Role of peripheral clock genes in the progression， prevention， and treatment of nonalcoholic steatohepatitis

Siyan LIU; Jingyi TIAN; Yuyang HUANG; Tianqi GU; Mingyue DENG; Pan YANG

doi:10.12449/JCH241222

Volume 40 Issue 12

Dec. 2024

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Abstract

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Article Navigation > Journal of Clinical Hepatology > 2024 > 40(12): 2505-2512

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Role of peripheral clock genes in the progression， prevention， and treatment of nonalcoholic steatohepatitis

DOI: 10.12449/JCH241222

1.
International Medical College，Chongqing Medical University，Chongqing 401331，China
2.
Emergency Department，The Second Affiliated Hospital of Chongqing Medical University，Chongqing 400000，China

Research funding:

Natural Science Foundation of Chongqing (cstc2020jcyj-bshX0068);

2021 Chongqing Municipal Middle and Young Age High-end Talent Project (yxgdrc20210101);

2022 National Level Innovation Training Project (202210631001)

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Corresponding author: YANG Pan， tmmu_yp@126.com （ORCID： 0000-0003-3962-6281）
Received Date: 2024-04-07
Accepted Date: 2024-05-23
Published Date: 2024-12-25

Abstract

Abstract

As a severe clinical manifestation of nonalcoholic fatty liver disease， nonalcoholic steatohepatitis （NASH） is characterized by lipid deposition and inflammatory damage in the liver. At present， clinical medications for NASH are still in the exploratory phase， and it is urgent to make progress. Recent studies have shown that the pathogenesis of NASH is associated with circadian rhythm disorders in the liver， with the specific manifestation of dysregulated expression of liver clock genes such as BMAL1， which increases hepatic lipogenesis， reduces fatty acid oxidation， and activates pro-inflammatory factors. Therefore， improving circadian rhythm of the liver and regulating the expression of liver clock genes are feasible strategies for the prevention and treatment of NASH. Currently， some medications for NASH via activating the proteins encoded by clock genes have been applied in animal experiments， for example， the REVERB full-agonist SR9009 can inhibit the development of liver inflammation， which confirms the possibility of NASH treatment by targeting the proteins encoded by clock genes. This article summarizes the role of hepatic clock genes in regulating lipid metabolism and the development and progression of inflammation in the liver and elaborates on the recent advances in medications targeting clock genes and the proteins encoded by clock genes， in order to provide new targets for the treatment of NASH.
- Non-alcoholic Fatty Liver Disease,
- Circadian Rhythm,
- CLOCK Proteins

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肝星状细胞（HSC）作为肝细胞癌（HCC）微环境中最主要的窦周细胞，在HCC的发生发展中发挥了重要作用^［1-2］。体外研究^［3-4］表明，在HSC与HCC细胞的共培养体系中，HSC通过分泌生长因子、细胞外基质蛋白和金属基质蛋白酶诱导肿瘤细胞向恶性表型转化，同时增强肝癌细胞的侵袭与迁移能力。在皮下移植异种裸鼠肝癌模型^［5］中发现，HSC通过促进HCC细胞增殖和抑制细胞凋亡，从而促进肿瘤生长。研究^［6-7］还发现，HSC通过分泌血管内皮生长因子、血小板源性生长因子和转化生长因子等生长因子，促进肿瘤的血管生成。硫化氢（hydrogen sulfide， H₂S）是继一氧化碳和一氧化氮后第3种重要的内源性气体信号分子，参与氧化应激、细胞增殖与凋亡、血管扩张、血管再生、炎症等多种病理生理过程^［8］。内源性H₂S由半胱氨酸和同型半胱氨酸经胱硫醚γ-裂解酶（cystathionine γ-lyase， CSE）、胱硫醚β-合酶（cystathionine β-synthase， CBS）及3-巯基丙酮酸转硫酶（3-mercapto-pyruvate sulphurtransferase， MPST）合成产生^［9］。前期研究^［10］发现，外源性H₂S供体——NaHS，通过PI3K/Akt/mTOR信号通路诱导肝癌细胞自噬，从而促进了细胞凋亡，抑制细胞增殖与迁移。然而，在肝癌微环境中HSC是否通过H₂S参与调控HCC的生物学过程尚不明确。因此，本研究通过HSC与肝癌细胞系共培养，探讨HSC通过分泌H₂S参与调控肝癌细胞凋亡的作用及其机制。

1. 材料与方法

1.1 材料

DMEM细胞培养基、胎牛血清、胰蛋白酶、青霉素-链霉素、CSE抑制剂——炔丙基甘氨酸（propargylglycine， PPG）及JNK/JunB抑制剂——SP600125试剂购自赛默飞世尔科技（中国）有限公司；H₂S检测ELISA试剂盒购自南京金益柏生物科技有限公司；RNA提取、逆转录及SYBR等RT-qPCR试剂购自宝日医生物技术（北京）有限公司；Transwell小室购自美国康宁公司；CCK-8试剂盒购自美国Abmole奥默生物公司；Annexin V-FITC/PI细胞凋亡试剂盒购自上海翊圣生物科技有限公司；人源β- actin、GAPDH、JNK、JunB、p-JNK/JunB抗体和二抗、ChIP试剂盒购自美国Cell Signaling technology公司；肿瘤坏死因子超家族成员-14（tumor necrosis factor superfamily member-14，TNFSF14）多克隆抗体购于美国Affinity Biosciences公司。

1.2 细胞培养

肝星状细胞系（LX-2）及肝癌细胞系（HepG2、PLC/PRF/5），用含10%胎牛血清及1%的青霉素-链霉素混合液的DMEM培养基，置于37 ℃，5%（体积分数）CO₂恒温细胞孵育箱内培养。Transwell小室建立共培养体系，上室接种LX-2，下室接种HepG2或PLC/PRF/5，按照说明书操作。所有细胞实验重复3次。

1.3 ELISA定量测定H₂S

LX-2产生的H₂S浓度及NaHS释放的H₂S，按照说明书进行操作。在酶标板对应的孔中分别加入标品及待检测的LX-2培养上清液和NaHS，随后进行孵育和洗板，加入显色液37 ℃孵育15 min，加入终止液终止反应。酶标仪450 nm波长测定，根据标准曲线，计算所测样本中H₂S浓度。

1.4 RNA提取、二代测序及RT-q PCR

Trizol法提取总RNA。取1 μg总RNA，按照Takara逆转录试剂盒合成cDNA，SYBR Green Master Mix说明书配置20 μL RT PCR体系，ABI 7500进行RT-q PCR。所需的引物序列见表1。LX-2与HepG2共培养（LX-2共培养组）、NaHS处理HepG2（NaHS处理组）及对照组（单独HepG2培养）提取的HepG2细胞总RNA送至北京诺禾致源科技股份有限公司进行转录组二代测序分析，对差异基因［以|log2（fold change）|≥1为差异表达基因的标准］进行聚类分析和Veen分析。

表 1 RT-qPCR和ChIP-PCR所需要的引物序列

Table 1. Primers for Real-Time PCR and ChIP-PCR

项目	寡核苷酸序列
RT-qPCR引物
TNFSF14	F：5'-CGTGAGACCTTCGCTCTTGTAT-3'
	R：5'-CCCTCAGTGTTTGTGGTGGAT-3'
CSE	F：5'-AAGACGCCTCCTCACAAGGT-3'
	R：5'-ATATTCAAAACCCGAGTGCTGG-3'
GAPDH	F：5'-TGAAGGTCGGAGTCAACGGA-3'
	R：5'-CCTGGAAGATGGTGATGGGAT-3'
CBS	F：5'-AATGGTGACGCTTGGGAA-3' R：5'-TGAGGCGGATCTGTTTGA-3'
MPST	F：5'-GACCCCGCCTTCATCAAG-3' R：5'-CATGTACCACTCCACCCA-3'
ChIP-qPCR引物
TNFSF14	F：5'-TTGTTCATTGCTGCATCCCC-3'
	R：5'-CTCCTCTTCTTCCGGTACCC-3'

下载: 导出CSV

| 显示表格

1.5 蛋白提取及Western Blot （WB）检测

取LX-2共培养组及NaHS处理组的HepG2细胞，PBS清洗2次，随后在10 cm²培养皿中加入1 mL RAPI裂解液，冰上裂解20 min，转移裂解液到EP管中，4 ℃，12 000×g离心30 min，上清即为细胞总蛋白。BCA试剂盒对提取的蛋白进行定量分析，加入对应体积的蛋白上样缓冲液后置于100 ℃金属浴5 min，使蛋白变性，用于后续WB检测。SDS-PAGE胶用于分析所提取的蛋白，取20 μg蛋白上样电泳，转膜，牛奶封闭，随后加入对应的抗体4 ℃孵育过夜，将孵育好的膜放到对应的二抗中，室温孵育1 h后，加入化学发光液测定和分析。

1.6 CCK-8检测细胞活力

将生长状态良好的细胞接种在96孔板，LX-2分别与HepG2、PLC/PRF/5共培养1、2、3、4天，单独HepG2、PLC/PRF/5培养分别作为对照组。按照说明书，每孔加入10 μL CCK-8试剂，随后置于37 ℃培养箱内孵育1 h后，酶标仪450 nm测定吸光度值。细胞活力计算：细胞活力（%）=［A₄₅₀（样本－空白）］/［A₄₅₀（对照－空白）］×100%。

1.7 流式细胞术测定细胞凋亡

将处理好的细胞制备成为单细胞悬液，按照Annexin V-FITC/PI细胞凋亡试剂盒说明书，加入5 μL Annexin V孵育，再加入5 μL PI避光孵育，FACS流式细胞仪（BD公司）进行检测。

1.8 细胞免疫荧光染色

细胞均匀接种到已放入无菌盖玻片的24孔板中，NaHS处理HepG2细胞0、1、2、8、24 h。取处理好的细胞，加入1 mL 4%多聚甲醛固定，随后进行透膜处理，加入1% BSA稀释抗体p-JunB（1∶500），4 ℃孵育过夜。1% BSA稀释FITC标记的荧光二抗（1∶500）避光条件下，室温孵育1 h。DAPI染核及封片，荧光显微镜下采集图像，Image J软件分析目的蛋白平均免疫荧光强度。

1.9 染色质免疫共沉淀（chromatin immunoprecipitation， ChIP）

LX-2分别与HepG2和PLC/PRF/5共培养，以及NaHS分别处理HepG2和PLC/PRF/5细胞后，ChIP方法测定p-JunB与TNFSF14启动子结合力。WB测定HepG2和PLC/PRF/5细胞核中p-JunB表达，应用4'，6-二脒基-2-苯基吲哚（4'，6-diamidino-2-phenylindole，DAPI）对细胞核DNA染色（蓝色）和FITC标记的抗体测定细胞核p-JunB表达（绿色）。LX-2与NaHS处理后的HepG2和PLC/PRF/5细胞用4%多聚甲醛固定10 min，核酸酶将DNA特异性的消化为DNA片段，随后加入JunB、p-JunB抗体，从而特异性的富集目的蛋白质和DNA复合体，通过对复合体进行富集纯化，收集DNA用于PCR检测。

1.10 统计学方法

采用SPSS 21.0统计软件进行数据分析。计量资料以x¯±s表示，两组间比较采用成组t检验；多组间比较采用单因素方差分析或重复测量方差分析，进一步两两比较采用Dunnett-t检验。P<0.05为差异有统计学意义。

2. 结果

2.1 LX-2通过CSE产生H₂S

LX-2培养上清液中H₂S浓度随着时间延长逐渐增加［（22.89±0.08）pg/mL vs （28.29±0.15）pg/mL vs （36.19±1.90） pg/mL，F=79.63，P<0.05］（图1a）。LX-2中CSE mRNA水平显著高于CBS mRNA和MPST mRNA（1.008±0.13 vs 0.320±0.014 vs 0.05±0.02， F=80.84，P<0.05）（图1b）。在LX-2中加入不同剂量PPG后，随着PPG浓度增加，H₂S浓度下降（P<0.05）（图1c）。PPG特异性、浓度依赖性的降低CSE mRNA及蛋白水平（图1d、e）。

注： a，LX-2细胞产生H₂S的浓度；b，CSE、CBS和MPST的mRNA在LX-2细胞中的表达倍数；c，PPG处理后LX-2产生H₂S的浓度；d，CSE的mRNA表达；e，CSE蛋白的表达水平。

图 1 LX-2通过CSE途径产生H₂S

Figure 1. LX-2 produce H₂S mainly via the CSE

下载: 全尺寸图片幻灯片

2.2 LX-2抑制肝癌细胞活力、促进凋亡

LX-2分别与HepG2、PLC/PRF/5共培养，随着培养时间延长，HepG2（87.48%±0.82% vs 70.48%±0.641% vs 52.89%±0.57% vs 45.20%±0.69%，F=1 517.13，P<0.001）和PLC/PRF/5（92.41%± 0.48% vs 74.10%±0.73% vs 53.70%±0.60% vs 44.00%± 0.27%，F=2 626.21，P<0.001）细胞活力降低（图2a、b）；凋亡增加（HepG2：12.88%±0.64% vs 15.5%±0.16% vs 18.43%±0.37% vs 13.01%±0.58%，F=142.15，P<0.001；PLC/PRF/5：8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30，F=676.40，P<0.001），第3天最显著（图2c、d）。

注： a，HepG2细胞活力；b，PLC/PRF/5细胞活力；c，HepG2细胞凋亡；d，PLC/PRF/5细胞凋亡。a、c，对照组为单独HepG2培养；b、d，对照组为单独PLC/PRF/5培养。与第1天比较，*P<0.05。

图 2 LX-2与肝癌细胞系共培养对肝癌细胞活力及凋亡的影响

Figure 2. Effect of LX-2 co-culture with hepatoma cell on viability and apoptosis

下载: 全尺寸图片幻灯片

2.3 内源性和外源性H₂S调控肝癌细胞多种基因

LX-2与HepG2共培养及NaHS处理HepG2后，转录组测序显示分别上调1 047和1 183个基因，共同上调659个基因；分别下调997和1 144个基因，共同下调342个基因（图3a）。其中，30个变化最大的基因，包括TGM2、PDE2A、AQP7P1、SOCS3、B3GNT3、LGALS7B、CPNE7、TCAF2、AXL、CD3D、BEAN1、KIF26B、SLC1A7、TFF1、IGFBP3、ADAMTS16、ACTA1、SLC2A5、SERPINE2、KCNMB4、LSP1、A4GALT、TNFSF14、NPPB、MUC5B、NKAIN4、TRAF5、PFKP、EHD2、MTMR11（图3b）。选择促凋亡基因TNFSF14进行分析，与对照组相比，NaHS处理组TNFSF14基因增加约2.8倍，LX-2共培养组TNFSF14基因增加约2倍（P<0.05）（图3c）。

注： a，聚类分析；b，差异倍数值变化最大的30个差异基因；c，TNFSF14基因表达变化。对照组，单独HepG2培养。

图 3 RNA-seq分析内源性和外源性H₂S对HepG2细胞转录基因的影响

Figure 3. RNA-seq to analyse the transcriptome of endogenous and exogenous of H₂S on HepG2

下载: 全尺寸图片幻灯片

2.4 H₂S通过激活JNK/JunB信号通路调控TNFSF14转录

ChIP实验发现，与对照组相比，NaHS处理的HepG2和PLC/PRF/5细胞中p-JunB与TNFSF14基因转录调控区域结合增加（图4a）；p-JunB定位于细胞核中，NaHS作用1 h荧光强度开始升高，8 h达到高值（图4b）；且与WB结果一致（图4c）。PPG干预后，HepG2和PLC/PRF/5胞核蛋白p-JunB表达明显下降（图4d）；经SP600125（JNK/JunB抑制剂）处理后，显示JNK/JunB-TNFSF14信号通路被抑制。NaHS处理也显示了相似结果（图4e）。延长NaHS处理时间，细胞核p-JunB表达增加，但JunB表达无明显变化。结果提示，LX-2和NaHS通过释放H₂S，激活JNK/JunB信号通路调控TNFSF14基因转录。

注： a，ChIP显示，在HepG2和PLC/PRF/5细胞中，p-JunB与TNFSF14启动子结合力增加；b，NaHS分别处理HepG2和PLC/PRF/5 0、1、2、8、24 h后，HepG2和PLC/PRF/5细胞核中p-JunB表达及核转位，DAPI（蓝色）和FITC（绿色）分别为对细胞核DNA及p-JunB进行染色；c，NaHS不同处理时间后，HepG2和PLC/PRF/5细胞核与细胞质中p-JunB的表达；d，NaHS处理组和LX-2共培养组HepG2及PLC/PRF/5细胞的p-JunB蛋白变化；e，PPG和SP600125抑制了H₂S及NaHS对HepG2与PLC/PRF/5中JNK/p-JNK、JunB/p-JunB、TNFSF14蛋白水平。

图 4 内源性H₂S和NaHS通过激活JNK/JunB信号通路调控TNFSF14转录

Figure 4. Endogenous H₂S and NaHS upregulated TNFSF14 via JNK/JunB signaling pathway

下载: 全尺寸图片幻灯片

3. 讨论

第3个重要的内源性气体信号分子H₂S自1992年被发现，较多研究^［11-12］证实了H₂S及其合成酶的多种功能，参与了血管扩张、血管生成、炎症免疫、氧化应激和细胞凋亡与自噬的调控等过程，在肿瘤和非肿瘤性疾病，特别是心血管疾病的病理生理中具有重要作用。因此，H₂S及其合成酶可能是治疗的靶点。但是，CSE/H₂S在肝癌发生发展中的作用研究较少^［13］。由肝细胞及肝窦周细胞，包括HSC、肝窦内皮细胞、Kuffer细胞、肝癌细胞和细胞外基质等组成的复杂肿瘤微环境（tumor microenvironment， TME）中，HSC及信号分子H₂S促进肝癌细胞凋亡的机制尚未阐明^［14］。本研究发现，HSC主要通过CSE产生信号分子H₂S，激活了肝癌细胞中的JNK/JunB信号通路，上调TNFSF14基因，TNFSF14表达增加，从而促进了肝癌细胞的凋亡，发挥抗肿瘤作用。Han等^［15］发现，HepG2中TNFSF14下调Bcl-2和survivin，通过线粒体及p53独立途径，诱导肝癌细胞凋亡。体外研究^［16］表明，在HepG2和SMMC-7721细胞中，TNFSF14与LT-βR结合，激活caspase-9和caspase-3，抑制STAT3磷酸化下调Bcl-XL表达，进而诱导肝癌细胞凋亡，提示线粒体途径也是TNFSF14诱导细胞凋亡的关键因素。由此可见，TNFSF14通过线粒体凋亡途径和死亡受体凋亡途径等，促进肝癌细胞凋亡。此外，TNFSF14还表达于免疫细胞，如活化T淋巴细胞，未成熟树突状细胞和活化自然杀伤细胞，TNFSF14信号转导对T淋巴细胞激活和诱导细胞凋亡具有重要作用^［17］。TNFSF14也通过激活抗肿瘤免疫效应，间接杀伤肿瘤细胞和触发肿瘤细胞凋亡，表现出强大的免疫抗肿瘤活性，在多种不同的肿瘤中发挥抗瘤作用^［18］。因此推测，在肝癌微环境中，H₂S通过激活肝癌细胞中凋亡基因TNFSF14表达及多种细胞凋亡信号途径，促进肝癌细胞凋亡。

TNFSF14诱导肝癌细胞凋亡的分子机制尚不清楚。本研究ChIP实验证明，无论是在HepG2还是在PLC/PRF/5中，转录因子p-JunB均可与TNFSF14基因的转录调控区域结合，且H₂S增强了p-JunB与TNFSF14基因启动子的结合能力。H₂S生成酶CSE抑制剂——PPG和p-JNK抑制剂SP600125，均显著抑制了H₂S对JNK/JunB信号通路的激活，导致TNFSF14表达降低。上述结果提示H₂S在调节肝癌细胞TNFSF14表达中具有重要作用。细胞免疫荧光和WB也证明了外源性H₂S供体NaHS及HSC合成的内源性H₂S，促进了肝癌细胞p-JunB的表达及核转位。JunB作为Jun家族的重要成员之一，是转录因子AP-1（转录激活蛋白-1）的主要成分，对各种刺激如辐射、应激及生长信号等作出生理或病理应答，参与细胞增殖与分化、转化、凋亡、炎症等过程，在肿瘤的形成、转移和侵袭中发挥重要作用^［19］。也有研究^［20］发现，JNK/JunB促进HepG2凋亡并抑制了其增殖与侵袭能力。因此，本研究首次阐明了TNFSF14启动子的转录活性受到转录因子JunB的调控，H₂S可激活转录因子JunB，促进p-JunB的核转位，进而上调肝癌细胞凋亡基因TNFSF14。CSE/H₂S可能是防治HCC具有潜力的新靶点。

总之，在TME中活化HSC合成分泌信号分子H₂S，通过激活肝癌细胞中的JNK/JunB-TNFSF14信号通路，从而促进肝癌细胞凋亡、抑制肿瘤细胞活力，可能是HSC在TME中调节肝癌细胞生物学功能的新机制之一。

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