sirolimus and Arthritis

To investigate the mechanism of microRNA-100-5p (miR-100-5p) on mammalian target (mTOR) of rapamycin in temporomandibular arthritis.. Sixty SD rats were randomly divided into group A, group B, group C, group D, and group E, with 12 rats in each group. Rat models of temporomandibular arthritis were prepared by injecting sodium iodoacetate solution into the bilateral spaces of temporomandibular joint. After establishment, group C was injected pcDNA3.1-miR-100-5p recombinant plasmid, group D was injected mTOR inhibitor rapamycin, group E was injected with pcDNA3.1-miR-100-5p recombinant plasmid and rapamycin, and group A was injected same amount of normal saline in the same way. Various indexes were observed in each group, including morphological changes of temporomandibular joint tissues, matrix metalloproteinase-3 (MMP-3), MMP-1, MMP-13, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miR-100-5p, mTOR expression. The data were processed using SPSS 22.0 software package.. In group B, the structure of temporomandibular joint was fuzzy, with synovial hyperplasia, vascular dilatation, clustered cells and a large amount of inflammatory infiltration. Histopathological changes of temporomandibular joint in each interventional group were improved to different degrees compared with group B, among which group E showed the most obvious improvement. The levels of MMP-3, MMP-1, MMP-13, IL-6, IL-1β and TNF-α in group B were significantly higher than those in group A(P＜0.05). The levels of MMP-3, MMP-1, MMP-13, IL-6, IL-1β and TNF-α in group C, group D and group E were significantly lower than those in group B(P＜0.05). The levels of MMP-3, MMP-1, MMP-13, IL-6, IL-1β and TNF-α in group D were not significantly different from those in group C (P＜0.05). The levels of MMP-3, MMP-1, MMP-13, IL-6, IL-1β and TNF-α in group E were significantly lower than those in group D (P＜0.05). The expression level of miR-100-5p in group E was significantly higher than that in group B (P＜0.05). The expression level of mTOR protein in group E was significantly lower than that in group B (P＜0.05).. MicroRNA-100-5p may alleviate temporomandibular arthritis by down-regulating the expression of mTOR.

Topics: Animals; Arthritis; Interleukin-1beta; Interleukin-6; Mammals; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; MicroRNAs; Rats; Rats, Sprague-Dawley; Sirolimus; Temporomandibular Joint Disorders; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune cells. But the combined inhibition of both pathways was rarely investigated. This study investigated how inhibiting both glutamine metabolism with 6-diazo-5-oxo-L-norleucine (DON) and mTOR with rapamycin affects immune cells and the arthritis in a mouse model. We revealed that rapamycin and DON additively suppressed CD4

Topics: Animals; Arthritis; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Dendritic Cells; Diazooxonorleucine; Female; Glutamine; Immunosuppression Therapy; Macrophages; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Sirolimus; Th17 Cells; TOR Serine-Threonine Kinases

Interleukin 22 (IL-22), a relatively new cytokine has been found to induce significant proliferation of human keratinocytes and fibroblast like synoviocytes (FLS) and thus plays an important role in the pathogenesis of autoimmune diseases like psoriasis and rheumatoid arthritis (RA) which are characterized by hyperproliferation of keratinocytes and FLS respectively. PI3K/Akt/mTOR signaling cascade plays crucial role in cell growth and survival. Therefore our objective was to see the regulatory role of PI3K/Akt/mTOR signaling cascade in IL-22 induced proliferation of keratinocytes and FLS.. Normal human epidermal keratinocytes (NHEK) and FLS were isolated from skin of healthy volunteer's undergone plastic surgery and synovial tissue of psoriatic arthritis (PsA) and RA patients respectively. IL-22 induced proliferation of NHEK and FLS was measured by MTT assay. Phosphorylation of Akt/mTOR was determined by western blot assay and further confirmed by real time polymerase chain reaction (RT-PCR).. We observed that IL-22 induced significant proliferation of NHEK and FLS which was effectively inhibited by dual kinase (PI3K/mTOR) inhibitor, NVP-BEZ235 and specific mTOR inhibitor, Rapamycin. In NHEK and FLS, IL-22 significantly induced phosphorylation of Akt and mTOR which was effectively blocked by Rapamycin and NVP-BEZ235. Further we did RT-PCR in NHEK and found that IL-22 significantly upregulated AKT1 and MTOR gene.. These results show that IL-22 induced proliferation of NHEK and FLS is dependent on PI3K/Akt/mTOR signaling pathway. This novel observation provides the scope to develop new therapeutics targeting PI3K/Akt/mTOR signaling pathway in autoimmune diseases like psoriasis and rheumatoid arthritis.

Topics: Arthritis; Arthritis, Rheumatoid; Blotting, Western; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Imidazoles; Interleukin-22; Interleukins; Keratinocytes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Psoriasis; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Synovial Membrane; TOR Serine-Threonine Kinases

To assess the effects of epigallocatechin-3-gallate (EGCG) on cytokine-induced Cyr61 synthesis in human osteoblastic cells and the associated signalling pathways. The therapeutic effect of EGCG on CIA in rats was also studied.. The expression of Cyr61 and NF-κB pathway molecules was examined by western blotting. CCL2 expression was assessed by northern blotting and ELISA. Interaction between NF-κB and Cyr61 promoter was evaluated by electrophoretic mobility shift assay. In rat CIA, osteoblastic expression of Cyr61 was examined by immunohistochemistry and disease progression was assessed by clinical, radiographic and histological examinations.. EGCG inhibited Cyr61 expression stimulated by cytokines in primary human osteoblasts and human osteoblastic cell line U2OS. In U2OS, oncostatin M (OSM) induced IκB-α degradation through the mTOR/rictor/Akt pathway, and EGCG attenuated the action. Electrophoretic mobility shift assay revealed that the OSM-enhanced NF-κB/DNA binding was reduced by EGCG, possibly through abrogating nucleus localization of p65 and p50. Cyr61 enhanced OSM-induced expression of CCL2. Moreover, EGCG diminished OSM-stimulated CCL2 expression at least partially via suppressing Cyr61 induction. Co-distribution of CD68(+) macrophages and Cyr61(+) osteoblasts in osteolytic areas was obvious in the CIA model. Clinical, radiographic and immunohistochemical analyses revealed that administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the number of Cyr61-synthesizing osteoblasts.. By modulating the mTOR/rictor/Akt/NF-κB pathway, EGCG attenuated Cyr61 production in osteoblastic cells and in turn diminished macrophage chemotaxis. Our data support the therapeutic potential of EGCG on arthritis.

Topics: Adult; Animals; Arthritis; Catechin; Cells, Cultured; Chemokine CCL2; Chromones; Cysteine-Rich Protein 61; Cytokines; Enzyme Inhibitors; Humans; Inositol Phosphates; Male; Morpholines; NF-kappa B; Osteoblasts; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

Topics: Administration, Oral; Animals; Arthritis; Arthritis, Experimental; Autoimmune Diseases; Blood Glucose; Diabetes Mellitus, Type 1; Drinking; Encephalomyelitis, Autoimmune, Experimental; Female; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Male; Mice; Mice, Inbred DBA; Mice, Inbred NOD; Polyenes; Rats; Rats, Inbred Lew; Sirolimus

SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in animal models of autoimmune diseases such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The effect of SK&F 105685 on the proliferation of rat lymphoid cells was examined in vitro. The compound inhibited the proliferative response of spleen, thymus and lymph node cells to the mitogen concanavalin A (Con A) in a dose-dependent manner but had little or no effect on the mitogenic response of peripheral blood lymphocytes. Although less potent than cyclosporin A, SK&F 105685 was able to inhibit the proliferation of spleen cells stimulated with PMA and ionomycin or the mitogens phytohemagglutinin (PHA), Con A and pokeweed mitogen (PWM). Relatively early event(s) in cell proliferation were affected by SK&F 105685 since delaying addition of the drug by 24 to 48 hours after Con A stimulation of rat spleen cells resulted in reduced levels of suppression. The mode of action of SK&F 105685 appeared to differ from that of cyclosporin A or rapamycin. Unlike cyclosporin A, SK&F 105685 did not affect IL-2 production by Con A-stimulated spleen cells or the IL-2-producing Jurkat cell line, but, like rapamycin, the compound significantly reduced the IL-2-induced proliferation of rat ConA blasts. These results suggest that inhibition of lymphocyte proliferation by SK&F 105685 may require the activity of an intermediate effector cell(s) present in susceptible populations such as cells from the spleen, thymus, lymph nodes and Con A blast preparations but absent or present in low numbers in resistant populations such as peripheral blood cells. Indomethacin and NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of nitric oxide synthase, were both unable to relieve SK&F 105685-induced suppression of splenic Con A responses thereby ruling out a role for the production of prostaglandins or nitric oxide by macrophages as an intermediate in drug-mediated suppression. In summary, SK&F 105685 was unable to inhibit lymphoproliferative responses by a mechanism distinct from that of cyclosporin A or rapamycin and which appears to involve regulation of cellular interactions rather than a direct effect on responding lymphocytes.

Topics: Animals; Arginine; Arthritis; Concanavalin A; Cyclosporine; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Indomethacin; Interleukin-2; Lymphocyte Activation; Male; omega-N-Methylarginine; Polyenes; Rats; Rats, Inbred Lew; Sirolimus; Spiro Compounds; Spleen; Time Factors; Tumor Cells, Cultured