Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

N-Acetylcysteine overcomes epalrestat-mediated increase of toxic 4-hydroxy-2-nonenal and potentiates the anti-arthritic effect of epalrestat in AIA model.

Epalrestat, an aldose reductase inhibitor (ARI), has been clinically adopted in treating diabetic neuropathy in China and Japan. Apart from the involvement in diabetic complications, AR has been implicated in inflammation. Here, we seek to investigate the feasibility of clinically approved ARI, epalrestat, for the treatment of rheumatoid arthritis (RA). The mRNA level of AR was markedly upregulated in the peripheral blood mononuclear cells (PBMCs) of RA patients when compared to those of healthy donors. Besides, the disease activity of RA patients is positively correlated with AR expression. Epalrestat significantly suppressed lipopolysaccharide (LPS) induced TNF-α, IL-1ß, and IL-6 in the human RA fibroblast-like synoviocytes (RAFLSs). Unexpectedly, epalrestat treatment alone markedly exaggerated the disease severity in adjuvant induced arthritic (AIA) rats with elevated Th17 cell proportion and increased inflammatory markers, probably resulting from the increased levels of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA). Interestingly, the combined treatment of epalrestat with N-Acetylcysteine (NAC), an anti-oxidant, to AIA rats dramatically suppressed the production of 4-HNE, MDA and inflammatory cytokines, and significantly improved the arthritic condition. Taken together, the anti-arthritic effect of epalrestat was diminished or even overridden by the excessive accumulation of toxic 4-HNE or other reactive aldehydes in AIA rats due to AR inhibition. Co-treatment with NAC significantly reversed epalrestat-induced upregulation of 4-HNE level and potentiated the anti-arthritic effect of epalrestat, suggesting that the combined therapy of epalrestat with NAC may sever as a potential approach in treating RA. Importantly, it could be regarded as a safe intervention for RA patients who need epalrestat for the treatment of diabetic complications.

Tricin promoted ATG-7 dependent autophagic degradation of α-synuclein and dopamine release for improving cognitive and motor deficits in Parkinson's disease.

Tricin, a natural nontoxic flavonoid distributed in grasses and euphorbia plants, has been reported to scavenge free radicals, possess anti-inflammatory and antioxidative effects. However, its autophagic effect on Parkinson's disease (PD) has not been elucidated. By adopting cellular and C. elegans models of PD, the autophagic effect of tricin was identified based on the level of autophagy markers (LC3-II and p62). Besides, the pharmacological effects on neurotransmitters (dopamine), inflammatory cytokines (IFN γ, TNFα, MCP-1, IL-10, IL-6 and IL-17A), histology (hematoxylin & eosin and Nissl staining) and behavioural pathology (open-field test, hindlimb clasping, Y-maze, Morris water-maze and nest building test) were also confirmed in the A53T-α-synuclein transgenic PD mouse model. Further experiments demonstrated that tricin induced autophagic flux and lowered the level of α-synuclein through AMPK-p70s6K- and ATG7-dependent mechanism. Compared to the existing clinical PD drugs, tricin mitigated pathogenesis and symptoms of PD with no observable side effects. In summary, tricin is proposed as a potential adjuvant remedy or nutraceutical for the prevention and treatment of PD.

Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.

GSH/ROS Dual-Responsive Supramolecular Nanoparticles Based on Pillar[6]arene and Betulinic Acid Prodrug for Chemo-Chemodynamic Combination Therapy.

Chemodynamic therapy (CDT) based on intracellular Fenton reactions is attracting increasing interest in cancer treatment. A simple and novel method to regulate the tumor microenvironment for improved CDT with satisfactory effectiveness is urgently needed. Therefore, glutathione (GSH)/ROS (reactive oxygen species) dual-responsive supramolecular nanoparticles (GOx@BNPs) for chemo-chemodynamic combination therapy were constructed via host-guest complexation between water-soluble pillar[6]arene and the ferrocene-modified natural anticancer product betulinic acid (BA) prodrug, followed by encapsulation of glucose oxidase (GOx) in the nanoparticles. The novel supramolecular nanoparticles could be activated by the overexpressed GSH and ROS in the tumor microenvironment (TME), not only accelerating the dissociation of nanoparticles-and, thus, improving the BA recovery and release capability in tumors-but also showing the high-efficiency conversion of glucose into hydroxyl radicals (·OH) in succession through intracellular Fenton reactions. Investigation of antitumor activity and mechanisms revealed that the dramatic suppression of cancer cell growth induced by GOx@BNPs was derived from the elevation of ROS, decrease in ATP and mitochondrial transmembrane potential (MTP) and, finally, cell apoptosis. This work presents a novel method for the regulation of the tumor microenvironment for improved CDT, and the preparation of novel GSH/ROS dual-responsive supramolecular nanoparticles, which could exert significant cytotoxicity against cancer cells through the synergistic interaction of chemodynamic therapy, starvation therapy, and chemotherapy (CDT/ST/CT).

The effect of disaggregated nano-hydroxyapatite on oral biofilm in vitro.

OBJECTIVE: Agglomeration is a common problem facing the preparation and application of nanomaterials, and whether nano-hydroxyapatite (nano HA) can modulate oral microecology left to be unclear. In this study, nano HA was disaggregated by sodium hexametaphosphate (SHMP) and ultrasonic cavitation to observe whether agglomeration would affect its effect on oral bacterial biofilm. METHODS: Dynamic light scattering (DLS) and scanning electronic microscope (SEM) were used to observe the treatment solutions. Single-species biofilms and multi-species biofilms were treated with 10% nano HA, 10% disaggregated nano HA, 10% micro hydroxyapatite (micro HA) and deionized water (DDW) for 30min and analyzed via MTT assay, lactic acid measurement, SEM and confocal laser scanning microscope (CLSM). Real-time polymerase chain reaction was performed to analyze the biofilm composition. RESULTS: Ultrasonic cavitation combined with SHMP could significantly reduce the degree of agglomeration of nano HA. Disaggregated nano HA could inhibit bacterial growth and reduce the ability of bacterial biofilm to produce lactic acid and extracellular polysaccharides. There was no significant difference on composition of multi-species biofilms between nano HA and disaggregated nano HA. SIGNIFICANCE: The disaggregated nano-hydroxyapatite could inhibit the metabolism and acid production of oral bacterial biofilm, but did not significantly affect the composition of multi-species biofilms.