Anti-Inflammatory and Immunomodulatory Effects of 0.1 Sub-Terahertz Irradiation in Collagen-Induced Arthritis Mice.

The primary emphasis of photoimmunology is the impact of nonionizing radiation on the immune system. With the development of terahertz (THz) and sub-terahertz (sub-THz) technology, the biological effects of this emerging nonionizing radiation, particularly its influence on immune function, remain insufficiently explored but are progressively attracting attention. Here, we demonstrated that 0.1 sub-THz radiation can modulate the immune system and alleviate symptoms of arthritis in collagen-induced arthritis (CIA) mice through a nonthermal manner. The application of 0.1 sub-THz irradiation led to a decrease in proinflammatory factors within the joints and serum, reducing the levels of blood immune cells and the quantity of splenic CD4+ T cells. Notably, 0.1 sub-THz irradiation restored depleted Treg cells in CIA mice and re-established the Th17/Treg equilibrium. These findings suggested that sub-THz irradiation plays a crucial role in systemic immunoregulation. Further exploration of its immune modulation mechanisms revealed the anti-inflammatory properties of 0.1 sub-THz on LPS-stimulated skin keratinocytes. Through the reduction in NF-κB signaling and NLRP3 inflammasome activation, 0.1 sub-THz irradiation effectively decreased the production of inflammatory factors and immune-active substances, including IL-1ß and PGE2, in HaCaT cells. Consequently, 0.1 sub-THz irradiation mitigated the inflammatory response and contributed to the maintenance of immune tolerance in CIA mice. This research provided significant new evidence supporting the systemic impacts of 0.1 sub-THz radiation, particularly on the immune system. It also enhanced the field of photoimmunology and offered valuable insights into the potential biomedical applications of 0.1 sub-THz radiation for treating autoimmune diseases.

Suppression of NLRP3 Inflammasome by Dihydroarteannuin via the HIF-1α and JAK3/STAT3 Signaling Pathway Contributes to Attenuation of Collagen-Induced Arthritis in Mice.

Dihydroarteannuin (DHA), the primary element of artemisinin extracted from the traditional Chinese herb Artemisia annua L., has been used in malaria treatment for a long time. Recently, many studies have indicated that DHA also exhibits potent anti-rheumatoid arthritis (RA) activity. In this study, collagen-induced arthritis (CIA) in DBA/1J mice and inflammatory model in THP-1 cells were established to evaluate the modulatory effects of DHA on joint destruction and to explore the underlying mechanisms. Our results showed that DHA decreased the serum levels of IL-1ß and IL-6, alleviated paw oedema, and reduced bone destruction in DBA/1J mice with CIA. Further exploration with the inflammatory model in THP-1 cells indicated that DHA reduced the protein expression of hypoxia-inducible factor (HIF)-1α and the phosphorylation in Janus kinase (JAK) 3 and signal transducer and activator of transcription (STAT) 3 protein, which resulted in a decrease in NOD-like receptor protein (NLRP) 3 expression and interleukin (IL)-1ß release. Consequentially, the inflammatory activation in THP-1 cells was inhibited. Therefore, we concluded that DHA efficiently alleviated the inflammation and arthritic symptoms in CIA mice and downregulated inflammation in part by inhibiting NLRP3 expression via the HIF-1α and JAK3/STAT3 signaling pathway. Thus, DHA may be considered as a potential therapeutic agent in RA treatment.

Dihydroarteannuin Ameliorates Collagen-Induced Arthritis Via Inhibiting B Cell Activation by Activating the FcγRIIb/Lyn/SHP-1 Pathway.

Background: Dihydroarteannuin (DHA), which is extracted from the traditional Chinese herb Artemisia annua L, exhibits potent immunosuppressive activity in rheumatoid arthritis (RA). Strong evidence indicates that B cells act as an essential factor in the pathogenesis of RA, but research on the immunosuppressive function of DHA in regulating B cells is limited. Objective: To investigate the modulatory effects of DHA on joint destruction, proinflammatory cytokine production, activation, apoptosis and proliferation of B cells and to explore the possible associated mechanism in RA treatment. Methods: Collagen-induced arthritis (CIA) model was established. Weight and joint oedema were record weekly, and joint damage was detected by micro-CT scan. Human Burkitt B lymphoma cells lacking endogenous Fc gamma receptor b (FcγRIIb) gene were transfected with a 232Thr loss-of-function mutant to construct a mutant cell model ST486. The proliferation of ST486 cells was assessed with Cell Counting Kit-8. Apoptosis and activation were tested by flow cytometry. The effects of DHA on the activation of FcγRIIb, protein tyrosine kinases (Lyn), and SH2-containing tyrosine phosphatase-1 (SHP-1) signaling pathways were determined by western blotting. Results: In comparison to model group, bone volume/tissue volume (BV/TV) and bone mineral density (BMD) were increased, whereas joint oedema was decreased in both of the DHA and MTX group. The mRNA and protein expression levels of Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-α) were decreased after treatment with DHA. In addition, DHA treatment promoted the apoptosis, inhibited the activation and proliferation of ST486 cells. Furthermore, the protein expression levels of FcγRIIb, SHP-1, and Lyn were increased after treatment with DHA. Moreover, the expression of phosphorylated CD19 was also inhibited by DHA. Conclusion: We provide the first evidence that DHA may alleviate collagen-induced arthritis by activating the FcγRIIb/Lyn/SHP-1 signaling pathway in B cell, indicating that DHA is a novel and valuable candidate for RA therapy.

Glaucocalyxin A suppresses inflammatory responses and induces apoptosis in TNF-a-induced human rheumatoid arthritis via modulation of the STAT3 pathway.

The pathogenesis of rheumatoid arthritis (RA) is characterized by synoviocyte hyperplasia and proinflammatory cytokine secretion, as well as the destruction of cartilage and bone. Glaucocalyxin A (GLA) is an alkaloid derived from a Chinese medicinal plant that exhibits anti-inflammatory, anti-tumor and neuroprotective properties. We investigated the effects of GLA on RA-fibroblast-like synoviocytes (FLS cells), and collagen-induced arthritis (CIA), and further explored the underlying mechanisms. GLA inhibited TNF-a-induced RA-FLS proliferation, increased apoptotic ratios and upregulated levels of caspase-3, cleaved PARP, and Bax. GLA also inhibited the expression of IL-10, IL-1ß, and IL-6 in vitro. Levels of p-STAT3 were downregulated in a dose-dependent manner. Over-expression of STAT3 partly neutralized the GLA-mediated elevation of caspase-3 and cleaved PARP levels as well as the downregulation of IL-10, IL-1B and IL-6 expression levels. This suggests that GLA inactivated the STAT3 pathway. Furthermore, the production of inflammatory cytokines in RA-FLS and a CIA rat model were inhibited effectively by GLA. Taken together, our data suggest that GLA is a potential long-term therapeutic agent for patients with RA.

Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice / 中国实验方剂学杂志

Objective::Duanteng Yimu decoction(DTYMD)is effective in treatment of rheumatoid arthritis (RA) by relieving joint inflammation and down-regulating some inflammatory factors in a short period of time, but the mechanism is still unclear. We aimed to investigate upstream kinase of mitogen activated protein kinases(MAPK) and define the anti-inflammatory mechanism of DTYMD. Method::Fibroblasts-like synovial cells(FLSs) were divided into blank group, model group (IL-1β), high-dose DTYMD group (1 000 mg·L-1), medium-dose DTYMD group (800 mg·L-1), low-dose DTYMD group (600 mg·L-1) and armour ammonia butterfly(MTX) group (20 μmol·L-1). The protein and mRNA expressions of mitogen-activated protein kinase kinase kinase 2 (MEKK2) were analyzed by real-time fluorescence quantitative PCR(Real-time PCR). Totally 42 male DBA/1J mice were randomly divided into 6 groups, with 7 mice in each group, namely normal group, model group and MTX group (2 mg·kg-1), low-dose DTYMD group (6.25 mg·kg-1), medium-dose DTYMD group (12.5 mg·kg-1), and high-dose DTYMD group (25 mg·kg-1). Except for the normal group, the other five groups were included in collagen-induced arthritis(CIA) model by secondary immunoassay. After administration, the posterior limbs and ankle joints were stained with htoxylin-eosin(HE), and the pathological scores of the joints were evaluated. Result::Compared with the model group, DTYMD inhibited the activity of FLSs in a concentration-dependent manner (P<0.01). Compared with the blank control group, the cell proliferation rate of the model group increased (P<0.01). Compared with the model group, high and middle-dose DTYMD groups could inhibit protein and mRNA expressions of MEKK2 (P<0.01), but there was no significant difference in low-dose group. However, the expression of DTYMD protein in high/medium/low-dose groups was significantly higher than that in blank group (P<0.01), but there was no significant difference in MTX group. Compared with the model group, the expressions of matrix metalloprotease-1 (MMP-1), tumor necrosis factor-α(TNF-α) and interleukin(IL)-6 were negatively regulated in different DTYMD groups(P<0.01), and the expressions of MMP-1, IL-6, TNF-α in the model group were significantly higher than those in the blank group (P<0.05, P<0.01). In the animal experiment, compared with the model group, high/middle-dose DTYMD groups could decrease the degree of joint swelling in CIA mice (P<0.01), but there was no significant difference in the low dose group, and the joint swelling in the model group was significantly higher than that in the blank group (P<0.05). In HE staining of ankle joint of CIA mice, the pathological scores of high/small-dose DTYMD groups were significantly lower those of model group (P<0.05, P<0.01), and the pathological score of model group was higher than that of blank group (P<0.01). Conclusion::DTYMD might down-regulate MEKK2 to negatively regulate inflammatory cytokines IL-6, TNF-α and MMP-1, thereby alleviating the inflammatory response in rheumatoid arthritis.

Therapeutic effect of long-interval repeated intravenous administration of human umbilical cord blood-derived mesenchymal stem cells in DBA/1 mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen-induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long-interval repeated intravenous administration of human umbilical cord blood-derived (hUCB)-MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long-interval repeated intravenous administration of hUCB-MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB-MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin-O staining, and toluidine blue staining. We used real-time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB-MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB-MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB-MSCs also significantly decreased IL-1 beta protein levels in multimedium and multihigh groups and IL-6 protein levels in all hUCB-MSCs-treated groups. Furthermore, mRNA levels of IL-1 beta and IL-6 were decreased significantly in all hUCB-MSCs-treated groups, whereas the expression of anti-inflammatory cytokine IL-10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long-interval repeated intravenous administration of hUCB-MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose-dependent manner.

The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis mice.

HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and ß2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B.

Treatment of CIA Mice with FGF21 Down-regulates TH17-IL-17 Axis.

Recently, FGF21 was reported to play an important role in anti-inflammation. The aim of the study is to explore the mechanism for FGF21 alleviating inflammation of CIA. CIA mice were injected with FGF21 once a day for 28 days after first booster immunization. The results showed that FGF21 alleviates arthritis severity and decreases serum anti-CII antibodies levels in CIA mice. Compared with CIA model, the number of the splenic TH17 cells was significantly decreased in FGF21-treated mice. FGF21 treatment reduced the mRNA expression of IL-17, TNF-α, IL-1ß, IL-6, IL-8, and MMP3 and increased level of IL-10 in the spleen tissue. The expression of STAT3 and phosphorylated STAT3 was suppressed in FGF21-treated group. The mRNA expression of RORγt and IL-23 also decreased. In conclusion, these findings suggest that the beneficial effects of FGF21 on CIA mice were achieved by down-regulating Th17-IL-17 axis through STAT3/RORγt pathway. Modulating of Th17-mediated inflammatory response may be one of the mechanisms for FGF21 attenuating inflammation in CIA.

Expression of bone morphology proteins in mice with collagen-induced arthritis / 重庆医学

Objective To establish collagen‐induced arthritis (CIA) model used of DBA/1J mouse ,a preliminary study of the expression levels of BM Ps in mononuclear cells of peripheral blood and the joints tissue of CIA mice .Methods We induced DBA/1J mice and developed arthritis pathology by using of bovine type Ⅱ ;then ,we detected mRNA and protein expression levels of BMPs by using quantitative PCR and immunohistochemical staining .Results We successfully established CIA mouse model .The expres‐sion of BMP4 and BMP9 mRNA was significantly down‐regulated in peripheral blood mononuclear cells and in the pathogenesis of joint tissues of CIA mice (P<0 .05) .It showed that BMP9 protein significantly decreased in joint tissues of CIA mice by immuno‐fluorescence technique (P=0 .002) .Conclusion At the genetic level ,the expression of BMP4 and BMP9 could be significantly down‐regulated in the CIA mouse .At the protein level ,BMP9 could be significantly down‐regulated in the CIA mouse .

Roles of BCG in establishing collagen-induced arthritis mice / 中国免疫学杂志

Objective:To determine the correlation of BCG concentration in Freund complete adjuvant ( FCA) and severity of arthritis during arthritis establishment in DBA 1/j male mice induced by bovine type Ⅱcollagen.Methods:CIA was induced by the im-munization of DBA1/j mice with bovine type Ⅱcollagen and BCG of various concentrations dissolved in FCA.To ascertain the effects administering the collagen booster,CIA-related features(including body weight,clinical scoring of arthritis),TNF-αin serum and the histopathological changes in the spleen and the joints regions were measured.Results:4 mg/ml BCG induced more serious arthritis than 1 mg/ml in DBA1/j mice after collagen exposure.Conclusion:There is a positive correlation of arthritis severity with BCG.Which indicates that selection of adjuvant with suitable BCG concentration could determine pathological outcome in CIA mouse model .