Dectin-1 Acts as a Non-Classical Receptor of Ang II to Induce Cardiac Remodeling.

BACKGROUND: Cardiac remodeling in heart failure involves macrophage-mediated immune responses. Recent studies have shown that a PRR (pattern recognition receptor) called dectin-1, expressed on macrophages, mediates proinflammatory responses. Whether dectin-1 plays a role in pathological cardiac remodeling is unknown. Here, we identified a potential role of dectin-1 in this disease. METHODS: To model aberrant cardiac remodeling, we utilized mouse models of chronic Ang II (angiotensin II) infusion. In this model, we assessed the potential role of dectin-1 through using D1KO (dectin-1 knockout) mice and bone marrow transplantation chimeric mice. We then used cellular and molecular assays to discover the underlying mechanisms of dectin-1 function. RESULTS: We found that macrophage dectin-1 is elevated in mouse heart tissues following chronic Ang II administration. D1KO mice were significantly protected against Ang II-induced cardiac dysfunction, hypertrophy, fibrosis, inflammatory responses, and macrophage infiltration. Further bone marrow transplantation studies showed that dectin-1 deficiency in bone marrow-derived cells prevented Ang II-induced cardiac inflammation and dysfunction. Through detailed molecular studies, we show that Ang II binds directly to dectin-1, causing dectin-1 homodimerization and activating the downstream Syk (spleen tyrosine kinase)/NF-κB (nuclear factor kappa B) signaling pathway to induce expression of inflammatory and chemoattractant factors. Mutagenesis studies identified R184 in the C-type lectin domain to interact with Ang II. Blocking dectin-1 in macrophages suppresses Ang II-induced inflammatory mediators and subsequent intercellular cross talk with cardiomyocytes and fibroblasts. CONCLUSIONS: Our study has discovered dectin-1 as a new nonclassical receptor of Ang II and a key player in cardiac remolding and dysfunction. These studies suggest that dectin-1 may be a new target for treating hypertension-related heart failure.

OTUD1 promotes hypertensive kidney fibrosis and injury by deubiquitinating CDK9 in renal epithelial cells.

Hypertensive renal disease (HRD) contributes to the progression of kidney dysfunction and ultimately leads to end-stage renal disease. Understanding the mechanisms underlying HRD is critical for the development of therapeutic strategies. Deubiquitinating enzymes (DUBs) have been recently highlighted in renal pathophysiology. In this study, we investigated the role of a DUB, OTU Domain-Containing Protein 1 (OTUD1), in HRD models. HRD was induced in wild-type or Otud1 knockout mice by chronic infusion of angiotensin II (Ang II, 1 µg/kg per min) through a micro-osmotic pump for 4 weeks. We found that OTUD1 expression levels were significantly elevated in the kidney tissues of Ang II-treated mice. Otud1 knockout significantly ameliorated Ang II-induced HRD, whereas OTUD1 overexpression exacerbated Ang II-induced kidney damage and fibrosis. Similar results were observed in TCMK-1 cells but not in SV40 MES-13 cells following Ang II (1 µM) treatment. In Ang II-challenged TCMK-1 cells, we demonstrated that OTUD1 bound to CDK9 and induced CDK9 deubiquitination: OTUD1 catalyzed K63 deubiquitination on CDK9 with its Cys320 playing a critical role, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells. Administration of a CDK9 inhibitor NVP-2 significantly ameliorated Ang II-induced HRD in mice. This study demonstrates that OTUD1 mediates HRD by targeting CDK9 in kidney epithelial cells, suggesting OTUD1 is a potential target in treating this disease.

Britannin as a novel NLRP3 inhibitor, suppresses inflammasome activation in macrophages and alleviates NLRP3-related diseases in mice.

Overactivation of the NLRP3 inflammasomes induces production of pro-inflammatory cytokines and drives pathological processes. Pharmacological inhibition of NLRP3 is an explicit strategy for the treatment of inflammatory diseases. Thus far no drug specifically targeting NLRP3 has been approved by the FDA for clinical use. This study was aimed to discover novel NLRP3 inhibitors that could suppress NLRP3-mediated pyroptosis. We screened 95 natural products from our in-house library for their inhibitory activity on IL-1ß secretion in LPS + ATP-challenged BMDMs, found that Britannin exerted the most potent inhibitory effect with an IC50 value of 3.630 µM. We showed that Britannin (1, 5, 10 µM) dose-dependently inhibited secretion of the cleaved Caspase-1 (p20) and the mature IL-1ß, and suppressed NLRP3-mediated pyroptosis in both murine and human macrophages. We demonstrated that Britannin specifically inhibited the activation step of NLRP3 inflammasome in BMDMs via interrupting the assembly step, especially the interaction between NLRP3 and NEK7. We revealed that Britannin directly bound to NLRP3 NACHT domain at Arg335 and Gly271. Moreover, Britannin suppressed NLRP3 activation in an ATPase-independent way, suggesting it as a lead compound for design and development of novel NLRP3 inhibitors. In mouse models of MSU-induced gouty arthritis and LPS-induced acute lung injury (ALI), administration of Britannin (20 mg/kg, i.p.) significantly alleviated NLRP3-mediated inflammation; the therapeutic effects of Britannin were dismissed by NLRP3 knockout. In conclusion, Britannin is an effective natural NLRP3 inhibitor and a potential lead compound for the development of drugs targeting NLRP3.

Damage Identification for Underground Structure Based on Frequency Response Function.

Damage identification that is based on modal analysis is widely used in traditional structural damage identification. However, modal analysis is difficult in high damping structures and modal concentrated structures. Unlike approaches based on modal analysis, damage identification based on the frequency response function allows for the avoidance of error and easy verification through other test points. An updating algorithm is devised is this study by utilizing the frequency response function together with the dynamic reduction with respect to the selected design parameters. Numerical results indicate that the method can be used to define multiple parameters with large variation and incomplete measurement data and is robust against measurement noise. With the purpose of avoiding the occurrence of resonance and gaining additional information, the trial and error method has been used to choose a proper frequency. Furthermore, an experimental scale model in a soil box is subjected to the excitation of moving load to validate the effectiveness of the damage identification approach. The improved damage identification method for underground structures, which is based on the analysis of the frequency response function, can be adopted as an efficient and functional damage identification tool.

Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.

BACKGROUND: Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored. METHODS: This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells. RESULTS: Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling. CONCLUSIONS: These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF. KEY POINTS: Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.

Inhibition of MD2 by natural product-drived JM-9 attenuates renal inflammation and diabetic nephropathy in mice.

Diabetic kidney disease (DKD) is one of the severe complications of diabetes mellitus-related microvascular lesions, which remains the leading cause of end-stage kidney disease. The genesis and development of DKD is closely related to inflammation. Myeloid differentiation 2 (MD2) mediates hyperlyciemia-induced renal inflammation and DKD development and is considered as a potential therapeutic target of DKD. Here, we identified a new small-molecule MD2 inhibitor, JM-9. In vitro, JM-9 suppressed high glucose (HG) and palmitic acid (PA)-induced inflammation in MPMs, accompanied by inhibition of MD2 activation and the downstream TLR4/MyD88-MAPKs/NFκB pro-inflammatory signaling pathway. Macrophage-derived factors increased the fibrotic and inflammatory responses in renal tubular epithelial cells, which were inhibited by treating macrophages with JM-9. Then, we investigated the therapeutic effects against DKD in streptozotocin-induced type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) mouse models. Treatment with JM-9 prevented renal inflammation, fibrosis, and dysfunction by targeting MD2 in both T1DM and T2DM models. Our results show that JM-9, a new small-molecule MD2 inhibitor, protects against DKD by targeting MD2 and inhibiting MD2-mediated inflammation. In summary, JM-9 is a potential therapeutic agent for DKD.

Corynoline protects ang II-induced hypertensive heart failure by increasing PPARα and Inhibiting NF-κB pathway.

Heart failure is a fairly common outcome of hypertension. Recent studies have highlighted the key role of the non-hemodynamic activity of angiotensin II (Ang II) in hypertensive heart failure via inducing cardiac inflammation. Drugs that disrupt Ang II-induced cardiac inflammation may have clinical utility in the treatment of hypertensive heart failure. A naturally occurring compound, corynoline, exhibit anti-inflammatory activities in other systems. C57BL/6 mice were injected with Ang II via a micro-osmotic pump for four weeks to develop hypertensive heart failure. The mice were treated with corynoline by gavage for two weeks. RNA-sequencing analysis was performed to explore the potential mechanism of corynoline. We found that corynoline could inhibit inflammation, myocardial fibrosis, and hypertrophy to prevent heart dysfunction, without the alteration of blood pressure. RNA-sequencing analysis indicates that the PPARα pathway is involved Ang II-induced cardiac fibrosis and cardiac remodeling. Corynoline reversed Ang II-induced PPARα inhibition both in vitro and in vivo. We further found that corynoline increases the interaction between PPARα and P65 to inhibit the NF-κB pro-inflammatory pathway in H9c2 cells. Our studies show that corynoline relieves Ang II-induced hypertensive heart failure by increasing the interaction between PPARα and P65 to inhibit the NF-κB pathway.

Mesencephalic astrocyte-derived neurotrophic factor ameliorates steatosis in HepG2 cells by regulating hepatic lipid metabolism.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a global metabolism-associated liver disease. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a newly discovered secreted protein that is involved in metabolic homeostasis. However, much remains to be discovered about its function in hepatic lipid metabolism; thus, we assessed whether MANF could regulate hepatic metabolism. AIM: To establish in vivo and in vitro NAFLD models to explore the role of MANF in hepatic lipid metabolism. METHODS: HepG2 cells treated with free fatty acids (FFAs) and ob/ob mice were used as NAFLD models. Liver tissues collected from wild type and ob/ob mice were used to detect MANF expression. Cells were treated with FFAs for different durations. Moreover, we used lentiviral constructs to establish overexpression and knockdown cell models in order to interfere with MANF expression levels and observe whether MANF influences hepatic steatosis. Western blot analysis and quantitative real-time PCR were used to detect protein and gene expression, and oil red O staining was used to visualize intracellular lipid droplets. RESULTS: Hepatic MANF protein and mRNA expression in wild type mice were 10-fold and 2-fold higher, respectively, than those in ob/ob mice. The MANF protein was temporarily increased by 1.3-fold after stimulation with FFAs for 24 h and gradually decreased to 0.66-fold that of the control at the 72 h time point in HepG2 cells. MANF deficiency upregulated the expression of genes involved in fatty acid synthesis, cholesterol synthesis, and fatty acid uptake and aggravated HepG2 cell steatosis, while MANF overexpression inhibited fatty acid synthesis and uptake and cholesterol synthesis, and rescued HepG2 cells from FFAs-induced steatosis. Furthermore, a significant decrease in triglyceride levels was observed in the MANF overexpression group compared with the control group (0.4288 ± 0.0081 mmol/g vs 0.3746 ± 0.0121 mmol/g, P < 0.05) upon FFAs treatment. There was also a 17% decrease in intracellular total cholesterol levels between the MANF overexpression group and the control group (0.1301 ± 0.0059 mmol/g vs 0.1088 ± 0.0009 mmol/g, P < 0.05) upon FFAs treatment. Moreover, MANF suppressed lipid deposition in HepG2 cells. CONCLUSION: Our findings indicate that MANF improves the phenotype of liver cell steatosis and may be a potential therapeutic target in hepatic steatosis processes.

[Lycorine inhibits the proliferation and promotes apoptosis of SGC-7901 gastric cancer cells by downregulating expression of cyclin D1].

Objective To investigate the effect of lycorine on the proliferation and apoptosis of gastric cancer SGC-7901 cells. Methods Effect of lycorine on the proliferation of SGC-7901 cells and 50% inhibitory concentration (IC50) of lycorine were determined by CCK-8 assay. SGC-7901 cells were treated with IC50 lycorine, and 48 hours later, cell cycle and apoptosis were detected by flow cytometry, and the level of cyclin D1 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blot analysis. Results Lycorine obviously inhibited the proliferation of SGC-7901 gastric cancer cells in a concentration- or time-dependent manner. The IC50 of 24 hours and 48 hours to gastric cancer cells treated with lycorine was (20.85±2.36) µmol/L and (13.29±1.28) µmol/L, respectively. The cell cycle was arrested in G0/G1 phase, the apoptosis rate of cells increased, and the level of cyclin D1 mRNA and protein decreased significantly 48 hours after the treatment with 13 µmol/L lycorine. Conclusion Lycorine can inhibit the proliferation of SGC-7901 gastric cancer cells, induce cell cycle arrest in G0/G1 phase and promote cell apoptosis. The mechanism may be related to the down-regulation of cyclin D1 expression.

Plasma Exosomal miRNA-122-5p and miR-300-3p as Potential Markers for Transient Ischaemic Attack in Rats.

Background: Differentiation of transient ischaemic attack (TIA) from ischaemic stroke within the thrombolysis time window is difficult. Although TIA may be diagnosed within this window, the latest imaging technologies are complex and costly. Serum markers, which are non-invasive, rapid and economic, are used for diagnosis and prognosis of various diseases. Exosome-derived miRNA markers for TIA are unknown. Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery (MCAo) for 5 min, 10 min, and 2 h in rats. Exosomal miRNAs with consistent trends in cerebrospinal fluid (CSF) and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic (ROC) curve were used to evaluate the diagnostic accuracy of these miRNAs for TIA in rats. Results: Rno-miR-122-5p and rno-miR-300-3p were selected. Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with control and 5 min plasma. Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with control, 10 min and 2 h rats. Plasma and CSF levels of these miRNAs were correlated. ROC analysis showed high AUC values for rno-miR-122-5p (0.960) and rno-miR-300-3p (0.970) in the 10 and 5 min rats, respectively, compared with controls. Conclusions: Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be blood-based TIA biomarkers.

Transcription factor glioma-associated oncogene homolog 1 is required for transforming growth factor-ß1-induced epithelial-mesenchymal transition of non-small cell lung cancer cells.

Epithelial­mesenchymal transition (EMT) is the process by which epithelial cells depolarize and acquire a mesenchymal phenotype, and is a common early step in the process of metastasis. Patients with lung cancer frequently already have distant metastases when they are diagnosed, highlighting the requirement for early and effective interventions to control metastatic disease. Transforming growth factor­ß1 (TGF­ß1) is able to induce EMT, however the molecular mechanism of this remains unclear. In the current study, TGF­ß1 was reported to induce EMT and promote the migration of non­small cell lung cancer (NSCLC) cells. A notable observation was that EMT induction was accompanied by the upregulation of human glioma­associated oncogene homolog 1 (Gli1) mRNA and protein levels. Furthermore, Gli1 levels were depleted by small interfering RNA, and the Gli1 inhibitor GANT 61 attenuated the TGF­ß1­mediated induction of EMT and cell migration. The results of the current study suggest that Gli1 regulates TGF­ß1­induced EMT, which may provide a novel therapeutic target to inhibit metastasis in patients with NSCLC.

A case of Hashimoto's encephalopathy misdiagnosed as viral encephalitis.

PATIENT: Male, 61 FINAL DIAGNOSIS: Hashimoto's encephalopathy Symptoms: Neuropsychiatric or neurological manifestations Medication: Steroids and immunoglobulins Clinical Procedure: Immunoglobulin combined with corticosteroid therapy Specialty: Neurology. OBJECTIVE: Mistake in diagnosis. BACKGROUND: Hashimoto's encephalopathy is a rare autoimmune syndrome characterized by various neuropsychiatric or neurological manifestations and associated with Hashimoto's thyroiditis, responsive to steroids. Until now, misdiagnosis and delay of treatment of Hashimoto's encephalopathy are very common because of the diversity of the symptoms. CASE REPORT: This recent case of a 61-year-old man presented with unconsciousness, spasms and a previous misdiagnosis as viral encephalitis. Response to anti-viral and steroid therapy was unsatisfactory, but treatment with immunoglobulin combined with corticosteroid therapy achieved rapid and complete recovery. CONCLUSIONS: Any patient presenting with acute or subacute unexplained encephalopathy should be considered Hashimoto's encephalopathy, even if the thyroid function is normal. Thyroid antibody testing should be performed because this may be the most important clue to diagnosis. As soon as the diagnosis is made, steroid therapy is the first choice. If the steroid therapy does not lead to immediate improvement, IVIG is an effective alternative treatment.