Paw Thickness Research Articles

FORMULATION AND EVALUATION OF HERBAL CAPSULE CONTAINING CURCUMA MANGGA VALETON and ZIJP. EXTRACT AND ITS IMMUNOSTIMULATORY EFFECTS

Objective: The present study was carried out to formulate and evaluate C. mangga extract as granule mass in a capsule dosage form as well as investigate its immunostimulatory effects. Methods: The granule mass was evaluated for its bulk density, tapped density, Carr’s index, Hausner ratio, angle of repose, and flow ability. Meanwhile, capsule weight and disintegration time were determined to evaluate the C. mangga capsule. The immunostimulatory effects of extract in capsule dosage form were investigated by measuring delayed-type hypersensitivity (DTH) response and IgG production. There were six formula prepared (F1-F6). Formula F1-F6 were vary according to the component of adsorbent (Corn starch/polyvinyl pyrrolidone) and filler (maltodextrin/Avicel/lactose) to obtain the optimum formula. Results: The granule mass of all formula had a good flow ability and free-flowing properties according to the Carr’s index (10.26±3.38 to 17.54±1.60) and flowing time (1.05±0.05 to 1.60±0.08 seconds). Of all the capsule formula, F2 capsule containing dry extract of C. mangga with Avicel as the filler met the standard requirement in terms of disintegration time (2.15±0.76 min) and weight uniformity (the deviation was below than 7.5%). The capsule of C. mangga extract enhanced the paw thickness as compared to those of the negative control (P<0.05). The level of Immunoglobulin G (IgG) was also increased after treatment with the capsule of C. mangga extract. Conclusion: The results suggest that the capsule of C. mangga extract has immunostimulatory effects, emphasizing its potential to be used for the treatment of infectious and immunodeficiency diseases.

The Effect of Evogliptin Tartrate on Controlling Inflammatory Pain.

Background: Evogliptin tartrate inhibits dipeptidyl peptidase-4 (DPP-4), boosting glucagon-like peptide 1 (GLP-1) secretion and improving insulin release and glucose tolerance, while also exerting anti-inflammatory effects. We investigated its anti-inflammatory and analgesic effects. Methods: Forty male Sprague Dawley rats were divided into (N = 10 in each): (1) naïve, (2) complete Freund's adjuvant (CFA) inflammation + evogliptin tartrate (once for 10 mg/kg) (CFAE), (3) CFA + vehicle (same volume with normal saline with evogliptin tartrate/once) (CFAV), and (4) CFA + indomethacin (5 mg/mL/kg/1 time) (CFAI) groups. CFA was injected subcutaneously into rat plantar regions, and medications (evogliptin tartrate, vehicle, and indomethacin) were administered orally for 5 days. Post treatment, blood from the heart and plantar inflammatory tissue were collected to assess inflammatory cytokines. Evogliptin tartrate effects on controlling inflammation and pain were evaluated by measuring rat plantar paw thickness, paw withdrawal threshold, dorsal root ganglion (DRG) resting membrane potential, DRG action potential firing, and cytokine (TNF-α and IL-1β) levels. Results: Compared with the naïve group, plantar paw thickness, cytokine (TNF-α and IL-1β) levels, DRG resting membrane potential, and DRG action potential firing increased, whereas the paw withdrawal threshold decreased in all CFA groups. However, CFAE and CFAI rats showed recovery. The degree of CFAE recovery resembled that observed in the CFAI group. Conclusions: Evogliptin tartrate mirrored the anti-inflammatory pain relief of indomethacin. We aim to broaden its use as an anti-inflammatory drug or pain relief drug.

Phytochemical characterization and anti-arthritic potential of Croton bonplandianus leaves extract: In-vivo and in-silico approach

Croton bonplandianus, a natural source traditionally used for treating various illnesses, including rheumatoid arthritis, was evaluated in this study. The effects of ethanolic extracts (CBEE) and aqueous fractions (CBAF) of C. bonplandianus leaves on arthritis-induced inflammation were studied using an albino rat model of inflammation induced by Freund's complete adjuvant. Eight test groups (n = 5 per group) and one vehicle control were used to evaluate the antiarthritic effects of different doses of CBEE and CBAF (125 mg.kg−1, 250 mg.kg−1, and 500 mg.kg−1) on days 5, 10, 15, and 20 compared to arthritic and vehicle controls. Arthritis severity was assessed using macroscopic arthritis grading, histological analysis, body weights, and paw thickness. CBEE and CBAF were found to reduce the prevalence of arthritis, increase body weight, and decrease paw inflammation compared to the vehicle control group by the 23rd day. In addition, they showed no effect on biochemical parameters, but a significant difference (p < 0.05) in hematological parameters compared to the arthritic control group. The study identified Hentriacontane compound as a potential contributor to the anti-inflammatory effect of C. bonplandianus, as it showed the lowest dock score for IL-1β and IL-6. Palmitoylethanol amide was identified as a potential contributor to the anti-inflammatory effect of TNF-α. Gene expression of IL-6, IL-1β, and TNF-α was down-regulated significantly (p < 0.05) in a dose-dependent manner in all treatment groups compared to the arthritic control group. In conclusion, this study validated the anti-arthritic and anti-inflammatory properties of CBEE and CBAF in a time and dose-dependent manner.

Anti-inflammatory potential of a Thai traditional remedy called Prabchompoothaweep in an animal model of acute and sub-acute inflammation

Ethnopharmacological relevancePrabchompoothaweep (PCT) is a Thai remedy which is composed of 23 herbs and has been added onto the National List of Essential Medicines (NLEM) of Thailand. This remedy has been used to treat allergic rhinitis and asthma in Thai traditional medicine for many years. Furthermore, a few studies have reported anti-allergic, anti-malarial, anti-inflammatory and antioxidant activities. Aim of the studyThis study aims to evaluate anti-inflammatory activity of PCT extract in an animal model. Materials and methodsThe animal model of acute inflammation was studied over a 24-h period, utilizing the method of carrageenan-induced paw oedema. In addition, sub-acute inflammation was examined over 7 days, using the formalin-induced paw oedema method. The treatment groups received PCT extracts, via the oral route, at 1-h prior to injection and then the sub plantar of the rat right paw was injected with the named substances to generate paw oedema. The paw thickness was measured by vernier caliper at regular intervals after injection. At the end of experiment, the blood and paw tissues were collected for measurement of pro-inflammatory cytokines and histological examination respectively. ResultsIn acute inflammation, all doses of PCT extract (250, 500 and 1000 mg/kg p.o.) significantly reduced paw thickness after the first 3 h in a dose-dependent manner and the percentage of inhibition was 38.7%, 47.8% and 49.5% respectively. The pro-inflammatory cytokines, including TNF-α and IL-1β, statistically decreased with all doses of the extracts. However, the histological examination did not reveal significant results due to the short time duration. As regards to sub-acute inflammation, all doses of PCT extract significantly reduced paw thickness with 12.78%, 23.64% and 35.78%, in a dose dependent manner. Also, the pro-inflammatory cytokines (TNF-α and IL-1β) significantly decreased at day 7. Interestingly, the histological examination of paw tissue demonstrated reductions of mononuclear infiltrations of inflammatory cells, this was observed in the group receiving PCT extracts, also in a dose-dependent manner. ConclusionTherefore, PCT exerted anti-inflammatory activity in an animal model of acute and sub-acute inflammation, suggesting that it could be used as a new source for treatment of inflammatory diseases.

Evaluation of the anti-arthritic activity of Capparis spinosa L. roots extracts incomplete Freund’s adjuvant-induced arthritis in mice.

This study aimed to estimate the ant-arthritic activity of Capparis spinosa L. roots extract in complete Freund’s adjuvant (CFA)-induced arthritis mice by assessment of paw thickness, serological detection of inflammatory markers [rheumatoid factor (RF) and tumor necrosis factor-alpha (TNF-)] using ELISA, and histopathology. In total, 30 male mice were selected, prepared, and divided equally into five groups, including 2 control (NC and PC)and 3 experimental: EG1 (arthritis mice treated total effective dose of extract), EG2(arthritis mice treated total effective dose of Diclofenac Sodium), and EG3 (arthritis mice treated a half effective dose of each the extract and Diclofenac). Clinically, the extract administration lowered the paw thickness from day 7th onwards, the 21st day of study, while the extract administration lowered the paw thickness in EG1 from day 7th onwards compared to EG2 and EG3. For hematology, significant decreases were reported in RBCs, HCT, Hb, WBCs and neutrophils due to arthritis, and significant amelioration was seen obviously in mice of EG1 as a result of therapy. Significant increases in platelets, lymphocytes and monocytes were observed in PC and experimentally groups, significantly improving the values of treated groups due to therapy, in particular, EG1. All study groups ' values of MCV, MCH, MCHC, basophils and eosinophils have differed insignificantly. Concerning RF, the highest significant value was reported in PC while the lowest in NC, but without significant differences between EG1, EG2 and EG3 values. For TNF-, elevation had been recorded in PC mice, whereas reduction in NC, EG1 and EG3. Also, the value of EG2 was significantly higher than recorded in mice of EG1 and EG3. Concerning histology, the findings of EG1 revealed a significant amelioration compared to other groups. In conclusion, C. spinosa L. root extract demonstrated anti-inflammatory and anti-arthritic activity. Furthermore, studies to detect the therapeutic effects of the root extract on other systemic or local diseases are needed. Keywords: Paw thickness; Rheumatoid factor; Inflammatory markers; Diclofenac Sodium; Iraq

Protective effect of Cordia myxa leaves extract in FCA-induced arthritis in rats

Background: Arthritis, an age-related chronic inflammatory autoimmune disease primarily characterized by joint pain, swelling, stiffness, cartilage destruction, is increasing tremendously at an alarming rate and also becoming a major risk factor for other health complications. Cordia myxa, family Boraginaceae is an herbal plant used since ancient times for its various significant pharmacological activities, including anti-inflammatory effects. Scientific studies reporting anti-arthritic potential of leaves extract of this plant are still lacking. Therefore, the present work has been designed to investigate the protective effect of ethanolic extract of Cordia myxa leaves in Freund’s complete adjuvant (FCA)-induced arthritis in rats. Methods: Ethanolic extract (100mg/Kg, 200mg/Kg and 400mg/Kg) was investigated for their efficacy in the management of FCA induced arthritis in rats. Various parameters like paw volume (using plethysmograph), paw thickness (using Vernier calliper), body weight, hematological and histopathological profile were observed. The protocol was approved by Institutional Animal Ethical Committee (MMCP/IAEC/94) and was according to CPCSEA guidelines. Results: The phytochemical screening reveals the presence of flavonoids, glycosides, alkaloids, saponins and carbohydrates in the ethanolic extract of Cordia myxa leaves. Promising results with significant improvement were discovered as evident from all determined parameters when compared with standard drug methotrexate, which might be due to the presence of flavonoids, glycosides, alkaloids in the ethanolic extract of Cordia myxa leaves. Conclusion: The present study supports the traditional use of Cordia myxa leaves as potential anti-arthritic agent which can be effective in Freund’s complete adjuvant (FCA)-induced arthritis in rats, however its mode of action is still need to be studied.

Unveiling the Chemical Profiling Antioxidant and Anti-Inflammatory Activities of Algerian Myrtus communis L. Essential Oils, and Exploring Molecular Docking to Predict the Inhibitory Compounds against Cyclooxygenase-2.

Considering the large spectrum of side effects caused by synthetic drugs and the development of natural alternatives utilizing Algerian flora, this study aimed to place a spotlight on the chemical profile and antioxidant and anti-inflammatory activities of Myrtus communis L. essential oils (MCEOs). In this study, essential oils (EOs) were collected via hydro-distillation of the plant's leaves, and a chemical constituent analysis was performed using gas chromatography-mass spectrophotometry (GC-MS). The in vitro antioxidant activity was evaluated using DPPH, ABTS, and hydroxyl radical scavenging tests. The in vitro anti-inflammatory capacity was estimated by studying the antidenaturation effect using bovine serum albumin (BSA) as a protein model. The in vivo anti-inflammatory activity was carried out by utilizing the classical model of carrageenan-induced paw edema in rats, using diclofenac (DCF) as the reference drug. Moreover, the molecular interaction of the compounds obtained from the GC-MS analysis was studied within the binding site of cyclooxygenase-2 (COX-2) using an in silico approach as the confirmatory tool of the in vitro and in vivo experiments. The GC-MS analysis revealed that MCEOs were mainly composed of oxygenated monoterpenes (70.56%), oxygenated sesquiterpenes (3.1%), sesquiterpenes (4.17%), and monoterpenes (8.75%). Furthermore, 1,8-cineole was the major compound (19.05%), followed by cis-geranyl acetate (11.74%), methyl eugenol (5.58%), α-terpineol (4.62%), and β-myrcene (4.40%). MCEOs exhibited remarkable concentration-dependent free radical scavenging activity, with an IC50 of 15.317 ± 0.340 µg/mL, 18.890 ± 2.190 µg/mL, and 31.877 ± 0.742 µg/mL for DPPH, ABTS, and hydroxyl radical, respectively. The significant in vitro anti-inflammatory activity due to the inhibition of BSA denaturation was proportional to the EO concentration, where the highest value was recorded at 100 μg/mL with an approximately 63.35% percentage inhibition and an IC50 of 60.351 ± 5.832 μg/mL. MCEOs showed a good in vivo anti-inflammatory effect by limiting the development of carrageenan-induced paw thickness. The in silico study indicated that, among the 60 compounds identified by the GC-MS analysis, 9 compounds were observed to have a high binding energy to cyclooxygenase-2 as compared to diclofenac. Our study revealed that EOs from Algerian Myrtus communis L. can be considered to be a promising candidate for alleviating many debilitating health problems and may provide new insights in the fields of drug design, agriculture, and the food industry.

High-Sucrose Diet Accelerates Arthritis Progression in a Collagen-Induced Rheumatoid Arthritis Model.

High dietary sugar and sweeteners are suspected to cause the development of rheumatoid arthritis (RA) symptoms through the induction of proinflammatory cytokine release. However, the mechanisms by which increased dietary sugar affects RA etiology are not yet fully understood. The study uses a mouse model of collagen-induced RA (CIA) to investigate the relationship between excessive sugar consumption and RA risk. RA-associated pathological features are assessed in the nonimmunized (NI) control group, the CIA-positive control group, and the CIA + high-sucrose diet (CIA+HS, 63% calories from sucrose) group. Compared with the CIA group, the CIA+HS group shows a greater increase in paw thickness and clinical scores, as well as, a higher degree of pannus formation and inflammation in the knee, ankle, and sole tissues. Moreover, the infiltration of immune cells is increased in the CIA+HS group. Although the expression of hepatic lipogenic genes, is not altered, that of toll-like receptor (TLR4) and IL-1β is considerably elevated in the CIA+HS group. These findings suggest that excessive sucrose consumption causes hepatic fibrosis and inflammation, contributing to the pathophysiology of RA.

Melittin acupoint injection in attenuating bone erosion in collagen-induced arthritis mice via inhibition of the RANKL/NF-κB signaling pathway.

Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. Bone erosion is the most serious pathological condition of RA and the main cause of joint deformities and disability. Melittin acupoint injection (MAI) is an effective traditional Chinese medicine (TCM) method for RA treatment. This study aimed to investigate the effect of MAI on RA bone erosion and to elucidate the underlying mechanism. A collagen-induced arthritis (CIA) mouse model was established as the experimental subject. MAI was administrated once every other day for 28 days to mice with CIA. The effects of MAI on joint diseases were assessed by body weight, arthritis index (AI) score, swollen joint count (SJC) score, and hind paw thickness. Ankle radiological changes were captured by micro-computed tomography (micro-CT) and histological changes were observed by pathological staining. Organ histological changes, spleen index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Crea) levels of serum were tested to evaluate the toxicity of MAI. Cytokine expression levels were confirmed by enzyme-linked immunosorbent assay (ELISA) to evaluate the immunity of CIA mice. MAI administration markedly improved the clinical signs of CIA in mice, including hind paw thickness, AI, and the number of swollen paw joints (most of them P<0.05 or even <0.01). According to histopathological analysis, MAI ameliorated inflammatory cell infiltration, synovial hyperplasia, pannus formation, and bone erosion (all P<0.01). Micro-CT and tartrate-resistant acid phosphatase (TRAP) staining (P<0.01) also revealed that MAI could relieve bone erosion via reducing the formation of osteoclasts. Not only could MAI relieve the immunological boost [P<0.05 for the high-dose MAI (HM) group], but also it had no liver or kidney side effects (P>0.05). In addition, it decreased the serum levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) and increased the serum levels of IL-4 and IL-10 (the majority of P<0.05 or even <0.01). Transcriptome sequencing results indicated that MAI affected the expression of osteoclast differentiation pathway genes, which was connected with the receptor activator of the nuclear factor κB ligand/nuclear factor kappa B (RANKL/NF-κB) pathway. Based on our findings, MAI could suppress joint inflammation and inhibit RANKL/NF-κB-mediated osteoclast differentiation to rescue bone erosion in CIA mice, suggesting that MAI can be a potentially therapeutic substance for RA.

Ganoderma lucidum polysaccharide peptide (GLPP) attenuates rheumatic arthritis in rats through inactivating NF-κB and MAPK signaling pathways

BackgroundNot many drugs with fewer side effects are available for the treatment of rheumatoid arthritis (RA). Ganoderma lucidum polysaccharide peptide (GLPP) has good immunomodulatory effects, but whether it is effective in managing RA is not clear. PurposeThis study was conducted to examine the anti-RA activity and possible mechanisms of GLPP in collagen-induced arthritis (CIA) rats. MethodsMale Wistar rats were intradermally injected with bovine type II collagen in the tail base to establish the CIA model and were orally administered 100 or 200 mg/kg GLPP for 35 days. Paw thickness, clinical arthritis scores, gait analysis, organ index determination, blood cell counts, micro-CT imaging and pathological staining were performed on the rats. Liver and kidney function were measured by commercial kits, and antibody levels were measured by ELISA kits. RA-related protein levels were detected by Western blotting. ResultsGLPP effectively alleviated CIA symptoms and reduced immune organ indexes, antibody levels and systemic organ injury. GLPP decreased the protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)2, MMP9, MMP13, BCL-2, OPN, β-Catenin, and hypoxia inducible factor (HIF)-1α and increased the protein expression of BAX in the joint tissues of CIA rats. Moreover, GLPP decreased the phosphorylation levels of p65, IκB-α and ERK1/2. ConclusionGLPP effectively alleviated RA symptoms in CIA rats by inhibiting the NF-κB and MAPK pathways. This study suggests a promising therapeutic effect of mushroom-derived polysaccharide peptides on RA.

Laggera alata Attenuates Inflammatory Response by Regulating Macrophage Polarization in Rheumatoid Arthritis Mice.

Rheumatoid arthritis (RA) is a type of joint injury, which can induce the activation of inflammatory factors and polarization of tissue macrophages. Total phenolics from Laggera alata (TPLA) has been reported to exhibit anti-inflammatory effect in various diseases. However, its specific function in RA is still unknown. Here, the protective properties of TPLA were studied in collagen-induced arthritis (CIA)-induced RA mice. RA mouse model was established through the CIA induction. Arthritis score, hind paw thickness, and the body weight of the RA mice were evaluated in each group. H&E staining was conducted in hind paw and joint tissues for histopathological staining. The distal femur was analyzed by microCT, and bone loss-related indicators were assessed. The expression of macrophage polarization markers was detected by immunofluorescence staining in RA mice. The serum levels of inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA). TPLA reduced the CIA-induced arthritis score and hind paw thickness in mice. The body weight of the CIA mouse was significantly increased by TPLA treatment. TPLA improved the CIA-induced histopathological changes in the hind paw and joint tissues from the mice. TPLA inhibited the bone loss and alleviated bone destruction in CIA mouse model. TPLA altered the macrophage phenotype from M1 macrophages into M2 in CIA mice. TPLA suppressed the levels of inflammatory markers both in the serum and joint tissues of the CIA mice. TPLA mitigated RA development by suppressing inflammatory reaction through the inhibition of M1 microphage polarization.

Experimental Study of the Prevention and Treatment of Lymphoedema in a Rat Hindlimb Model by Applying Cardioperitoneal Catheters and Lacrimal Intubation Tubes in the Inguinoperitoneal Region

Secondary lymphoedema (LE) is a chronic condition with limited surgical treatment options for restoring extremity form and function. This study aims to establish a reproducible model of secondary LE and evaluate the preventive and corrective effects of fenestrated catheters (FC) and capillary tubes (CT). A total of 35 rats underwent left hindlimb inguinal and popliteal node dissection, followed by radiotherapy after two weeks. The right hindlimb served as the control. Rats were divided into five groups: sham, two preventive (Group 2 - EFC, Group 3 - ECT), and two corrective (Group 4 - LFC, Group 5 - LCT). Measurements of ankle circumference (AC) and paw thickness (PT) were taken weekly, and imaging modalities were performed. After a 16 week follow up, rats were euthanised for histological examination. Data include paw thickness (PT) and ankle circumference (AC) ratios for hindlimbs. In the sham group, AC ratio was 1.08 (p = .002) and PT ratio was 1.11 (p = .020), confirming successful lymphoedema model establishment. Early catheter and tube placement in Groups 2 and 3 prevented AC and PT increase until the 16th week. Group 2: AC ratio was 0.98 (p = .93), PT ratio was 0.98 (p = .61). Group 3: AC ratio was 0.98 (p = .94) and PT ratio was 0.99 (p = .11). From the 10th to the 16th week, Groups 4 and 5 exhibited reduced measurements after insertion of catheters and tubes. Computed tomography imaging as an objective examination supported the results obtained from the measurements. The histological findings confirmed the benefits of both FC and CT. The insights gained from the present study provide a basis for further exploration and refinement of drainage system designs, ultimately leading to improved treatment approaches for individuals suffering from lymphoedema in the future.

Naru-3 inhibits inflammation, synovial hyperplasia, and neovascularization in collagen-induced arthritis in rats.

Naru-3 is a prescribed formulation based on the theory of Mongolian medicine for the treatment of rheumatoid arthritis (RA). Naru-3 consists of three medicinal agents: Aconitum kusnezoffii Reichb (caowu), Terminalia chebula Retz (hezi), and Piper longum L (biba). These medicinal agents are widely distributed in the Mongolian area of China and have been used to treat rheumatism for centuries. Mongolian medicine Naru-3 is commonly prescribed to treat RA, but its mechanism of action is unknown. A rat collagen-induced arthritis (CIA) model was established to investigate the mechanism of Naru-3. Rats were treated with Naru-3, Etanercept (ETN), and sodium carboxymethylcellulose (CMC) for four weeks. After treatment was terminated, paw thickness, ankle diameter, and arthritis index (AI) were scored. Synovial hyperplasia was evaluated using hematoxylin and eosin (H&E) staining and two-dimensional ultrasonography. Synovitis and neovascularization were assayed using power Doppler imaging (PDI) and contrast-enhanced ultrasonography (CEUS). Levels of vascular endothelial growth factor (VEGF), interleukin (IL)-1, and CD31 in the serum or synovium were detected using ELISA and immunohistochemistry analyses. Naru-3 and ETN alleviated the symptoms of CIA as evidenced by diminished paw thickness, ankle diameter, and AI scores. Mechanistically, Naru-3 inhibited synovial hyperplasia, synovitis, and neovascularization by diminishing systemic and local inflammation, as indicated by the relative expression of VEGF and IL-1 in the serum and synovium. After four weeks of treatment, no significant neovascularization was observed in the Naru-3 group, but neovascularization and synovitis occurred in the ETN group, as demonstrated by H&E staining, PDI, and CEUS examination. Naru-3 inhibited inflammation, synovial hyperplasia, and neovascularization and alleviates RA in our CIA rat model. No symptom recurrence was observed four weeks after drug treatment.

Du Huo Ji Sheng Tang inhibits Notch1 signaling and subsequent NLRP3 activation to alleviate cartilage degradation in KOA mice

BackgroundKnee osteoarthritis (KOA) has a complex pathological mechanism and is difficult to cure. The traditional medicine Du Huo Ji Sheng Tang (DHJST) has been used for the treatment of KOA for more than one thousand years, but its mechanism for treating KOA has not been revealed. In our previous study, we confirmed that DHJST inhibited the activation of NLRP3 signaling in rats and humans. In the current study, we aimed to determine how DHJST inhibits NLRP3 to alleviate knee cartilage damage.MethodsMice were injected with NLRP3 shRNA or Notch1-overexpressing adenovirus into the tail vein to construct systemic NLRP3 low-expressing or Notch1 high-expressing mice. Mice were injected with papain into the knee joint to replicate the KOA model. DHJST was used to treat KOA model mice with different backgrounds. The thickness of the right paw was measured to evaluate toe swelling. The pathohistological changes and the levels of IL-1β, MMP2, NLRP3, Notch1, collagen 2, collagen 4, HES1, HEY1, and Caspase3 were detected by HE staining, ELISA, immunohistochemical staining, western blotting, or real-time qPCR.ResultsDHJST reduced tissue swelling and serum and knee cartilage IL-1β levels, inhibited cartilage MMP2 expression, increased collagen 2 and collagen 4 levels, decreased Notch1 and NLRP3 positive expression rates in cartilage, and decreased HES1 and HEY1 mRNA levels in KOA model mice. In addition, NLRP3 interference decreased cartilage MMP2 expression and increased collagen 2 and collagen 4 levels without affecting the expression levels of notch1, HES1 and HEY1 mRNA levels in the synovium of KOA mice. In KOA mice with NLRP interference, DHJST further reduced tissue swelling and knee cartilage damage in mice. Finally, Notch1-overexpressing mice not only showed more severe tissue swelling and knee cartilage degradation but also abolished the therapeutic effect of DHJST on KOA mice. Importantly, the inhibitory effects of DHJST on the mRNA expression of NLRP3, Caspase3 and IL-1β in the knee joint of KOA mice were completely limited after Notch1 overexpression.ConclusionDHJST significantly reduced inflammation and cartilage degradation in KOA mice by inhibiting Ntoch1 signaling and its subsequent NLRP3 activation in the knee joint.

Chemical compositions of Souliea vaginata (Maxim) Franch rhizome and their potential therapeutic effects on collagen-induced arthritis in rats.

Rheumatoid arthritis (RA) is a global prevalent chronic autoimmune inflammatory disease and acceptable safety drugs are lack for its treatment. The rhizomes of Souliea vaginata (Maxim) Franch (SV) possess anti-inflammatory functions and are used as substitution of Coptis chinensis Franch. SV is also traditional Chinese medicine and Tibetan medicine for the treatment of conjunctivitis, enteritis and rheumatic. For searching complementary and alternative anti-RA drugs, it is necessary to characterize the potential anti-arthritic activity of SV and underlying mechanism involved. The aim of the study was to test the chemical compositions, evaluate the anti-arthritic effects and underlying mechanisms of SV. The chemical compositions of SV were analyzed using liquid chromatography-ion trap-time of flight tandem mass spectrometry (LCMS-IT-TOF). From day 11 to day 31, SV (0.5, 1.0 and 1.5 g/kg body weight) and Tripterygium glycosidorum (TG, 10 mg/kg body weight) were administered orally to the CIA model rats once a day. Thickness of paw and body weights were measured once every two days from day 1 to day 31. Histopathological changes were measured using hematoxylin-eosin (HE) staining. Effects of SV on the levels of IL-2, TNF-α, IFN-γ, IL-4 and IL-10 in serum of CIA rats were measured by enzyme-linked immunosorbent assay (ELISA) kits. CD3+, CD4+, CD8+ and CD4+CD25+ T cells populations were measured using flow cytometric analysis. To evaluate the possible hepatotoxicity and nephrotoxicity, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (UREA) and creatinine (CREA) in CIA rats were also tested using blood auto analyzer. 34 compounds were identified from SV based on LCMS-IT-TOF, and triterpenoids are major anti-arthritic compositions. SV significantly relieved CIA rats' paw swelling without obvious influence on the body weight growth. SV decreased the serum levels of IL-2, TNF-α and IFN-γ in CIA rat, and increased the serum levels of IL-4 and IL-10. SV significantly increased and decreased the percentages of CD4+ and CD8+, with no significant effects on CD3+ in lymphocytes of CIA model rats. Moreover, SV simultaneously decreased thymus and spleen indexes and no hepatotoxicity and nephrotoxicity was observed after short-term treatment. These findings suggest that SV possesses preventive and therapeutic effect on RA by modulating the inflammatory cytokines, T-lymphocyte, thymus and spleen indexes and shows no hepatotoxicity and nephrotoxicity.

ON104, a novel antibody targeting oxidized Macrophage Migration Inhibitory Factor (oxMIF) shows efficacy in preclinical models of chronic inflammation

Abstract Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic inflammatory cytokine, ubiquitously present in various tissues and the circulation of healthy subjects. MIF is a primary counter-actor of glucocorticoids that emerged as a pivotal regulator of chronic inflammation. During inflammation, MIF converts into oxidized (ox)MIF which was described as the pathogenic and druggable isoform of MIF. ON104, a fully human antibody, was engineered to specifically neutralize oxMIF without triggering immune cell effector functions. ON104 was tested in three experimental models of chronic inflammatory diseases (CIDs): Glomerulonephritis (GN) in WKY rats, Collagen-Induced Arthritis (CIA) in DBA/1j mice; and adoptive T-cell transfer colitis in SCID mice. At disease onset, ON104 was administered at different dose levels twice a week for 1, 3, and 8 weeks in GN, CIA, and colitis, respectively. Clinical evaluations, blood and tissue sampling were done for all individual animals. In the GN model, ON104 significantly reduced proteinuria, hematuria, and CD68 +macrophage infiltration within the inflamed kidneys. During CIA, ON104 ameliorated the severity of arthritis as indicated by reduced paw thickness, and decreased disease score. Furthermore, treatment with ON104 substantially attenuated clinical signs of colitis by preventing body weight loss and restoring stool consistency compared to the vehicle-treated controls. Our findings substantiate the role of oxMIF in the pathogenesis of various CIDs. Thus, ON104 has the potential to become a well-tolerated therapeutic antibody for treating patients with CIDs.

Antinociceptive and anti-inflammatory activity of DW-1021, the ionic complex of pelubiprofen and tramadol, in rodents

Although drugs such as acetaminophen, opioids, and nonsteroidal anti-inflammatory drugs (NSAIDs), are commonly used for pain management, the side effects of these drugs such as hepatotoxicity, nephrotoxicity, nausea, and vomiting, can not be neglected. Therefore, combinations of analgesics with different mechanisms raise the possibility of developing novel analgesics. Therefore, the aim of the present study was to evaluate whether DW-1021, the ionic complex of pelubiprofen (NSAID) and tramadol (opioid), has synergic antinociceptive and anti-inflammatory effects in nociceptive as well as inflammation-induced nociceptive models compared to pelubiprofen- or tramadol-only administration. Strong synergistic antinociceptive efficacy of DW-1021 was observed in the mouse writhing test and von Frey paw withdrawal threshold test in the carrageenan-induced rats. The hot plate test in mice and the Randall-Selitto mechanical paw pressure test in carrageenan-induced rats revealed that DW-1021 had a preferable effect on relieving pain to pelubiprofen, but not as much as tramadol. In the carrageenan-induced rats, DW-1021 had a more potent effect on reducing paw inflammation (paw volume, width, and thickness) via the suppression of PGE2 production than tramadol, but less than that of pelubiprofen. Taken together, our results suggest that the administration of DW-1021, a combination of pelubiprofen and tramadol, exerted a potent effect and can be used as a potential therapeutic agent for relieving pain and inflammation.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis

PurposeThe goal of this study was to develop an imaging probe—IRDye-680RD-OX40 mAb—that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA.MethodsOX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups.ResultsNIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring.ConclusionThe results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.Graphical

UHPLC-MS and GC-MS phytochemical profiling, amelioration of pain and inflammation with chloroform extract of Funaria hygrometrica Hedw. via modulation of inflammatory biomarkers.

People of Pakistan have undisturbed customs for the employment of medicinal plants for healthcare requisites. Chloroform extract of F. hygrometrica (CE FH) was examined for its ability to reduce inflammation and to produce analgesia. Carrageenan and formalin-induced paw edema model for inflammatory activity, hot-plate and tail-flick methods to assess analgesic activity were executed. Phytochemical analysis was done by UHPLC-MS and GC-mass spectrometer. The results demonstrated that in carrageenan-induced paw edema, maximum reduction in inflammation was observed at 5th hour at the dose 100mg/kg; while at doses 250 and 500mg/kg, maximum response was observed at 5th and 6th hours. Analgesic activity results indicated that maximum analgesia was observed up to 120min at 100mg/kg, while up to 90min in case of 250 and 500mg/kg doses. The formalin-induced rat paw edema showed significant (p < 0.05) anti-inflammatory activity after 5days treatment. After, testing period of 10days, the biochemical parameters such as CBC, CRP, serum enzymes like CAT, SOD, GSH and inflammatory mediators like TNF-α, IL-6, IL-4 and IL-10 were estimated. The administration of formalin resulted in an increase in the level of leucocytes, total WBC, CRP, serum enzymes and in the diameters of paw thickness, while pre-treatment with CE FH at dose levels of 100, 250 and 500mg/kg exhibited a diminution in the levels of SOD, GSH, CAT, total RBC and HB. Acute inflammatory mediators such as TNFα, IL -6 and IL-4 were reduced, and IL-10 was upregulated in the treated group as compared to the control. Many phytoconstituents, i.e., chitobiose, chlorovulone III, γ-tocotrienol, emmotin, cassine, hexacosanedioic acid, neophytadiene, fumaric acid, neophytadiene, hexadecanoic acid, phytol and stigmasterol were detected during UHPLC-MS and GC-MS analysis seems to be responsible for the said activity in correlation with the already reported data about these compounds. The results concluded that CE FH possess noteworthy anti-inflammatory and central analgesic action at different doses (100, 250 and 500mg/kg).

New natural pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and iNOS inhibitors identified from Penicillium polonicum through in vitro and in vivo studies.

Overexpression of pro-inflammatory cytokines and iNOS have been found to be concomitant with several chronic inflammatory diseases and hence targeting their inhibition would be a useful therapy for inflammation. In view of this, study on discovery of natural pro-inflammatory cytokines inhibitory lead molecules from Penicillium polonicum, an endophytic fungus isolated from the fresh fruits of Piper nigrum was performed. When the culture broth extract of P. polonicum (EEPP) was subjected to LPS-induced cytokines expression (ELISA in RAW 264.7 cells), it exhibited inhibition of TNF-α, IL-6 and IL-1β and this encouraged us to do chemical investigation on EEPP to explore the bioactive components. Four compounds isolated and characterised as 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) were tested for their effect on the production of TNF-α, IL-1β and IL-6 in RAW 264.7 cells (ELISA). All the compounds exhibited a highly significant (P<0.0001) inhibition effect, particularly against IL-1β (IC50: 4-0.91μM, 1-2.81μM, 3-4.38μM, and 2-5.54μM). Tyrosol (4) was most active with IC50 values of 0.91, 2.67 and 4.60μM against IL-1β, IL-6 and TNF-α, respectively. On observing the potential activity of the compounds, two compositions C1 and C2 were prepared by mixing equimolar concentrations of compounds 1, 2, 3 & 4 (C1) and compounds 1, 2, 3, 4 & piperine (C2) in equal ratio. A synergistic effect was observed with C1 exhibiting potential suppression of IL-6 secretion (IC50 1.91μM) and C2 against IL-1β (IC50 5.98μM). Also, the individual compounds and C1 were effective in controlling iNOS expressions in RAW 264.7 cells (RTPCR). Further, the in vivo performance of the compounds and compositions were studied under two in vivo inflammatory models (LPS-induced endotoxaemia and carrageenan-induced paw oedema). Compounds 1, 2, 3, 4, C1 and C2 at 50mg/kg oral dose showed a significant control over the LPS-stimulated TNF-α, IL-1β and IL-6 levels in plasma. C1, C2 and 1 exhibited>50% pan-cytokine inhibition effect. Under the carrageenan-induced anti-inflammatory model, a significant reduction in the paw oedema measured in terms of the difference in the paw thickness was observed. Further, attenuation of pro-inflammatory cytokines levels following ELISA and RT-PCR experiments in the paw tissue homogenate was in agreement with paw thickness results. All compounds and C1 decreased the iNOS gene expression levels, and also the MPO activity and NO production in the paw tissue homogenate with tyrosol (4) as the most active molecule. Further, the mechanism of action was explored by testing the effect of the compounds on the expression of inflammatory markers using western blot analysis (in vitro). They were found to regulate the expression of pro-form and matured-form of IL-1β by inhibiting NFκB. Also, the compounds reduced the translocation of the NF-κB subunit p65 to the nucleus. Thus, compounds 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) are reported as new natural multiple pro-inflammatory cytokines inhibitory leads. The interesting results of C1 might lay a footing for the development of a new anti-inflammatory composition.