Huangqin Qingre Chubi Capsule inhibits JAK/STAT-driven synovial angiogenesis in rheumatoid arthritis by suppressing the LncRNA EBLN3P/miR-369-3p/NFIX axis

doi:10.12122/j.issn.1673-4254.2026.03.21

Abstract

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

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.

Figures/Tables 7

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

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%

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.

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.

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.

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.

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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