Qing-Luo-Yin Alleviated Experimental Arthritis in Rats by Disrupting Immune Feedback Between Inflammatory T Cells and Monocytes: Key Evidences from Its Effects on Immune Cell Phenotypes

Abstract

Background

Qing-Luo-Yin (QLY) is an anti-rheumatic herbal formula. Despite the well-investigated therapeutic efficacy of QLY, its immune regulatory properties are largely unknown. CD4⁺ T cells and monocytes are two key parameters in rheumatoid arthritis (RA). This study investigated the changes in these cells in QLY-treated RA animal models.

Materials and Methods

RA models were induced in male SD rats and were orally treated with QLY. Dynamic metabolic changes in collagen-induced arthritis (CIA) rats were monitored by ¹H NMR approach. The immunity profiles of CIA and adjuvant-induced arthritis (AIA) rats were evaluated using immunohistochemical, PCR, ELISA, cytokine chip, flow cytometry, and immunofluorescence experiments. The bioactive components in QLY were identified by bioinformatic-guided LC-MS analyses. The compounds with high abundance in QLY decoction and easily absorbed were taken as key anti-rheumatic components and used to treat blood-derived immune cells using in vitro experiments.

Results

The results indicated that QLY decreased Th17 cells frequency and T cells-released IL-6, IL-17 and GM-CSF in CIA rats, which was attributed to the impaired lymphocyte maturation and altered differentiation. QLY inhibited lactic acid production and inflammatory polarization in the monocytes during the peak period of AIA and CIA. AIA monocytes elicited significant increase in Th17 cells counts, IL-6 and IL-1β secretion in co-cultured splenocytes, which was abrogated by QLY. QLY-containing serum suppressed the phosphorylation of JNK and p65 in AIA lymphocyte-stimulated normal monocytes and consequently inhibited iNOS and IL-1β expression as well as IL-6 and IL-1β production. Matrine, sinomenine and sophocarpine were identified as major bioactive compounds in QLY. These identified compounds effectively inhibited the development of inflammatory T cells using concentrations detected in QLY-treated rats. At higher concentrations (20-fold increase), the chemical stimuli significantly suppressed the production of IL-1β in AIA monocytes by inhibiting JNK and p65 pathways.

Conclusion

By targeting inflammatory T cells and monocytes as well as disrupting their interplay, QLY improved immune environment in RA models especially during the active stages of disease.

Keywords:

• traditional Chinese medicine
• rheumatoid arthritis
• innate immunity
• T cells
• chronic inflammation
• metabolomics

Abbreviations

MTX, methotrexate; CII, bovine type II collagen; IFA, incomplete Freund’s adjuvant; PMA, phorbol 12-myristate 13-acetate; BFA, brefeldin A; FBS, fetal bovine serum; HRP, horseradish peroxidase; PLS-DA, partial least squares-discrimination analysis; VIP, variable importance in projection; CFA, complete Freund’s adjuvant; WB, western-blot; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; SCF, stem cell factor; PPP, pentose phosphate pathway; LPS, lipopolysaccharide; RA, rheumatoid arthritis; CIA, collagen-induced arthritis; QLY, Qing-Luo-Yin; AIA, adjuvant-induced arthritis; cDMARDs, conventional disease modifying anti-rheumatic drugs; bDMARDs, biological disease modifying anti-rheumatic drugs; WBCs, white blood cells; ELISA, enzyme linked immunosorbent assay; BCG, Bacillus Calmette-Guerin; WB, western-blot; SIN, sinomenine; MT, matrine; SCA, sophocarpine; GM-CSF, granulocyte-macrophage colony stimulating factor; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin 1 beta; IL-17α, interleukin 17 alpha; IL-6, interleukin 6; SCF, stem cell factor; TCM, traditional Chinese medicine.

Acknowledgment

This work was supported by National Natural Science Foundation of China (81973828), Key Project of Natural Science Foundation of Anhui Province for College Scholar (KJ2019A0416 and KJ2020A0868), and Major Project of Natural Science Foundation of the Department of Education of Anhui province under (KJ2019ZD32).

Author Contributions

All authors made a significant contribution to conception and design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval for the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest.