Circ_0004712 Silencing Suppresses the Aggressive Changes of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting miR-633/TRAF6 Axis.

Circular RNA_0004712 (circ_0004712) is reported to be up-regulated in rheumatoid arthritis (RA) patients. Nevertheless, its role and mechanism in RA pathology remain to be clarified. RNA and protein expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Cell viability, proliferation, apoptosis, migration, and inflammation were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 5-ethynyl-20-deoxyuridine assay, flow cytometry, scratch test, and enzyme-linked immunosorbent assay. The target correlation between microRNA-633 (miR-633) and circ_0004712 or TNF receptor associated factor 6 (TRAF6) was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Circ_0004712 was up-regulated in RA synovial tissues and RA fibroblast-like synoviocytes (RA-FLSs). Circ_0004712 silencing suppressed the viability, proliferation, migration and inflammatory response and facilitated the apoptosis of RA-FLSs. miR-633 was confirmed to be a direct target of circ_0004712, and miR-633 knockdown reversed circ_0004712 silencing-mediated protective effects on the dysfunction and inflammation of RA-FLSs. TRAF6 was a direct target of miR-633, and miR-633 overexpression suppressed the aggressive changes of RA-FLSs by down-regulating TRAF6. Circ_0004712 could up-regulate TRAF6 expression by sponging miR-633 in RA-FLSs. Circ_0004712 interference inactivated nuclear factor (NF)-κB signaling by targeting miR-633/TRAF6 axis. Circ_0004712 silencing inhibited the aggressive changes of RA-FLSs by targeting miR-633/TRAF6 axis and NF-κB signaling, which provided new targets for RA therapy.

Design, synthesis and biological evaluation of novel 2,4-disubstituted quinazoline derivatives targeting H1975 cells via EGFR-PI3K signaling pathway.

In order to find new and highly effective anti-tumor drugs with targeted therapeutic effects, a series of novel 4-aminoquinazoline derivatives containing N-phenylacetamide structure were designed, synthesized and evaluated for antitumor activity against four human cancer cell lines (H1975, PC-3, MDA-MB-231 and MGC-803) using MTT assay. The results showed that the compound 19e had the most potent antiproliferative activity against H1975, PC-3, MDA-MB-231 and MGC-803 cell lines. At the same time, compound 19e could significantly inhibit the colony formation and migration of H1975 cells. Compound 19e also arrested the H1975 cell cycle in the G1 phase and mediated cell apoptosis, promoted the accumulation of ROS in H1975 cells. Furthermore, compound 19e exerted antitumor effect in vitro by reducing the expression of anti-apoptotic protein Bcl-2 and increasing the pro-apoptotic protein Bax and p53. Mechanistically, compound 19e could significantly decreased the phosphorylation of EGFR and its downstream protein PI3K in H1975 cells. Which indicated that compound 19e targeted H1975 cell via interfering with EGFR-PI3K signaling pathway. Molecular docking showed that compound 19e could bind into the active pocket of EGFR. Those work suggested that compound 19e would have remarkable implications for further design of anti-tumor agents.

CTLA-4 regulates T follicular regulatory cell differentiation and participates in intestinal damage caused by spontaneous autoimmunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is a co-inhibitory molecule expressed by T cells and is required for immune regulation and inflammation prevention. In clinical patients, the CTLA-4 mutation causes spontaneous immune-related early-onset Crohn's disease; however, its potential mechanism is still unknown. In the current study, we found that defects in CTLA-4 in CD4 cells lead to limited differentiation of T follicular regulatory (Tfr) cells and relatively increased T follicular helper (Tfh) cells and spontaneous B cell germinal centres (GCs) responses that trigger the accumulation of autoantibodies in intestinal epithelial cells. In addition, the deficiency of Tfr cells caused by defects in CTLA-4 causes these cells to lose their function of inhibiting the non-specific immune response produced during the specific humoural immune response induced by MCMV (mouse cytomegalovirus), resulting in acute intestinal injury and death in mice. The lack of Tfr cells may be responsible for the immunosuppressive disorder of inflammatory bowel disease caused by CTLA-4 deficiency. In conclusion, we verified that CTLA-4 may be required for Tfr cell differentiation and production. Tfr cells inhibit B cell responses and prevent humoural autoimmune-mediated intestinal damage by regulating Tfh-dependent GC responses.

Exploration of leech therapy in treating gouty rats and its uric acid lowering mechanism.

BACKGROUND: Gout is a hyperuricemia (HUA)-related inflammatory reaction in the joints. Leech therapy has been effective in the gout, but the exact mechanism is unclear. OBJECTIVES: In this study, an exploration of the therapeutic mechanism of leech therapy in HUA and gouty arthritis (GA) rats was done. MATERIAL AND METHODS: HUA and GA construction utilizing sodium urate crystal, the potassium form of oxygen oxazine acid, and adenine. Serum and tissues were collected to measure uric acid (UA), creatinine (Cr), and urea nitrogen (UN). Enzyme linked immunosorbent assay was executed to evaluate the levels of xanthine oxidase (XOD), interleukin-6 (IL-6)and tumor necrosis factor α (TNF-α). The expression of glucose transporter 9 (GLUT9), organic anion transporter 3 (OAT3), adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) and the nuclear factor kappa B (NF-kB), interleukin-1ß (IL-1ß), Toll-like Receptor 2 (TLR2) were assessed by Western blot and visualized in immunohistochemistry staining. RESULTS: Leech therapy reduces the levels of UA, Cr, and UN as well as the liver and serum levels of XOD activity, increasing the expressions of GLUT9, ABCG2, and OAT3 in the kidney. Meanwhile, it reduces joint swelling and lowers the levels of TNF-α, IL-6, IL-1ß, TLR2, and NF-kB. CONCLUSIONS: Leech therapy regulates the metabolism of uric acid and treats gouty arthritis with an anti-inflammatory effect.

SGMS1-AS1/MicroRNA-106a-5p/CPT2 Axis as a Novel Target for Regulating Lactate Metabolism in Colon Cancer.

PURPOSE: The malignant transformation of cells can lead to aerobic glycolysis, an important form of metabolic reprogramming in colon cancer cells, which can cause the accumulation of lactate and accelerate the proliferation of tumor cells also enhance their chemotherapy drug resistance. The aim of this study was to investigate the possible molecular mechanisms responsible for the increased lactate expression in colon cancer. METHODS: Several bioinformatics methods, including differential analysis, gene ontology enrichment, univariate and multivariate Cox regression analysis were used to find the lactic acid-related gene carnitine palmitoyltransferase 2. We analyzed the relationship between carnitine palmitoyltransferase 2 and clinical features as well as immune microenvironment. To further explore the mechanism of carnitine palmitoyltransferase 2 in colon cancer, we performed methylation analysis and constructed a competitive endogenous RNA network, which was validated in cell lines and clinical specimens. RESULTS: We used bioinformatics to select the lactic acid-related gene carnitine palmitoyltransferase 2 and found low expression of carnitine palmitoyltransferase 2 was associated with poor prognosis in colon cancer. An inhibitory tumor microenvironment was created when carnitine palmitoyltransferase 2 expression was reduced, with decreased CD4 T cells, CD8 T cells, dendritic cells, and B cells but increased cancer-associated fibroblasts. Methylation analysis showed that the abnormal decrease in carnitine palmitoyltransferase 2 might be caused by hypermethylation. We constructed a network of SGMS1-AS1/microRNA-106a-5p/carnitine palmitoyltransferase 2 and verified their expression in cell lines and clinical specimens. CONCLUSION: Our work revealed the possible mechanism of lactate accumulation in colon cancer and explored a new potential treatment for colon cancer by cutting off aerobic glycolysis in tumor cells.

Saikosaponin D Inhibits Peritoneal Fibrosis in Rats With Renal Failure by Regulation of TGFß1/ BMP7 / Gremlin1/ Smad Pathway.

Peritoneal dialysis (PD) can improve the quality of life of patients with kidney disease and prolong survival. However, peritoneal fibrosis can often occur and lead to PD withdrawal. Therefore, it is imperative to better understand how to inhibit and slow down progression of peritoneal fibrosis. This study aimed to investigate the regulatory effect of Saikosaponin d (SSD), a monomer extracted from the plant Bupleurum, on peritoneal fibrosis and the contribution of TGFß1/BMP7/Gremlin1 pathway cross-talk in this process. To this aim, we used a model 5/6 nephrectomy and peritoneal fibrosis in rats. Rats were divided into four groups, namely a control group (saline administration); a model group (dialysate administration; group M); a SSD group (dialysate and SSD administration); and a positive drug group (dialysate and Benazepril Hydrochloride administration; group M + A). Histological analysis indicated that peritoneal fibrosis occurred in all groups. WB, ELISA, and PCR essays suggested that TGFß1 and Gremlin1 levels in group M were significantly higher than those in group C, whereas BMP7 expression was significantly lower. TGFß1, Gremlin1 and BMP7 levels were significantly lower in the group where SSD was administered than in the other groups. The expression of BMP7 in SSD group was significantly increased. In addition, levels of Smad1/5/8 as assessed by PCR, and levels of p-Smad1/5/8 expression as assessed by WB were also significantly higher in the SSD group than in the M group. Expression of vimentin and α-SMA, two important markers of fibrosis, was also significantly decreased. Our study suggests a role for the TGFß1/BMP7/Gremlin1/Smad pathway in peritoneal fibrosis with potential therapeutic implications. Finally, our results also suggest that the monomer SSD may be able to reverse peritoneal fibrosis via regulation of the TGFß1/BMP7/Gremlin1/Smad pathway.

Epithelium- and endothelium-derived exosomes regulate the alveolar macrophages by targeting RGS1 mediated calcium signaling-dependent immune response.

Alveolar macrophages (AM) maintain airway immune balance; however, the regulation of heterogeneity of AMs is incompletely understood. We demonstrate that RGS1 coregulates the immunophenotype of AM subpopulations, including pro- and anti-inflammatory, injury- and repair-associated, and pro- and antifibrotic phenotypes, through the PLC-IP3R signal-dependent intracellular Ca2+ response. Flt3+ AMs and Tie2+ AMs had different immune properties, and RGS1 expression in the cells was targeted by exosomes (EXOs) containing miR-223 and miR-27b-3p that were derived from vascular endothelial cells (EnCs) and type II alveolar epithelial cells (EpCs-II), respectively. Imbalance of AMs was correlated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis (PF) caused a lack of secretion of CD31+ and CD74+ EXOs derived from EnCs and EpCs-II. Timely treatment with EXOs significantly improved endotoxin-induced ALI/ARDS and bleomycin-induced PF in mice. Thus, EnC- and EpC-II-derived EXOs regulate the immune balance of AMs and can be used as potential therapeutic drugs.

Downregulation of Bach1 protects osteoblasts against hydrogen peroxide-induced oxidative damage in vitro by enhancing the activation of Nrf2/ARE signaling.

Oxidative-stress-induced osteoblast dysfunction plays an important role in the development and progression of osteoporosis. BTB and CNC homology 1 (Bach1) has been suggested as a critical regulator of oxidative stress; however, whether Bach1 plays a role in regulating oxidative-stress-induced osteoblast dysfunction remains unknown. Thus, we investigated the potential role and mechanism of Bach1 in regulating oxidative-stress-induced osteoblast dysfunction. Osteoblasts were treated with hydrogen peroxide (H2O2) to mimic a pathological environment for osteoporosis in vitro. H2O2 exposure induced Bach1 expression in osteoblasts. Functional experiments demonstrated that Bach1 silencing improved cell viability and reduced cell apoptosis and reactive oxygen species (ROS) production in H2O2-treated cells, while Bach1 overexpression produced the opposite effects. Notably, Bach1 inhibition upregulated alkaline phosphatase activity and osteoblast mineralization. Mechanism research revealed that Bach1 inhibition increased the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling and upregulated heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 mRNA expression. The Bach1 inhibition-mediated protective effect was partially reversed by silencing Nrf2 in H2O2-exposed osteoblasts. Taken together, these results demonstrate that Bach1 inhibition alleviates oxidative-stress-induced osteoblast apoptosis and dysfunction by enhancing Nrf2/ARE signaling activation, findings that suggest a critical role for the Bach1/Nrf2/ARE regulation axis in osteoporosis progression. Our study suggests that Bach1 may serve as a potential therapeutic target for treating osteoporosis.

MiR-155 controls follicular Treg cell-mediated humoral autoimmune intestinal injury by inhibiting CTLA-4 expression.

High expression levels of miR-155 are involved in the pathogenesis of inflammatory bowel disease (IBD). We observed an increase in miR-155 in peripheral regulatory T (Treg) cells from IBD patients. Mice that specifically overexpress miR-155 in Foxp3+ Treg cells exhibit spontaneous autoimmunity and more severe dextran sulfate sodium (DSS)-induced intestinal injury. MiR-155 overexpression can lead to a lack of follicular Treg (Tfr) cells and central Treg (cTreg), whereas DSS treatment further depletes the Tfr cells. Furthermore, miR-155 can target the expression of CTLA-4 in cTreg and Tfr, directly inhibiting Tfr cell production and promoting enhanced germinal center (GC) B cell activation and autoantibody overproduction. This outcome may be the cause of severe intestinal injury in patients with autoimmune IBD.

Blocking Smad2 signalling with loganin attenuates SW10 cell cycle arrest induced by TNF-α.

The activity of Schwann cells (SWCs) is very important in trauma-induced nerve repair, and tumour necrosis factor-α (TNF-α) produced during tissue injury inhibits the viability of SWCs, which delays the repair of peripheral nerves. Loganin is an iridoid glycoside that has been shown to alleviate a variety of cytotoxic effects. In the current study, we evaluated the potential efficacy and the mechanism of action of loganin in TNF-α-induced cytotoxicity in SW10 cells. The experimental results indicated that loganin blocked TNF-α-mediated Smad2 activation, downregulated the expression of the G1 phase cell cycle inhibitor p15IN4KB, and upregulated the expression of the G1 phase cell cycle activator cyclin D1-CDK4/6, which upregulated E2F-1-dependent survivin expression and relieved TNF-α-induced apoptosis in SW10 cells. The protective effect of loganin on SWCs has potential medicinal value in the promotion of peripheral nerve repair and is significant for studies in the field of tissue regeneration.

Ginsenoside Rb2 inhibits osteoclast differentiation through nuclear factor-kappaB and signal transducer and activator of transcription protein 3 signaling pathway.

Ginsenoside-Rb2 (Rb2) is a 20(S)-protopanaxadiol glycoside extracted from ginseng possessing various bioactivities which has drawn considerable interest regarding the area of bone metabolism. However, the effect of Rb2 on osteoclast differentiation remains unknown. In this study, we aimed to investigate the potential role of Rb2 in regulating osteoclast differentiation and the underlying molecular mechanisms. Osteoclast differentiation was induced by receptor activator nuclear factor-kappaB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in mouse RAW 264.7 cells. The results showed that Rb2 dose-dependently inhibited the formation of the tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP expression. Furthermore, Rb2 promoted osteoprotegerin expression and bone resorption. The expression of osteoclast marker genes including nuclear factor of activated T cells c1 (NFATc1), c-Fos, OSCAR, and cathepsin K were also markedly inhibited by Rb2 treatment. Moreover, Rb2 significantly inhibited the RANKL-induced NF-κB activation. In addition, Rb2 also markedly suppressed the activation of signal transducer and activator of transcription protein 3 (STAT3) signaling pathway. Interestingly, the knockdown of STAT3 significantly strengthened the inhibitory effect of Rb2 on osteoclast differentiation. Taken together, our study suggests that Rb2 inhibits osteoclast differentiation associated with blocking NF-κB and STAT3 signaling pathways.

Activation of NRF2/ARE by isosilybin alleviates Aß25-35-induced oxidative stress injury in HT-22 cells.

Aß-mediated oxidative stress damage is considered a direct cause of Alzheimer's disease (AD). Therefore, drugs that have been developed to block oxidative stress are considered effective for AD treatment. Isosilybin is a flavonoid compound extracted from Silybum marianum, and it has been confirmed to have many pharmacological activities. This study aimed to verify that isosilybin could alleviate the Aß25-35-induced oxidative stress damage in HT-22 hippocampal cells and to investigate the specific targets of isosilybin. A non-toxic dose of isosilybin significantly inhibited the production of reactive oxygen species (ROS), the release of malondialdehyde (MDA) and lactate dehydrogenase (LDH), and the Aß25-35-stimulated reduction in total antioxidant capacity (T-AOC). Subsequent studies showed that isosilybin significantly increased the protein and mRNA expression of antioxidases, including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), and aldo-keto reductases 1C1 and 1C2 (AKR1C2). Moreover, isosilybin stimulated the activity of an antioxidant-response element (ARE)-driven luciferase reporter gene. Further studies showed that isosilybin induced the expression of NFR-2 in a time- and dose-dependent manner and promoted its translocation to the nucleus. This result indicated that the antioxidant function of isosilybin might be achieved through the activation of NRF2/ARE signalling. Subsequent studies showed that the NRF2-specific agonist t-BHQ effectively inhibited ROS, MDA and LDH release and T-AOC reduction under Aß25-35 stimulation. In addition, t-BHQ induced the expression of HO-1, GST, and AKR1C2, as well as the activity of ARE luciferase reporter plasmids. NRF2 siRNA blocked the antioxidative stress damage function of isosilybin. Therefore, NRF2 is likely to be a key mediator of isosilybin's anti-Aß25-35-mediated oxidative stress damage function. Overall, our results confirmed that isosilybin regulates the expression of HO-1, GST, and AKR1C2 through the activation of NRF2/ARE signalling, inhibiting ROS accumulation and ultimately alleviating Aß25-35-induced oxidative stress damage in HT-22 cells.