND ND 0.591 12.845 5-Hydroxymethylfurfural 67-47-0 745 786 2.141 ND ND 13.404 Naphthalene,1,2-dihydro-4-methyl- 4373 -13-1843 879 ND ND 0.316 13.433 Hydroquinone 123-31-9 893 908 ND 5.873 1. 402 13.645 Cinnamaldehyde, (E)- 14371 -10-9892 910 3.759 1.733 ND 13.78 1H-Indene,2,3-dihydro-4,7-dimethyl- 6682 -71-9809 835 ND ND 0.753 13.98 Tridecane 629-50-5 858 900 ND ND 0.884 14.086 2-Propen-1-ol, 3-phenyl- 104-54-1 939 939 12.898 6.157 ND 14.41 1H-Indene,2,3-dihydro-1,1,5-trimethyl- 40650 -41-7829 831 ND ND 1.408 15.08 2-Propadienylmesitylene 29555 -07-5800 912 ND ND 0.517 15.351 Tetradecane 629-59-4 816 907 ND ND 1.113 15.456 Diphenyl ether 101-84-8 831 856 0.447 0.254 ND 15.562 Naphthalene, 1,8-dimethyl- 569-41-5 830 869 ND ND 1.117 15.692 Ethanone, 1-(3-hydroxyphenyl)- 121-71-1 909 909 0.193 ND ND 15.792 Naphthalene, 2,6-dimethyl- 581-42-0 872 928 ND ND 1.261 16.574 Benzene, 1,2-dimethoxy-4-propenyl-, (E)- 6379 -72-2871 894 0.919 1.740 ND 16.815 1-Naphthalenol 96-15-3 800 816 ND 0.253 ND 17.056 Naphthalene, 2-ethoxy- 93-18-5 938 947 2.280 1.707 ND 17.515 Naphthalene, 1,6,7-trimethyl- 2245 -38-7914 929 ND ND 2.292 17.845 Naphthalene,2,3,6-trimethyl- 829-26-5 892 898 ND ND 1.703 18.156 (+)-Cedrol 77-53-2 817 834 ND 1.461 ND 19.521 Tetramethylnaphthalene 2717 -39-7851 851 ND ND 0.596 20.509 Neophytadiene 504-96-1 800 800 ND 0.286 ND
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