Metabolic effects of quercetin on inflammatory and autoimmune responses in rheumatoid arthritis are mediated through the inhibition of JAK1/STAT3/HIF-1α signaling

Abstract

Background: Rheumatoid arthritis, a chronic autoimmune disease, is characterized by synovial hyperplasia and cartilage erosion. Here, we investigated the potential mechanism of action of quercetin, the main component of flavonoids, in treating rheumatoid arthritis.

Object: To examine the anti-arthritic effects of quercetin and elucidate the specific mechanisms that differentiate its metabolic effects on autoimmune and inflammatory responses at the synovial cell level.

Methods: We created a collagen-induced arthritis (CIA) model in Wistar rats, which were administered quercetin (50 or 100 mg/kg) continuously for four weeks via stomach perfusion. The arthritis score, histopathological staining, radiological assessment, and serum biochemical parameters were used to study the impact of quercetin on disease improvement. Additionally, immunofluorescence was employed to detect JAK1/STAT3/HIF-1α expression in rat joints. Moreover, the effects of quercetin (20, 40, and 80 µmol/L) on the properties and behavior of synovial fibroblasts were evaluated in an in vitro MH7A cell model using flow cytometry, CCK8, and transwell assays. Further, the mRNA expression levels of inflammatory cytokines IL1β, IL6, IL17, and TNFα were assessed by quantitative real-time PCR. Glucose, lactate, lactate dehydrogenase, pyruvate, pyruvate dehydrogenase, and adenosine triphosphate assay kits were employed to measure the metabolic effects of quercetin on synovial fibroblasts. Finally, immunoblotting was used to examine the impact of quercetin on the JAK1/STAT3/HIF-1α signaling pathway in synovial fibroblasts.

Results: In vivo experiments confirmed the favorable effects of quercetin in CIA rats, including an improved arthritis score and reduced ankle bone destruction, in addition to a decrease in the pro-inflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α in serum. Immunofluorescence verified that quercetin may ameliorate joint injury in rats with CIA by inhibiting JAK1/STAT3/HIF-1α signaling. Various in vitro experiments demonstrated that quercetin effectively inhibits IL-6-induced proliferation of MH7A cells and reduces their migratory and invasive behavior, while inducing apoptosis and reducing the expression of the pro-inflammatory cytokines IL1β, IL6, IL17, and TNFα at the mRNA level. Quercetin caused inhibition of glucose, lactate, lactate dehydrogenase, pyruvate, and adenosine triphosphate and increased pyruvate dehydrogenase expression in MH7A cells. It was further confirmed that quercetin may inhibit energy metabolism and inflammatory factor secretion in MH7A cells through JAK1/STAT3/HIF-1α signaling.

Conclusions: Quercetin's action on multiple target molecules and pathways makes it a promising treatment for cartilage injury in rheumatoid arthritis. By reducing joint inflammation, improving joint metabolic homeostasis, and decreasing immune system activation energy, quercetin inhibits the JAK1/STAT3/HIF-1α signaling pathway to improve disease status.

Keywords: Inflammatory pathways; Metabolic effects; Molecular docking; Quercetin; Rheumatoid arthritis.

Fig. 1

Quercetin impedes the proliferation of MH7A cells. (A) Molecular docking of quercetin at HIF-1α. (B) Antiproliferative effect of 5, 10, 20, 40, 80, and 100 µM quercetin on MH7A cells following the addition of 50 ng/mL IL6 (n = 3) (C) IC₅₀ of quercetin. Data are expressed as the mean ± SD. Results indicate a significant improvement in comparison with the arthritis group (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Quercetin impedes migration and invasion and induces apoptosis in MH7A cells. (A, B) Assessment of MH7A cell migration and invasion using a transwell assay following quercetin treatment (n = 5). (C) MH7A cells in active apoptosis were detected by flow cytometry following annexin V-FITC/PI staining (n = 3). (D) IL-1β, IL-6, IL-17, and TNF-α mRNA expression as assessed by RT-qPCR (n = 3). Data are expressed as the mean ± SD. Results indicate a significant improvement in comparison with the arthritis (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) or control (*P < 0.05, **P < 0.01, ***P < 0.001) groups

Fig. 3

Quercetin decreases IL6-induced oxidative stress and energy metabolism in MH7A cells. (A) Quercetin decreases the expression levels of HIF-1α in MH7A cells at both the protein and mRNA levels (n = 3). (B) Effect of quercetin on glucose, LDH, LA, PA, PDH and ATP levels in MH7A cells (n = 4). Data are expressed as the mean ± SD. Results indicate a significant improvement in comparison with the arthritis (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) or control (*P < 0.05, **P < 0.01, ***P < 0.001) groups

Fig. 4

Effect of quercetin on JAK1/STAT3/HIF-1α signaling in MH7A cells. (A) Crosstalk in the JAK1/STAT3/HIF-1α signaling pathway. Protein expression levels as assessed using immunoblotting (n = 3). (B) JAK1, STAT3, and HIF-1α protein expression levels were assessed using immunoblotting (n = 3). Data are expressed as the mean ± SD. Results indicate a significant improvement in comparison with the arthritis (*P < 0.05, **P < 0.01, ***P < 0.001) or control (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) group

Fig. 5

Quercetin improves the destruction of bone in CIA rats. (A) The schedule for establishing the CIA model and performing the drug treatment regimen (Que and Tofacitinib) is depicted as a schematic diagram. (B) Comparison of the changes in the scores indicating arthritis, paw swelling, and global assessment among the groups of rats (n = 7). (C) Representative appearance of each ankle. (D) Comparison of the histopathology of rodent ankle joints among the groups (n = 7). (E) Comparison of the micro-CT images of rodent ankle joints among the groups (n = 7). (F) Histopof the ankle joints among the groups of rats (n = 3). (G) Morphometric data of TMD (g/mm³), BMD (mm²) and BV/TV (%) in knee and ankle joints (n = 6). Data are expressed as the mean ± SD. Results indicate a significant improvement in comparison with the arthritis (*P < 0.05, **P < 0.01, ***P < 0.001) or control (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) group

Fig. 6

Quercetin decreases inflammatory factor expression in CIA rats. (A) Pro-inflammatory cytokine levels, including IL-1β, IL-6, IL-17, and TNF-α (n = 4), were assessed using ELISA. (B) Effects of quercetin on liver function parameters in CIA rats. (C) Quercetin’s effects on renal function parameters. Data are expressed as the mean ± SD. Results indicate a marked improvement in comparison with the arthritis (*P < 0.05, **P < 0.01, ***P < 0.001) or control (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) group

Fig. 7

Immunofluorescence of JAK1/STAT3/HIF-1α in the ankle joints of CIA rats. (A) Effects Levels of Immunofluorescence in the ankle joints of CIA rats, including JAK1, STAT3, and HIF1α. (B-D) Levels of Immunofluorescence in the ankle joints of CIA rats, including JAK1, STAT3, and HIF1α (n = 3). Data are expressed as the mean ± SD. Results indicate a marked improvement in comparison with the arthritis (*P < 0.05, **P < 0.01, ***P < 0.001) or control (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) group

Fig. 8

Quercetin’s effect on JAK1/STAT3/HIF-1α signaling in rheumatoid arthritis. The regulatory effect of quercetin on synoviocytes in rheumatoid arthritis mainly involves JAK1/STAT3/HIF-1α signaling and its downstream metabolic and molecular regulation