转录组测序筛选大鼠滑膜炎差异表达基因及秦皮素治疗靶点的体外验证

doi:10.12300/j.issn.1674-5817.2022.100

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (1): 11-20.DOI: 10.12300/j.issn.1674-5817.2022.100

• 人类疾病动物模型：药学专题 • 上一篇下一篇

转录组测序筛选大鼠滑膜炎差异表达基因及秦皮素治疗靶点的体外验证

杨凌¹(), 庄迪², 金立伦¹()()

^1.上海交通大学医学院附属新华医院中医科, 上海 200092
^2.上海交通大学医学院附属新华医院麻醉科, 上海 200092

收稿日期:2022-07-04 修回日期:2022-10-02 出版日期:2023-02-25 发布日期:2023-03-09
通讯作者: 金立伦（1967—），男，硕士，主任医师，研究方向：膝骨关节炎的发病机制与中药干预。E-mail：jinlilun@xinhuamed.com.cn。ORCID： 0000-0001-9840-836X
作者简介:杨凌（1996—），女，硕士研究生，研究方向：膝骨关节炎的发病机制与中药干预。E-mail： yangll4141@163.com
基金资助:
上海市科学技术委员会科研计划项目“消瘀散新组方治疗KOA的临床规范化研究”(19401932000);上海市进一步加快中医药传承创新发展三年行动计划项目“北部区域中医骨关节病专病联盟建设”［ZY（2021-2023）-03-02-25］

Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin

Ling YANG¹(), Di ZHUANG², Lilun JIN¹()()

^1.Department of Traditional Chinese Medicine, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^2.Department of Anesthesiology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received:2022-07-04 Revised:2022-10-02 Published:2023-02-25 Online:2023-03-09
Contact: JIN Lilun (ORCID: 0000-0001-9840-836X), E-mail: jinlilun@xinhuamed.com.cn

摘要/Abstract

摘要：

目的基于转录组测序分析碘乙酸钠（monosodium iodoacetate，MIA）诱导的膝骨关节炎（knee osteoarthritis，KOA）大鼠相比于正常组大鼠滑膜组织中的表达差异基因，并初步筛选出秦皮素（fraxetin）治疗滑膜炎的靶点。方法将SD大鼠分为KOA模型组和对照组，采用MIA膝关节注射法制备大鼠右膝KOA模型。造模4周后，取各组大鼠右膝滑膜组织进行转录组测序，获得了KOA模型组与对照组大鼠滑膜组织中mRNA差异表达情况，然后进行差异基因表达分析、GO富集分析、KEGG功能富集分析和PPI蛋白网络互作分析。同时将巨噬细胞Raw264.7分为对照组、脂多糖（lipopolysaccharide，LPS）干预组和LPS+60 μmol/L秦皮素联合干预组，并采用实时荧光定量PCR法对转录组测序后的差异基因分析结果进行验证。结果转录组测序后的差异基因表达分析显示，KOA模型组与对照组相比，共有1 730个上调基因和1 546个下调基因，其中显著上调的基因有mmp12、Acod1、Acan、Col2a1和Atp6v0d2等［|log2（FoldChange）|≥1，校正后P＜0.01］。KEGG聚类分析和GO聚类分析显示，差异基因主要参与调节炎症及免疫代谢过程，例如三羧酸循环、线粒体功能。LPS干预后的Raw264.7细胞中Acod1与Atp6v0d2显著高表达（P＜0.000 1），而且相较于LPS干预组，LPS+秦皮素联合干预后的Raw264.7细胞中Atp6v0d2表达水平显著降低（P＜0.001）。结论 MIA诱导KOA建模后，大鼠膝关节滑膜组织中介导炎症及免疫代谢的巨噬细胞相关基因mmp12、Acod1和Atp6v0D2等高表达，提示KOA的发生、发展过程中可能存在着由滑膜巨噬细胞介导的免疫代谢改变。LPS干预后巨噬细胞Raw264.7中Acod1和Atp6v0d2表达增高可初步证实这一结果。其中Atp6v0D2可能是秦皮素治疗滑膜炎的一个潜在靶点，这为KOA治疗提供了新的思路。

关键词: 骨关节炎, 转录组测序, 免疫代谢, 滑膜巨噬细胞, 秦皮素

Abstract:

Objective Using transcriptome sequencing to screen the differentially expressed genes between the synovial tissue of rats with knee osteoarthritis (KOA) induced by monosodium iodoacetate (MIA) and that of normal rats, and then screen the target of fraxetin in the treatment of synovitis. Methods SD rats were divided into KOA group and the negative control (NC) group. Rat right knee KOA model was prepared by MIA knee joint injection in KOA group and none treatments in NC group. Four weeks after modeling, the right knee synovial tissue of rats in each group was taken for transcriptome sequencing. Then the differential gene expression analysis, GO enrichment analysis, KEGG function enrichment analysis and PPI protein network interaction analysis were performed. The synovial macrophage Raw264.7 cells were divided into the control group, lipopolysaccharide (LPS) intervention group and LPS+60 μmol/L fraxetin intervention group, then RNA-sequencing results were verified by qRT-PCR in the three groups. Results The results of differential gene-expression analysis showed that there were 1 730 up-regulated genes and 1 546 down-regulated genes in the KOA group compared with the control group, among which the significantly up-regulated genes were mmp12, Acod1, Acan, Col2a1, Atp6v0d2 (|log2(FoldChange)|≥1, adjusted P<0.01). KEGG cluster analysis and GO cluster analysis showed that differential genes were mainly involved in the regulation of inflammation and immune metabolism, such as tricarboxylic acid cycle and mitochondrial function. The expressions of Acod1 and Atp6v0d2 in Raw264.7 cells after LPS intervention were significantly higher. Compared with the LPS intervention group, the expression level of Atp6v0d2 in Raw264.7 cells after LPS+fraxetin combined intervention was significantly lower. Conclusion After modeling KOA induced by MIA, macrophage-related genes mmp12, Acod1 and Atp6v0D2, which mediate inflammation and immune metabolism, were highly expressed in the synovial tissue of rats, suggesting that there might be immune metabolism changes mediated by synovial macrophages during the occurrence and development of KOA. The increased expression of Acod1 and Atp6v0d2 in macrophages Raw264.7 after LPS intervention can preliminarily confirm this result. Among them, Atp6v0d2 may be a potential target of fraxetin in the treatment of synovitis, which provides a new idea for KOA treatment.

Key words: Osteoarthritis, Transcriptome sequencing, Immunometabolism, Synovial macrophage, Fraxetin

中图分类号:

Q95-33

杨凌, 庄迪, 金立伦. 转录组测序筛选大鼠滑膜炎差异表达基因及秦皮素治疗靶点的体外验证[J]. 实验动物与比较医学, 2023, 43(1): 11-20.

Ling YANG, Di ZHUANG, Lilun JIN. Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin[J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 11-20.

图/表 6

图1 样品间基因表达量相关性检验（A）和主成分分析（B）注：A为相关性检验，相关系数越接近1表明样本相似性越高，图中对照组（NC）与膝骨关节炎模型组（OA）的各组内系数均接近于1，表明组内更为相近而组间差异较为显著。B为主成分（PC）分析，图中同一分组中的样本聚在一起，表明测序数据质量可靠。

Figure 1 Gene expression correlation analysis （A） and principal components analysis （B） between the samplesNote： A, showed the correlation test, the closer the correlation coefficient is to 1, the higher the sample similarity is. In the negative control group (NC) and knee osteoarthritis model group (OA), the intra-group samples are more similar than inter-group samples. B, performed a principal component (PC) analysis, samples from the same group packed together in the figure, which indicates that the sequencing data are of reliable quality.

表1 碘乙酸钠诱导的膝骨关节炎模型大鼠滑膜组织中显著上调基因和显著下调基因

Table 1 Genes up-regulated and down-regulated in synovial tissues of knee osteoarthritis model rats iduced by monosodium iodoacetate

差异Difference	基因Gene	log2FC	P值P value	校正P值P_adj value
上调Up-regulated	Mmp12	9.933 625 864	9.132 91×10^-11	3.537 83×10^-9
	Acod1	9.272 106 280	0.000 127 507	0.000 802 842
	Col2a1	9.153 847 197	7.690 69×10^-9	1.807 62×10^-7
	Atp6v0d2	8.282 894 807	5.603 80×10^-6	5.607 99×10^-5
	Fbn2	7.973 227 345	2.921 38×10^-37	3.387 43×10^-34
	Acan	7.718 643 189	4.203 11×10^-20	1.015 34×10^-17
	Ocstamp	7.591 893 578	0.001 876 497	0.007 726 778
	LOC103690326	7.546 217 455	7.903 40×10^-5	0.000 529 726
	Cxcl2	7.422 908 965	1.713 86×10^-5	0.000 145 198
	Mmp9	7.188 248 072	2.374 04×10^-11	1.027 150×10^-9
下调Down-regulated	Plekha2	-1.000 555 825	1.696 39×10^-5	0.000 143 999
	Nudt7	-1.001 123 965	5.602 09×10^-5	0.000 395 268
	Cdc42bpg	-1.002 206 901	0.000 106 010	0.000 684 033
	Mtus1	-1.002 219 197	0.000 139 053	0.000 863 768
	Ppp1r37	-1.002 289 468	4.948 870×10^-20	1.179 120×10^-17
	Oard1	-1.003 517 453	0.000 375 535	0.002 019 066

图2 表达差异基因火山图（A）和聚类热图（B）注：A，横坐标为基因表达倍数变化，纵坐标为表达量变化差异的统计显著性，红色点和绿色点分别表示上调与下调基因，而无显著性差异表达的基因用蓝点表示。B，横坐标为样品名称，纵坐标为基因名称，红色表示高表达基因，绿色表示低表达基因。NC，对照组；OA，膝骨关节炎模型组。

Figure 2 Volcano plots （A） and heat map （B） of differentially expressed genesNote：A， the abscissa is log2FC， the ordinate is -log10（adjusted P）. Red or green dots indicate significantly up-regulated and down-regulated genes， respectively. While， genes with no significantly differentially expressed are indicated with blue dots. B， Gene heat map， the abscissa is sample names， the ordinate is gene names， red indicates high gene expression， while green indicates low expression. NC， negative control group； OA， knee osteoarthritis model group.

图3 表达差异基因的GO富集分析（A~C）和KEGG通路富集分析（D）注：A~C分别为GO富集分析的3个本体“分子功能”（A）、“细胞组分”（B）和“生物过程”（C）。横坐标为富集倍数，纵坐标为富集条目，图中气泡大小表示富集于条目上的基因数，越大则表示富集的基因数目越多。颜色表示对应P值的大小，越红则P值越显著。

Figure 3 Gene ontology （GO） enrichment analysis （A-C） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis （D） of differentially expressed genesNote：A-C，GO enrichment analysis showed three ontologies “molecular function”（A）， “cellular component”（B） and “biological process”（C）. The abscissa is Fold Enrichment， the ordinate is term， the size of the bubbles indicates the number of term-enriched genes. The bigger the bubble is， the more genes enriched under the term. The colors of different bubbles correspond to P-value， the increasingly reddish colors means the more significant of P-value.

图4 差异表达基因的蛋白网络互作图注：圆点大小代表节点（node）的度（degree），与此节点连接数目越多，节点越大；点内填充红色代表该节点基因表达量上调，绿色代表该节点基因表达量下调。

Figure 4 The protein-protein interaction （PPI） networks of differentially expressed genesNote：The size of the points means the degree of the node. The more protein connects with the node, the bigger the node will become. The red notes mean the up-regulated genes, the green ones mean down-regulated genes.

图5 实时荧光定量PCR检测脂多糖（LPS）和秦皮素联合干预后巨噬细胞RAW264.7中Atp6v0d2和Acod1基因的表达情况注：与未处理对照组（Control）相比，****P<0.000 1；与LPS干预组相比，###P<0.001。ns表示差异无统计学意义。n=3。

Figure 5 Atp6v0d2 and Acod1 gene expression in synovial macrophage RAW264.7 cells treated with lipopolysaccharide （LPS） and Fraxetin detected by real-time fluorescence quantitative PCRNote：Compared with the control group，****P<0.000 1； Compared with the LPS group， ###P<0.001. ns indicated none statistically significant differences. n=3。

参考文献 24

1	VINA E R, KWOH C K. Epidemiology of osteoarthritis: literature update[J]. Curr Opin Rheumatol, 2018, 30(2):160-167. DOI: 10.1097/BOR.0000000000000479 .
2	ABRAMOFF B, CALDERA F E. Osteoarthritis: pathology, diagnosis, and treatment options[J]. Med Clin North Am, 2020, 104(2):293-311. DOI: 10.1016/j.mcna.2019.10.007 .
3	EYMARD F, PIGENET A, CITADELLE D, et al. Knee and hip intra-articular adipose tissues (IAATs) compared with autologous subcutaneous adipose tissue: a specific phenotype for a central player in osteoarthritis[J]. Ann Rheum Dis, 2017, 76(6):1142-1148. DOI: 10.1136/annrheumdis-2016-210478 .
4	NEOGI T, GUERMAZI A, ROEMER F, et al. Association of joint inflammation with pain sensitization in knee osteoarthritis: the multicenter osteoarthritis study[J]. Arthritis Rheumatol, 2016, 68(3):654-661. DOI: 10.1002/art.39488 .
5	KEMBLE S, CROFT A P. Critical role of synovial tissue-resident macrophage and fibroblast subsets in the persistence of joint inflammation[J]. Front Immunol, 2021, 12:715894. DOI: 10.3389/fimmu.2021.715894 .
6	LOPES E B P, FILIBERTI A, HUSAIN S ALI, et al. Immune contributions to osteoarthritis[J]. Curr Osteoporos Rep, 2017, 15(6):593-600. DOI: 10.1007/s11914-017-0411-y .
7	马振源, 莫蕙. 中医贴敷治疗膝骨关节炎的研究进展[J]. 南京中医药大学学报, 2020, 36(6):926-930. DOI: 10.14148/j.issn.1672-0482.2020.0926 .
	MA Z Y, MO H. Research progress of traditional Chinese medicine sticking therapy in the treatment of knee osteoarthritis[J]. J Nanjing Univ Tradit Chin Med, 2020, 36(6):926-930. DOI: 10.14148/j.issn.1672-0482.2020.0926 .
8	侯成志, 李秋月, 魏戌, 等. 独活寄生汤治疗膝骨关节炎的研究[J]. 中国中医基础医学杂志, 2021, 27(11):1843-1846. DOI: 10.19945/j.cnki.issn.1006-3250.2021.11.036 .
	HOU C Z, LI Q Y, WEI X, et al. Study on Duhuo Jisheng Decoction in the treatment of knee osteoarthritis[J]. J Basic Chin Med, 2021, 27(11):1843-1846. DOI: 10.19945/j.cnki.issn.1006-3250.2021.11.036 .
9	王倩, 杨凯, 马强, 等. 中医药治疗膝骨关节炎研究进展[J]. 陕西中医药大学学报, 2022, 45(3):130-134. DOI: 10.13424/j.cnki.jsctcm.2022.03.029 .
	WANG Q, YANG K, MA Q, et al. Research progress on treatment of knee osteoarthritis with traditional Chinese medicine[J]. J Shaanxi Univ Chin Med, 2022, 45(3):130-134. DOI: 10.13424/j.cnki.jsctcm.2022.03.029 .
10	HUSSAIN S M, NEILLY D W, BALIGA S, et al. Knee osteoarthritis: a review of management options[J]. Scott Med J, 2016, 61(1):7-16. DOI: 10.1177/0036933015619588 .
11	WANG Q, ZHUANG D, FENG W, et al. Fraxetin inhibits interleukin-1β-induced apoptosis, inflammation, and matrix degradation in chondrocytes and protects rat cartilage in vivo [J]. Saudi Pharm J, 2020, 28(12):1499-1506. DOI: 10.1016/j.jsps.2020.09.016 .
12	金灵璐, 朱尉东, 仲卫红, 等. 升麻素苷治疗膝骨关节炎的作用机制研究[J]. 风湿病与关节炎, 2021, 10(12):45-48.
	JIN L L, ZHU W D, ZHONG W H, et al. On the mechanism of cimicifugin in the treatment of knee osteoarthritis[J]. Rheum Arthritis, 2021, 10(12):45-48.
13	高嘉美, 潘赐明, 郗域江, 等. 当归治疗骨关节炎作用机制的研究进展[J]. 风湿病与关节炎, 2021, 10(11):77-80.
	GAO J M, PAN C M, XI Y J, et al. Research progress on the mechanism of Angelica sinensis in the treatment of osteoarthritis[J]. Rheum Arthritis, 2021, 10(11):77-80.
14	DESHMUKH V, O'GREEN A L, BOSSARD C, et al. Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment[J]. Osteoarthritis Cartilage, 2019, 27(9): 1347-1360. DOI: 10.1016/j.joca.2019. 05. 006 .
15	LI Z H, HUANG Z Y, ZHANG H, et al. P2X7 receptor induces pyroptotic inflammation and cartilage degradation in osteoarthritis via NF-κB/NLRP3 crosstalk[J]. Oxidative Med Cell Longev, 2021, 2021:1-16. DOI: 10.1155/2021/8868361 .
16	ZHENG L L, ZHANG Z J, SHENG P Y, et al. The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis[J]. Ageing Res Rev, 2021, 66: 101249. DOI: 10.1016/j.arr.2020.101249 .
17	MOBASHERI A, RAYMAN M P, GUALILLO O, et al. The role of metabolism in the pathogenesis of osteoarthritis[J]. Nat Rev Rheumatol, 2017, 13(5):302-311. DOI: 10.1038/nrrheum.2017.50 .
18	朱璐丹. 秦皮素对嗜水气单胞菌的抗感染机制研究[D].上海:上海海洋大学, 2020. DOI: 10.27314 /d.cnki.gsscu. 2020.000028 .
	ZHU L D. Anti-infective mechanism of fraxetin against aeromonas hydrophila[D]. Shanghai: Shanghai Ocean University, 2020. DOI: 10.27314 /d.cnki.gsscu. 2020.000028 .
19	曹桂云, 宁波, 秦金淼, 等. 基于标准汤剂的秦皮(白蜡树)配方颗粒质量标准分析[J/OL]. 中国实验方剂学杂志:1-11(2022-11-22)[2023-02-22]. .
	CAO G Y, NING B, QIN J M, et al. Quality standard analysis of fraxinus fraxinus dispensing granules based on standard decoction[J/OL]. Chin J Exp Formul: 1-11(2022-11-22)[2023-02-22]. .
21	WU R, CHEN F, WANG N, et al. ACOD1 in immunometabolism and disease[J]. Cell Mol Immunol, 2020, 17(8):822-833. DOI: 10.1038/s41423-020-0489-5 .
22	GRIFFIN T M, SCANZELLO C R. Innate inflammation and synovial macrophages in osteoarthritis pathophysiology[J]. Clin Exp Rheumatol, 2019, 37 Suppl 120(5):57-63.
23	RU L B. Synovium and the innate inflammatory network in osteoarthritis progression[J]. Curr Rheumatol Rep, 2013, 15(5):323. DOI: 10.1007/s11926-013-0323-5 .
24	XIA Y, LIU N, XIE X X, et al. The macrophage-specific V-ATPase subunit ATP6V0D2 restricts inflammasome activation and bacterial infection by facilitating autophagosome-lysosome fusion[J]. Autophagy, 2019, 15(6):960-975. DOI: 10.1080/15548627.2019.1569916 .

[1]	张渊, 李晗, 张成芳. 奥氮平诱导体重增加小鼠的全脑转录组学分析[J]. 实验动物与比较医学, 2023, 43(3): 262-270.
[2]	崔若琳, 王庆, 杨玲, 冯文昌, 刘智伟, 滕蔚然, 马碧涛, 王乐杨, 秦丽萍, 金立伦. 消瘀散新组方修复膝骨关节炎模型兔的软骨损伤及MMP-13表达[J]. 实验动物与比较医学, 2023, 43(1): 30-38.
[3]	郑杰, 张国忠, 胡艳梅, 伍学一, 杨宗富, 字向东. 基于RNA-Seq技术的犏牛囊胚冷冻前后单核苷酸多态性和可变剪切分析[J]. 实验动物与比较医学, 2020, 40(4): 279-.
[4]	王慧, 卓书洪, 高雨. 胡椒根提取物对大鼠骨关节炎模型的治疗作用[J]. 实验动物与比较医学, 2017, 37(1): 64-67.

转录组测序筛选大鼠滑膜炎差异表达基因及秦皮素治疗靶点的体外验证

Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 24

相关文章 4

编辑推荐

Metrics

本文评价