Complete Adjuvant Research Articles

Design of chondroitin sulphate coated proglycosomes for localized delivery of tofacitinib for the treatment of rheumatoid arthritis

Long-term oral tofacitinib (TOF) administration has been linked to serious side effects majorly immunological suppression. The aim of this work was to enhance the therapeutic efficacy of TOF by chondroitin sulphate (CS) coated proglycosomes through the anchoring of high-affinity CS to CD44 receptors on immune cells in the inflammatory region. The CS was coated onto the TOF-loaded proglycosomes (CS-TOF-PG) formulations and they were evaluated for in vitro drug release, ex vivo (permeation, dermatokinetics) studies. In vivo efficacy studies were carried out in Freund's complete adjuvant (CFA) induced arthritis model. The optimized CS-TOF-PG showed particle sizes of 181.13 ± 7.21 nm with an entrapment efficiency of 78.85 ± 3.65 %. Ex-vivo studies of CS-TOF-PG gel exhibited 1.5-fold high flux and 1.4-fold dermal retention compared to FD-gel. The efficacy study revealed that CS-TOF-PG showed a significant (P < 0.001) reduction in inflammation in arthritic rat paws compared to the TOF oral and FD gel. The current study ensured that the CS-TOF-PG topical gel system would provide a safe and effective formulation for localization and site-specific delivery of TOF at the RA site and overcome the adverse effects associated with the TOF.

The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

Effects of heat-reinforcing needling on synovial inflammation and miR-155/TLR4/NF-κB signaling axis in rheumatoid arthritis rabbits with cold syndrome

To observe the effects of heat-reinforcing needling on synovial inflammation and microRNA-155 (miR-155)/Toll-like receptor 4 (TLR4)/nuclear transcription factor-κB (NF-κB) signaling axis, so as to investigate its anti-inflammatory mechanism in rabbits with cold syndrome of rheumatoid arthritis (RA). A total of 36 rabbits were randomly divided into normal, model, agonist, inhibitor, heat-reinforcing needling (HRN) and agonist+heat-reinforcing needling (A+HRN) groups, with 6 rabbits in each group. The RA with cold syndrome model was induced by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Rabbits in agonist group were intraperitoneally injected with miR-155 agomir 4.5 OD; rabbits in the inhibitor group were intraperitoneally injected with miR-155 antagomir 6.1 OD; rabbits in HRN group received heat-reinforcing needling at bilateral "Zusanli" (ST36) for 30 min；rabbits in A+HRN group received the same treatment as agonist group, and 30 min later, received the same treatment as the HRN group; rabbits in the normal and model groups were grasped and fixed in the same way, all groups received continuous treatment once a day for 7 d. After modeling, the knee joints of rabbits were examined by ultrasound, the pain threshold and the circumference were determined. After the interventions, the pain threshold and knee circumference were measured; the pathological morphology of synovial tissue of the knee joints were observed by HE staining; the mRNA levels of miR-155 and suppressor of cytokine signaling protein 1 (SOCS1), the expression levels of SOCS1, TLR4, NF-κB p65, interleukin (IL)-1β and IL-17A proteins in synovial tissue of knee joints were detected by real-time PCR and Western blot respectively. Compared with the normal group, the pain threshold was significantly decreased (P<0.05), and the knee circumference was significantly increased (P<0.05); the synovial tissue of knee joints showed significant hyperplasia, abundant blood flow signal, joint cavity effusion and obvious inflammatory invasion, the pathological score was significantly increased (P<0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly increased (P<0.05), the expressions of SOCS1 mRNA and protein were significantly decreased (P<0.05) in the model group. Compared with model group, the pain threshold was significantly increased (P<0.05), the circumference of knee joint was significantly decreased (P<0.05); in synovial tissue, the pathological score was decreased (P<0.05), the expression levels of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly decreased (P<0.05), and the expressions of SOCS1 mRNA and protein were significantly increased (P<0.05) in inhibitor group and HRN group, while the above changes in agonist group were reversed (P<0.05). Compared with the agonist group, the pain threshold was significantly increased (P<0.05), the knee circumference was significantly decreased (P<0.05), the synovial pathological score was significantly decreased (P<0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB proteins in synovial tissue were significantly decreased (P<0.05), and the expression levels of SOCS1 mRNA and protein were significantly increased (P<0.05) in A+HRN group. The heat-reinforcing needling can increase the pain threshold, reduce the knee circumference and inhibit the inflammatory response in rabbits with RA cold syndrome. The possible mechanism is related to the regulation of miR-155/TLR4/NF-κB signaling axis.

Patient perceptions of benefits and risks of adjuvant therapy in renal cell carcinoma.

663 Background: Adjuvant treatment with the immune checkpoint inhibitor (CPI) pembrolizumab and the tyrosine kinase inhibitor (TKI) sunitinib are currently standard of care options for patients with high-risk localized renal cell carcinoma (RCC). However, treatment decisions need to be based on anticipated benefit, toxicity risk, and patient goals and values, particularly since multiple trials testing CPIs and TKIs in the adjuvant setting did not meet their primary endpoints. To improve patient selection and communication, we sought to assess how patients perceive the benefits and risks of adjuvant therapy. Methods: The survey was developed by the Kidney Cancer Research Alliance (KCCure), with multidisciplinary representation from urologic surgeons, medical oncologists and patient advocates. The survey was broadcast between 07/2022 and 09/2022 to patients via website, mailing lists and social media platforms. Out of 1,062 participants 639 patients self-identified with localized RCC and 223 patients had stage 3 RCC at initial diagnosis. 113 patients were offered adjuvant therapy by their doctor, 74 patients reported being treated with or having received treatment with adjuvant therapy (18% treated with TKI, 82% with CPI). Results: When asked to assess risk of recurrence, patients with stage 3 disease assessed their average personal risk to be 47%, 10% higher than what they reported their doctor assessed risk to be (37%). When asked to identify the most important factors related to adjuvant decision making, patients ranked risk of dying from cancer (76%) over severity of possible side effects (43%), chance of having side effects (35%), time required for treatment (24%), and cost of treatment (7%). 49% of patients offered adjuvant therapy believe that treatment reduces the risk of recurrent disease by more than 30% and 25% believe it reduces risk by more than 50%. Among patients receiving or having received adjuvant therapy, 46% completed adjuvant therapy, 17% stopped early due to side effects and 14% recurred while on therapy. Side effects were asymptomatic or mild in 38%, moderate without invasive intervention or hospitalization in 48% severe, requiring hospitalization in 8% and life-threatening in 7%. 22% of patients were told that their side effects would be life-long. Conclusions: Patients with high-risk localized disease estimate their recurrence risk to be higher than what their doctors assess their risk to be. When weighing decisions about adjuvant therapy, patients rank risk of dying from cancer over risks related to toxicity. An overestimation of benefits of adjuvant therapy is noted with one quarter of patients believing that adjuvant therapy will reduce their risk of recurrence by 50 percent or more. The findings highlight the importance of effective communication between patients and providers regarding the risks and benefits of adjuvant therapy to promote an informed and shared decision.

Electrical Stimulation of the Auricular Branch Vagus Nerve Using Random and Alternating Frequencies Triggers a Rapid Onset and Pronounced Antihyperalgesia via Peripheral Annexin A1-Formyl Peptide Receptor 2/ALX Pathway in a Mouse Model of Persistent Inflammatory Pain.

This study evaluated the antihyperalgesic and anti-inflammatory effects of percutaneous vagus nerve electrical stimulation (pVNS) by comparing the effects of alternating and random frequencies in an animal model of persistent inflammatory hyperalgesia. The model was induced by Freund's complete adjuvant (CFA) intraplantar (i.pl.) injection. Mice were treated with different protocols of time (10, 20, or 30min), ear laterality (right, left or both), and frequency (alternating or random). Mechanical hyperalgesia was evaluated, and some groups received i.pl. WRW4 (FPR2/ALX antagonist) to determine the involvement. Edema, paw surface temperature, and spontaneous locomotor activity were evaluated. Interleukin-1β, IL-6, IL-10, and IL4 levels were verified by enzyme-linked immunosorbent assay. AnxA1, FPR2/ALX, neutrophil, M1 and M2 phenotype macrophage, and apoptotic cells markers were identified using western blotting. The antihyperalgesic effect pVNS with alternating and random frequency effect is depending on the type of frequency, time, and ear treated. The pVNS random frequency in the left ear for 10min had a longer lasting antihyperalgesic effect, superior to classical stimulation using alternating frequency and the FPR2/ALX receptor was involved in this effect. There was a reduction in the levels of pro-inflammatory cytokines and an increase in the immunocontent of AnxA1 and CD86 in mice paw. pVNS with a random frequency in the left ear for 10min showed to be optimal for inducing an antihyperalgesic effect. Thus, the random frequency was more effective than the alternating frequency. Therefore, pVNS may be an important adjunctive treatment for persistent inflammatory pain.

Chondroprotective Effects of Grapefruit (Citrus paradisi Macfad.) Juice in a Complete Freund's Adjuvant Rat Model of Knee Osteoarthritis.

Osteoarthritis (OA) is a common disorder that can affect any joint in the human body. This study aimed to examine the anti-arthritic properties of high and low doses of grapefruit juice (GFJ), as grapefruit appears to contain anti-inflammatory biochemicals. Forty male Sprague-Dawley rats weighing 170-180 g were divided into five groups. These groups comprised the untreated control group and osteoarthritic (Osteo) rats administered intra-articular injections of Freund's complete adjuvant (CFA; 0.5 mL; 1 mg/mL) as follows: OA rats administered low doses of GFJ (Osteo+GFJ (low); 5 mL/kg body weight (BW)); OA rats administered high doses of GFJ (Osteo+GFJ (high); 27 mL/kg BW); and OA rats administered diclofenac sodium (Osteo+Diclo) as a reference drug. Injections of CFA induced OA, as indicated by a significant increase in the serum levels of the inflammatory biomarkers C-reactive protein (CRP), interleukin-1β (IL-1β), and (prostaglandin (PGE2), as well as matrix metalloproteinases (MMP-1) and cathepsin K. The synovial levels of glycosaminoglycans (GAGs), tumor necrosis factor (TNF-α), and interleukin 6 (IL-6) also increased, with a concomitant reduction in osteocalcin levels. The administration of either high or low doses of GFJ reduced CRP, IL-1β, PGE2, MMP-1, cathepsin K, and osteocalcin while increasing the synovial levels of GAGs, TNF-α, and IL-6, slowing cartilage degradation and boosting joint function. The results showed comparable histopathological and biochemical responses. A comparison of the treatments showed that high-dose GFJ had a greater chondroprotective effect than low-dose GFJ.

Use of adjuvant ISA VG 71 to produce neutralizing egg yolk antibodies against bothropic venom.

The use of egg yolk antibodies-IgY technology-represents an alternative to the production of mammalian immunoglobulins and has several advantages regarding animal welfare and lower costs of production. The use of adjuvants to achieve the hyperimmunization of laying hens plays a key role in the success of the production of high levels of the antibodies. In the present work, two different adjuvant systems (Freund's adjuvants and MontanideTM ISA 71 VG) were compared to produce IgY anti-Bothrops alternatus. For the first immunization, formalin-inactivated Salmonella was added to MontanideTM ISA 71 VG to emulate Freund's complete adjuvant which includes a mycobacteria antigen. After eight immunizations, IgY produced by using either adjuvant was able to neutralize the lethal activity of the venom in a mouse model, but differences were found regarding the recognition of components of the venom between the two adjuvants tested. Overall, MontanideTM adjuvant used in this work could be a good alternative choice to produce antibodies capable of neutralizing the lethality of complex antigens. This adjuvant is commercially available and used in the formulation of several poultry vaccines and could be used for the IgY technology instead of traditional immunomodulators such as Freund's adjuvants. Key points • IgY extracts recognized major components of the venom.• Avidity indexes of the IgY extracts increased after the successive immunizations.• IgY obtained by two adjuvant systems neutralized the lethal activity of the venom.

A qualitative study of sleep in young breast cancer survivors: "No longer able to sleep through the night".

Sleep disturbance is common among women with breast cancer and is associated with greater symptom distress and poorer outcomes. Yet, for the unique subgroup of young women with breast cancer (YWBC), there is limited information on sleep. To address the gap in our understanding of sleep health in YWBC, we explored their perspective on sleep quality, sleep changes over time, contributing factors, and any strategies used to promote sleep. As part of an explanatory sequential mixed method study, we recruited a sub-sample of 35 YWBC (≤ 50years of age at the time of diagnosis) from the larger quantitative study phase. These participants were within the first 5years since diagnosis and completed primary and systemic adjuvant therapy. We conducted virtual semi-structured interviews, transcribed them verbatim, and analyzed data with an interpretive description approach. YWBC experience difficulty falling asleep, waking up at night, and not feeling refreshed in the morning. They attributed interrupted sleep to vasomotor symptoms, anxiety/worry, ruminating thoughts, everyday life stressors, and discomfort. The sleep disturbance was most severe during and immediately after treatment but persisted across the 5years of survivorship. The participants reported trying pharmacologic and non-pharmacologic strategies to improve the quantity and quality of their sleep. Future research would benefit from longitudinal designs to capture temporal changes in sleep and develop interventions to improve sleep health. Clinically, assessment of sleep health is indicated for YWBC related to the prevalence of disturbed sleep. IMPLICATIONS FOR CANCER SURVIVORS: Early access to sleep assessment and management, ideally before cancer treatment, would be beneficial for youngbreast cancer survivors. In addition, cancer treatment plans should include physical and psychological symptoms,especially those reported by women in this study: vasomotor symptoms, anxiety and worry, discomfort, and pain.

Complete Tolerogenic Adjuvant Stimulates Regulatory T Cell Response to Immunization.

We have determined in mice the minimum composition required for forming a vaccine adjuvant that stimulates a regulatory T (Treg) cell response to immunization, and we named the adjuvant "complete tolerogenic adjuvant." This new kind of adjuvant may let us use the well-proven "Ag with adjuvant" form of immunization for inducing Treg cell-mediated Ag-specific immunosuppression. The minimum composition consists of dexamethasone, rapamycin, and monophosphoryl lipid A at a mass ratio of 8:20:3. By dissecting the respective role of each of these components during immunization, we have further shown why immunosuppressive and immunogenic agents are both needed for forming true adjuvants for Treg cells. This finding may guide the design of additional, and potentially more potent, complete tolerogenic adjuvants with which we may form numerous novel vaccines for treating immune diseases.

Preoperative therapy confers survival advantage in early-stage pancreatic cancer

Background: Five-year overall survival (OS) for resected pancreatic ductal adenocarcinoma (PDAC) remains poor at 16.3%. In clinical stage (CS) I-II PDAC, CA19-9 levels < 98U/mL portend a favorable prognosis, while surgical resection improves survival irrespective of CA19-9 levels. Currently, 40% of patients with PDAC are unable to complete adjuvant therapy. We evaluated the role of therapy for early-stage resectable PDAC based on pre-operative CA19-9 levels.

Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization.

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.

Antigen B modulates anti-inflammatory cytokines in the EAE model of multiple sclerosis.

Multiple sclerosis (MS) is characterized by the destruction of the blood-brain barrier, loss of myelin sheath, and contribution of inflammatory interleukins such as TNF-alpha, interleukin-17, and interleukin-6. The current study investigated the effect of antigen B of hydatid cyst fluid on the reduction of anti-inflammatory cytokines and nerve conduction velocity in rats with experimental autoimmune encephalomyelitis (EAE)-induced MS. After isolation of antigen B from sterile cyst fluid, the rats were randomly divided into four groups: saline, EAE, EAE + teriflunomide (EAE + TF), and EAE + antigen B (EAE + AngB). The EAE model was induced using cow spinal cord homogenization, in combination with Freund's complete adjuvant. The serum concentration of cytokines including IL-1B and IL-17, IL-10, IL-6, and TNF-X was measured by the ELISA method, and real-time PCR was performed to study gene expression. Electrophysiological, behavioral, and neuropathological tests were also conducted. Nerve conduction velocity and IL-10 concentration were increased in the antigen B group. The results of this study showed that antigen B reduced the inflammatory component of the EAE MS animal model by modulating the immune system compared to teriflunomide, which eventually led to a reduction in symptoms at the behavioral and electrophysiological level. It seems that antigen B plays a critical role in regulating immunity and it can be used as a possible therapeutic agent to modulate the immune system in MS patients. It might be rational to consider hydatid cyst fluid antigen as a modifier in MS.

Effect of moxibustion on JAK2/STAT3 signal pathway in synovial tissues of rabbits with adjuvant arthritis under the overexpression of NLRP3 inflammatory bodies

To observe the effect of moxibustion on Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2-STAT3) signal pathway and interleukin (IL)-1β and IL-18 in synovial fluid of adjuvant arthritis (AA) rabbits, so as to explore the mechanism of moxibustion in the treatment of rheumatoid arthritis. Twenty-eight rabbits were randomized into control, model, moxibustion, and NLRP3 overexpression groups, with 7 rabbits in each group. AA rabbit model was established by injection of Freund's complete adjuvant (FCA, 0.5 mL/kg) into the rabbits' bilateral hind-knee joint cavities. On the third day after modeling, the NLRP3 lentiviral vector (40 μL) were injected into the bilateral hind-knee joint cavities of rabbits in NLRP3 overexpression group. Moxibustion was used to bilateral "Shenshu" (BL23) and "Zusanli" (ST36), 5 cones every time, once daily, 6 days a week for 3 weeks in the moxibustion and NLRP3 overexpression groups. The perimeters of rabbits' hind legs were measured after modeling and after the intervention. The contents of IL-1β, IL-18 in synovial fluid were detected by ELISA and the expression levels of NLRP3, JAK2 and STAT3 mRNAs in synovial tissue were detected by real-time PCR. In comparison with the control group, the perimeters of bilateral knee joints were significantly increased at each time point (P<0.05),and the contents of IL-1β, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNA in synovial tissue were significantly increased (P<0.05) in the model group. Compared with the model group, the perimeters of bilateral knee joints were significantly decreased (P<0.05), and the contents of IL-1β, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNAs in synovial tissue were significantly decreased (P<0.05) in the moxibusion group. Compared with the moxibustion group, the above indexes were higher in the NLRP3 overexpression group (P<0.05). Moxibustion may play an anti-inflammatory and detumescent role in AA rabbits by regulating JAK2-STAT3 signal pathway, and its therapeutic effect may be closely related to the expression of NLRP3.

EFFICIENCY OF OILY AND POLYELECTROLYTE ADJUVANTS IN CASE OF RABBIT HYPERIMMUNIZATION WITH DIMETHYLSULFOXIDE ANTIGEN OF PSEUDOTUBERCULOSIS MICROORGANISM

A comparative evaluation of two adjuvants: polyazolidinammonium modified with iodine hydrate ions (PAAH) and complete Freud's adjuvant (CFA) was conducted. The assessment was carried out in the process of hyperimmunization of rabbits with a mixture of an adjuvant and dimethyl sulfoxide antigen (DA) of a pseudotuberculous microbe. Both adjuvants stimulated antibody genesis and allowed to obtain hyperimmune sera with high antibody titers. However, CFA showed a slightly higher activity (1:819200) than PAAH (1:409600). The most pronounced stimulating effect of adjuvants was expressed at low doses of DA (0.2-1 mg/rabbit). Large doses of the antigen (16.0 mg/rabbit) enabled to obtain sera with a high antibody titer without adjuvants. The stimulating effect of DA-PAAH and DA-CFA complexes on antibody genesis is associated with the rate and quality of lymphocyte differentiation, and to a lesser extent with an increase of their number. PAAH showed a weak irritating effect on body tissues at the injection spot, and CFA had a strong irritating effect. The application of CFA led to formation of many painful indurations in the subcutaneous tissue of rabbits, which did not resolve between immunizations. Thus, usage of PAAH as an adjuvant in hyperimmunization of rabbits with DA of the pseudotuberculous microbe is potentially productive and without CFA disadvantages.

Quercetin-loaded chitosan nanoparticles ameliorate adjuvant-induced arthritis in rats by regulating anti-oxidant enzymes and downregulating pro- and inflammatory cytokines.

Rheumatoid arthritis (RA) is an inflammatory condition and associated with the symmetrical synovitis of the joints and cause joint pain. The use of anti-rheumatic drugs is associated with many adverse effects. Quercetin, an important polyphenolic flavonoid, possess anti-inflammatory and anti-rheumatic effects. Quercetin use is limited due to poor absorption and bioavailability. Nanomedicines are used for the targeted drug delivery, hence it reduces the adverse effects of the drug. Based upon these factors, quercetin-loaded chitosan nanoparticles (Q-NPs) were prepared by solvent evaporation method, characterized and their better anti-rheumatic effect with mechanistic insights was validated in Freund's complete adjuvant (FCA)-induced arthritic rats along with safety studies. The animals were divided into five groups, each containing 5 animals. Group I was normal control, group II was arthritic control, while groups III, IV and V were administered with quercetin (15mg/Kg) and Q-NPs (10 and 20mg/Kg), respectively. The reduction in ankle diameter, serum oxidative stress markers as well as pro- and inflammatory cytokines, e.g., tumor necrosis factor (TNFα), interleukin (IL-6) were determined. The prepared Q-NPs showed hydrodynamic size of 83.9nm, polydispersity index of 0.687, entrapment efficiency 90.5% as well as no interaction between quercetin and chitosan in Fourier transform infrared spectroscopy (FTIR). A significant reduction (p < 0.001) in ankle diameter was observed after administration of high-dose Q-NPs (4.32 ± 0.14cm to 5.13 ± 0.62cm). There was also reduction (p < 0.001) in levels of TNFα and IL-6 following high-dose Q-NPs (72.56 ± 2.30 and 308.19 ± 11.5pg). The effect on biochemical tests, hematological parameters and oxidative stress parameters was also found to be significant. Histopathological changes of kidney, liver and ankle also confirmed the anti-rheumatic effect of high-dose Q-NPs. The study concludes that administration of Q-NPs (20mg/Kg) may be used for the treatment of FCA-induced RA in rats.

Litsea salicifolia Roxb. bark ethanolic extract attenuates rheumatoid arthritis by inhibiting TLR4/NF-κB and activating SIRT1-Nrf2/HO-1 signaling axis: In-vitro and in-vivo evaluations

BackgroundLitsea salicifolia Roxb. (LS) is a pioneer medicinal plant that can be found in tropical and subtropical countries like India, China and Japan. LS is traditionally used for treating chronic pulmonary inflammatory diseases like asthma and wound healing. Also, LS contains an essential oil with powerful anti-oxidant and excite-repellency properties. However, no research findings have been reported about its protective activity on chronic inflammatory disease like rheumatoid arthritis (RA) till yet. PurposeThis study aims to investigate the anti-rheumatic potential of ethanolic extract of LS (LSE) in-vitro on RAW 264.7 cells for its anti-oxidant and anti-inflammatory activities and in-vivo CFA-induced adjuvant arthritis in the rat model. MethodsThe chemical characterization of LSE was identified by using LC-MS/MS. The in-vitro effects of LSE on RAW 264.7 cells stimulated with LPS 1 µg/ml were investigated to its potential therapeutic effects. Freund's complete adjuvant (CFA) arthritis was induced by 100 µl of CFA intradermally administered to rats. LSE 100 and 200 mg/kg were administered orally from day1 to day 28. Parameters such as paw volumes measured, X-ray radiography analysis, anti-oxidant status, Gene expression studies, western blot analysis and histological analysis were performed to check the anti-oxidant and anti-inflammatory effects of LSE. ResultsOur in-vitro results revealed that LSE at 5 μg/ml and 10 μg/ml reduced the LPS-induced oxidative stress burden evidenced by reduced ROS levels and improved endogenous anti-oxidants Nrf2 and SIRT1 in RAW 264.7 cells. Also, LSE inhibited the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, PGE2, COX-2 and NO levels induced by LPS in murine macrophage cells at 5 μg/ml and 10 μg/ml in a dose dependent manner. Our in-vivo results revealed that LSE significantly attenuated the CFA-induced inflammation by restoring body weight, arthritis index, nitrosative and lipid peroxidation assays. LSE treatment significantly reduced the pannus formation and bone erosion evidenced by histopathology of joint tissues. Additionally treatment with LSE significantly reduced inflammation-mediated protein expression levels like TLR4, NF-κB, COX-2 and iNOS levels and improved the Nrf2/HO-1/SIRT1 levels in paw tissues evidenced by immunoblotting. ConclusionLSE alleviated RA through its anti-oxidant and anti-inflammatory properties by inhibiting TLR4-mediated NF-κB nuclear translocation and by activating the Nrf2/SIRT1 signaling pathways, according to our in-vitro and in-vivo findings.

Efficacy of Moxa-burning heat stimulating Zusanli (ST36) and Shenshu (BL23) on expressions of macrophage migration inhibitory factor and macrophage apoptosis in rabbits with adjuvant-induced arthritis.

To evaluate the effects of moxa-burning heat stimulating acupoints Zusanli (ST36) and Shenshu (BL23) on macrophage migration inhibitory factor (MIF) and its related molecules which can provide scientific experimental basis for the clinical application of moxibustion treatment of rheumatoid arthritis (RA). Thirty rabbits were randomly assigned to control group, RA model (established by injecting Freund's Complete Adjuvant) group (RA group) and RA model with moxibustion group [Moxa group, Zusanli (ST36) and Shenshu (BL23), 5 moxa pillars/day, 6 d × 3]. The expressions of MIF mRNA were evaluated with reverse transcription polymerase chain reaction; the apoptosis rates of macrophages were detected by erminal deoxynucleotidyl transferase-mediated dTUP nick end labeling; the expressions of related signal molecules were detected with immunohistochemical S-P method and the levels of IL-2 were detected with enzyme-linked immunosorbent assay. The expressions of MIF mRNA, extracellular regulated protein kinases 2, p38 mitogen-activated protein kinase and nuclear factor-κ-gene binding p65 in synovial tissue of RA group were significantly increased when compared with control group, which were lower remarkably in moxa group than those in RA group. The apoptosis rates of macrophages in RA group were significantly down-regulated as compared with the control group, which were up-regulated in moxa group compared with the RA group. The levels of IL-2 in synovial fluid from the RA group were elevated significantly as compared with that from control group, but those of the moxa group were reduced when compared with those from RA group. Moxibustion may simultaneously regulate the expressions of MIF and its related signaling pathways molecules, the apoptosis rate of macrophages in synovial tissue, as well as the level of inflammatory factors in synovial fluid. The results suggest that the anti-inflammatory effect of moxibustion on RA may be related to inhibit the expression of MIF in synovial tissue, the molecules of some related signaling pathways and promote the apoptosis of macrophage.

Saussurea involucrata oral liquid regulates gut microbiota and serum metabolism during alleviation of collagen-induced arthritis in rats.

Saussurea involucrata oral liquid (SIOL) can clinically relieve symptoms, such as joint pain and swelling, and morning stiffness, in patients with rheumatoid arthritis (RA). However, the mechanism of action remains unclear. This study used a combination of gut microbiota and serum metabolomics analysis to investigate the effects and potential mechanisms of SIOL intervention on rats with RA induced by type II bovine collagen and Freund's complete adjuvant. Results showed that SIOL treatment consequently improved the degree of ankle joint swelling, joint histopathological changes, joint pathological score, and expression of serum-related inflammatory cytokines (interleukin (IL)-1β, IL-4, IL-6, IL-10, and tumor necrosis factor-α) in RA model rats. 16 S rRNA sequencing results showed that SIOL increased the relative richness of the Lactobacillus and Bacteroides genus and decreased the relative richness of Romboutsia, Alloprevotella, Blautia, and Helicobacter genus. Serum nontargeted metabolomic results indicated that SIOL could regulate metabolites related to metabolic pathways, such as glycine, serine, threonine, galactose, cysteine, and methionine metabolism. Spearman correlation analysis showed that the regulatory effects of SIOL on the tricarboxylic acid (TCA) cycle, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and glyoxylate and dicarboxylate metabolism pathways were correlated with changes in the richness of the Lactobacillus, Romboutsia, Bacteroides, and Alloprevotella genus in the gut microbiome. In conclusion, this study revealed the ameliorative effects of SIOL on RA and suggested that the therapeutic effects of SIOL on RA may be related to the regulation of the community richness of the Lactobacillus, Romboutsia, Bacteroides, and Alloprevotella genus, thereby improving the TCA cycle; phenylalanine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis, and glyoxylate and dicarboxylate metabolism-related pathways.

Effect of moxibustion on AMPK/ULK1 signaling pathway in synovium tissue of toes in rats with rheumatoid arthritis

To observe the effect of moxibustion on AMP-activated protein kinase (AMPK)/UNC-51-like kinase 1 (ULK1) signaling pathway in the synovial tissue of toes in rheumatoid arthritis rats, so as to explore the mechanism of mo-xibustion in the treatment of rheumatoid arthritis (RA). A total of 45 SD rats were randomly divided into blank, model， moxibustion, methotrexate and rapamycin groups, with 9 rats in each group. RA rat model was established by injection of Freund's complete adjuvant. Moxibustion was applied to "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 3 weeks. Methotrexate group was given methotrexate (0.35 mg/kg) by gavage, twice a week for 3 weeks. Rapamycin group was intraperitoneally injected with rapamycin (1 mg/kg)，once every other day for 3 weeks. The toe volume of the left hind limb of rats was measured by the toe volume measuring instrument. The content of AMP in toe synovium was detected by ELISA. The expression of AMPK and VPS34 protein in toe synovium was detected by Western blot.The expression of ULK1 and Atg13 mRNA in toe synovium was detected by RT-PCR. Compared with the blank group, the volume of toe in the model group was increased (P<0.01)，while the content of AMP, the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly decreased(P<0.01).Compared with the model group, the volume of toe in the moxibustion，methotre-xate and rapamycin groups was decreased (P<0.05); the content of AMP, the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly increased(P<0.05) in the moxibustion group; the content of AMP, the expression of VPS34 protein, the expression of Atg13 mRNA were significantly increased(P<0.05) in the methotrexate group; the expression of AMPK and VPS34 proteins, the expression of ULK1 and Atg13 mRNAs were significantly increased (P<0.05, P<0.01) in the rapamycin group. Compared with the moxibustion group, the expression of AMPK protein in the methotrexate group and the content of AMP in the rapamycin group were significantly decreased (P<0.05). Moxibustion can improve joint swelling in RA rats, and the mechanism may be related to promoting the activity of AMPK/ULK1 signaling pathway.

Improving safety of oil adjuvant-based vaccines

High adjuvant reactogenicity is the main limitation for increasing the effectiveness of vaccine therapy. The aim was to reduce the immunotoxicity effects of complete Freunds adjuvant (CFA) in warm-blooded animals. Materials and methods. The study examined Wistar rats by dividing animals into negative control (solvents); positive control (single subcutaneous CFA injection of 0.1 ml/200 g body weight (b.w.)); the minimum and maximum (per os administration of 1:4 citric and succinic acids in ratio of 17 and 88 mg/kg b.w. during 4 weeks after immunization of CFA) experiment. Body weight, hematological (complete blood count) and biochemical (hydroperoxides, malondialdehyde, catalase activity, mitochondrial dehydrogenase activity) parameters were dynamically investigated. At the end of the experiment, necropsy was performed and the relative internal organ mass coefficients were calculated. The spleen and connective tissue (knee joint) were examined histologically. The median, C25C75 quartiles, MannWhitney U-test were calculated. Results and discussion. it was found that parameters examined were within normal range in animals of negative control group. Immunization of warm-blooded animals with CFA was accompanied by transition of acute-to- chronic inflammatory reaction (week 3 and week 7, respectively). The total leukocyte count increased from 12.5 109 (negative control) up to 26.6 109/L (P = 0.01) on week 3 followed by its decline down to 19.2 109/L (P = 0.01) by week 7. Platelet count also increased significantly: from 506 109 (negative control) up to 656 109/L (P = 0.01, week 3) followed by decrease down to 610 109/L by week 7 (P = 0.01). Activation of lipid peroxidation was manifested by malondialdehyde (MDA) level elevated by 55.861.8% (P = 0.01); the general CFA-related toxic effect resulted in 11.7% weight loss (P = 0.01), spleen swelling and thymus reduction. Administration of antioxidant acids led to a dose-dependent decline in inflammation (leukocyte count at the minimum dosage 19.6 10920.9 109/L; at the maximum 16.6 10916.0 109/L), as well as normalized the platelet/leukocyte index up to 29.536.3 (positive control 24.6, negative control 40.5). The acid-related protective effect was also manifested as maintained body weight, activated catalase and inhibited lipid peroxidation. The therapeutic effect in alleviated degenerative changes in the spleen and connective tissue were revealed: reduced hemorrhagic focuses and swelling as well as preserved histoarchitectonics. Conclusion. The use of citric and succinic acids contributes to profoundly lowered CFA toxicity due to increased total antioxidant status, inhibited lipid peroxidation, improved mitochondrial metabolic activity, which ultimately lead to a decline in general systemic inflammation and allows to recommend such acids as immunoprotectors from oil adjuvant-coupled effects.