Xinfeng Capsule Inhibits Pyroptosis and Ameliorates Myocardial Injury in Rats with Adjuvant Arthritis via the GAS5/miR-21/TLR4 Axis.

Purpose: This study probed the mechanism of action of Xinfeng Capsule (XFC) in myocardial injury in rats with adjuvant arthritis (AA) via the growth arrest-specific transcript 5 (GAS5)/microRNA-21 (miR-21)/Toll-like receptor 4 (TLR4) axis. Methods: Rats were injected with Freund's complete adjuvant to establish a rat model of AA. Then, some modeled rats were given normal saline or drugs only, and some modeled rats were injected with adeno-associated viruses or necrosulfonamide (NSA; a pyroptosis inhibitor) before drug administration. Toe swelling and arthritis index (AI) were calculated. Pathological and morphological changes in synovial and myocardial tissues were analyzed with hematoxylin-eosin staining, and pyroptotic vesicles and the ultrastructural changes of myocardial tissues were observed with transmission electron microscopy. The serum levels of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor (TNF)-α were detected, and lactate dehydrogenase (LDH) release was measured in myocardial tissues, accompanied by the examination of GAS5, miR-21, TLR4, nuclear factor-kB (NF-κB) p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) expression in myocardial tissues. Results: After AA modeling, rats presented with significantly increased toe swelling and AI scores, synovial and myocardial tissue damage, elevated pyroptotic vesicles, and markedly enhanced serum levels of IL-1ß, IL-18, IL-6, and TNF-α, accompanied by significantly diminished GAS5 expression, substantially augmented miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression, greatly increased LDH release in myocardial tissues. XFC treatment significantly declined toe swelling, AI scores, synovial and myocardial tissue damage, and the serum levels of IL-1ß, IL-18, IL-6, and TNF-α in AA rats. Additionally, XFC treatment markedly elevated GAS5 expression and substantially lowered LDH release and miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression in myocardial tissues of AA rats. Moreover, the above effects of XFC in AA rats were further promoted by GAS5 overexpression or NSA treatment. Conclusion: XFC alleviated myocardial injury in AA rats by regulating the GAS5/miR-21/TLR4 axis and inhibiting pyroptosis and pro-inflammatory cytokine secretion.

A STUDY OF THE INFLUENCE OF JUVENILE ADJUVANT ARTHRITIS ON DENTAL HARD TISSUES CONDITION IN EXPERIMENTAL ANIMALS.

OBJECTIVE: The aim: To study the intensity and depth of carious tooth lesions in rats with experimental juvenile adjuvant-induced arthritis. PATIENTS AND METHODS: Materials and methods: An experimental study on a model of juvenile adjuvant arthritis (JAA) in 10 one-month-old rats induced by method of A.M. Bendele was carried out. 10 rats of the same age were intact. Injection of adjutant in rats of experimental group led to the development of acute local reaction and then caused generalized joint reaction of autoimmune origin. The performed basic therapy of JAA promoted transition of acute autoimmune process to chronic. Rats were withdrawn from the experiment in 58 days and the dental-jaw blocks were made, in which the intensity and depth of carious lesions of the masticatory group of teeth were determined. RESULTS: Results: The course of JAA was accompanied by the development of dental caries in 100% of experimental animals. It was found that the intensity of carious teeth lesions in terms of the number of carious teeth and cavities is probably higher than in intact rats (respectively 4.3 ± 0.3 vs. 2.2 ± 0.6 and 4.5 ± 0.3 vs. 2.3 ± 0.7, p <0.001). In rats with JAA, mostly middle and deep carious cavities were revealed, at the same time in intact rats - superficial and middle carious cavities were observed. CONCLUSION: Conclusions: It has been established that adjuvant arthritis is accompanied by 100% prevalence of dental caries, high intensity of carious process, presence of middle and deep carious cavities, that confirm the negative influence of autoimmune disease on the condition of the hard tooth tissues.

Moxibustion intervention improves synovitis by down-regulating NLRP3/Caspase-1/IL-1ß signaling of synovial tissue in rats with adjuvant arthritis. / 基于NLRP3/Caspase-1/IL-1ßä¿¡å·é€šè·¯æŽ¢è®¨è‰¾ç¸æ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ æ»‘è†œç‚Žçš„æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion on activities of NOD-like receptor family protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1)/interleukin-1ß (IL-1ß) signaling pathway in rats with adjuvant arthritis (AA), so as to explore its mechanisms underlying improvement of rheumatoid arthritis (RA). Me-thods Thirty male Wistar rats were randomly divided into normal control, AA model and moxibustion groups, with 10 rats in each group. The AA model was replicated by raising in wind, cold and damp environment combined with complete Freund's adjuvant injection. In the moxibustion group, moxibustion was applied to bilateral "Shenshu" (BL23) and "Zusanli"(ST36) for 20 min each time, once daily for 21 days. Changes of joint swelling degree (JSD) and arthritis index (AI) in each group were observed. The ultrastructural changes of synovial cells in each group were observed by transmission electron microscopy. The protein expression levels of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, tumor necrosis factor-α (TNF-α) and IL-1ß in the synovial tissues of the knee joint were measured by Western blot. RESULTS: Compared with the normal control group, JSD, AI and the protein expressions of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß in the synovial tissues were significantly increased (P<0.01) in the model group. In comparison with the model group, JSD, AI and the protein expression levels of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß were significantly decreased (P<0.01) in the moxibustion group. Results of transmission electron microscope showed an irregular and vague nuclear membrane of synovial cells, and unclear mitochondrial membrane boundary with sparse, swelling crests in the model group, which was relatively milder in the damage degree in the moxibustion group. CONCLUSIONS: Moxibustion can relieve the inflammatory response in the synovial membrane of AA rats, which may be related to its function in down-regulating synovial NLRP3/Caspase-1/IL-1ß inflammatory signaling.

Carboxyamidotriazole alleviates pannus formation and cartilage erosion in rats with adjuvant arthritis.

Carboxyamidotriazole (CAI) was initially considered a non-cytotoxic anticancer agent. However, recently, pronounced anti-inflammatory properties of CAI have been reported. Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by aberrant activation of signaling pathways. Therefore, this study explored the therapeutic effects and potential mechanism of action of CAI on RA in the adjuvant arthritis (AA) model. The results showed that CAI reduced the severity of arthritis in AA rats as demonstrated by inhibited hind paw swelling, reduced body weight, and decreased infiltration of joint pathological inflammatory cells. Importantly, pathological scoring of new blood vessels and immunohistochemical assays revealed that CAI inhibited pannus formation. CAI decreased the expression of pro-angiogenic growth factors, such as vascular epidermal growth factor, basic fibroblast growth factor, and metalloproteinases (MMPs), namely, MMP-1 and MMP-3 in the synovium of AA rats. Furthermore, CAI significantly reduced the increased levels of phosphorylated p38, c-Jun N-terminal kinase (JNK)1/2, and extracellular signal-regulated kinase (ERK)1/2 proteins in AA rats. In addition, the proliferation of fibroblast-like synoviocytes (FLS) was downregulated by CAI both in vivo and in vitro. In conclusion, this investigation illustrates the therapeutic effect of CAI on synovitis and erosion of articular cartilage in RA. Furthermore, the mechanism might involve inhibition of aberrantly activated mitogen-activated protein kinase signaling, as well as a decrease in pro-angiogenic factors, MMP expression, and FLS proliferation.

Rhein methotrexate-decorated solid lipid nanoparticles altering adjuvant arthritis progression through endoplasmic reticulum stress-mediated apoptosis.

Methotrexate (MTX) and diacerein (DIA) are two of the most potent disease-modifying anti-rheumatic drugs used for the treatment of rheumatoid arthritis (RA). DIA has reflected some GIT and hepatobiliary manifestations in numerous cases. It undergoes biotransformation in the liver into the active metabolite rhein (RH) which is characterized by its excellent anti-inflammatory activity and lower side effects. However, RH's hydrophobic nature and low bioavailability do not encourage its use in RA. The current study aims to use RH in combination with MTX in targeted solid lipid nanoparticles (RH-MTX-SLNs) for better effectiveness and shadowing light on its possible mechanistic pathways. RH-MTX-SLNs were prepared and assessed for their quality attributes. The effect of the formulation was assessed in-vivo in an adjuvant arthritis animal model investigating the role of the endoplasmic reticulum stress (ERS)-induced apoptosis. Results revealed that RH-MTX-SLNs were in the suitable nanosized range with high negative zeta potential indicating good stability. In-vivo, RH-MTX-SLNs significantly improved all measured inflammatory and arthritic markers, confirmed by electron microscopy and histology examination of the joints. Besides, the formulation was able to alter the ERS-mediated apoptosis. In conclusion, RH-MTX-SLNs can represent a promising therapeutic approach for RA showing significant anti-arthritic activity.

Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats.

Lung injury is one of the common extra­articular lesions in rheumatoid arthritis (RA). Due to its insidious onset and no obvious clinical symptoms, it can be easily dismissed in the early stage of diagnosis, which is one of the reasons that leads to a decline of the quality of life and subsequent death of patients with RA. However, its pathogenesis is still unclear and there is a lack of effective therapeutic targets. In the present study, tandem mass tag­labeled proteomics was used to research the lung tissue proteins in RA model (adjuvant arthritis, AA) rats that had secondary lung injury. The aim of the present study was to identify the differentially expressed proteins related to RA­lung injury, determine their potential role in the pathogenesis of RA­lung injury and provide potential targets for clinical treatment. Lung tissue samples were collected from AA­lung injury and normal rats. The differentially expressed proteins (DEPs) were identified by tandem mass spectrometry. Bioinformatic analysis was used to assess the biological processes and signaling pathways associated with these DEPs. A total of 310 DEPs were found, of which 244 were upregulated and 66 were downregulated. KEGG anlysis showed that 'fatty acid degradation', 'fatty acid metabolism', 'fatty acid elongation', 'complement and coagulation cascades', 'peroxisome proliferator­activated receptor signaling pathway' and 'hypoxia­inducible factor signaling pathway' were significantly upregulated in the lung tissues of AA­lung injury. Immunofluorescence staining confirmed the increased expression of clusterin, serine protease inhibitors and complement 1qc in lung tissue of rats with AA lung injury. In the present study, the results revealed the significance of certain DEPs (for example, C9, C1qc and Clu) in the occurrence and development of RA­lung injury and provided support through experiments to identify potential biomarkers for the early diagnosis and prevention of RA­lung injury.

The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components.

Diclofenac is the most prescribed nonsteroidal anti-inflammatory drug worldwide and is used to relieve pain and inflammation in inflammatory arthritis. Diclofenac is associated with serious adverse effects, even in regular-dose regimens. Drug delivery systems can overcome this issue by reducing adverse effects and optimizing their efficacy. This study evaluated the activity of lipid-core nanocapsules loaded with diclofenac (DIC-LNCs) in an experimental model of adjuvant-induced arthritis. The diclofenac nanoformulation was obtained via self-assembly. A stereological analysis approach was applied for the morphological quantification of the volume, density, and cellular profile count of the metatarsophalangeal joints of rats. Proinflammatory cytokines and biochemical profiles were also obtained. Our results showed that the diclofenac nanocapsule DIC-LNCs were able to reduce arthritis compared with the control group and the DIC group. DIC-LNCs efficiently reduced proinflammatory cytokines, C-reactive protein, and xanthine oxidase levels. Additionally, DIC-LNCs reduced the loss of synoviocytes and chondrocytes compared with the DIC (p < 0.05) and control groups (p < 0.05). These data suggest that DIC-LNCs have anti-arthritic activity and preserve joint components, making them promising for clinical use.

The Utility of Peptide Ligand-Functionalized Liposomes for Subcutaneous Drug Delivery for Arthritis Therapy.

Liposomes and other types of nanoparticles are increasingly being explored for drug delivery in a variety of diseases. There is an impetus in the field to exploit different types of ligands to functionalize nanoparticles to guide them to the diseased site. Most of this work has been conducted in the cancer field, with relatively much less information from autoimmune diseases, such as rheumatoid arthritis (RA). Furthermore, in RA, many drugs are self-administered by patients subcutaneously (SC). In this context, we have examined the attributes of liposomes functionalized with a novel joint-homing peptide (denoted ART-1) for arthritis therapy using the SC route. This peptide was previously identified following phage peptide library screening in the rat adjuvant arthritis (AA) model. Our results show a distinct effect of this peptide ligand on increasing the zeta potential of liposomes. Furthermore, liposomes injected SC into arthritic rats showed preferential homing to arthritic joints, following a migration profile in vivo similar to that of intravenously injected liposomes, except for a less steep decline after the peak. Finally, liposomal dexamethasone administered SC was more effective than the unpackaged (free) drug in suppressing arthritis progression in rats. We suggest that with suitable modifications, this SC liposomal treatment modality can be adapted for human RA therapy.

[Effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis rats].

OBJECTIVE: To observe the effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis (AA) rats, and to explore the potential mechanism of moxibustion in treatment of rheumatoid arthritis. METHODS: Forty-five Wistar male rats were randomly divided into a normal group, a model group and a moxibustion group, with 15 rats in each group. In the model group and the moxibustion group, the AA model was replicated under wind, cold and humid environment and by injection with complete freund's adjuvant. In the moxibustion group, moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) was used, 20 min each time, once daily, for consecutive 21 days. In the normal group and the model group, no intervention was processed. The scores of the knee joint swelling degree (JSD) and arthritis index (AI) were compared among groups. The ultrastructure of synovial cells of knee joint were observed under transmission electron microscope (TEM). The levels of serum cytokines such as tumor necrosis factor-α (TNF-α), interieukin (IL)-1ß, IL-6 and IL-10 were detected using ELISA method. RESULTS: Compared with the normal group, JSD and AI scores, the levels of TNF-α, IL-1ß and IL-6 were increased (P<0.01), while IL-10 was reduced (P<0.01) in the model group after intervention. JSD and AI scores, and the levels of TNF-α, IL-1ß and IL-6 were lower (P<0.05, P<0.01), while the level of IL-10 was higher (P<0.01) in the moxibustion group compared with the model group. Compared with the normal group, the ultrastructure of synovial cell was obviously damaged in the model group, and the damage was attenuated in the moxibustion group compared with the model group. CONCLUSION: Moxibustion can reduce the symptoms of arthritis in AA rats, which may be related to the improvement of the ultrastructure of synovial cells and the regulation of cytokines.

Effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis rats / 中国针灸

OBJECTIVE@#To observe the effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis (AA) rats, and to explore the potential mechanism of moxibustion in treatment of rheumatoid arthritis.@*METHODS@#Forty-five Wistar male rats were randomly divided into a normal group, a model group and a moxibustion group, with 15 rats in each group. In the model group and the moxibustion group, the AA model was replicated under wind, cold and humid environment and by injection with complete freund's adjuvant. In the moxibustion group, moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) was used, 20 min each time, once daily, for consecutive 21 days. In the normal group and the model group, no intervention was processed. The scores of the knee joint swelling degree (JSD) and arthritis index (AI) were compared among groups. The ultrastructure of synovial cells of knee joint were observed under transmission electron microscope (TEM). The levels of serum cytokines such as tumor necrosis factor-α (TNF-α), interieukin (IL)-1β, IL-6 and IL-10 were detected using ELISA method.@*RESULTS@#Compared with the normal group, JSD and AI scores, the levels of TNF-α, IL-1β and IL-6 were increased (P<0.01), while IL-10 was reduced (P<0.01) in the model group after intervention. JSD and AI scores, and the levels of TNF-α, IL-1β and IL-6 were lower (P<0.05, P<0.01), while the level of IL-10 was higher (P<0.01) in the moxibustion group compared with the model group. Compared with the normal group, the ultrastructure of synovial cell was obviously damaged in the model group, and the damage was attenuated in the moxibustion group compared with the model group.@*CONCLUSION@#Moxibustion can reduce the symptoms of arthritis in AA rats, which may be related to the improvement of the ultrastructure of synovial cells and the regulation of cytokines.

Combination Therapy of Carnosic Acid and Methotrexate Effectively Suppressed the Inflammatory Markers and Oxidative Stress in Experimental Arthritis.

BACKGROUND: Combination therapy with methotrexate (MTX) is the most common therapeutic strategy used for the treatment of patients with rheumatoid arthritis (RA). In this study, we combined the natural compound carnosic acid (CA) with MTX to reduce inflammation and oxidative stress in adjuvant arthritis (AA). METHODS: AA was induced in 6-8 rats per group. MTX was administrated twice a week at a dose of 0.3 mg/kg b.w., while CA was administered daily at a dose of 100 mg/kg both in monotherapy and in combination with MTX. Plasma samples were collected on the 14th, 21st, and 28th day. Body weight and hind paw volume were measured once a week. RESULTS: We found that, mainly, the CA + MTX combination significantly reduced the hind paw swelling, the levels of IL-17A, MMP-9, and MCP-1 in plasma, and GGT activity in joint homogenates. The mRNA expression of HO-1, catalase, and IL-1ß in the liver were significantly improved by CA + MTX only. Our results indicate that adding CA to MTX treatment could be a good therapeutic option for patients suffering from RA. CONCLUSIONS: The addition of CA to methotrexate treatment significantly improved its efficacy in decreasing the development of AA by inhibiting the markers of inflammation and oxidative stress.

Bone-Targeted Delivery of Novokinin as an Alternative Treatment Option for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune inflammatory bone destructive disorder that is orchestrated by multiple systems in the body, including Renin-Angiotensin System (RAS) and arachidonic acid (ArA) pathway. Current therapeutic options are not highly effective and are associated with severe side effects, including cardiovascular complications. Therefore, new safe and effective disease modulators are seriously needed. In this study, we investigate the anti-inflammatory effects of a synthetic peptide, novokinin, through Angiotensin Type (II) receptor (AT2R). Peptide drugs like novokinin suffer from plasma instability and short half-life. Thus, we developed a novel bone targeting novokinin conjugate (Novo Conj). It uses the bone as a reservoir for sustained release and protection from systemic degradation, improving stability and enhancing pharmacological efficacy. We tested Novo Conj's anti-inflammatory effects in adjuvant-induced arthritis (AIA) rat model to prove our hypothesis by measuring various RAS and ArA pathway components. We observed that inflammation causes a significant imbalance in cardioprotective RAS components like ACE2, AT2R, and Ang 1-7 and increases the ArA inflammatory metabolites like hydroxyeicosatetraenoic acids (HETEs). Treatment with novokinin or Novo Conj restores balance in the RAS and favors the production of different epoxyeicosatrienoic acids (EETs), which are anti-inflammatory mediators. This study demonstrated that the bone-targeted delivery improved the stability and enhanced the anti-inflammatory effects of the parent peptide novokinin in AIA. These observations offer an efficacious alternative therapy for managing RA.

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

Inroduction: The 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 methods: Adjuvant 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. Results: In 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. Conclusions: Cell 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.

[Effects of moxibustion on p53， SLC7A11， and GPX4 expression in synovial tissues of rats with adjuvant arthritis].

OBJECTIVE: To observe the effects of moxibustion on p53, cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in synovial tissues of rats with adjuvant arthritis (AA), so as to explore the mechanism of moxibustion in alleviating rheumatoid arthritis. METHODS: Eighty rats were randomly divided into normal, model, moxibustion, and medication groups (n=20 in each group). The AA model was established by exposure to wind, cold, and damp environmental factors combined with injection of complete Freund's adjuvant. Rats in the moxibustion group received suspended moxibustion at "Shenshu" (BL23) and "Zusanli" (ST36) alternately, while those in the medication group were treated with tripterygium glycoside tablet suspension (8 mg/kg) by gavage, once a day, for 15 successive days. The pathological change in synovial tissue of rat right knee joint was observed by HE staining. The protein and mRNA expression levels of p53, SLC7A11, and GPX4 in the synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. The se-rum glutathione (GSH) and reactive oxygen species (ROS) contents were measured by colorimetry and fluorescence probe me-thod. RESULTS: Compared with the normal group, the model group exhibited synovial hyperplasia of the right knee joint, massive inflammatory cell infiltration, up-regulated mRNA and protein expression of p53 in synovial tissue, elevated serum ROS content (P<0.01), down-regulated mRNA and protein expression of SLC7A11 and GPX4 in synovial tissue, and lowered serum GSH content (P<0.01). Comparison with the model group showed that the synovial injuries in the moxibustion and medication groups were obviously alleviated. The mRNA and protein expression levels of p53 in the synovial tissues and the serum ROS content declined significantly (P<0.01), while the mRNA and protein expression of SLC7A11 and GPX4 in the synovial tissues and the se-rum GSH content increased (P<0.01). There was no significant difference in histopathological change of synovial tissue between the moxibustion group and medication group. However, the p53 protein expression in the synovial tissue and the level of serum ROS were significantly higher in the medication group than in the moxibustion group (P<0.05), while the GPX4 protein expression and serum GSH content were down-regulated (P<0.05). CONCLUSION: Moxibustion improves the inflammatory response in synovial tissue of AA model rats, which may be closely related to its regulation of the expression of ferroptosis-related factors.

Enhanced Anti-Inflammatory Effect of the Combination of Lactiplantibacillus plantarum LS/07 with Methotrexate Compared to Their Monotherapies Studied in Experimental Arthritis.

The gut microbiome (GM) of rheumatic arthritis (RA) patients is often altered in composition and function. Moreover, methotrexate (MTX), one of the most frequently used disease-modifying antirheumatic drugs, is known to negatively affect GM composition. The modulation of immune system activity is one of the therapeutic benefits of probiotics. The aim of the current investigation was to determine the impact of MTX therapy combined with one of the Lactobacillus strains, Lactoplantibacillus plantarum LS/07 (LB), on adjuvant arthritis (AA) in rats. Methods focused on biometric and inflammatory parameters in AA, particularly on plasmatic levels of IL-17A, MMP-9, and MCP-1, and the activities of gamma-glutamyl transferase in the spleen and joints were applied. Enhancing the effect of MTX, LB positively influenced all biometric and inflammatory parameters. The findings of the present study may be of help in proposing novel therapeutic strategies for RA patients.

Effectof Yishen-Qingluo-Huoxue prescription on OPG/RANKL pathway in CFA rats and its mechanism / 中国药理学通报

To investigate the effect of Xinan Fang Yisheng-Qingluo-Huoxie prescription(YSF)on OPG/RANKL pathway in rats with rheumatoid arthritis induced by complete Freund's adjuvant(CFA)and its mechanism. Methods Thirty-two male SD rats were randomly divided into normal group, AIA group, QLY group and YSF group, with 8 rats in each group. AIA rats were induced except for the normal group. QLY and YSF were administered intragastrically on the first day after the model was successful for 30 days. The body weight and AI score of rats were observed. HE staining was used to observe the histomorphological changes of the ankle joint of rats. The expression of MMP3 and P-P65 was observed by immunohistochemistry. Serum levels of TNF-α, IL-1β, OPG and RANKL were detected by ELISA. The expression of OPG and RANKL proteins in synovium of rats was detected by Western blot and RT-PCR. Results Compared with AIA model, the body weight of the rats gradually returned to normal, and inflammation score decreased. HE staining showed that the bone was well preserved, no synovial infiltration was observed after YSF administration, and MMP3 and P-P65 showed low expression. The levels of TNF-α, IL-1β and RANKL in serum were significantly down-regulated, while the levels of OPG and OPG/RANKL were significantly up-regulated. In addition, Western blot and RT-PCR showed that OPG and OPG/RANKL were highly expressed in synovium of rats, while RANKL was on the contrary. Conclusions YSF may decrease inflammatory score and expression of TNF-α and IL-1β in serum by inhibiting inflammatory response. What's more, the increase of OPG/RANKL ratio can regulate the environment of joint bone destruction, thus alleviating RA.

Allopurinol regulates abnormal kynurenine pathway metabolism and plays a role in treatment of adjuvant arthritis in rats / 中国药理学通报

Aim To observe the therapeutie effeet of (AA) rats and its effect on the aetivity of tryptophan allopurinol ( ALLO) on adjuvant induced arthritis 2,3 dioxygenase (TDO).Methods SD rats were randomly divided into normal group, AA model group, ALLO group (10,20 , 40 mg • kg 1 ) and methotrexate group (0.5 mg • kg 1 ).The AA rats were established by intracutaneous injection of complete Freund's adju¬vant into the right toes of rats.The body weight,joint swelling number, joint pathology, spleen index and fi¬broblast like synoviocytes ( FLS) proliferation of the rats were observed to explore the therapeutic effect of ALLO.Flow cytometry detected the number of CD68 ∗ macrophages and the ratio of Thl7/Treg of spleen.The concentration of tryptophan ( Trp) and kvnurenine ( Kyn) in the liver and the supernatant of FLS were de¬termined by high performance liquid chromatography.Results Compared with model group, ALLO adminis¬ tration significantly increased the body weight of AA rats, reduced the number of joint swelling, improved joint pathological injury,decreased spleen index,inhib¬ited the proliferation of FLS, reduced the number of macrophages in the spleen,decreased Thl7/Treg ratio, inhibited the metabolism of Tip and the production of Kyn in liver and FLS culture supernatant, and de¬creased the liver Kyn/Trp ratio (TOO activity).Con¬clusion ALLO has therapeutic effect on AA rats, which may be related to its regulation of TDO-mediated kyn metabolism pathway.

[Acupuncture regulates PI3K/Akt/mTOR signaling and up-regulates cell autophagy to protect sy-novial tissue of adjuvant arthritis rats].

OBJECTIVE: To observe the effect of acupuncture of "Yinlingquan"(SP9) and "Sanyinjiao"(SP6) on expression of phosphatidylinositol-3 kinase/protein kinase B/mammalian target protein of rapamycin (PI3K/Akt/mTOR) signaling in adjuvant arthritis (AA) rats, so as to explore its mechanism underlying improvement of AA. METHODS: Forty-eight male Wistar rats were randomly divided into normal control, AA model, acupuncture and medication (tripterygium wilfordii) groups, with 12 rats in each group. The AA model was established by putting the rats in a windy, cold and wet environment for 12 h, once every day for 21 days and injection of Freund's complete adjuvant (CFA) into the sole of the right hindlimb on the 21st day. Manual acupuncture stimulation was applied at SP9 and SP6 for 30 min/time, once a day for 21 days. Rats of the medication group received gavage of tripterygium wilfordii tablets solution (8 mg/kg), once a day for 21 days, and those of the normal control group and model group received gavage of the same amount of normal saline, once a day for 21 days. The degree of joint swelling and arthritis index (AI) were detected 1 day before modeling, 3 days after modeling, and 21 days after the treatment. Twenty-four hours after the last treatment, the contents of serum cytokines interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA); and changes of synovial ultrastructure were observed under electron microscope. Western blot was used to detect the expression levels of PI3K, Akt, phosphorylated protein kinase B (p-Akt), phosphorylated mammalian rapamycin target protein (p-mTOR), mTOR, microtubule associated protein 1 light chain 3B (LC3-Ⅱ) and mammalian atg6 homologous protein (Beclin-1) in the synovial membrane tissue. RESULTS: Compared with the normal control group, the joint swel-ling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in the synovium were increased in the model group (P<0.05), while the expression levels of LC3-Ⅱ and Beclin-1 proteins in the synovium were significantly decreased (P<0.05). Compared with the model group, the joint swelling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR were significantly decreased in the acupuncture and medication groups (P<0.05), while the expresion levels of LC3-Ⅱ and Beclin-1 proteins were significantly increased （P<0.05）. Comparison between the two treatment groups showed that the therapeutic effects of acupuncture were obviously weaker than those of medication in down-regulating TNF-α and IL-6, Akt, p-Akt and mTOR levels (P<0.05) and in up-regulating Beclin-1 expression (P<0.05). Outcomes of electron microscope displayed widened nuclear membrane space, some fractured mitochondrial cristae with vacuoles, expanded rough endoplasmic reticulum, reduction of autophagosomes in the cytoplasm and ruptured synovial cell membrane in the model group, and increase of autophagosomes, deformed organelles in the acupuncture and medication groups. CONCLUSION: Acupuncture can relieve the inflammatory reactions and joint synovial injury of the affected joint in AA rats which may be associated with its effects in inhibiting PI3K/Akt/mTOR signaling and increasing the auto-phagy level of synovial cells.

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

Qingluoyin granules protect against adjuvant-induced arthritis in rats via downregulating the CXCL12/CXCR4-NF-κB signalling pathway.

CONTEXT: Qingluoyin (QLY) is a traditional Chinese medicine (TCM) formula which has been used in treating human rheumatoid arthritis (RA) for years in China. OBJECTIVE: This study investigates the effect of QLY granules on adjuvant arthritis (AA) and the possible mechanism. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were injected with Complete Freund's adjuvant (CFA) to induce the AA model. After the onset of arthritis, rats received intragastric administrations of the QLY granules (1.35, 2.70, and 5.40 g/kg) or Tripterygium glycosides (TG) tablets (positive drug, 10 mg/kg) for 14 d. After 28 d immunization, the symptoms, inflammatory parameters and molecular mechanisms were investigated. RESULTS: In the QLY granule (1.35, 2.70, and 5.40 g/kg) therapy groups, the arthritis index decreased to 6.30 ± 2.06, 5.80 ± 1.55, 5.30 ± 1.16 compared with the model (9.00 ± 3.01), paw swelling decreased to 1.56 ± 0.40, 1.28 ± 0.38, 1.12 ± 0.41 mL compared with the model (2.22 ± 0.73 mL). QLY granules (1.35, 2.70 and 5.40 g/kg) significantly reduced the thymus and the spleen indexes, inhibited the production of pro-inflammatory cytokines, and alleviated the pathological changes of joints compared with the model group. Furthermore, the treatment of QLY granules (2.70 and 5.40 g/kg) markedly inhibited CXCL12, CXCR4 (in spleen and synovium) and p-NF-κB p65 (in synovium) protein expression of AA rats. CONCLUSIONS: QLY granules have obvious therapeutic effects on AA rats, which may be associated with downregulating the CXCL12/CXCR4-NF-κB signalling pathway. QLY granules can be used as a candidate for the treatment of RA, which deserves further study.