Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation.

CONTEXT: Rheumatoid arthritis (RA) is an autoimmune disease with aberrant Th17 cell differentiation. Panax notoginseng (Burk.) F. H. Chen (Araliaceae) saponins (PNS) have an anti-inflammatory effect and can suppress Th17 cell differentiation. OBJECTIVE: To investigate mechanisms of PNS on Th17 cell differentiation in RA, and the role of pyruvate kinase M2 (PKM2). MATERIALS AND METHODS: Naive CD4+T cells were treated with IL-6, IL-23 and TGF-ß to induce Th17 cell differentiation. Apart from the Control group, other cells were treated with PNS (5, 10, 20 µg/mL). After the treatment, Th17 cell differentiation, PKM2 expression, and STAT3 phosphorylation were measured via flow cytometry, western blots, or immunofluorescence. PKM2-specific allosteric activator (Tepp-46, 50, 100, 150 µM) and inhibitor (SAICAR, 2, 4, 8 µM) were used to verify the mechanisms. A CIA mouse model was established and divided into control, model, and PNS (100 mg/kg) groups to assess an anti-arthritis effect, Th17 cell differentiation, and PKM2/STAT3 expression. RESULTS: PKM2 expression, dimerization, and nuclear accumulation were upregulated upon Th17 cell differentiation. PNS inhibited the Th17 cells, RORγt expression, IL-17A levels, PKM2 dimerization, and nuclear accumulation and Y705-STAT3 phosphorylation in Th17 cells. Using Tepp-46 (100 µM) and SAICAR (4 µM), we demonstrated that PNS (10 µg/mL) inhibited STAT3 phosphorylation and Th17 cell differentiation by suppressing nuclear PKM2 accumulation. In CIA mice, PNS attenuated CIA symptoms, reduced the number of splenic Th17 cells and nuclear PKM2/STAT3 signaling. DISCUSSION AND CONCLUSIONS: PNS inhibited Th17 cell differentiation through the inhibition of nuclear PKM2-mediated STAT3 phosphorylation. PNS may be useful for treating RA.

Triptolide inhibits Th17 differentiation via controlling PKM2-mediated glycolysis in rheumatoid arthritis.

CONTEXT: Rheumatoid arthritis (RA) is an autoimmune disease with the aberrant differentiation of T helper 17 (Th17) cells. Pyruvate kinase M2 (PKM2), a key enzyme of glycolysis, was associated with Th17 cell differentiation. AIM: To investigate the potential therapeutic effects of triptolide (TP) in collagen-induced arthritis (CIA) and Th17 cell differentiation, and elucidated the underlying mechanisms. METHODS: PKM2 expression and IL-17A production in peripheral blood of RA patients were detected by RT-qPCR or ELISA. Flow cytometry and ELISA were employed to assess the effect of Th17 cell differentiation by TP. PKM2 expression and other glycolysis-related factors were detected using RT-qPCR and Western Blot. PKM2 specific inhibitor Compound 3 K was used to verify the mechanisms. Male DBA/1J mice were divided into control, model, and TP (60 µg/kg) groups to assess the anti-arthritis effect, Th17 cell differentiation and PKM2 expression. RESULTS: PKM2 expression positively correlated with IL-17A production in RA patients. PKM2 expression was increased upon Th17 cell differentiation. Down-regulating PKM2 expression could strongly reduce Th17 cell differentiation. Molecular docking analysis predicted that TP targeted PKM2. TP treatment significantly reduced Th17 cell differentiation, PKM2 expression, pyruvate, and lactate production. In addition, compared with down-regulating PKM2 alone (Compound 3 K treatment), co-treatment with TP and Compound 3 K further significantly decreased PKM2-mediated glycolysis and Th17 cell differentiation. In CIA mice, TP repressed the PKM2-mediated glycolysis and attenuated joint inflammation. CONCLUSION: TP inhibited Th17 cell differentiation through the inhibition of PKM2-mediated glycolysis. We highlight a novel strategy for the use of TP in RA treatment.

Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels.

PURPOSE: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, and Th17 cells are key factors in the pathogenesis of human inflammatory conditions, such as RA. Catalpol (CAT), a component in Rehmanniae Radix (RR), has been found to regulate human immunity. However, the effects of CAT on Th17 cell differentiation and improvement of RA are not clear. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) mice were constructed to detect the effects of CAT on arthritis and Th17 cells. The effect of CAT on Th17 differentiation was evaluated with let-7g-5p transfection experiments. Flow cytometry was used to detect the proportion of Th17 cells after CAT treatment. Levels of interleukin-17 and RORγt were assessed by qRT-PCR and enzyme-linked immunosorbent assay. The expression of signal transducer and activator of transcription 3 (STAT3) was determined by qRT-PCR and Western blot. RESULTS: We found that the proportion of Th17 cells was negatively associated with let-7g-5p expression in CIA mice. In in vitro experiments, CAT suppressed traditional differentiation of Th17 cells. Simultaneously, CAT significantly decreased Tregs-to-Th17 cells transdifferentiation. Our results demonstrated that CAT inhibited Tregs-to-Th17 cells transdifferentiation by up-regulating let-7g-5p and that the suppressive effect of CAT on traditional differentiation of Th17 cells is not related with let-7-5p. CONCLUSION: Our data indicate that CAT may be a potential modulator of Tregs-to-Th17 cells transdifferentiation by up-regulating let-7g-5p to reduce the expression of STAT3. These results provide new directions for research into RA treatment.

Zishen Tongluo formula ameliorates collagen-induced arthritis in mice by modulation of Th17/Treg balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Tongluo formula (ZTF) is simplified from the Qingluo Tongbi formula, which has been applied to treat rheumatoid arthritis (RA) in clinical practices for several decades. Our previous studies have verified the effects of ZTF on arthritis animal models. However, its mechanism of treating RA is not clear. AIM OF THE STUDY: The present study was designed to investigate the effects of ZTF on the Th17/Treg balance in RA mice and the role of the different herb groups with the effect of Zishen yangyin (YY), Huatan quyu (HT), or Qufeng chushi (QF) in ZTF. MATERIALS AND METHODS: A mouse model of collagen-induced arthritis (CIA) was established. The animals were randomly divided into the normal, model, positive drug, YY, QF, HT, and the whole compound (ZTF) groups. After oral administration for one-month, cytokine levels in the plasma and histopathological changes of the joint were measured by ELISA and hematoxylin-eosin staining, respectively. Meanwhile, the balance of Th17/Treg cells in blood, spleen or lymph nodes was detected using flow cytometry and qPCR. RESULTS: ZTF or the different functional groups could improve the joint inflammation, decrease the levels of proinflammatory cytokines, restore the balance of Th17 and Treg cells in CIA mice. However, there were some differences in each functional group: YY mainly promoted the responses of Treg cells while QF inhibited the functions of Th17 cells. Besides, HT regulated both Th17 and Treg cells to keep the immune balance. CONCLUSIONS: ZTF could notably ameliorate CIA mice by restoring the balance of Th17/Treg cells. Each functional group could target Th17 and/or Treg cells to produce synergistic/enhancement effects, and ZTF had a better holistic effect in RA treatment.

Th17 cell pathogenicity and plasticity in rheumatoid arthritis.

CD4+ Th cells play an important role in the development of rheumatoid arthritis (RA) by regulating adaptive immune response. As major subsets of CD4+ Th cells, Th17 cells can produce a large number of hallmark cytokines such as IL-17A and IL-17F, which participate in host defense and immune homeostasis. However, increasing researches have shown that Th17 cells are unstable and exhibit a certain degree of plasticity, which aggravates their pathogenicity. Furthermore, the plasticity and pathogenicity of Th17 cells are closely related with the disease activity in RA. In this paper, the characteristics including phenotype, differentiation, plasticity, and pathogenicity of Th17 cells in RA will be systematically summarized. This will contribute to clarify the immunologic mechanism of RA and further provide a novel strategy for the clinical treatment of autoimmune diseases.

Effects of Qingluo Tongbi Decoction on Gut Flora of Rats with Adjuvant-Induced Arthritis and the Underlying Mechanism.

Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease. Recent studies show that gut flora plays an important role in regulating the systemic immune response, and gut dysbacteria are linked with systemic chronic inflammation in the development of RA. Our previous results found that Qingluo Tongbi decoction (QLT) can treat RA effectively. The present study explored the effect of QLT on gut flora in an adjuvant-induced arthritis (AA) rat model. Thirty rats were divided randomly into three groups: a control group, a model group, and a treatment group (n = 10 per group). The rats in the model group were injected with complete Freund's adjuvant (FCA), while the treatment group received FCA combined with QLT treatment. After 27 days, the gut flora was profiled by 16S rRNA gene sequencing. The levels of cadherin-11, IL-17α, TLR2, and TLR4 proteins in the synovial tissues were detected by western blotting (WB). The results showed that QLT treatment significantly inhibited raw swelling during the 15-27 d period compared with the model group. QLT treatment reversed the ten altered bacterial genera in the model group, and three families (Lachnospiraceae, Eubacteriaceae, and Leuconostocaceae) were closely related to QLT treatment based on linear discriminant analysis (LDA). Functional prediction showed seven types of predicted functions were related to the QLT treatment, and WB results showed that QLT treatment reversed the increased expression levels of cadherin-11, IL-17α, TLR2, and TLR4 in synovial tissues significantly. The expression levels of cadherin-11, IL-17α, and TLR2 correlated negatively with the abundance of Staphylococcus and Candidatus_Saccharimonas. Therefore, RA development was related to gut dysbiosis, and QLT effectively ameliorated RA with decreased inflammatory responses regulated by the gut flora.

MicroRNA­143­3p contributes to the regulation of pain responses in collagen­induced arthritis.

Patients with rheumatoid arthritis (RA) suffer from pain, which is associated with inflammation, peripheral and central pain processing, and joint structure damage. The aim of the present study was to investigate a key microRNA (miR) and its target genes that are involved in the pain responses of RA, and to clarify the mechanism of pain regulation. Collagen­induced arthritis (CIA) was induced in DBA/1 and C57BL/6 mice. The paw swelling, mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and expression levels of tumor necrosis factor (TNF)­α and prostaglandin (PG)E2 in the sera were investigated. Decreased MWT and TWL, and increased TNF­α and PGE2, in the CIA model group were observed in DBA/1 and C57BL/6 mice. DBA/1 mice exhibited greater hyperalgesia and higher levels of inflammatory mediators. miR­143­3p expression in the blood and the dorsal root ganglion (DRG) were detected, and low miR­143­3p expression was demonstrated in the blood and DRG tissue of CIA mice. The target genes of miR­143 were predicted and analyzed. A total of 1,305 genes were predicted and 55 pain­associated genes were obtained. Prostaglandin­endoperoxide synthase 2 (Ptgs2), MAS related GPR family member E (Mrgpre), prostaglandin D2 receptor and Tnf were selected as target genes of miR­143. DRG cells were cultured and transfected with miR­143­3p inhibitor or mimic. The expression of Mrgpre, Ptgs2 and Tnf was significantly inhibited following miR­143­3p mimic transfection, while the expression of Mrgpre, Ptgs2 and Tnf was increased following inhibitor transfection. Additionally, the expression of pain­associated genes in the DRG of mice was investigated and the expression of Ptgs2, Mrgpre and Tnf in the DRG of CIA mice was also significantly upregulated. These results revealed that CIA mice exhibited marked hyperalgesia and high levels of inflammatory pain mediators. Low expression of miR­143­3p negatively regulated the pain­associated target genes, including Mrgpre, Ptgs2 and Tnf, thereby affecting chronic inflammatory pain and neuropathic pain in RA.