黄芩清热除痹胶囊调控LncRNA EBLN3P/miR-369-3p/NFIX轴抑制JAK/STAT通路介导的类风湿关节炎滑膜血管新生

doi:10.12122/j.issn.1673-4254.2026.03.21

摘要/Abstract

摘要：

目的研究黄芩清热除痹胶囊（HQC）调控LncRNA EBLN3P/miR-369-3p/NFIX 轴干预类风湿关节炎（RA）中JAK/STAT驱动的滑膜血管生成的作用机制。方法通过优化干预策略构建类风湿关节炎成纤维样滑膜细胞/人脐静脉内皮细胞（RA-FLS/HUVEC）协同培养模型，采用梯度浓度含HQC血清（2.5%~30%，干预24~72 h）、LncRNA EBLN3P过表达及基因-药物联合处理，利用CCK-8法筛选共培养体系的最佳血清浓度与干预时长。通过EdU增殖实验、Transwell侵袭实验及细胞划痕实验评估HQC对RA-FLS病理活化的抑制作用，ELISA定量分析血管生成因子（VEGF、FGF2）及基质金属蛋白酶（MMP9、MMP2）表达水平，管腔形成实验联合免疫荧光技术检测RA-FLS诱导的HUVEC血管生成能力及内皮标志物（CD34、CD105）表达变化，qRT-PCR及Western blotting解析信号通路关键分子（LncEBLN3P、miR-369-3p、NFIX、JAK2、STAT3、p-JAK2、p-STAT3）的mRNA或蛋白表达动态。结果共培养体系优化作用条件（细胞比例5：1，时间48 h）模型中，与对照组相比，模型组RA-FLS的恶性行为（增殖、侵袭及迁移能力）显著增强（P<0.01），促血管生成因子（VEGF、FGF2）与基质降解酶（MMP2、MMP9）表达显著上调（P<0.01），LncEBLN3P/miR-369-3p/NFIX轴异常激活，miR-369-3p表达受抑制（P<0.01），而LncEBLN3P、NFIX及下游JAK2/STAT3通路关键分子（JAK2、STAT3、p-JAK2、p-STAT3）表达均显著升高（P<0.01）。经HQC干预后，模型组RA-FLS恶性行为受抑（P<0.01），促血管因子及基质降解酶表达下调（P<0.01），且LncEBLN3P/miR-369-3p/NFIX-JAK/STAT轴被抑制（miR-369-3p表达升高，P<0.01；LncEBLN3P、NFIX、JAK2、STAT3、p-JAK2、p-STAT3表达均下调，P<0.01）。在LncRNA EBLN3P过表达（OE-Lnc）模型中，HQC仍可部分逆转其诱导的病理表型及通路激活（P<0.01 vs OE-Lnc组），但与单纯HQC处理组相比，OE-Lnc+HQC联合处理组对细胞恶性行为及通路活化的抑制作用显著减弱（P<0.01）。结论 HQC含药血清可能通过靶向LncRNA EBLN3P/miR-369-3p/NFIX轴抑制JAK/STAT通路，从而抑制RA-FLS的促血管生成功能。

关键词: 类风湿关节炎, 黄芩清热除痹胶囊, 滑膜血管新生, 竞争性内源RNA

Abstract:

Objective To investigate the mechanism by which Huangqin Qingre Chubi Capsule (HQC) inhibits synovial angiogenesis mediated by the JAK/STAT pathway in rheumatoid arthritis (RA). Methods An optimized co-culture model of RA-derived fibroblast-like synoviocytes (RA-FLS) and human umbilical vein endothelial cells (HUVECs) was treated with gradient concentrations of HQC-medicated serum with or without plasmid transfection for LncRNA EBLN3P overexpression. The inhibitory effects of HQC on pathological behaviors of RA-FLS were assessed using EdU assay, Transwell invasion assay, and scratch wound healing assay. Cellular secretions of pro-angiogenic factors (VEGF and FGF2) and matrix metalloproteinases (MMP2 and MMP9) were measured by ELISA. HUVEC tube formation capacity and expressions of the endothelial markers CD34 and CD105 were evaluated, and the expressions of molecules in the LncEBLN3P/miR-369-3p/NFIX axis and the JAK2/STAT3 pathway were detected using qRT-PCR and Western blotting. Results The RA-FLS exhibited significantly enhanced proliferation, invasion, and migration with upregulated expressions of VEGF, FGF2, MMP2, and MMP9 and dysregulation of tthe LncEBLN3P/miR-369-3p/NFIX axis, as shown by decreased miR-369-3p and increased expressions of LncEBLN3P, NFIX, JAK2, STAT3, p-JAK2, and p-STAT3. Treatment with HQC-medicated serum effectively reversed these pathological changes, suppressed malignant phenotype of the cells, downregulated angiogenic and matrix-degrading factors, and inhibited the axis and pathway activity. In the LncRNA EBLN3P overexpression (OE-Lnc) model, HQC treatment less effectively reversed the pathological phenotype and pathway activation in the cells as compared to its effect in the non-overexpression setting. Conclusion HQC inhibits pro-angiogenic function of RA-FLS likely by targeting the LncRNA EBLN3P/miR-369-3p/NFIX axis, thereby suppressing the downstream JAK/STAT signaling pathway.

Key words: rheumatoid arthritis, Huangqin Qingre Chubi Capsules, synovial neovascularization, competitive endogenous RNAs

孙梦雨, 汪元, 刘菲菲. 黄芩清热除痹胶囊调控LncRNA EBLN3P/miR-369-3p/NFIX轴抑制JAK/STAT通路介导的类风湿关节炎滑膜血管新生[J]. 南方医科大学学报, 2026, 46(3): 675-685.

Mengyu SUN, Yuan WANG, Feifei LIU. Huangqin Qingre Chubi Capsule inhibits JAK/STAT-driven synovial angiogenesis in rheumatoid arthritis by suppressing the LncRNA EBLN3P/miR-369-3p/NFIX axis[J]. Journal of Southern Medical University, 2026, 46(3): 675-685.

图/表 7

表1 引物序列

Tab.1 Primer sequences

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
Hu-β-actin	96	CCCTGGAGAAGAGCTACGAG	GGAAGGAAGGCTGGAAGAGT
Hu-U6	94	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
Hu-Linc-EBLN3P	151	GTCCAGTCTTTGAGGACCGA	TGGTTCCTATGCCCAGATCG
Hu-NFIX	80	ACGGCTGCGATAGAACATGG	GGTGGAACTCATCACACGCT
hsa-miR-369-3p		CCGCGCAATACATGGTTG	AGTGCAGGGTCCGAGGTATT
hsa-miR-369-3p RT		GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAGAT
Hu-STAT3	124	GGAGAAACAGGATGGCCCAA	ATCCAAGGGGCCAGAAACTG
Hu-JAK2	88	TGAGTTCGAAGCTAGCAGGGC	ACAGTTGTCTCCACCCTCTCC

表2 Western blotting所用抗体信息

Tab.2 Antibodies for Western blotting

Product name	Manufacturer	Product number	Batch number	Theoretical molecular weight	Antibody source	Dilution ratio	Separation gel concentration
β-actin	Zs-BIO	TA-09	19AW0505	42 000	mouse	1:1000	10%
Goat anti-mouse IgG antibody	Zs-BIO	ZB-2305	249760203			1:10000
Goat anti-rabbit IgG antibody	Zs-BIO	ZB-2301	247860216			1:10000
JAK2	Affinity	AF6022	22V9316	131 000	rabbit	1:1000	10%
P-JAK2	Affinity	AF3024	75d7669	131 000	rabbit	1:1000	10%
STAT3	Affinity	AF6294	15X8824	88 000	rabbit	1:1000	10%
P-STAT3	Affinity	AF3293	74M1478	88 000	rabbit	1:1000	10%
NFIX	HUABIO	ER1913-79	H650014015	55 000	rabbit	1:1000	10%

图1 RA-FLS/HUVEC共培养体系建立及HQC给药参数优化

Fig. 1 Establishment of RA-FLS/HUVEC co-culture system and optimization of HQC treatment protocol.A: CCK-8 assay for determining optimal co-culture time (n=6). ****P<0.0001 vs the same time point in other groups. B: Dose-time effect analysis of HQC-medicated serum (n=6). ****P<0.0001 vs all other groups.

图2 HQC抑制RA-FLS的病理活化、血管生成因子及基质金属蛋白酶表达

Fig.2 HQC inhibits pathological activation, angiogenesis factor, and matrix metalloproteinase expression in RA-FLS. A, B: EdU assay for assessing cell proliferation (n=3) using Click Chemistry fluorescent staining (Original magnification, ×200). C, D: Scratch assay for assessing cell migration ability (n=3,×100). E, F: Transwell assay for assessing cell invasion ability (n=3) with crystal violet staining (×200). G-I: Detection of the concentrations of angiogenic factors and matrix-degrading enzymes by ELISA (n=6). ****P<0.0001, ***P<0.001, **P<0.01, * P<0.05.

图3 HQC抑制RA-FLS诱导的HUVEC血管生成和血管内皮标志物表达

Fig.3 HQC inhibits RA-FLS-induced HUVEC angiogenesis and vascular endothelial marker expression. A, B: Angiogenesis assay of testing HUVEC tubule formation ability (n=3,×200). C, D: CD34 immunofluorescence quantification (n=3) using a dual-color indirect immunofluo-rescence staining method (×200). E, F: CD105 immunofluorescence quantification (n=3) using a dual-color indirect immunofluorescence staining method (×200). ****P<0.0001.

图4 HQC下调RA-FLS诱导的HUVEC信号转导通路关键分子的表达

Fig.4 HQC downregulates expressions of key molecules in the RA-FLS-induced HUVEC signal transduction pathway. A-E: qRT-PCR detection of molecular expression of regulatory axes and their targeted signaling pathways (n=6). ****P<0.0001, ***P<0.001, **P<0.01.

图5 HQC抑制RA-FLS诱导的HUVEC信号转导相关蛋白活性

Fig.5 HQC inhibits RA-FLS-induced HUVEC signal transduction-related protein activity. A: Western blots of the proteins. B-F: Expression levels of the pathway-related proteins (n=3).****P<0.0001, ***P<0.001, **P<0.01.

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