Glutathione regulates CIA-activated splenic-lymphocytes via NF-κB/MMP-9 and MAPK/PCNA pathways manipulating immune response.

Reduced glutathione (GSH) is an antioxidant involved in redox homeostasis, and recently regarded as an inducer of Reductive stress. Its immune-regulatory effects on lymphocytes have not been extensively studied. This study is based on the finding that much increased GSH level in collagen-induced arthritis (CIA) rat spleen, and aimed to investigate the effects of GSH (0, 1, 10, 100 mM) on normal and immune-stimulated spleen lymphocytes respectively. The elevated GSH level is associated with the increased levels of inflammatory factors; especially the increased DPP1 activity indicated immune-granulocytes activation in CIA rat spleen. Exogenous GSH had different influences on normal and CIA lymphocytes, affecting intracellular levels of GSH, Glutathione-S-transferases (GSTs) and Reactive oxygen species (ROS); as well as the expressions of NF-κB, MMP-9, Bcl-2, GST, P38, PCNA and TLR4. The increased extracellular GSH level disturbed redox homeostasis and induces reductive stress to spleen lymphocytes, which decreased intracellular GSH concentration and influenced the MAPK/PCNA and NF-κB/MMP-9 signaling pathways, as well as cell cycles respectively, leading to cell senescence/ferroptosis/apoptosis. This study also revealed the multiple faces of GSH in regulating spleen lymphocytes, which depended on its levels in tissue or in cells, and the activation status of lymphocytes. These findings indicate the immune-regulatory role of GSH on spleen-lymphocytes, and the high level GSH in CIA rat spleens may contribute to CIA development.

[Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type â ¡ collagen-induced rheumatoid arthritis].

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1ß(IL-1ß) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1ß, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

[Comparative study on dose-toxicity-effect of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets on CIA model rats].

The aim of this paper was to compare the properties of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets from dose-effect-toxicity on type Ⅱ collagen-induced arthritis( CIA) in rats. SD rats were randomly divided into eight groups,including normal group,model group,Tripterygium Glycosides Tablets groups( 1 times equivalent dose 0.009 g·kg-1,4 times equivalent dose 0.036 g·kg-1,16 times equivalent dose 0.144 g·kg-1),Tripterygium wilfordii Tablets groups( 1 times equivalent dose 0.007 5 mg·kg-1,4 times equivalent dose 0.030 mg·kg-1,16 times equivalent dose 0.120 mg·kg-1). Beginning on the first immunization,Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets administered intraperitoneally once a day. After the second immunization,the symptoms such as redness and swelling of joints were observed,and the clinical score and incidence of arthritis were evaluated. HE and Masson staining were used to examine the histopathological changes of joints. The expression level of anti-type Ⅱ collagen antibody Ig G in serum was detected by ELISA,routine testing of blood components,the concentration of ALP( alkaline phosphatase),ALT( alanine aminotransferase),AST( aspartate aminotransferase),GGT( gamma-glutamyltransferase),TBi L( total bilirubin),CRE( creatinine) and UREA( urea) in serum were detected by enzymatic assay. The rate of sperm deformity in the epididymis was evaluated under light microscope. The extent of damage to the testis and ovarian tissue was assessed by HE staining. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets attenuated the inflammation,redness,swelling and deformity of joints and reduced the clinical score and incidence of arthritis in CIA rats. Meanwhile,it also exhibited obvious reduction in all pathological features such as joint synovitis,pannus,cartilage erosion and bone destruction. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets reduced Ig G in a dose-dependent manner,and Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets( P<0.05 or P<0.01). The high doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase the organ coefficient of liver and spleen and reduced RBC and HGB in CIA rats( P<0.01),and severity leading to death. Gastric mucosal injury and morphological changes of liver and kidney were not observed in CIA rats of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets treatment group. The 4 and 16 times doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase serum ALT,GGT and decrease CRE( P<0.05 or P<0.01). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could increase the sperm deformity rate and damage the testicular seminiferous tubules of CIA male rats. Severity increased with dose and time increasing. The effect of Tripterygium Glycosides Tablets( 16 times) is more significant than Tripterygium wilfordii Tablets( 16 times). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets significantly delayed onset of arthritis and inhibited the paw edema and arthritic score. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets also caused male reproductive damage,high dose affected hematopoiesis,and maximum dose leading to death. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets all depended on dose-effect-toxicity manner. Anti-arthritis effect of Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets,but the toxicity of Tripterygium Glycosides Tablets maximum dose is more obvious. The relevant conclusions of our study will provide experimental references for clinical rational use of drugs,and further clinical studies are needed to confirm our conclusions.

Anti-rheumatoid arthritis potential of different fractions derived from of Coluria longifolia.

Coluria longifolia Maxim (C. longifolia) is a Chinese folk medication commonly used to treat arthritis and joint pain. Literatures have reported that C. longifolia has significant anti-inflammatory, analgesic and antipyretic effects. The aim of this research was to assay the effective fractions of C. longifolia (EFCL) against rheumatoid arthritis (RA) and to elucidate its anti-RA mechanism on a preliminary basis. The rat model of collagen-induced arthritis (CIA) was established. The therapeutic effects of different fractions in vivo were evaluated by body weight changes, a foot swelling score, inflammatory factors and histopathological examination. The mechanism of EFCL was investigated by activity of oxidative stress related enzyme, qPCR and Western blotting tests. In vivo results showed that total extraction (TE) and n-butanol fraction (NF) could significantly alleviate the symptoms of RA, decrease the levels of IL-6 and TNF-α (P < 0.01), and improve histopathological injury. The mechanism study showed that SOD level was significantly increased with MDA level decreased in the NF group. The upregulated proteins and mRNA expression levels of Nrf2, HO1 and NQO1 after TE and NF administration suggested that the anti-arthritic effect may be related to the Nrf2 signaling pathway and downstream HO1 and NQO1. In conclusion, this study confirmed that C. longifolia is capable of treating RA with NF as the main effective fraction. Its anti-RA action may be associated with Nrf2 signaling pathway and downstream HO1 and NQO1.

An alcohol extract prepared from the male flower of Eucommia ulmoides Oliv. promotes synoviocyte apoptosis and ameliorates bone destruction in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease dominated by synovial hyperplasia and bone destruction. The male flower of Eucommia ulmoides Oliv. (EF) has been shown to exert effects on the inflammation caused by RA. However, how EF affects synoviocyte apoptosis and bone destruction on RA have not been investigated thoroughly. The effects of EF on apoptosis of human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) cells, osteoclast differentiation of RAW264.7 cells, and bone destruction in a collagen-induced arthritis (CIA) model in rats were explored. METHODS: First, the main components of EF were identified by high-performance liquid chromatography. In vitro, we investigated the anti-proliferative and pro-apoptotic effects of EF on HFLS-RA cells by immunofluorescence assays, flow cytometry, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting; we also investigated how EF influenced the differentiation of RAW264.7 cells into osteoclasts. In vivo, we used a rat model of CIA to investigate the effects of EF on anti-arthritis activity, toe swelling, Arthritis Score, serum levels of metabolic bone factors, and pathologic conditions. Micro-computed tomography was used to scan ankle joints. mRNA and protein expression of factors related to the nuclear factor-kappa B (NF-κB) pathway were determined by RT-qPCR and western blotting, respectively. RESULTS: EF inhibited synoviocyte proliferation and promoted apoptosis in a dose-dependent manner. EF inhibited osteoclast differentiation by inhibiting activation of the NF-κB pathway. EF reduced articular inflammation in CIA rats, inhibited the expression of pro-angiogenic factors, and delayed the destruction of articular cartilage and bone. Our data indicated that EF acted via a mechanism related to bone metabolism induced by the NF-κB pathway. CONCLUSIONS: EF exerts a potential therapeutic effect upon RA. Our research will help to elucidate the potential pharmacologic mechanisms associated with the beneficial effects of EF, and provide an experimental basis for EF application in clinical treatments.

Naltrexone (NTX) relieves inflammation in the collagen-induced- arthritis (CIA) rat models through regulating TLR4/NFκB signaling pathway.

OBJECTIVE: Our aim was to study the efficacy and mechanism by which NTX alleviate arthritis in CIA rat models in vivo. METHODS: Female Wistar rats were randomly divided into 6 groups, their weights were observed and the severity of arthritis and pathological changes were evaluated by HE staining. T lymphocyte subsets were detected by flow cytometry. The expression of cytokines was detected in peripheral serum by ELISA. Real time PCR, immunohistochemical staining and western blot analysis were utilized to detect the mRNA and protein expression of opioid receptors, TLR4, RANKL and /NF-κB in synovial tissue and the spleen. RESULTS: The weight of the rats in the 10 mg/kg NTX group decreased the least, and had the least severe arthritis. CD4+ T cells, Th1 cells and Treg cells increased, and CD8+T cells, Th1 cells and Th17 cells decreased in the splenic lymphocytes. The expression of proinflammatory cytokines decreased, and the expression of anti-inflammatory cytokines increased. MOR and DOR were strongly expressed in the spleen, whereas KOR and DOR were strongly expressed in synovial tissue. The expression of TLR4, NF-κB and RANKL was reduced in the spleen and synovium in the NTX group. CONCLUSIONS: NTX relieved the severity of arthritis in the CIA rat models at a concentration of 10 mg/kg by regulating T lymphocyte subsets and the expression of cytokines. NTX affected opioid receptors to inhibit the TLR4/NF-κB signaling pathway, regulating the systemic immune response and decreasing osteoclast differentiation, thereby alleviating inflammation and the erosion of articular cartilage along with bone tissue.

Effect of a cathepsin K inhibitor on arthritis and bone mineral density in ovariectomized rats with collagen-induced arthritis.

OBJECTIVES: Cathepsin K is expressed by osteoclasts and synovial fibroblasts and degrades key components of bone and cartilage. Inhibition of cathepsin K protease activity may be beneficial for the prevention of bone erosion and cartilage degradation in rheumatoid arthritis (RA). The collagen-induced arthritis (CIA) rat model is well established for studying the pathology and treatment of RA. We investigated the effect of ONO-KK1-300-01, a cathepsin K inhibitor (CKI), on arthritis and bone mineral density (BMD) in rats with CIA. METHODS: Seven-month-old female Sprague Dawley rats were divided into 5 groups: rats without CIA (CNT); CIA rats that underwent ovariectomy (OVX) and were treated with CKI; CIA rats that underwent OVX and were treated with vehicle (Veh); CIA rats that underwent sham surgery and were treated with CKI; and CIA rats that underwent sham surgery and were treated with Veh. CKI was orally administered at a dose of 15 mg/kg, thus initiating collagen sensitization, until death at 4 weeks. We evaluated hind paw thickness and the arthritis score every week until death. Radiographs of the resected left foot were obtained with a soft X-ray apparatus. Destruction of bone and cartilage was classified and scored as previously described by Engelhardt et al. BMD was measured by bone densitometry at the halfway point between the distal metaphysis and the diaphysis of the resected right femur. We also performed histomorphometry of the proximal left tibia, histological evaluation of arthritis, and a bone strength test. RESULTS: CKI administration significantly reduced hind paw thickness and the arthritis score, and prevented a decrease in BMD. The radiographic score was significantly lower in the CKI group than in the Veh group. In the histomorphometric analysis, bone-resorption parameters were significantly lower in the CKI groups than in the Veh groups. CKI significantly inhibited synovial proliferation in the CIA rats. In the bone strength test, the ultimate stress was significantly higher in the CKI groups than in the Veh groups. CONCLUSION: Our findings indicate that cathepsin K inhibitors may inhibit systemic and local bone loss, ameliorate arthritis, and attenuate the decrease of bone strength in an animal model of arthritis.

Liposomes encapsulated dimethyl curcumin regulates dipeptidyl peptidase I activity, gelatinase release and cell cycle of spleen lymphocytes in-vivo to attenuate collagen induced arthritis in rats.

Rheumatoid arthritis (RA) is an autoimmune disease and characterized by the excessive cell proliferation, abnormal cell cycle of lymphocytes and synovial cells. The therapeutic effects of curcumin in active RA patients were reported, but limited by its insolubility and rapid systemic elimination. Dimethyl curcumin (DiMC) is a metabolically stable analogue of curcum with anti-inflammatory property. In this study, liposomes encapsulated dimethyl curcumin (Lipo-DiMC) was prepared to improve the bioavailability and metabolic-stability; collagen induced arthritis (CIA) rat model was employed to investigate the effects of Lipo-DiMC treatments during CIA progress. Physical assessments and routine-blood-test were performed. Fresh spleen lymphocytes were isolated from normal, CIA and Lipo-DiMC-treated CIA rats; flow-cytometry for cell-cycle analysis, western-blotting for intracellular signal pathway protein expressions, gelatin-zymography for matrix-metalloproteases 2/9 (MMP-2/9) and GF-AFC for dipeptidyl-peptidase I (DPPI) activity assay. Compared with untreated CIA rats, Lipo-DiMC treatments relieved paw-swellings, suppressed the increments of immunocytes numbers and inhibited DPPI and MMP-2/9 over-activity in blood. Lipo-DiMC adjusted CIA-induced cell cycle dysfunction at G0/G1-phase and S-phase of spleen lymphocytes for CIA rats. The intracellular expression-trends of P38, P21, Bcl-2, JNK-1 and DPPI of spleen lymphocytes were observed during CIA progress with and without Lipo-DiMC administrations. Lipo-DiMC exhibited its therapeutic functions by attenuating CIA development in rats, associated with down-regulating CIA-induced lymphocytes numbers, inhibiting over-expressed of DPPI and MMP-2/9, and adjusting cell cycles. These findings provide a new insight into the mechanism of Lipo-DiMC treatment in CIA rat model and suggest that Lipo-DiMC could be considered as a potential drug for RA treatment.

Inhibitory effect of Triperygium wilfordii polyglucoside on dipeptidyl peptidase I in vivo and in vitro.

BACKGROUD: Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease is derived from granule immune cells including mast cell, neutrophils, and toxicity T cells. DPPI can activate serine proteases by removal of dipeptides from N-termini of the pro-proteases, resulting in granule immune cells activation which involved in physiological or pathological responses. Triperygium Wilfordii Polyglucoside (TWP) is one of the traditional Chinese medicines, and commonly used in rheumatoid arthritis (RA) treatment. The present study intended to evaluate the effects of TWP on DPPI activity. METHODS: In vivo and in vitro studies were carried out to investigate the functions of TWP or triptolide (TP) on DPPI activities in serum, tissues of CIA rats. Rats were divided into five groups randomly: normal group, untreated CIA rat group, TWP treatment CIA groups (the low dose 2.5mg/100g body-weight and high dose 5mg/100g body-weight), and TP treatment CIA group (4µg/100g body-weight). Arthritis development was monitored visually, and joint pathology was examined radiologically. Total protein concentrations in synovial fluids (SFs) were determined by BCA method. Serums and tissue homogenates from CIA rats were collected and DPPI activities were detected using fluorescence substrate GF-AFC. The in vitro interactions between DPPI in serums or in tissue homogenates and TWP or TP were assessed. RESULTS: TWP-treated CIA rats showed a significant improvement in bone erosion. TWP significantly suppressed paw swelling and total protein concentration in the SFs of CIA rats compared with untreated CIA rats. The elevated activities of DPPI in serums or tissues of CIA rats were significantly inhibited by TWP, but not by TP in vivo. The inhibitory effects of TWP on DPPI activities were also confirm by in vitro study. CONCLUSION: One of the therapeutic functions of TWP in RA treatment could be inhibiting DPPI activity in serums and synovial tissue produced during RA development, and then reducing inflammatory serine proteases activities and further recovering CIA rats from RA symptoms.