Adjuvant Arthritis Research Articles

Effect of Saffron Extract, Astaxanthin, and Carnosic Acid on the Levels of Matrix Metalloproteinase-9 and on Body Weight Changes in Arthritis Experiments

Abstract AIM The aim of this study was to explore the potential effect of natural compounds and their combination with methotrexate (M) on levels of matrix metalloproteinase-9 (MMP-9) as a key biochemical parameter in rat adjuvant arthritis. Further change of body weight was selected as one of clinical parameters monitored in this animal model. MATERIALS AND METHODS Adjuvant arthritis (AA) was induced in Lewis rats. Methotrexate (M) was administrated twice a week in a dose of 0.3 mg/kg b.w. The saffron extract was administrated daily in two doses: 25 mg/kg b.w. (SF1) and 50 mg/kg b.w. (SF2). Both doses were administrated alone and in combination with M. Astaxanthin was administrated also daily in two doses: 1 mg/kg b.w. (AS1) and 5 mg/kg b.w. (AS2) only as monotherapy. Carnosic acid was administrated daily in one dose: 100 mg/kg (C) in monotherapy and in combination with M. All compounds and M were administrated orally. Plasma samples were collected on the 21st experimental day and used for ELISA determination. The 21st experimental day was used also for the analysis of body weight changes. RESULTS We observed a significant decrease of MMP-9 plasmatic levels in SF1 and SF2 monotherapy in AA animal groups. The decrease in levels of MMP-9 in combined therapy of SF1 and M had higher significance than the effect of M only in AA. The same decreasing effect on the levels of MMP-9 was observed in the combined therapy of C and M. Astaxanthin and saffron extract had a very similar effect on clinical parameters and the change in body weight: both have significantly increased body weight in monotherapy in both doses used. The combined therapy of M and saffron extract doses showed no significant difference from M itself. Carnosic acid did not affect the change of body weight, and the combination of C with M reached the same level as M alone. CONCLUSION Astaxanthin in monotherapy and saffron extract in monotherapy and in combined therapy with M have significantly decreased plasmatic levels of MMP-9 and increased body weight in animals suffering from AA. Lower doses were more efficient for both experiments: astaxanthin and saffron extract. Carnosic acid has no effect in monotherapy in both parameters, but a combination with M has a significant effect with respect to the improvement of cachexia as well as the inhibition of inflammation.

Geniposide restricts angiogenesis in experimentary arthritis via inhibiting Dnmt1-mediated PTEN hypermethylation

Neovascularization in rheumatoid arthritis (RA) is a key bridge between malignant proliferative synovial tissue and pannus. In view of previous studies on the efficacy of Geniposide (GE) in experimentary arthritis, the purpose of this study was to investigate the possible mechanism of GE inhibiting angiogenesis by regulating the gene of phosphate and tension homology deleted on chromosome ten (PTEN). In this study, human umbilical vein endothelial cells (HUVEC) and adjuvant arthritis (AA) rat models were performed to research in vitro and in vivo. The results showed that GE treatment significantly reduced synovitis and angiogenesis in AA rats, which may be associated with the increased expression of PTEN with GE treatment. Meanwhile, the hypermethylation of PTEN accompanied by the over-expression of DNA methyltransferases (Dnmts) was demonstrated in TNF-α-induced HUVEC and AA rats. Knockdown of Dnmt1 by Dnmt1- siRNA significantly inhibited the tube formation of HUVEC in vitro. GE significantly restricted the angiogenesis of HUVEC by inhibiting DNA methylation, which was attributed to the down-regulation of Dnmt1 rather than Dnmt3a and Dnmt3b. The anti-angiogenesis effect of GE was further verified in AA model by the inhibition of Dnmt1. These results indicate that GE exhibited anti-angiogenesis effects in experimentary arthritis by inhibiting Dnmt1-mediated PTEN gene hypermethylation, which may brings new insights for the prevention and research of RA.

Metabonomic analysis of abnormal sphingolipid metabolism in rheumatoid arthritis synovial fibroblasts in hypoxia microenvironment and intervention of geniposide.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by a joint hypoxia microenvironment. Our previous untargeted metabolomics study found that sphingolipid (SPL) metabolism was abnormal in the joint synovial fluid samples from adjuvant arthritis (AA) rats. Geniposide (GE), an iridoid glycoside component of the dried fruit of Gardenia jasminoides Ellis, is commonly used for RA treatment in many Asian countries. At present, the mechanism of GE in the treatment of RA, especially in the joint hypoxia microenvironment, is not entirely clear from the perspective of SPL metabolism. The purpose of this research was to explore the potential mechanism of abnormal SPL metabolism in RA joint hypoxia microenvironment and the intervention effect of GE, through the untargeted metabolic analysis based on the ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Arthritis index, foot swelling and histopathology were used to assess whether the AA rat model was successfully established. The SPLs extracts collected from AA rats’ synovial tissue, serum and rheumatoid arthritis synovial fibroblasts (RASFs, MH7A cells, hypoxia/normoxia culture) were analyzed by metabolomics and lipdomics approach based on UPLC-Q-TOF/MS, to identify potential biomarkers associated with disorders of GE regulated RA sphingolipid metabolism. As a result, 11 sphingolipid metabolites related to RA were screened and identified. Except for galactosylceramide (d18:1/20:0), GE could recover the change levels of the above 10 sphingolipid biomarkers in varying degrees. Western blotting results showed that the changes in ceramide (Cer) level regulated by GE were related to the down-regulation of acid-sphingomyelinase (A-SMase) expression in synovial tissue of AA rats. To sum up, this research examined the mechanism of GE in the treatment of RA from the perspective of SPL metabolism and provided a new strategy for the screening of biomarkers for clinical diagnosis of RA.

TRPM7 channel inhibition attenuates rheumatoid arthritis articular chondrocyte ferroptosis by suppression of the PKCα-NOX4 axis

A role for ferroptosis in articular cartilage destruction associated with rheumatoid arthritis (RA) has not been identified. We previously reported transient receptor potential melastatin 7 (TRPM7) expression was correlated with RA cartilage destruction. Herein, we further characterized a role for TRPM7 in chondrocyte ferroptosis. The expression of TRPM7 was found to be elevated in articular chondrocytes derived from adjuvant arthritis (AA) rats, human RA patients, and cultured chondrocytes treated with the ferroptosis inducer, erastin. TRPM7 knockdown or pharmacological inhibition protected primary rat articular chondrocytes and human chondrocytes (C28/I2 cells) from ferroptosis. Moreover, TRPM7 channel activity was demonstrated to contribute to chondrocyte ferroptosis by elevation of intracellular Ca2+. Mechanistically, the PKCα-NOX4 axis was found to respond to stimulation with erastin, which resulted in TRPM7-mediated chondrocyte ferroptosis. Meanwhile, PKCα was shown to directly bind to NOX4, which could be reduced by TRPM7 channel inhibition. Adeno-associated virus 9-mediated TRPM7 silencing or TRPM7 blockade with 2-APB alleviated articular cartilage destruction in AA rats and inhibited chondrocyte ferroptosis. Collectively, both genetic and pharmacological inhibitions of TRPM7 attenuated articular cartilage damage and chondrocyte ferroptosis via the PKCα-NOX4 axis, suggesting that TRPM7-mediated chondrocyte ferroptosis is a promising target for the prevention and treatment of RA.

Role of cryopreserved multipotent mesenchymal stromal cells in modulation of some indices of cell immunity in adjuvant arthritis.

InroductionThe results of experimental and clinical studies in recent years indicate that the transplantation of multipotent mesenchymal stromal cells (MMSCs) is a possible approach for the “restoration” of the immune system of patients with autoimmune diseases, in particular, rheumatoid arthritis. However, the strength and duration of the effect vary greatly, which indicates incomplete correction of the tested parameters, thereby opening up the prospect of improving this method of treatment by choosing dose-time parameters and methods of their administration. The aim of this research was to determine the indices of cellular immunity in animals with adjuvant arthritis and therapy with cryopreserved MMSCs derived from adipose and cartilage tissues.Material and methodsAdjuvant arthritis in male rats was modeled by subplantar administration of Freund’s complete adjuvant. On day 7 of modeling, experimental animals were administered with saline (control group) or cryopreserved MMSCs from adipose or cartilaginous tissue locally or generalized. On day 28 after therapy the body weight, spleen index and cellularity, and content of CD3+, CD4+, CD8+, CD4+CD25+ cells in the spleen were determined.ResultsIn the control group of animals, the inflammation was pronounced, as evidenced by a significant increase in the studied parameters throughout the observation period. The use of cryopreserved MMSCs from adipose and cartilaginous tissues led to the restoration of T regulatory cells (Treg) on day 28. Generalized administration of cells had a more pronounced therapeutic effect compared to the animals with local administration. These data can be used to justify and develop a therapeutic approach to rheumatoid arthritis in clinical practice.ConclusionsCell therapy with cryopreserved MMSCs from investigated sources provided by both local and generalized administration to animals with adjuvant arthritis has a correcting effect on the cellular immunity.

Correction of Cytokine Profile in Autoimmune Diseases with Cryopreserved Embryofetoplacental Complex Products

In this work, we experimentally substantiated the possibility of correcting the cytokine profile to reduce the inflammation in mice with adjuvant arthritis (AA) via combined use of lyophilized human cord blood leukoconcentrate (lHCBL) and cryopreserved human placental extract (cHPE). Adjuvant arthritis was modelled in CBA/H mice. To day 7 after pathology induction, the animals were injected with lHCBL and cHPE singly or in combination. To days 14 and 28 of observation, all the introduced products showed a therapeutic effect. To 28 day of observation, the efficiency of combined use of lHCBL and cHPE was 3.1 and 2.5 times higher vs. the monotherapy (respectively), likely resulting from their synergy in respect to a decrease in arthritis index, TNFα concentration, impact of lHCBL and cHPE on IFNγ and IL-6 levels, correspondingly.

Anti-inflammatory properties of the extract of maruna virgin (Tanacetum parthenium L.) on the model of chronic inflammation modeled by the Freund’ adjuvant in rats

Еhe aim of the study was to investigate the effectiveness of a thick extract of feverfew (Tanacetum parthenium L.) herb in chronic inflammation (adjuvant arthritis) in rats.
 The anti-inflammatory properties of the water-alcohol thick extract of feverfew (Tanacetum parthenium L.) herb (ETP) were studied in the model of adjuvant arthritis (ADA) (0.1 ml of Freund's adjuvant (PAF), once subcutaneously at the base of the tail of white nonlinear male rats (body weight 200–220 g). After 7 days, the introduction of PAF was repeated (subcutaneously in the right hind paw of rats). The animals were divided into 4 groups of 9 rats each, in the intact group – 6 animals. Distilled water (1 ml/100 g) was administered to positive control animals (PCs) on the background of pathology. An aqueous solution of ETP (50 mg/kg) and diclofenac sodium (1% starch suspension, 8 mg/kg) were administered from day 1 of the experiment and for four weeks daily intragastrically once daily. The volume of the affected foot (V) was measured using a LE7500 plethysmometer (PANLAB, Italy) in dynamics – at 1, 5, 10, 15, 20, 25 and 30 days after administration of a permissive dose of PAF. The intensity of inflammation in rats was assessed by peripheral blood parameters, biochemical indicators - markers of the inflammatory process. Statistical analysis of data was performed using the standard software package «Statistica v. 6.0». Differences between groups were considered statistically significant at p <0.05.
 The course of ADA was characterized by the development of systemic inflammation, as evidenced by an increase in the number of leukocytes, increased erythrocyte sedimentation rate (ESR) and the content of C-reactive protein (CRP). A statistically significant increase in the level of LF and sialic acids indicates membrane-destructive processes in connective tissue. Against the background of ETP significantly decreased the level of the main indicator of inflammation CRP 2 times, the content of markers of destruction of joint tissue – sialic acid and LF decreased by 1.9 times relative to PC animals, indicating a positive effect on local and systemic manifestations of inflammation in ADA. The effectiveness of ETP was not inferior to diclofenac sodium (DS).
 Based on the data obtained, it can be concluded that the studied ETP at a dose of 50 mg/kg in the ADA model in rats has a pronounced anti-inflammatory effect and is not inferior to diclofenac sodium, which makes it promising further research as an effective anti-inflammatory agent.

Ethanolic Extracts of leaves of Alstonia scholaris Linn. Modulates Chronic Inflammatory Immune Response in FCA Induced Arthritis

AIM- The aim of the present investigation is scientifically establishing the anti-arthritic activity of selected medicinal plant so that we are able to come up with a more effective and potent bioactive phytoconstituents with less side effects in comparison with existing synthetic drugs. MATERIAL & METHODS- The leaves of Alstonia scholaris were collected from campus of College of Pharmacy. All the plant materials were taxonomically identified by Dr. Gyanendra Tiwari, Senior Scientist, KNK College of Horticulture. All the plant materials were dried under shade and subjected to coarse powder for extraction process. Accurately weighed quantity of leaf powder of Alstonia scholaris were extracted using 95 % ethanol by soxhlet apparatus for 72 h. The ethanolic extracts were dried under the reduced pressure to get crude ethanolic extracts. The Wistar albino rats were divided into 10 groups of six animals in each. For the induction of chronic inflammatory response, FCA (0.1 ml) was injected through intra-articular injection in left ankle joint of rats on 0 day. The severity of adjuvant arthritis was quantified by measuring the volume of the hind paw using Plethysmograph. Body weight was measured of all groups at zero days before immunization and at 21st day after treatments over by using a single pan weighing balance. RESULTS- The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds. The assessment made on the 21st day showed that the A. scholaris treatments at both doses (low and high) had moderately significant and highly significant effect and reduced (p< 0.01& p<0.001) the adjuvant-induced lesions in the respective treatment groups as compared with the arthritis control group. However, standard, A. scholaris had highly significant effects (p<0.001) in recovery of RBCs and haemoglobin. They also showed highly significant effects on decrease in WBCs and ESR treated groups, also showed moderately significant effects as compared to arthritic group. CONCLUSION- Besides from the obvious therapeutic importance, these components would be useful in understanding the mechanism of diseases with higher levels of cellular and molecular level. These components could serve as lead molecules for development of prospective anti-arthritic agents. Keywords: Anti-arthritic agent, Lead molecules, Alstonia scholaris, Shronic inflammatory response, Synthetic drugs, Plethysmograph

Effect of Buyi Pishen acupuncture on inflammatory factor and cartilage matrix in adjuvant arthritis rats

To observe the effect of Buyi Pishen acupuncture (acupuncture for invigorating spleen and kidney) on inflammatory factor and synovial cartilage matrix in adjuvant arthritis (AA) rats, and to explore the mechanism of acupuncture for rheumatoid arthritis (RA). A total of 60 clean male Wistar rats were randomized into a normal group, a model group, a tripterygium wilfordii polyglycoside tablet (TWP) group and an acupuncture group, 15 rats in each group. Rats in the model group, the TWP group and the acupuncture group received intradermal injection of Freund's complete adjuvant (FCA) at right hind foot pad to induce the AA model. TWP suspension of 8 mg/kg was given by gavage in the TWP group. Acupuncture was applied at "Shenshu" (BL 23), "Pishu" (BL 20) and right "Housanli" (ST 36), "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34) in the acupuncture group, 15 min a time, once a day. The intervention was given 15 days in both TWP group and acupuncture group. The foot-pad swelling degree before modeling, before and after intervention and the arthritis index (AI) score before and after intervention were calculated; the serum levels of interleukin (IL)-1β, IL-4, IL-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA method; the ultrastructure and histomorphological changes of synovium issue were observed by transmission electron microscope and HE staining; the positive expression of matrix metalloproteinase (MMP)-3 and MMP-9 in synovium issue was detected by immunohistochemistry method. Before intervention, foot-pad swelling degree of the model group, the TWP group and the acupuncture group was increased compared with the normal group (P<0.01). After intervention, foot-pad swelling degree and AI score were increased compared with the normal group (P<0.01), foot-pad swelling degree and AI scores in the TWP group and the acupuncture group were lower than the model group (P<0.05), and those in the acupuncture group were decreased compared with the TWP group (P<0.05). The model group exhibited unclear nuclear membrane of synovial cells, chromatin pyknosis, massive inflammatory cell infiltration and hyperplasia in synovial tissue; the TWP group and the acupuncture group exhibited clear and smooth nuclear membrane of synovial cells, inapparent chromatin pyknosis, less inflammatory cell infiltration and hyperplasia in synovial tissue, the acupuncture group exhibited less matrix destruction as well. Compared with the normal group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were increased (P<0.01), while serum levels of IL-4 and IL-10 were decreased (P<0.01) in the model group. Compared with the model group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05, P<0.01), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the TWP group and the acupuncture group; compared with the TWP group, serum level of TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the acupuncture group. Buyi Pishen acupuncture can effectively improve the injury of articular cartilage in AA rats, its mechanism maybe related to reducing the inflammatory reaction in synovium and inhibiting the degradation of articular cartilage matrix.

Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway

Background Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway. Materials and Methods Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and p-GSK-3β (Ser9) protein were detected by immunofluorescence and Western blot. Results We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b, β-catenin, c-Myc, cyclin D1, and p-GSK-3β/GSK-3β mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway.

Effect of Er Miao San on peritoneal macrophage polarisation through the miRNA-33/NLRP3 signalling pathway in a rat model of adjuvant arthritis

Context Er Miao San (EMS) is a formulation that contains Atractylodis Rhizoma and Phellodendri Cortex in 1:1 ratio, and is commonly used to treat rheumatoid arthritis (RA) and other inflammatory diseases. Objective We investigated the mechanism of action and effects of EMS on peritoneal macrophage differentiation in a rat model of adjuvant arthritis (AA). Materials and methods EMS (3, 1.5 and 0.75 g/kg; once daily) and methotrexate (0.5 mg/kg; once every 3 days) were administered orally from days 21 to 35 after immunisation. Paw swelling and arthritis index were measured; pathological changes in the ankle joint were observed using x-ray and haematoxylin eosin staining. The ratio of CD86/CD206 in macrophages was detected by flow cytometry. Examination of the miRNA-33/NLRP3 signalling pathway was examined by RT-qPCR and western blotting. The levels of cytokines in the serum and cell supernatants were tested by ELISA. Results EMS significantly reduced the AA index in rats (from 11.0 to 9.3) and pathological changes in the ankle joint (from 3.8 to 1.4). The ratio of CD86/CD206 was reduced, and polarisation to M1 improved (from 0.9 to 0.6) in macrophages of EMS-treated rats. EMS downregulated the miRNA-33/NLRP3 pathway. Furthermore, EMS treatment increased IL-10 and TGF-β levels in the serum and supernatant of macrophages of AA rats and simultaneously decreased the levels of IL-1β and TNF-α. Discussion and conclusions Our results suggest that EMS may reduce macrophage polarisation to the M1 inflammatory phenotype by downregulating the miRNA-33/NLRP3 pathway in AA rats. These findings may provide new insights into the treatment of RA.

Anti‐inflammatory effects of Angiotensin‐(1‐7) through Renin‐Angiotensin System and Arachidonic Acid Pathway

ObjectiveRheumatoid arthritis (RA) is a prevalent chronic inflammatory disease that affects 1‐2% of the population worldwide. Renin‐angiotensin system (RAS) and Arachidonic acid (ArA) pathway are associated with the pathogenesis of inflammatory diseases (Fig. 1). Inflammatory conditions such as RA disrupt the balance between pro‐ and anti‐inflammatory components of these two pathways. The RAS is composed of two opposing axes. Ang II is one of its components that is implicated in the up‐regulation of pro‐inflammatory mediators and contributes to the pathogenesis of RA. On the other hand, Ang‐(1‐7), counteracts the inflammatory effects of Ang II. Several studies have shown a cross‐talk between the RAS and ArA metabolites, especially eicosanoids such as 20‐hydroxyeicosatetraenoic acid (20‐HETE) and epoxyeicosatrienoic acids (EETs). Ang II stimulates 20‐HETE synthesis in microvessels and decreases EET levels by increasing their degradation. In this study, using Ang‐(1‐7) and its long‐acting bone‐targeting conjugate, we studied the impact of Ang‐(1‐7) anti‐inflammatory effects on the RAS, ArA pathways and their interrelation.MethodsTwenty‐four male Sprague‐Dawley rats were randomly divided into healthy control and adjuvant arthritic (AA) rats. The rats in the AA group were injected with Mycobacterium butyrium in squalene to induce AA. AA rats were further divided into and treated with four groups INF, Ang‐(1‐7), and Ang. Conj. After 21 days of treatment, animals’ plasma samples were collected for analysis of Ang II, Ang‐(1‐7), 20‐HETE, and different EETs. The quantification was done using an LC‐MS/MS system in positive ESI mode for Ang peptides and negative for HETE and EETs. The one‐way ANOVA with Tukey posthoc test was used to test the significance difference (p‐value &lt; 0.05).ResultsThe Ang peptide analysis shows that the level of Ang‐(1‐7) in control (0.75 ± 0.08) and INF (0.19 ± 0.05) rats was significantly different. However, treating with Ang‐(1‐7) peptide (0.90 ± 0.17) or Ang. Conj. peptide conjugate (1.45 ± 0.22) could increase the Ang‐(1‐7) peptide level in rats. In contrast to Ang‐(1‐7), Ang II levels in the INF group were significantly different from control, Ang‐(1‐7), and Ang. Conj. (Fig. 2A). Analyzing the level of total EETs and 20‐HETE in plasma reveals that the total EETs/20‐HETE ratio decreases in INF rats (0.10 ± 0.02) significantly, whereas this ratio increased in Ang‐(1‐7) (3.48 ± 0.65), and Ang. Conj. (7.73 ± 1.21) in comparison to the control (6.90 ± 1.03) group (Fig. 2B). Our finding reveals a positive correlation between the level of Ang II and 20‐HETE, Ang‐(1‐7), and total‐EETs. This trend was reversed when Ang II was correlated with total EETs or the ratio of total‐EETs/20‐HETE (Fig. 3B). Concentrations are reported as ng/mL.ConclusionThe physiological balance of CYP‐mediated ArA metabolites and the RAS components are disturbed by the inflammation, resulting in the augmentation of pro‐inflammatory arms of both pathways. Our approach of using Ang‐(1‐7) and its long‐acting bone targeting conjugate could reverse such an effect and promote anti‐inflammatory effect in the AA animal model.

Novel Treatment Option for Rheumatoid Arthritis through Bone‐Targeted Delivery of Novokinin

ObjectiveRheumatoid arthritis (RA) is an autoimmune inflammatory disorder affecting about 1‐2% of the world population. RA is known to be a major cause of disability which progresses eventually to systemic complexities with high chances of early morbidity. This complex dynamic situation of inflammation is orchestrated by multiple systems in the body, including the Renin‐Angiotensin System (RAS). Angiotensin II (Ang II), a main effector peptide in the RAS, predominantly binds to Angiotensin II Type (I) receptor (AT1R). When it overproduced can result in inflammation, vasoconstriction, and oxidative stress. Ang II also binds to Angiotensin II Type (II) receptor (AT2R), which provides functional antagonism to AT1R activation. Novokinin, a synthetic peptide, promotes beneficial effects through activation of AT2R, and can be considered a potential drug candidate for alternative therapy in RA if its plasma stability and half‐life could be improved. Here, we developed a novel bone‐targeting novokinin Conjugate (Novo Conj), which binds to the bone, utilizes it as a reservoir while protecting it from enzymatic and hydrolytic degradation. We hypothesized that this delivery approach would extend the peptide half‐life and provide sustained plasma levels to elicit anti‐inflammatory activity.MethodHealthy male, Sprague Dawley rats, were induced adjuvant arthritis (AA) by administering 0.2mL of 50mg/mL of Mycobacterium butyricum through the tail base injection. Arthritic rats were divided into three groups (n=5/group) and dosed with saline (Inflamed), novokinin of 400 µg/kg (Novokinin), or an equivalent dose of 400 µg/kg of novokinin (Novo Conj). A group of healthy rats (n=5) received saline and was taken as a healthy control (Control) group. After the emergence of arthritis (i.e., 10‐12 days after adjuvant arthritis induction), animals were dosed with assigned treatment every other day for two weeks. Animals were monitored three times a week for their body weight, paw, and joint diameters. At the end of the experiment, rats were euthanized, plasma/tissues were harvested and analyzed for plasma nitric oxide (NO) and the RAS components by western blot.ResultsThe induction of AA led to a significant reduction in percent body weight gain over time, increased paws and joints swelling, and elevated plasma NO levels. AA brought an imbalance in the RAS enzymatic inflammatory axis indicator, i.e., Angiotensin‐Converting Enzyme (ACE) and ACE2 causing a reduction in ACE2/ACE and AT2R/AT1R ratio. The treatment of arthritic rats by novokinin and Novo Conj restored their body weight gain, reduced paws and joints' swelling, lowered the plasma NO level, and reinstated the imbalanced ratios of enzymes and receptors. These observed anti‐inflammatory effects were significantly higher in the Novo Conj group.ConclusionThis result indicates that the novokinin and its bone‐targeted conjugate shows anti‐inflammatory effects through the RAS. The observed enhanced impacts could be attributed to improved stability and extension of the half‐life of novokinin through its conjugation and binding to the bone. Our novel bone‐targeting delivery of novokinin offers effective alternative therapy for managing RA signs and symptoms and could be further utilized for various RAS disorders.

Methotrexate-loaded biodegradable nanoparticles exert anti-arthritic effect by downregulating pro-inflammatory cytokines in Freund's complete adjuvant-induced arthritic rats.

Methotrexate (MTX), the first-line drug for the treatment of rheumatoid arthritis (RA), can cause considerable toxicity, which limits effective dosage regimens. Moreover, it has rapid clearance, which leads to poor patient compliance. To mitigate such challenges, this study aimed to validate the use of MTX-loaded chitosan nanoparticles (NPs) in treating Freund's complete adjuvant (FCA) arthritis in rats. Healthy Wistar rats (n = 30) were divided into five groups. The first group served as healthy control, while the second group served as arthritic control. Group 3 was administered methotrexate, while groups 4 and 5 were MTX-loaded NP-treated groups. NPs were prepared by solvent evaporation method and characterized by zeta size, potential, polydispersity index (PDI), and Fourier-transform infrared spectroscopy. NPs were 190nm in size, and PDI was 0.25, confirming the uniform distribution of NPs. A significant increase in paw thickness was noted up to the 21st day of the study, which was reversed by a high dose of MTX-loaded NPs. MTX NPs significantly reduced the level of pro-inflammatory markers, including TNF-α and IL-6, along with improving control of oxidative stress biomarkers. The findings of biochemical, haematological, radiological, and histopathological investigations further confirmed amelioration of necrosis and cellular infiltration. It can be concluded that MTX-loaded chitosan NPs are promising candidates for treating FCA-induced arthritis in a rat model.

The effect of an aminoguanidine derivative on adjuvant arthritis in rats

Aim. To study anti-inflammatory, analgesic, and possible ulcerogenic effects of a novel aminoguanidine derivative in adjuvant arthritis (a model of rheumatoid arthritis) in rats.Materials and methods. The experiments were carried out on 42 outbred male Sprague Dawley rats. After modeling arthritis (starting from day 7 after the administration of complete Freund’s adjuvant), intramuscular injections of the aminoguanidine derivative (code LIS-M) at a dose of 2.5, 5, and 10 mg / kg or the reference drug diclofenac at a dose of 4 mg / kg were performed once a day for 22 days. The volume of the inflamed limb was measured twice a week, pain threshold was measured every week. After finishing the administration of the compounds, the levels of interleukin (IL) 1, IL-6, and tumor necrosis factor-α (TNFα) were measured in rat plasma, the ankle joint was histologically studied, and the gastric mucosa was studied to detect damage, ulcers, and scarring.Results. The aminoguanidine derivative, an inhibitor of inducible nitric oxide synthase, was more effective at the dose of 10 mg / kg than diclofenac at the dose of 4 mg / kg. It had anti-inflammatory and analgesic effects in the joint affected by complete Freund’s adjuvant, promoted restoration of the histologic structure in the synovial membrane and articular cartilage, and reduced the plasma concentration of IL-1, IL-6, and TNFα by 1.4–1.5 times. The LIS-M compound did not damage the gastric mucosa in rats with adjuvant arthritis.Conclusion. The aminoguanidine derivative LIS-M exerts potent anti-inflammatory and analgesic effects in adjuvant arthritis in rats (a model of rheumatoid arthritis). LIS-M has no ulcerogenic effect on the gastric mucosa in rats.

Effect of moxibustion on inflammatory pain and N-methyl-D aspartic acid receptor-nitric oxide-cyclic GMP pathway in spinal cord of adjuvant arthritis rats

To observe the effect of moxibustion on pain and N-methyl-D aspartic acid receptor/nitric oxide/cyclic guanosine monophosphate (NMDA-NO-cGMP) signaling pathway in the spinal cord of rats with adjuvant arthritis (AA), so as to explore its underlying mechanisms in relieving inflammatory pain of rheumatoid arthritis (RA). SD rats were randomly divided into normal, model, moxibustion (Moxi), Moxi +NMDA receptor antagonist AP-5 (Moxi+AP-5) and Moxi +NMDA receptor agonist (NMDA) groups, with 20 rats in each group. The AA model was established by placing the rats in a wind, cold and damp environment for 12 h, once daily for 20 days and by injection of complete Freund's adjuvant into the right hind paw. Rats of the three Moxi groups received ignited moxa-stick stimulation of "Zusanli"(ST36) and "Shenshu"(BL23) alternately for 20 min, once a day for 15 days. The Moxi + AP-5 group and Moxi +NMDA group received intraperitoneal injection of AP-5 (0.7 mg·kg-1·d-1) and NMDA (5 mg·kg-1·d-1), respectively, once a day, for a total of 15 days. Mechanical pain thres-hold (MPT) was measured before and after modeling and interventions. The spinal cord tissue was sampled for detecting the expression of iNOS mRNA and protein, content of cGMP and NO, and the activity of NOS by using fluorescence quantitative PCR, Western blot, ELISA,nitrate reductase method and colorimetric method, respectively. Before modeling, there was no significant difference in MPT among all the 5 groups (P>0.05). After modeling, the MPT was remarkably decreased (P<0.01), the expression levels of iNOS mRNA and protein,the contents of cGMP and NO, the activity of NOS were significantly increased in the model group relevant to the normal group (P<0.01). After the interventions, the MPT was obviously increased (P<0.01), while the expression levels of iNOS mRNA and protein, the contents of cGMP and NO, the activity of NOS were significantly down-regulated in the Moxi, Moxi-AP-5 and Moxi+NMDA groups (P<0.05, P<0.01). The effect of Moxi+AP-5 group was significantly superior to that of Moxi group in raising MPT and down-regulating the expression levels of iNOS mRNA and protein, and the content of NO (P<0.05, P<0.01). The effect of Moxi+NMDA group was obviously inferior to that of Moxi group in up-regulating MPT and down-regulating the levels of iNOS mRNA and protein, and the contents of cGMP and NO, and the activity of NOS (P<0.01), suggesting a reduction of the therapeutic effects in raising MPT and down-regulating expression of iNOS mRNA and protein after administration of AP-5. Moxibustion can relieve RA inflammatory pain in AA rats, which may be related to its function in down-regulating the NMDA/NO/cGMP signaling pathway in the spinal cord.

Вплив клітинної терапії на динаміку маркерів запалення в крові щурів з ад’ювантним артритом

The purpose of the study was to investigate the dynamics of inflammation in blood of the rats with adjuvant arthritis under the conditions of generalized and local administration of cryopreserved multipotent mesenchymal stromal cells from adipose and cartilage tissues. Materials and methods. A model of adjuvant arthritis in male rats was induced by subplantar administration of Freund’s complete adjuvant. On the 7th day of modeling, experimental animals were administered: control group – saline; experimental group 1 and 2 – cryopreserved multipotent mesenchymal stromal cells from adipose or cartilaginous tissue locally; experimental group 3 and 4 – cryopreserved multipotent mesenchymal stromal cells from adipose or cartilaginous tissue generalized. Groups of intact animals served as control. On days 14, 21 and 28 erythrocyte sedimentation rate was determined in blood, and content of total protein, C-reactive protein and rheumatoid factor were measured in serum. Results and discussion. In the control group of animals, the inflammatory process was pronounced, as evidenced by a significant increase in the studied parameters throughout the observation period. The use of cryopreserved multipotent mesenchymal stromal cells from adipose and cartilaginous tissues led to the restoration of the total protein content and erythrocyte sedimentation rate on the 28th day of observation, as well as positive dynamics in the level of C-reactive protein and rheumatoid factor. Generalized administration of cells had a more pronounced therapeutic effect compared to the animals with local one. A comparative assessment of the use of cryopreserved multipotent mesenchymal stromal cells from adipose and cartilaginous tissues showed the absence of significant changes in the studied parameters both with studied ways of cell administration. These data can be used to justify and develop methods of treating arthritis in clinical practice. But important issue that needs further research is the determination of cell immunity in the animals with adjuvant arthritis and treatment of cryopreserved multipotent mesenchymal stromal cells from adipose and cartilage tissues. Conclusion. Cell therapy with the use of cryopreserved multipotent mesenchymal stromal cells from the investigated sources provided by both local and generalized administration to animals with adjuvant arthritis has a modulating effect on the course of inflammation

Geniposide alleviates VEGF-induced angiogenesis by inhibiting VEGFR2/PKC/ERK1/2-mediated SphK1 translocation

Rheumatoid arthritis (RA) is an angiogenesis-dependent disease caused by the imbalance of pro- and anti-angiogenic factors. More effective strategies to block synovial angiogenesis in RA should be studied. Geniposide (GE), a natural product isolated from the fruit of Gardenia jasminoides Ellis (GJ), is reported to have anti-inflammatory, anti-angiogenic and other pharmacological effects. However, the underlying mechanism through which GE affects synovial angiogenesis in RA remains unclear. In this research, we aimed to elucidate the effect and potential mechanisms of GE on angiogenesis in RA. Synovial angiogenesis in patients with RA and a rat model of adjuvant arthritis (AA) was detected by hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and western blottiing. The biological functions of vascular endothelial cells (VECs) and sphingosine kinase 1 (SphK1) translocation were checked by CCK-8, EdU, Transwell, tube formation, co-immunoprecipitation assays, and laser scanning confocal microscopy. The effect of the SphK1 gene on angiogenesis was assessed by transfection of SphK1-siRNA in cells and mices. The effect of GE on VEGF-induced angiogenesis was measured by Matrigel plug assay in a mouse model of AA. GE effectively inhibited synovial angiogenesis and alleviated the disease process. SphK1, as a new regulatory molecule, has a potentially important relationship in regulating VEGF/VEGFR2 and S1P/S1PR1 signals. SphK1 translocation was activated via the VEGFR2/PKC/ERK1/2 pathway and was closely linked to the biological function of VECs. GE significantly reduced SphK1 translocation, thereby ameliorating the abnormal biological function of VECs. Furthermore, after transfection of SphK1 siRNA in VECs and C57BL/6 mice, silencing SphK1 caused effectively attenuation of VEGF-induced VEC biological functions and angiogenesis. In vivo, the Matrigel plug experiment indicated that GE significantly inhibited pericyte coverage, basement membrane formation, vascular permeability, and fibrinogen deposition. Our findings suggest that GE inhibited VEGF-induced VEC biological functions and angiogenesis by reducing SphK1 translocation. Generally, studies have revealed that GE down-regulated VEGFR2/PKC/ERK1/2-mediated SphK1 translocation and inhibited S1P/S1PR1 signaling activation, thereby alleviating VEGF-stimulated angiogenesis. The above evidences indicated that angiogenesis inhibition may provide a new direction for RA treatment.

Anti-inflammatory effect of baicalin in rats with adjuvant arthritis and its autophagy- related mechanism.

BACKGROUND: It has been found that baicalin have anti-inflammatory effects since it reduces the elevated levels of pro-inflammatory cytokines. Meanwhile, it has also been shown that baicalin brings positive effects against rheumatoid arthritis (RA). However, little is observed on its beneficial effects on adjuvant arthritis.OBJECTIVE: To consider the anti-inflammatory influence of baicalin on adjuvant arthritis rats and its related autophagy mechanism.METHODS: In this research, there are six groups of rats, each has 10 rats in it. These groups are normal group (normal saline), model group (normal saline), dexamethasone group (0.125 mg/kg dexamethasone), low-dose baicalin group (50 mg/kg baicalin), medium-dose baicalin group (100 mg/kg baicalin) and high-dose baicalin group (200 mg/kg baicalin). The degrees of adjuvant-induced swelling in rats’ feet were measured every 4 days and the arthritis scores were calculated every 7 days. The inflamed joint tissues were taken after rats were sacrificed. The rat’ joints showed pathological changes, which were observed by HE staining. The relative expression levels of inflammatory factors IL-6, IL-1, IL-17, TNF-, COX2, and COX1 in the rats’ snovial tissues were detected by RT-PCR. As for the expression levels of autophagy markers Beclin1, Atg5, Atg7, Atg12, microtubule-associated protein-light chain3-II (LC3-II), Bcl-2, and Bax in the synovial tissue, they were discoverd by Western blot.RESULTS: Baicalin could significantly inhibit the inflammatory response of adjuvant arthritis rats.CONCLUSIONS: RT-PCR studies showed that the different doses of baicalin could inhibit the expression of TNF-a, IL-6, IL-1, IL-17, COX2 and COX1 in the synovial tissue ( 0.05 or 0.01). Western blot studies showed that the different doses of baicalin could reduce the expression of Atg5, Atg7, Atg12, LC3-II, Beclin1 and Bcl-2 proteins, and increase the expression of Bax proteins in the synovial tissue.

Nerolidol: a potential approach in rheumatoid arthritis through reduction of TNF-α, IL-1β, IL-6, NF-kB, COX-2 and antioxidant effect in CFA-induced arthritic model.

Rheumatoid arthritis is primarily associated with inflammation and increased level of proinflammatory cytokines which are released by immune cells, macrophages or activation of arachidonic acid metabolism. The expression of these cytokines, oxidative free radicals and the activation of COX-2 enzymes are crucial targets for chronic inflammation. On the basis of established anti-inflammatory efficacy of nerolidol, the primary study was further appraised to determine its approach against Freund's complete adjuvant (CFA) rheumatoid model. Arthritis was induced by inoculation of 0.1mL CFA injection into the left hind footpad of rats. Anti-arthritic potential of nerolidol (at 200, 400 and 800mg/kg doses) was assessed by measuring the paw volume, body weight, serum analysis, histopathological and radiographs of ankle joints. Expressions of cytokine's panels such as IL-10, IL-4, COX-2, NF-kB, TNF-α, IL-6, PGE-2 and IL-1β were determined by real-time qPCR. Antioxidant enzyme analyses were conducted by measuring the SOD, POD and catalase activity from serum and equated with arthritic control group. Nerolidol prevented body weight loss, stabilized biochemical and haematological homeostasis and significantly reduced the paw volume. Furthermore, X-ray and histopathological assessment of ankle joints showed an improvement in the joint structure of rats treated with nerolidol. Besides that, overexpression of gene pointers like TNF-α, IL-1β, IL-6, NF-kB, PGE-2 and COX-2 in CFA-treated control rats were also reversed with nerolidol. This anti-arthritic mechanism was further supported by the increased level of IL-10, IL-4 and serum antioxidant activity. The present findings demonstrate that nerolidol reduced adjuvant arthritis by downregulating the proinflammatory cytokines and upregulating the aforementioned anti-inflammatory cytokines and may be used as a therapeutic substance for the management of human rheumatoid arthritis.