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CN117959437A - Application of SHBG gene as target spot in inhibiting macrophage polarization in rheumatoid arthritis - Google Patents

  • ️Fri May 03 2024
Application of SHBG gene as target spot in inhibiting macrophage polarization in rheumatoid arthritis Download PDF

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Publication number
CN117959437A
CN117959437A CN202410099112.0A CN202410099112A CN117959437A CN 117959437 A CN117959437 A CN 117959437A CN 202410099112 A CN202410099112 A CN 202410099112A CN 117959437 A CN117959437 A CN 117959437A Authority
CN
China
Prior art keywords
shbg
expression
polarization
macrophages
macrophage
Prior art date
2024-01-24
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202410099112.0A
Other languages
Chinese (zh)
Inventor
蔡维维
文嫄媛
侯豹
邱丽颖
孙海建
朱雪雪
张仕杰
赵晨阳
孟鑫雨
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Jiangnan University
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Jiangnan University
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2024-01-24
Filing date
2024-01-24
Publication date
2024-05-03
2024-01-24 Application filed by Jiangnan University filed Critical Jiangnan University
2024-01-24 Priority to CN202410099112.0A priority Critical patent/CN117959437A/en
2024-05-03 Publication of CN117959437A publication Critical patent/CN117959437A/en
Status Pending legal-status Critical Current

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Abstract

本发明公开了SHBG基因作为靶点在抑制类风湿关节炎中巨噬细胞极化的应用，属于生物药和分子生物学技术领域。本发明通过转录组测序以及荧光共聚焦，确定了M1型极化的巨噬细胞中SHBG蛋白的高表达，进一步通过抑制SHBG的表达，实现了对巨噬细胞M1型极化的抑制，同时抑制了免疫因子iNOS、IL‑6的表达水平。本发明提供的技术方案在关节炎治疗中存在广泛应用前景。

The present invention discloses the application of SHBG gene as a target in inhibiting macrophage polarization in rheumatoid arthritis, and belongs to the technical field of biological medicine and molecular biology. The present invention determines the high expression of SHBG protein in M1 polarized macrophages through transcriptome sequencing and fluorescence confocal, and further inhibits the expression of SHBG to achieve the inhibition of M1 polarization of macrophages, and simultaneously inhibits the expression levels of immune factors iNOS and IL-6. The technical solution provided by the present invention has broad application prospects in the treatment of arthritis.

Description

SHBG基因作为靶点在抑制类风湿关节炎中巨噬细胞极化的 应用Application of SHBG gene as a target in inhibiting macrophage polarization in rheumatoid arthritis

技术领域Technical Field

本发明涉及SHBG基因作为靶点在抑制类风湿关节炎中巨噬细胞极化的应用，属于生物药和分子生物学技术领域。The invention relates to the application of SHBG gene as a target in inhibiting macrophage polarization in rheumatoid arthritis, and belongs to the technical field of biological medicine and molecular biology.

背景技术Background technique

类风湿关节炎(RA)是一种自身免疫性疾病，会引起关节疼痛、肿胀和骨侵蚀。类风湿关节炎在全球的发病率均较高，且可发生于任何年龄阶段。在病变组织部位，巨噬细胞浸润，其中促炎型M1细胞被大量激活，因其M1巨噬细胞能够释放出大量炎症因子，可使关节炎症持久存在，因此，基于抑制M1巨噬细胞激活开发新的靶点，设计新的靶向性药物，为RA的药物治疗提供了新的思路，也代表了关节炎症治疗策略的新方向。细胞代谢是巨噬细胞激活、功能和生物学的关键调节因素，其中脂质代谢对巨噬细胞功能有显著的控制作用。Rheumatoid arthritis (RA) is an autoimmune disease that causes joint pain, swelling, and bone erosion. The incidence of RA is high worldwide and can occur at any age. In the affected tissue, macrophages infiltrate, among which pro-inflammatory M1 cells are activated in large numbers. Because M1 macrophages can release a large number of inflammatory factors, they can cause persistent joint inflammation. Therefore, the development of new targets and the design of new targeted drugs based on inhibiting M1 macrophage activation provide new ideas for the drug treatment of RA and represent a new direction for the treatment of joint inflammation. Cell metabolism is a key regulator of macrophage activation, function, and biology, among which lipid metabolism has a significant control effect on macrophage function.

性激素结合球蛋白(SHBG)是血浆中性类固醇激素的主要转运结合蛋白，体内参与类固醇反应、脂质代谢等作用，而SREBP1(固醇调节元件结合蛋白1)是调控脂质合成的重要转录调控因子，有研究表明SREBP1诱导的脂肪酸合成耗尽巨噬细胞的抗氧化防御以调控巨噬细胞表型转化。文献Sex hormone-binding globulin and arthritis:a Mendelianrandomization study发现关节炎与患者SHBG蛋白水平存在关联。文献Salivarytestosterone in postmenopausal women with rheumatoid arthritis报道了以糖皮质激素治疗类风湿关节炎时，患者SHBG蛋白水平出现下降。但文献Bioavailabletestosterone in men with rheumatoid arthritis—high frequency of hypogonadism的研究表明类风湿关节炎患者SHBG水平没有明显变化。现有技术中没有SHBG如何参与RA发展进程的报道。Sex hormone binding globulin (SHBG) is the main transport binding protein of sex steroid hormones in plasma, and is involved in steroid response, lipid metabolism and other functions in vivo. SREBP1 (sterol regulatory element binding protein 1) is an important transcriptional regulator that regulates lipid synthesis. Studies have shown that SREBP1-induced fatty acid synthesis depletes macrophage antioxidant defense to regulate macrophage phenotypic transformation. The literature Sex hormone-binding globulin and arthritis: a Mendelian randomization study found that arthritis is associated with SHBG protein levels in patients. The literature Salivary testosterone in postmenopausal women with rheumatoid arthritis reported that when rheumatoid arthritis was treated with glucocorticoids, the SHBG protein level of patients decreased. However, the study of the literature Bioavailable testosterone in men with rheumatoid arthritis—high frequency of hypogonadism showed that there was no significant change in SHBG levels in patients with rheumatoid arthritis. There is no report in the prior art on how SHBG is involved in the development of RA.

发明内容Summary of the invention

为了解决现有技术的不足，本发明通过敲低SHBG基因表达，有效降低了M1巨噬细胞极化比例，进而抑制了炎症分子的分泌，本发明所述方案在类风湿关节炎的预防和/或治疗中存在显著的应用前景。In order to address the deficiencies of the prior art, the present invention effectively reduces the polarization ratio of M1 macrophages by knocking down SHBG gene expression, thereby inhibiting the secretion of inflammatory molecules. The scheme of the present invention has significant application prospects in the prevention and/or treatment of rheumatoid arthritis.

本发明提供了性激素结合球蛋白或其编码基因作为靶点在制备用于调控巨噬细胞M1极化的药物中的应用。The present invention provides the use of sex hormone binding globulin or its encoding gene as a target in the preparation of a drug for regulating macrophage M1 polarization.

在一种实施方式中，所述药物通过上调性激素结合球蛋白或其编码基因的表达，从而抑制固醇调节元件结合蛋白1或其编码基因的表达，进而促进巨噬细胞M1极化；或者，所述药物通过抑制性激素结合球蛋白或其编码基因的表达，从而促进固醇调节元件结合蛋白1或其编码基因的表达，进而抑制巨噬细胞M1极化。In one embodiment, the drug inhibits the expression of sterol regulatory element binding protein 1 or its encoding gene by up-regulating the expression of sex hormone binding globulin or its encoding gene, thereby promoting the polarization of macrophages M1; alternatively, the drug inhibits the expression of sex hormone binding globulin or its encoding gene, thereby promoting the expression of sterol regulatory element binding protein 1 or its encoding gene, thereby inhibiting the polarization of macrophages M1.

在一种实施方式中，所述应用包括：性激素结合球蛋白或其编码基因的激活剂在制备用于治疗和/或缓解激活巨噬细胞M1极化相关疾病的药物中的应用。In one embodiment, the application includes: use of sex hormone binding globulin or an activator of its encoding gene in the preparation of a drug for treating and/or alleviating diseases related to activated macrophage M1 polarization.

在一种实施方式中，所述应用包括：性激素结合球蛋白或其编码基因的抑制剂在制备用于治疗和/或缓解抑制巨噬细胞M1极化相关疾病的药物中的应用。In one embodiment, the application includes: use of an inhibitor of sex hormone binding globulin or a gene encoding it in the preparation of a drug for treating and/or alleviating diseases related to the inhibition of macrophage M1 polarization.

在一种实施方式中，所述抑制巨噬细胞M1极化相关疾病包括类风湿关节炎。In one embodiment, the disease associated with the inhibition of macrophage M1 polarization includes rheumatoid arthritis.

在一种实施方式中，所述抑制剂包括但不限于干扰RNA、基因敲除试剂或化学抑制剂。作为优选的例子，没有限定，所述抑制剂可以是siRNA。In one embodiment, the inhibitor includes but is not limited to interfering RNA, gene knockout reagents or chemical inhibitors. As a preferred example, without limitation, the inhibitor can be siRNA.

在一种实施方式中，所述siRNA含有以下(a)-(b)任一组所示的核苷酸序列：In one embodiment, the siRNA contains the nucleotide sequence shown in any one of the following groups (a)-(b):

(a)SHBG si-RNA-1：(a) SHBG si-RNA-1:

F：5’GACGCUGGAUAGAGUCAAATT 3’；F: 5′GACGCUGGAUAGAGUCAAATT 3′;

R：5’UUUGACUCUAUCCAGCGUCTT 3’；R: 5’UUUGACUCUAUCCAGCGUCTT 3’;

(b)SHBG si-RNA-2：(b) SHBG si-RNA-2:

F：5’CUCUGGAGCUAGGAUUUAATT 3’；F: 5′CUCUGGAGCUAGGAUUUAATT 3′;

R：5’UUAAAUCCUAGCUCCAGAGTT 3’。R: 5’UUAAAUCCUAGCUCCAGAGTT 3’.

在一种实施方式中，所述药物的剂型可以是医学上可接受的常规剂型，作为优选的例子，没有限定，可以是注射液、注射用冻干粉针、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂或口服液。In one embodiment, the dosage form of the drug can be a medically acceptable conventional dosage form, and as a preferred example, without limitation, it can be an injection, a lyophilized powder for injection, a suspension, an implant, an embolic agent, a capsule, a tablet, a pill or an oral solution.

在一种实施方式中，所述药物中含有药用辅料。In one embodiment, the drug contains pharmaceutical excipients.

在一种实施方式中，所述药用辅料包括溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂中的至少一种。In one embodiment, the pharmaceutical excipients include at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, and a wetting agent.

在一种实施方式中，所述药用辅料还包含渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、絮凝剂与反絮凝剂、助滤剂或释放阻滞剂中的至少一种。In one embodiment, the pharmaceutical excipients further include at least one of an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrator, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickener, a inclusion agent, a humectant, a flocculant and a deflocculating agent, a filter aid or a release retardant.

在一种实施方式中，所述药物中含有药用载体。In one embodiment, the drug contains a pharmaceutically acceptable carrier.

在一种实施方式中，所述药用载体选自微囊、微球、纳米粒或脂质体。In one embodiment, the pharmaceutically acceptable carrier is selected from microcapsules, microspheres, nanoparticles or liposomes.

本发明还提供了一种体外调节巨噬细胞M1极化水平的方法，其特征在于，所述方法包括将有效量的性激素结合球蛋白或其编码基因的激活/抑制剂与细胞接触，从而调节M1巨噬细胞极化水平。The present invention also provides a method for regulating the polarization level of M1 macrophages in vitro, characterized in that the method comprises contacting an effective amount of sex hormone binding globulin or an activator/inhibitor of its encoding gene with cells, thereby regulating the polarization level of M1 macrophages.

有益效果：Beneficial effects:

(1)本发明通过对经LPS和IFN-γ诱导的活化的巨噬细胞进行转录组测序以及免疫荧光共聚焦分析，结果显示活化的巨噬细胞中SHBG基因及蛋白水平出现了显著上调。进一步通过免疫共沉淀实验分析，表明活化的巨噬细胞中SHBG基因的上调进一步导致了SREBP1基因表达的下调，从而抑制了SREBP1下游脂肪酸代谢合成通路。(1) The present invention performs transcriptome sequencing and immunofluorescence confocal analysis on activated macrophages induced by LPS and IFN-γ, and the results show that the SHBG gene and protein levels in activated macrophages are significantly upregulated. Further immunoprecipitation experiments show that the upregulation of the SHBG gene in activated macrophages further leads to the downregulation of SREBP1 gene expression, thereby inhibiting the fatty acid metabolism synthesis pathway downstream of SREBP1.

(2)通过siRNA抑制LPS和IFN-γ诱导的活化的巨噬细胞中的SHBG基因表达水平后，SREBP1基因以及蛋白表达水平上调了50％，SREBP1基因下游的脂肪酸合酶FASN、SCD1、SCD2、Fads1、Fads2基因表达水平上调约50-60％。并且，通过siRNA抑制SHBG基因表达水平后，免疫因子iNOS、IL-6的表达的蛋白以及mRNA表达量也下调了约50％，此外，巨噬细胞的M1极化表型CD86分子表达量下调了约20％。表明SHBG基因可作为靶点，通过抑制SHBG基因表达，可有效巨噬细胞M1极化，进而抑制炎症以及免疫反应的发生，达到治疗类风湿关节炎的目的。(2) After inhibiting the SHBG gene expression level in activated macrophages induced by LPS and IFN-γ by siRNA, the SREBP1 gene and protein expression levels were upregulated by 50%, and the expression levels of fatty acid synthases FASN, SCD1, SCD2, Fads1, and Fads2 downstream of the SREBP1 gene were upregulated by about 50-60%. In addition, after inhibiting the SHBG gene expression level by siRNA, the protein and mRNA expression levels of immune factors iNOS and IL-6 were also downregulated by about 50%. In addition, the expression level of CD86 molecules, the M1 polarization phenotype of macrophages, was downregulated by about 20%. This shows that the SHBG gene can be used as a target. By inhibiting the expression of the SHBG gene, the M1 polarization of macrophages can be effectively inhibited, thereby inhibiting the occurrence of inflammation and immune response, and achieving the purpose of treating rheumatoid arthritis.

(3)通过构建关节炎(CIA)大鼠模型，验证了CIA大鼠中存在SHBG基因的活化、巨噬细胞M1极化以及免疫因子TNF-α、IL-6、IL-10的上调，进一步印证了本发明提供的SHBG基因表达抑制策略在治疗类风湿关节炎中的应用前景。(3) By constructing an arthritis (CIA) rat model, the activation of the SHBG gene, the polarization of macrophage M1, and the upregulation of immune factors TNF-α, IL-6, and IL-10 in CIA rats were verified, further confirming the application prospect of the SHBG gene expression inhibition strategy provided by the present invention in the treatment of rheumatoid arthritis.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

图1是本发明中SHBG基因在Raw264.7中表达情况。图A为差异基因热图；图B为差异基因的KEGG通路富集图，其中Y轴代表通路名称，X轴代表基因所占比例，气泡面积代表通路富集基因数，气泡颜色深浅代表P值的大小；图C-D通过免疫荧光共聚焦所拍摄SHBG在巨噬细胞中的定位及荧光强度统计图；图E为SHBG在Raw264.7细胞中的mRNA表达量；图F-G为SHBG在Raw264.7细胞中的蛋白表达量；图H为SHBG与SREBP1免疫共沉淀结果图。Figure 1 is the expression of SHBG gene in Raw264.7 in the present invention. Figure A is a differential gene heat map; Figure B is a KEGG pathway enrichment map of differential genes, where the Y axis represents the pathway name, the X axis represents the proportion of genes, the bubble area represents the number of pathway enriched genes, and the bubble color depth represents the size of the P value; Figures C-D are localization and fluorescence intensity statistics of SHBG in macrophages taken by immunofluorescence confocal; Figure E is the mRNA expression of SHBG in Raw264.7 cells; Figures F-G are the protein expression of SHBG in Raw264.7 cells; Figure H is the result of SHBG and SREBP1 immunoprecipitation.

图2是本发明中抑制SHBG的表达与M1促炎细胞的关系。图A-B为通过流式细胞术检测LPS-IFN诱导后M1表型标记CD86的表达量，可直接反映为M1巨噬细胞数量；图C-I为敲低SHBG基因后，实验组及对照组细胞SHBG、iNOS、IL-6的蛋白及mRNA表达量。Figure 2 is the relationship between the inhibition of SHBG expression and M1 proinflammatory cells in the present invention. Figures A-B are the expression of M1 phenotype marker CD86 after LPS-IFN induction detected by flow cytometry, which can directly reflect the number of M1 macrophages; Figures C-I are the protein and mRNA expression of SHBG, iNOS, and IL-6 in the experimental group and control group cells after knocking down the SHBG gene.

图3是SREBP1表达与促炎表型M1巨噬细胞极化的关系。图A-B为SREBP1蛋白WB图及表达量统计结果图；图C为敲低SHBG表达后的SREBP1基因的mRNA表达量；图D-H为敲低SHBG表达后SREBP1介导的脂肪酸合成途径中的脂肪酸合酶基因(FASN、SCD1/2、Fads1/2)的mRNA表达量及结果统计图；图I-J为使用SREBP1抑制剂Fatostatin干预Raw264.7中SREBP1的表达，检测M1极化数量流式结果图。Figure 3 shows the relationship between SREBP1 expression and polarization of pro-inflammatory phenotype M1 macrophages. Figures A-B show WB images of SREBP1 protein and statistical results of expression; Figure C shows the mRNA expression of SREBP1 gene after knocking down SHBG expression; Figures D-H show the mRNA expression of fatty acid synthase genes (FASN, SCD1/2, Fads1/2) in the fatty acid synthesis pathway mediated by SREBP1 after knocking down SHBG expression and statistical results; Figures I-J show the quantitative flow cytometry results of using SREBP1 inhibitor Fatostatin to intervene in the expression of SREBP1 in Raw264.7 and detect M1 polarization.

图4为高表达SHBG后M1巨噬细胞极化与巨噬细胞脂肪酸代谢合成的关系。图A-B为在巨噬细胞中高表达SHBG后M1巨噬细胞极化的表达量及结果统计图；图C-G为转染SHBG过表达质粒后SHBG、SREBP1、iNOS、IL-6在巨噬细胞的蛋白表达WB结果及数据统计图；图H为SHBG在Raw264.7细胞中转染效率结果图；图I-J为过表达SHBG基因后免疫因子(iNOS、IL-6)的mRNA表达量；图K-P为过表达SHBG基因后SREBP1以及其下游的脂肪酸合酶基因的(FASN、SCD1、SCD2、Fads1、Fads2)mRNA表达量图。Figure 4 shows the relationship between M1 macrophage polarization and macrophage fatty acid metabolism and synthesis after high expression of SHBG. Figures A-B show the expression level and statistical results of M1 macrophage polarization after high expression of SHBG in macrophages; Figures C-G show the WB results and statistical data of protein expression of SHBG, SREBP1, iNOS, and IL-6 in macrophages after transfection of SHBG overexpression plasmid; Figure H shows the results of SHBG transfection efficiency in Raw264.7 cells; Figures I-J show the mRNA expression of immune factors (iNOS, IL-6) after overexpression of SHBG gene; Figures K-P show the mRNA expression of SREBP1 and its downstream fatty acid synthase genes (FASN, SCD1, SCD2, Fads1, Fads2) after overexpression of SHBG gene.

图5为SHBG在CIA大鼠关节炎中表达与关节炎症关系。图A-B为通过免疫组化实验技术检测CIA关节炎大鼠膝关节滑膜组织部位中SHBG的表达量及统计图；图C-H为TNF-α、IL-6、IL-10在CIA大鼠膝关节的表达情况。Figure 5 shows the relationship between SHBG expression and joint inflammation in CIA rat arthritis. Figures A-B show the expression of SHBG in the synovial tissue of the knee joint of CIA rat arthritis detected by immunohistochemistry and statistical graphs; Figures C-H show the expression of TNF-α, IL-6, and IL-10 in the knee joint of CIA rats.

图6为CIA大鼠膝关节炎症浸润与膝关节滑膜中M1巨噬细胞表达的关系。图A为使用荧光双染对大鼠膝关节滑膜部位中巨噬细胞定位及CD86分子表达量检测；图B为CIA大鼠膝关节滑膜中血管翳增殖及炎性浸润情况。Figure 6 shows the relationship between inflammatory infiltration of the knee joint of CIA rats and the expression of M1 macrophages in the synovium of the knee joint. Figure A shows the detection of macrophage localization and CD86 molecule expression in the synovium of the rat knee joint using fluorescent double staining; Figure B shows the proliferation and inflammatory infiltration of pannus in the synovium of the knee joint of CIA rats.

具体实施方式Detailed ways

相关术语解释Explanation of relevant terms

现在来详细描述本发明的各种示例性实施例。应注意到：除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本发明的范围。Various exemplary embodiments of the present invention will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

术语“表达水平”是指在特定的时间点体内或样品中存在的基因产物的量。表达水平可以例如通过由基因表达的蛋白质或mRNA来测量/量化/检测。可以例如如下量化表达水平：用相同样品或参考样品(例如，在相同时间从同一个体得到的样品中相同类型基因产物的总量(总蛋白质或mRNA)归一化样品中存在的目的基因产物的量。可以通过本领域已知的任何方法来测量或检测表达水平，所述方法例如通常通过目的基因产物与对目的基因产物具有特异性的一种或更多种不同分子或检测装置(例如，引物、探针、抗体、蛋白质支架)结合而工作的用于间接检测和测量目的基因产物的方法。The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a specific time point. The expression level can be measured/quantified/detected, for example, by a protein or mRNA expressed by a gene. The expression level can be quantified, for example, as follows: the amount of the target gene product present in a normalized sample using the same sample or reference sample (for example, the total amount (total protein or mRNA) of the same type of gene product in a sample obtained from the same individual at the same time). Expression levels can be measured or detected by any method known in the art, such as, for example, a method for indirect detection and measurement of the target gene product, which is usually combined with one or more different molecules or detection devices (for example, primers, probes, antibodies, protein scaffolds) that are specific to the target gene product.

术语“蛋白质水平”指的是物质中蛋白质含量的高低；蛋白质水平可以通过WesternBlot进行检测。WesternBlot是一种常用于检测蛋白质的分子量和表达水平的实验方法。它可以通过蛋白质在电泳分离后的迁移行为，利用特异性抗体来识别目标蛋白质。WesternBlot广泛应用于分子生物学、细胞生物学、免疫学等领域，可用于检测蛋白质的表达、鉴定、定量、纯化以及亚细胞定位等The term "protein level" refers to the amount of protein in a substance; protein levels can be detected by Western Blot. Western Blot is an experimental method commonly used to detect the molecular weight and expression level of proteins. It can identify target proteins using specific antibodies based on the migration behavior of proteins after electrophoretic separation. Western Blot is widely used in the fields of molecular biology, cell biology, immunology, etc., and can be used to detect protein expression, identification, quantification, purification, and subcellular localization.

术语“指标”和“标志”在本发明中可互换使用，并且是指病症的体征或信号或者于监测病症。这样的“病症”是指细胞、组织或器官的生物状态，或者是指个体的健康和/或疾病状态。指标可以是包括但不限于肽、蛋白质和核酸的分子的存在或不存在，或者可以是细胞、或组织、器官或个体中这样的分子的表达水平或模式变化。指标可以是个体中疾病的生、发展或存在或者这样的疾病的进一步进展的体征。指标也可以是在个体中发生疾病的风险的体征。The terms "indicator" and "marker" are used interchangeably in the present invention and refer to a sign or signal of a disease or to monitor a disease. Such a "disease" refers to a biological state of a cell, tissue or organ, or refers to the health and/or disease state of an individual. An indicator can be the presence or absence of a molecule including but not limited to peptides, proteins and nucleic acids, or can be a change in the expression level or pattern of such a molecule in a cell, or tissue, organ or individual. An indicator can be a sign of the development, development or presence of a disease in an individual or the further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

术语指标的水平“上调”、“升高”、“促进”或“提高”是指与参照相比，样品中这样的指标的水平降低。The terms "upregulate", "increase", "promote" or "increase" the level of an indicator refer to a decrease in the level of such indicator in a sample compared to a reference.

术语指标的水平“下调”、“降低”、“抑制”或“下降”是指与参照相比，样品中这样的指标的水平降低。The terms "downregulate", "reduce", "inhibit" or "decrease" the level of an indicator refer to a decrease in the level of such indicator in a sample compared to a reference.

在原理上，可以通过应用标准统计学方法基于给定SHBG的平均值或中值来对本发明所指定的对象组或群组计算参考量。In principle, a reference amount can be calculated for a group or cohort of subjects specified in the present invention based on the mean or median value of a given SHBG by applying standard statistical methods.

实施例涉及的材料与方法Materials and methods involved in the examples

实验材料Experimental Materials

细胞Raw264.7购于武汉博士德生物工程有限公司；LPS和IFN-γ购于苏州近岸蛋白质科技股份有限公司；胎牛血清和DMEM培养基购于武汉博士德生物工程有限公司；CCK-8试剂盒购于生工生物工程(上海)股份有限公司；SREBP1、SHBG、iNOS抗体购于武汉三鹰生物技术有限公司。CD86、CD68抗体购于武汉爱博泰克生物科技有限公司；IL-10、IL-6抗体购于武汉博士德生物工程有限公司。CD86、CD206流式抗体购于美国BDPharmingen公司。转录组测序分析(RNA-seq)由上海欧易生物医学科技有限公司完成。Cell Raw264.7 was purchased from Wuhan Boster Biotechnology Co., Ltd.; LPS and IFN-γ were purchased from Suzhou Jinan Protein Technology Co., Ltd.; fetal bovine serum and DMEM culture medium were purchased from Wuhan Boster Biotechnology Co., Ltd.; CCK-8 kit was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; SREBP1, SHBG, and iNOS antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd. CD86 and CD68 antibodies were purchased from Wuhan Abotek Biotechnology Co., Ltd.; IL-10 and IL-6 antibodies were purchased from Wuhan Boster Biotechnology Co., Ltd. CD86 and CD206 flow cytometry antibodies were purchased from BD Pharmingen, USA. Transcriptome sequencing analysis (RNA-seq) was completed by Shanghai Ouyi Biomedical Technology Co., Ltd.

细胞实验验证方法Cell experiment validation method

1.细胞及培养：Raw264.7细胞使用10％胎牛血清和高糖DMEM培养基培养并在37℃和含5％二氧化碳的细胞培养箱中孵育。1. Cells and culture: Raw264.7 cells were cultured with 10% fetal bovine serum and high-glucose DMEM medium and incubated in a cell culture incubator at 37°C and 5% carbon dioxide.

2.活化巨噬细胞：取对数生长期Raw264.7细胞，以LPS-IFN-γ(10ng/mLLPS和20ng/mLIFN-γ)刺激24h使其向M1型极化。2. Activation of macrophages: Raw264.7 cells in the logarithmic growth phase were taken and stimulated with LPS-IFN-γ (10 ng/mL LPS and 20 ng/mL IFN-γ) for 24 h to polarize them to the M1 type.

3.qPCR法检测SHBG/SREBP1信号通路相关分子SCD1/2、Fads1/2、FASN的mRNA表达情况，检测M1巨噬细胞标志物促炎因子(iNOS、IL-6、IL-10、TNF-α)mRNA表达。3. The qPCR method was used to detect the mRNA expression of SHBG/SREBP1 signaling pathway-related molecules SCD1/2, Fads1/2, and FASN, and the mRNA expression of M1 macrophage markers proinflammatory factors (iNOS, IL-6, IL-10, TNF-α).

Trizol法提取总RNA，首先对样本进行反转录获得cDNA，然后进行qPCR扩增：以cDNA为模板，β-Actin为内参，预变性95℃10min；变性95℃20s，退火延伸60℃30s，40个循环。循环结束后，以2-ΔΔCt相对定量法计算SHBG、SREBP1、iNOS、IL-6、SCD1/2、FASN、Fads1/2的mRNA的表达情况。引物序列如下所示，见表1。Total RNA was extracted by Trizol method, and the samples were first reverse transcribed to obtain cDNA, and then qPCR amplification was performed: cDNA was used as template, β-Actin was used as internal reference, pre-denaturation was 95℃ for 10min; denaturation was 95 ℃ for 20s, annealing and extension was performed at 60℃ for 30s, and 40 cycles were performed. After the cycle, the expression of SHBG, SREBP1, iNOS, IL-6, SCD1/2, FASN, and Fads1/2 mRNA was calculated by 2-ΔΔCt relative quantitative method. The primer sequences are shown in Table 1.

表1引物、质粒及核酸序列Table 1 Primers, plasmids and nucleic acid sequences

4.流式细胞术4. Flow Cytometry

1)细胞重悬：无菌PBS缓冲溶液漂洗细胞两次，轻吹细胞至离心管，1000rpm，5min；1) Cell resuspension: Rinse the cells twice with sterile PBS buffer solution, and gently blow the cells into a centrifuge tube at 1000 rpm for 5 min;

2)对照设置：空白对照、单阳管、待测样本，用细胞洗液(含1％BSA的PBS)重悬细胞，使每管细胞数达到1×106/100μL；2) Control settings: blank control, single positive tube, and sample to be tested, resuspend the cells with cell washing solution (PBS containing 1% BSA) to make the number of cells in each tube reach 1×10 6 /100 μL;

3)封闭：每管加入0.25ug封闭抗体(156603，Biolegend)，4℃孵育10min；3) Blocking: Add 0.25ug blocking antibody (156603, Biolegend) to each tube and incubate at 4°C for 10min;

4)膜外抗体孵育：待测样本管加入PE-Cy7标记Anti-MouseCD86抗体，充分混匀，至冰浴30min，孵育期间每隔10min晃动一下反应管，使细胞和抗体充分反应；4) Extracellular antibody incubation: Add PE-Cy7 labeled Anti-MouseCD86 antibody to the sample tube to be tested, mix thoroughly, and place on ice for 30 minutes. During the incubation period, shake the reaction tube every 10 minutes to allow the cells and antibodies to react fully.

5)固定：加入适量细胞洗液，1000rpm离心5min，弃上清，每管细胞加入固定液(554722，BDPharmingen)冰浴30min后PBS清洗细胞2次；5) Fixation: add appropriate amount of cell washing solution, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fixative (554722, BD Pharmingen) to each tube of cells, bathe on ice for 30 min, and then wash the cells twice with PBS;

6)破膜+膜内抗体孵育：每管细胞加入250μL破膜剂(554723，BDPharmingen)配制的APC标记anti-mouseCD206(MMR)抗体冰浴40min细胞清洗液漂洗细胞2次，离心；6) Membrane permeabilization + incubation with intramembrane antibodies: add 250 μL of membrane permeabilization agent (554723, BD Pharmingen) prepared with APC-labeled anti-mouse CD206 (MMR) antibody to each tube of cells, place in an ice bath for 40 minutes, rinse the cells twice with cell washing solution, and centrifuge;

7)细胞重悬：使用100μL细胞洗液重悬细胞后上机检测。7) Cell resuspension: Use 100 μL of cell washing solution to resuspend the cells and then test on the instrument.

5.Westernblot5. Western blot

收获的细胞在RIPA细胞裂解缓冲液中用热变性进行裂解。蛋白溶解产物(20μg)进行分析，把蛋白质转移到聚乙二烯二氟化物薄膜(PVDF)。孵化主要抗体SHBG、SREBP1、iNOS等主要抗体，通过增强化学发光检测特异蛋白表达量。The harvested cells were lysed by heat denaturation in RIPA cell lysis buffer. The protein lysate (20 μg) was analyzed and the protein was transferred to polyvinylidene difluoride film (PVDF). The main antibodies SHBG, SREBP1, iNOS and other main antibodies were incubated and the expression of specific proteins was detected by enhanced chemiluminescence.

实施例1：SHBG基因对巨噬细胞RAW264.7细胞极化的影响Example 1: Effect of SHBG gene on polarization of macrophage RAW264.7 cells

(1)活化的巨噬细胞(即M1极化的巨噬细胞)Raw 264.7中SHBG基因表达水平，以及SHBG蛋白与SREBP1蛋白的关系。(1) The expression level of SHBG gene in activated macrophages (i.e., M1 polarized macrophages) Raw 264.7, and the relationship between SHBG protein and SREBP1 protein.

1.细胞及培养方法：Raw 264.7细胞使用10％胎牛血清和高糖DMEM培养基培养并在37℃和含5％二氧化碳的细胞培养箱中孵育培养。1. Cells and culture methods: Raw 264.7 cells were cultured with 10% fetal bovine serum and high-glucose DMEM medium and incubated in a cell culture incubator at 37° C. and 5% carbon dioxide.

2.活化巨噬细胞(炎症模型)：取对数生长期Raw264.7细胞，以10ng/mL LPS和20ng/mL IFN-γ刺激24h使其向M1型极化。对照组(Ctrl组)为将10ng/mL LPS和20ng/mLIFN-γ替换为等量培养基。2. Activated macrophages (inflammatory model): Raw264.7 cells in the logarithmic growth phase were stimulated with 10 ng/mL LPS and 20 ng/mL IFN-γ for 24 h to polarize to the M1 type. The control group (Ctrl group) was replaced with an equal amount of culture medium instead of 10 ng/mL LPS and 20 ng/mL IFN-γ.

3.收集细胞，转录组测序，转录组测序分析(RNA-seq)由上海欧易生物医学科技有限公司完成，对照组与实验组(活化巨噬细胞，也即LPS-IFN-γ组)差异表达的基因结果如图1A所示。利用KEGG数据库对差异基因进行富集分析，KEGG通路富集结果显示如图1B所示，对照组与模型组的差异基因在脂肪酸合成代谢通路中显现高富集。3. Cells were collected and transcriptome sequencing was performed. Transcriptome sequencing analysis (RNA-seq) was performed by Shanghai Ouyi Biomedical Technology Co., Ltd. The results of differentially expressed genes between the control group and the experimental group (activated macrophages, i.e., LPS-IFN-γ group) are shown in Figure 1A. The KEGG database was used to perform enrichment analysis on differentially expressed genes. The results of KEGG pathway enrichment are shown in Figure 1B. The differentially expressed genes between the control group and the model group were highly enriched in the fatty acid synthesis metabolism pathway.

通过对上述步骤2获得的活化巨噬细胞进行免疫荧光共聚焦，结果显示(图1C-E)，实验组SHBG蛋白表达量显著增加，且SHBG蛋白显示出了从胞质向胞膜转移的现象。此外，WB以及qPCR结果(图1E-G)进一步证明了，相较于对照组，经过LPS-IFN-γ诱导的巨噬细胞显示出了SHBG基因以及蛋白表达水平的增高。免疫共沉淀(CO-IP)结果表明，SHBG蛋白与SREBP1蛋白能够相互作用，且SHBG的上调抑制了SREBP1的表达。By performing immunofluorescence confocal on the activated macrophages obtained in step 2 above, the results showed (Figure 1C-E) that the expression of SHBG protein in the experimental group increased significantly, and SHBG protein showed a phenomenon of transfer from the cytoplasm to the cell membrane. In addition, WB and qPCR results (Figure 1E-G) further proved that compared with the control group, macrophages induced by LPS-IFN-γ showed increased expression levels of SHBG gene and protein. The results of co-immunoprecipitation (CO-IP) showed that SHBG protein can interact with SREBP1 protein, and the upregulation of SHBG inhibits the expression of SREBP1.

以上结果表明，经LPS和INF-γ诱导的炎症模型中，SHBG基因以及蛋白表达水平出现了显著的上调，且SHBG蛋白的上调会进一步抑制SREBP1蛋白的表达，从而显示了SHBG蛋白在抑制SREBP1所介导的脂肪酸合成代谢通路中的潜在作用。The above results indicate that in the LPS and INF-γ-induced inflammatory model, the SHBG gene and protein expression levels were significantly upregulated, and the upregulation of SHBG protein further inhibited the expression of SREBP1 protein, thus showing the potential role of SHBG protein in inhibiting the fatty acid anabolic pathway mediated by SREBP1.

(2)小干扰RNA(siRNA)抑制SHBG基因表达对巨噬细胞极化以及对SREBP1基因介导的脂肪酸合成代谢通路的影响。(2) The effect of small interfering RNA (siRNA) inhibiting SHBG gene expression on macrophage polarization and the fatty acid anabolic pathway mediated by the SREBP1 gene.

瞬时转染方法及步骤：Transient transfection method and steps:

将Raw 264.7以2.5×105/孔的密度接种与6孔板中，接种过夜后，将si-NC、si-SHBG-1、si-SHBG-2，2000(商用转染试剂)用不含双抗和FBS的DMEM高糖基础培养基稀释后室温静置5min，稀释比例为：小干扰RNA∶DMEM高糖基础培养基＝150nM∶150μL，/>2000∶DMEM高糖基础培养基＝6μL∶150μL，随后分别将小干扰RNA与稀释后的/>2000以1∶1的比例混合，室温静置20min；移除细胞培养上清液，PBS缓冲液充分淋洗细胞表面，加入合适体积无双抗、FBS高糖DMEM基础培养基，随后加入已混合好的小干扰RNA与转染试剂混合液，混匀后放置于37℃，5％ CO2，转染6h，去除转染试剂，PBS缓冲液充分洗涤细胞表面，更换完全培养基培养12-24h等待活化处理。Raw 264.7 was seeded in a 6-well plate at a density of 2.5×10 5 /well. After seeding overnight, si-NC, si-SHBG-1, and si-SHBG-2 were added. 2000 (commercial transfection reagent) was diluted with DMEM high-glucose basal medium without double antibody and FBS and then allowed to stand at room temperature for 5 min. The dilution ratio was: small interfering RNA: DMEM high-glucose basal medium = 150 nM: 150 μL, /> 2000: DMEM high glucose basal medium = 6 μL: 150 μL, then the small interfering RNA and the diluted 2000 in a ratio of 1:1, and stand at room temperature for 20 minutes; remove the cell culture supernatant, rinse the cell surface with PBS buffer, add an appropriate volume of DMEM basal medium without double antibody and FBS high glucose, and then add the mixed small interfering RNA and transfection reagent mixture, mix well and place at 37℃, 5% CO2 , transfect for 6 hours, remove the transfection reagent, wash the cell surface with PBS buffer, replace with complete medium and culture for 12-24 hours to wait for activation treatment.

实验分组：Experimental groups:

对照组：对照组1(si-Ctrl组)为：150nMsi-NC；对照组2(LPS-INF-γ+si-Ctrl组)为：LPS-INF-γ(10ng/mL+20ng/mL)+150nMsi-NCControl group: Control group 1 (si-Ctrl group): 150nM si-NC; Control group 2 (LPS-INF-γ+si-Ctrl group): LPS-INF-γ (10ng/mL+20ng/mL)+150nM si-NC

实验组：实验组1(LPS-INF-γ+si-SHBG-1)为LPS-INF-γ(10ng/mL+20ng/mL)+150nM si-SHBG-1；实验组2(LPS-INF-γ+si-SHBG-2)为：LPS-INF-γ(10ng/mL+20ng/mL)+150nM si-SHBG-2。Experimental groups: Experimental group 1 (LPS-INF-γ+si-SHBG-1) was LPS-INF-γ (10 ng/mL+20 ng/mL)+150 nM si-SHBG-1; Experimental group 2 (LPS-INF-γ+si-SHBG-2) was: LPS-INF-γ (10 ng/mL+20 ng/mL)+150 nM si-SHBG-2.

通过WB(图2C-D)以及qPCR(图2G)分别检测SHBG蛋白以及基因表达水平，结果显示小干扰RNA成功实现了对SHBG蛋白以及基因表达水平的抑制效果。The SHBG protein and gene expression levels were detected by WB (Figure 2C-D) and qPCR (Figure 2G), respectively. The results showed that small interfering RNA successfully achieved the inhibitory effect on the SHBG protein and gene expression levels.

通过流式细胞术检测M1极化巨噬细胞的数量，结果显示(图2A-B)，相较于对照组1，对照组2以及实验组中M1极化巨噬细胞均出现了明显的上调，转染了小干扰RNA后，相较于未转染的巨噬细胞，LPS-IFN-γ所诱导的M1表型CD86分子表达量下调了约20％。The number of M1 polarized macrophages was detected by flow cytometry, and the results showed (Figure 2A-B) that compared with control group 1, M1 polarized macrophages in control group 2 and experimental group were significantly upregulated. After transfection with small interfering RNA, the expression of M1 phenotype CD86 molecules induced by LPS-IFN-γ was downregulated by about 20% compared with untransfected macrophages.

进一步的，对与M1巨噬细胞极化相关的免疫因子(iNOS、IL-6)的基因及蛋白表达水平进行了检测，结果显示(图2C、图2E-F、图2H-I)，相较于对照组1，经过LPS和INF-γ诱导的巨噬细胞中上述免疫因子的基因以及蛋白表达水平出现了显著的升高，而通过小干扰RNA抑制SHBG基因的表达可以有效抑制iNOS、IL-6蛋白表达量约0.5倍，下调50％的mRNA表达量。Furthermore, the gene and protein expression levels of immune factors (iNOS, IL-6) related to M1 macrophage polarization were detected. The results showed (Figure 2C, Figure 2E-F, Figure 2H-I) that compared with the control group 1, the gene and protein expression levels of the above immune factors in macrophages induced by LPS and INF-γ increased significantly, and inhibiting the expression of SHBG gene by small interfering RNA can effectively inhibit the expression of iNOS and IL-6 proteins by about 0.5 times and reduce the mRNA expression by 50%.

此外，对SREBP1的基因以及蛋白表达水平检测结果显示(图3A-C)，通过小干扰RNA抑制SHBG的表达使得SREBP1的基因以及蛋白表达水平上调了50％。并且，通过小干扰RNA抑制SHBG的表达还使得SREBP1介导的脂肪酸合成途径中脂肪酸合酶(FASN、SCD1、SCD12、Fads1、Fads2)的基因表达水平上调约50-60％。(图3D-H)。In addition, the results of the detection of SREBP1 gene and protein expression levels (Figure 3A-C) showed that the inhibition of SHBG expression by small interfering RNA increased the gene and protein expression levels of SREBP1 by 50%. In addition, the inhibition of SHBG expression by small interfering RNA also increased the gene expression levels of fatty acid synthase (FASN, SCD1, SCD12, Fads1, Fads2) in the fatty acid synthesis pathway mediated by SREBP1 by about 50-60%. (Figure 3D-H).

为了进一步验证SREBP1信号通路的抑制对巨噬细胞的M1极化的促进效果，以不同浓度的Fatostatin(SREBP1抑制剂)分别处理巨噬细胞Raw 264.7以及活化的巨噬细胞Raw264.7，具体方法如下：In order to further verify the promoting effect of inhibition of SREBP1 signaling pathway on M1 polarization of macrophages, macrophages Raw 264.7 and activated macrophages Raw264.7 were treated with different concentrations of Fatostatin (SREBP1 inhibitor), and the specific methods are as follows:

将Raw 264.7以2.5×105/孔的密度接种与6孔板中，接种过夜后，按以下方法给予SREBP1抑制剂，按2步骤活化巨噬细胞后，收集细胞进行后续实验。Raw 264.7 was seeded in a 6-well plate at a density of 2.5×10 5 /well. After overnight seeding, SREBP1 inhibitor was administered as follows. After macrophages were activated according to step 2, cells were collected for subsequent experiments.

实验分组：Experimental groups:

对照组：对照组1(Ctrl组)为：不经过任何试剂干预组；对照组2(LPS-IFN-γ组)：为LPS-INF-γ(10ng/mL+20ng/mL)干预24h；Control group: Control group 1 (Ctrl group) was not intervened by any reagent; Control group 2 (LPS-IFN-γ group) was intervened by LPS-INF-γ (10ng/mL+20ng/mL) for 24h;

实验组：实验组1(1μM Fatostatin)：单独给予1μm Fatostatin干预24h；实验组2(LPS-IFN-γ+1μMFatostain)：1μMFatostatin+LPS-INF-γ(10ng/mL+20ng/mL)干预24h；实验组3(2μMFatostatin)：单独给予2μMFatostatin干预24h；实验组4(LPS-IFN-γ+2μMFatostain)：2μMFatostatin+LPS-INF-γ(10ng/mL+20ng/mL)干预24h；Experimental groups: Experimental group 1 (1μM Fatostatin): 1μM Fatostatin alone was given for 24h; Experimental group 2 (LPS-IFN-γ+1μM Fatostain): 1μM Fatostatin+LPS-INF-γ(10ng/mL+20ng/mL) was given for 24h; Experimental group 3 (2μM Fatostatin): 2μM Fatostatin alone was given for 24h; Experimental group 4 (LPS-IFN-γ+2μM Fatostain): 2μM Fatostatin+LPS-INF-γ(10ng/mL+20ng/mL) was given for 24h;

结果表明(图3I-J)，经过Fatostatin处理后，巨噬细胞Raw 264.7以及活化的巨噬细胞Raw 264.7中M1极化的巨噬细胞的数量会显著提升。The results showed (Figure 3I-J) that after treatment with Fatostatin, the number of M1 polarized macrophages in Raw 264.7 macrophages and activated Raw 264.7 macrophages was significantly increased.

以上结果表明，SREBP1的抑制会加据巨噬细胞的M1极化，而抑制SHBG在巨噬细胞中的表达可通过活化SREBP1介导的脂肪酸合成途径，治疗LPS-IFN-γ所诱导的巨噬细胞M1极化所导致的炎症情况，表明SHBG在RA中是一个潜在的治疗靶点。The above results indicate that inhibition of SREBP1 will increase the M1 polarization of macrophages, and inhibition of SHBG expression in macrophages can treat the inflammation caused by LPS-IFN-γ-induced macrophage M1 polarization by activating the SREBP1-mediated fatty acid synthesis pathway, indicating that SHBG is a potential therapeutic target in RA.

(3)过表达SHBG对巨噬细胞M1极化以及SREBP1介导的脂肪酸代谢通路的影响(3) Effects of SHBG overexpression on macrophage M1 polarization and SREBP1-mediated fatty acid metabolism pathway

将核苷酸序列如SEQ ID No.1所示SHBG通过基因合成并插入至pc-DNA3.1质粒的BamHI和EcoRI位点，构建SHBG表达质粒。质粒转染至巨噬细胞Raw 264.7中，得到过表达SHBG的巨噬细胞。转染方法同上述步骤(2)。The nucleotide sequence of SHBG shown in SEQ ID No. 1 was synthesized and inserted into the BamHI and EcoRI sites of the pc-DNA3.1 plasmid to construct an SHBG expression plasmid. The plasmid was transfected into macrophage Raw 264.7 to obtain macrophages overexpressing SHBG. The transfection method was the same as the above step (2).

实验分组：Experimental groups:

对照组：对照组1(pc-DNA3.1)：转染过表达质粒空载质粒pc-DNA3.1，浓度0.5ug/孔；对照组2(pc-DNA3.1+LPS-IFN-γ)：转染过表达质粒空载质粒pc-DNA3.1，0.5ug/孔后，使用LPS-IFN-γ(10ng/mL+20ng/mL)干预24h。Control group: Control group 1 (pc-DNA3.1): transfection of overexpression plasmid empty plasmid pc-DNA3.1, concentration 0.5ug/well; Control group 2 (pc-DNA3.1+LPS-IFN-γ): transfection of overexpression plasmid empty plasmid pc-DNA3.1, 0.5ug/well, and then intervention with LPS-IFN-γ (10ng/mL+20ng/mL) for 24h.

实验组：实验组1：转染SHBG过表达质粒pc-DNA3.1-SHBG，浓度为0.5ug/孔，使用LPS-IFN-γ(10ng/mL+20ng/mL)干预24h。Experimental group: Experimental group 1: transfection of SHBG overexpression plasmid pc-DNA3.1-SHBG at a concentration of 0.5ug/well, and intervention with LPS-IFN-γ (10ng/mL+20ng/mL) for 24h.

通过WB以及qPCR对SHBG蛋白以及基因的表达情况进行检测，结果显示(图4C-D、图4H)，SHBG的蛋白以及基因表达量相较于对照组，出现了明显上调，表明SHBG在巨噬细胞内成功实现了过表达。图4E-P的结果表明，SHBG的过表达会使得SREBP1的表达下调，进而破坏SREBP1介导的脂肪酸代谢通路，下调脂肪酸合酶(FASN、SCD1、SCD12、Fads1、Fads2)的基因表达水平，并使得免疫因子(iNOS、IL-6)出现上调。此外，SHBG的过表达还会使得M1极化巨噬细胞的数量显著增多(图4A-B)。以上结果可证明SHBG的表达与M1极化呈正相关，证实SHBG的高表达下调了SREBP1的表达，损害了由SREBP1介导的脂肪酸代谢通路，制使下游联级信号分子受到抑制，脂肪酸合成代谢受阻，干扰了巨噬细胞抗氧化防御状态，进而诱导了M1巨噬细胞的极化，促进炎症的发展。The expression of SHBG protein and gene was detected by WB and qPCR. The results showed (Figure 4C-D, Figure 4H) that the protein and gene expression of SHBG were significantly upregulated compared with the control group, indicating that SHBG was successfully overexpressed in macrophages. The results of Figure 4E-P show that overexpression of SHBG will downregulate the expression of SREBP1, thereby destroying the fatty acid metabolism pathway mediated by SREBP1, downregulating the gene expression level of fatty acid synthase (FASN, SCD1, SCD12, Fads1, Fads2), and upregulating immune factors (iNOS, IL-6). In addition, overexpression of SHBG will significantly increase the number of M1 polarized macrophages (Figure 4A-B). The above results prove that the expression of SHBG is positively correlated with M1 polarization, confirming that the high expression of SHBG downregulates the expression of SREBP1, damages the fatty acid metabolism pathway mediated by SREBP1, inhibits downstream cascade signaling molecules, hinders fatty acid anabolism, interferes with the antioxidant defense state of macrophages, and then induces the polarization of M1 macrophages and promotes the development of inflammation.

通过以上实验结果看出，LPS-IFN-γ促进了SHBG在M1表型巨噬细中的表达，SHBG的表达进一步促进M1巨噬细胞表型标记物CD86表达，诱导巨噬细胞进一步推动炎症发展。通过小干扰RNA抑制SHBG在巨噬细胞中的表达，能够抑制促炎因子iNOS、IL-6的分泌表达，可以挽救LPS-INF-γ所抑制的SREBP1相关下游信号通路，证实可通过调控SHBG/SREBP1信号通路，抑制巨噬细胞向M1极化，正调控免疫反应，促使炎症消退。The above experimental results show that LPS-IFN-γ promotes the expression of SHBG in M1 phenotype macrophages, and the expression of SHBG further promotes the expression of M1 macrophage phenotype marker CD86, inducing macrophages to further promote the development of inflammation. Inhibiting the expression of SHBG in macrophages by small interfering RNA can inhibit the secretion and expression of proinflammatory factors iNOS and IL-6, and can rescue the SREBP1-related downstream signaling pathway inhibited by LPS-INF-γ. It is confirmed that by regulating the SHBG/SREBP1 signaling pathway, the polarization of macrophages to M1 can be inhibited, the immune response can be positively regulated, and inflammation can be eliminated.

实施例2：胶原诱导性关节炎(CIA)大鼠动物模型验证Example 2: Validation of the collagen-induced arthritis (CIA) rat animal model

已有研究证实，类风湿关节炎患者血清及滑液中存在抗胶原抗体，且这种对胶原组织的自身免疫反应，可以解释类风湿关节炎所具有的系统性和慢性持续性发展的特点。因此，胶原诱导性关节炎大鼠动物模型在临床症状、病理学改变、免疫反应等方面都与人类特征高度相似，如对称关节受累、肢体远端关节侵犯、滑膜增生炎症、血管翳形成及软骨和骨的破坏。因此选择构建CIA大鼠关节炎模型验证SHBG与RA的作用。Studies have confirmed that anti-collagen antibodies exist in the serum and synovial fluid of patients with rheumatoid arthritis, and this autoimmune response to collagen tissue can explain the systemic and chronic persistent development characteristics of rheumatoid arthritis. Therefore, the collagen-induced arthritis rat animal model is highly similar to human characteristics in terms of clinical symptoms, pathological changes, and immune responses, such as symmetrical joint involvement, distal limb joint invasion, synovial hyperplasia and inflammation, pannus formation, and cartilage and bone destruction. Therefore, the CIA rat arthritis model was selected to verify the role of SHBG and RA.

CIA大鼠关节炎模型的构建及处理方法(模型组)：将牛Ⅱ二型胶原溶于0.1M的醋酸制成浓度2mg/ml的溶液，与等量的不完全弗氏佐剂混合，充分研磨成乳剂。第0天，对适龄大鼠采用四点皮内注射0.2ml牛II型胶原蛋白与不完全弗氏佐剂等体积乳化物。待7天后，制备相同的乳剂，再次对大鼠进行注射，待30天后，安乐死处死大鼠，剪取得到大鼠膝关节，多聚甲醛固定大鼠膝关节后，采用10％ EDTA脱钙液对大鼠膝关节进行脱钙处理，随后进行组织脱水、浸蜡、病理切片操作取得大鼠膝关节切片。Construction and treatment methods of CIA rat arthritis model (model group): Bovine type II collagen was dissolved in 0.1M acetic acid to prepare a solution with a concentration of 2mg/ml, mixed with an equal amount of incomplete Freund's adjuvant, and fully ground into an emulsion. On day 0, 0.2ml of an emulsion of bovine type II collagen and incomplete Freund's adjuvant was intradermally injected into rats of appropriate age at four points. After 7 days, the same emulsion was prepared and injected into the rats again. After 30 days, the rats were euthanized and the knee joints of the rats were cut and obtained. After the knee joints of the rats were fixed with paraformaldehyde, the knee joints of the rats were decalcified with 10% EDTA decalcification solution, and then tissue dehydration, wax immersion, and pathological sectioning were performed to obtain the rat knee joint sections.

对照组：处理方法同CIA大鼠关节炎模型，区别在于将乳化物换为等量生理盐水。Control group: The treatment method was the same as that of the CIA rat arthritis model, except that the emulsion was replaced with an equal amount of normal saline.

大鼠膝关节免疫组化分析：将切好的大鼠膝关节白片进行脱蜡复水后，使用柠檬酸缓冲液进行抗原修复，漂洗切片后使用5％的山羊血清进行封闭，随后在组织上加好SHBG、IL-6、TNF-α、IL-10一抗后于4℃冰箱中保存过夜，第二天后孵育二抗后进行显色。Immunohistochemical analysis of rat knee joints: The cut rat knee joint white slices were dewaxed and rehydrated, and then antigen repair was performed using citric acid buffer. After rinsing the slices, 5% goat serum was used for blocking. Then, SHBG, IL-6, TNF-α, and IL-10 primary antibodies were added to the tissues and stored in a 4°C refrigerator overnight. The secondary antibodies were incubated the next day for color development.

大鼠膝关节免疫荧光分析：将切好的大鼠膝关节白片进行脱蜡复水后，使用柠檬酸缓冲液进行抗原修复，漂洗切片后使用5％的山羊血清进行封闭，随后在组织上加好CD86、CD68一抗后于4℃冰箱中保存过夜，第二天后孵育荧光二抗后与正置荧光显微镜下拍照显色。Immunofluorescence analysis of rat knee joints: The cut rat knee joint white slices were dewaxed and rehydrated, and then antigen repair was performed using citric acid buffer. After rinsing the slices, 5% goat serum was used for blocking. Then, CD86 and CD68 primary antibodies were added to the tissues and stored in a 4°C refrigerator overnight. The next day, fluorescent secondary antibodies were incubated and the samples were photographed under an upright fluorescence microscope for color development.

CIA关节炎大鼠膝关节滑膜组织部位中SHBG的表达结果显示(图5)，与对照组相比模型组大鼠膝关节滑膜组织部位SHBG蛋白呈强阳性表达，免疫因子TNF-α、IL-6、IL-10的表达也显示出明显上调，提示CIA模型鼠膝关节中有明显炎症发生，炎症情况与SHBG的表达呈正相关。The results of SHBG expression in the synovial tissue of the knee joints of CIA arthritis rats showed (Figure 5) that compared with the control group, the SHBG protein in the synovial tissue of the knee joints of the rats in the model group was strongly positively expressed, and the expression of immune factors TNF-α, IL-6, and IL-10 also showed a significant upregulation, indicating that obvious inflammation occurred in the knee joints of CIA model rats, and the inflammation was positively correlated with the expression of SHBG.

免疫荧光分析结果显示(图6A-B)，在模型组中，CD68标记的巨噬细胞中M1型标记物CD86高表达，提示促炎M1巨噬细胞数量增加可加剧关节炎大鼠的病变情况。苏木精和伊红染色(HE)染色结果表明，与对照组相比，模型组大鼠半月板内血管内有明显的炎症细胞浸润，炎性血管脉络膜范围明显扩大。上述结果说明SHBG在大鼠膝关节中高表达，与大鼠关节炎严重程度成正相关，可以作为诊断、治疗或预防治疗类风湿关节炎的潜在靶点及预后靶点分子。The results of immunofluorescence analysis showed (Figure 6A-B) that in the model group, the M1 marker CD86 was highly expressed in CD68-labeled macrophages, suggesting that the increase in the number of pro-inflammatory M1 macrophages can aggravate the pathological changes in arthritis rats. The results of hematoxylin and eosin (HE) staining showed that compared with the control group, there was obvious inflammatory cell infiltration in the blood vessels in the meniscus of the model group rats, and the range of the inflammatory vascular choroid was significantly expanded. The above results indicate that SHBG is highly expressed in the knee joints of rats and is positively correlated with the severity of arthritis in rats. It can be used as a potential target and prognostic target molecule for the diagnosis, treatment or prevention of rheumatoid arthritis.

虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (10)

1.性激素结合球蛋白或其编码基因作为靶点在制备用于调控巨噬细胞M1极化的药物中的应用。1. Use of sex hormone binding globulin or its encoding gene as a target in the preparation of drugs for regulating macrophage M1 polarization. 2.根据权利要求1所述的应用，其特征在于，所述药物通过上调性激素结合球蛋白或其编码基因的表达，从而抑制固醇调节元件结合蛋白1或其编码基因的表达，进而促进巨噬细胞M1极化；2. The use according to claim 1, characterized in that the drug inhibits the expression of sterol regulatory element binding protein 1 or its encoding gene by up-regulating the expression of sex hormone binding globulin or its encoding gene, thereby promoting macrophage M1 polarization; 或者，所述药物通过抑制性激素结合球蛋白或其编码基因的表达，从而促进固醇调节元件结合蛋白1或其编码基因的表达，进而抑制巨噬细胞M1极化。Alternatively, the drug inhibits the expression of sex hormone binding globulin or its encoding gene, thereby promoting the expression of sterol regulatory element binding protein 1 or its encoding gene, thereby inhibiting the polarization of macrophage M1. 3.根据权利要求1所述的应用，其特征在于，所述应用包括：性激素结合球蛋白或其编码基因的激活剂在制备用于治疗和/或缓解激活巨噬细胞M1极化相关疾病的药物中的应用。3. The use according to claim 1, characterized in that the use comprises: use of an activator of sex hormone binding globulin or its encoding gene in the preparation of a drug for treating and/or alleviating diseases related to activated macrophage M1 polarization. 4.根据权利要求1所述的应用，其特征在于，所述应用包括：性激素结合球蛋白或其编码基因的抑制剂在制备用于治疗和/或缓解抑制巨噬细胞M1极化相关疾病的药物中的应用。4. The use according to claim 1, characterized in that the use comprises: use of an inhibitor of sex hormone binding globulin or its encoding gene in the preparation of a drug for treating and/or alleviating diseases related to inhibition of macrophage M1 polarization. 5.根据权利要求4所述的应用，其特征在于，所述抑制巨噬细胞M1极化相关疾病包括类风湿关节炎。5. The use according to claim 4, characterized in that the diseases associated with the inhibition of macrophage M1 polarization include rheumatoid arthritis. 6.根据权利要求1-5任一所述的应用，其特征在于，所述药物的剂型包括注射液、注射用冻干粉针、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂或口服液。6. The use according to any one of claims 1 to 5, characterized in that the dosage form of the drug includes injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill or oral solution. 7.根据权利要求6所述的应用，其特征在于，所述药物中含有药用辅料，所述药用辅料包括：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂或润湿剂中的至少一种。7. The use according to claim 6, characterized in that the drug contains pharmaceutical excipients, and the pharmaceutical excipients include: at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrant, a filler, a lubricant or a wetting agent. 8.根据权利要求7所述的应用，其特征在于，所述药物中含有药用载体。8. The use according to claim 7, characterized in that the medicine contains a pharmaceutical carrier. 9.根据权利要求8所述的应用，其特征在于，所述药用载体选自微囊、微球、纳米粒或脂质体。9. The use according to claim 8, characterized in that the pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles or liposomes. 10.一种体外调节巨噬细胞M1极化水平的方法，其特征在于，所述方法包括将有效量的性激素结合球蛋白或其编码基因的激活/抑制剂与细胞接触，从而调节M1巨噬细胞极化水平。10. A method for regulating the polarization level of M1 macrophages in vitro, characterized in that the method comprises contacting an effective amount of sex hormone binding globulin or an activator/inhibitor of its encoding gene with cells, thereby regulating the polarization level of M1 macrophages.

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