Blockade of myeloid differentiation protein 2 prevents obesity‐induced inflammation and nephropathy

Obesity is a major and independent risk factor of kidney diseases. The pathogenic mechanisms of obesity‐associated renal injury are recognized to at least involve a lipid‐rich and pro‐inflammatory state of the renal tissues, but specific mechanisms establishing causal relation remain unknown. Saturated fatty acids are elevated in obesity, and known to induce chronic inflammation in kidneys. Myeloid differentiation protein 2 (MD2) is an important protein in lipopolysaccharide‐induced innate immunity response and inflammation. We suggested that obesity‐associated renal injury is regulated by MD2 thereby driving an inflammatory renal injury. The used three mouse models for in vivo study: MD2 knockout mice (KO) maintained on high fat diet (HFD), wild‐type mice on HFD plus L6H21, a specific MD2 inhibitor and KO mice given palmitic acid (PA) by IV injection. The in vitro studies were carried out in cultured renal tubular epithelial cells, mouse mesangial cells and primary macrophages, respectively. The HFD mice presented with increased hyperlipidemia, serum creatinine and proteinuria. Renal tissue from HFD mice had increased fibrosis, inflammatory cytokines, macrophage infiltration, and activation of NF‐κB and MAPKs. This HFD‐induced renal injury profile was not observed in KO mice or L6H21‐treated mice. Mice given PA mimmicked the HFD‐induced renal injury profiles, which were prevented by MD2 knockout. The in vitro data further confirmed MD2 mediates PA‐induced inflammation. MD2 is causally related with obesity‐associated renal inflammatory injury. We believe that MD2 is an attractive target for future therapeutic strategies in obesity‐associated kidney diseases.     

Genistein‐3′‐sodium sulphonate protects against lipopolysaccharide‐induced lung vascular endothelial cell apoptosis and acute lung injury via BCL‐2 signalling

Under septic conditions, Lipopolysaccharide (LPS)‐induced apoptosis of lung vascular endothelial cells (ECs) triggers and aggravates acute lung injury (ALI), which so far has no effective therapeutic options. Genistein‐3′‐sodium sulphonate (GSS) is a derivative of native soy isoflavone, which has neuro‐protective effects through its anti‐apoptotic property. However, whether GSS protects against sepsis‐induced lung vascular endothelial cell apoptosis and ALI has not been determined. In this study, we found that LPS‐induced Myd88/NF‐κB/BCL‐2 signalling pathway activation and subsequent EC apoptosis were effectively down‐regulated by GSS in vitro. Furthermore, GSS not only reversed the sepsis‐induced BCL‐2 changes in expression in mouse lungs but also blocked sepsis‐associated lung vascular barrier disruption and ALI in vivo. Taken together, our results demonstrated that GSS might be a promising candidate for sepsis‐induced ALI via its regulating effects on Myd88/NF‐κB/BCL‐2 signalling in lung ECs.     

Autophagy maintains the integrity of endothelial barrier in LPS‐induced lung injury

Understanding the role and underlying regulation mechanism of autophagy in lipopolysaccharide‐induced lung injury (LPS‐LI) may provide potentially new pharmacological targets for treatment of acute lung injury. The aim of this study was to investigate the functional significance of autophagy in LPS‐LI. The autophagy of human pulmonary microvascular endothelial cells (HPMVECs) and mice was inhibited before they were challenged with LPS. In vitro, permeability, vitality, and the LDH release rate of the cells were detected, the zonula occluden‐1 (ZO‐1) expression and the stress fiber formation were determined. In vivo, the lung injury was assessed. We found LPS caused high permeability and increased lactate dehydrogenase (LDH) release rate, lowered viability of the cells, inhibited the ZO‐1 expression and induced stress fiber formation, these effects were further aggravated by prohibiting the level of autophagy. Consistently, in in vivo experiments, LPS‐induced serious lung injury, which was reflected as edema, leukocyte infiltration and hemorrhage in lung tissue, and the high concentration of pro‐inflammation cytokines tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β in bronchoalveolar lavage fluid (BALF). Inhibiting autophagy further exacerbated LPS‐LI. It appears that autophagy played a protective role in LPS‐LI in part through restricting the injury of lung microvascular barrier.     

Salidroside suppressing LPS‐induced myocardial injury by inhibiting ROS‐mediated PI3K/Akt/mTOR pathway in vitro and in vivo

The purpose of the present study was to investigate the effect of salidroside (Sal) on myocardial injury in lipopolysaccharide (LPS)‐induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg/kg), LPS plus dexamethasone (2 mg/kg), LPS plus Sal groups with different Sal doses (20, 40 mg/kg). Hemodynamic measurement and haematoxylin and eosin staining were performed. Serum levels of creatine kinase (CK), lactate dehydrogenase, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), glutathione, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) were measured after the rats were killed. iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on rat embryonic heart‐derived myogenic cell line H9c2 induced by LPS. Reactive oxygen species (ROS) in H9c2 cells was measured by flow cytometry, and the activities of the antioxidant enzymes CAT, SOD, GSH‐px, glutathione‐S‐transferase, TNF‐α, IL‐6 and IL‐1β in cellular supernatant were measured. PI3K/Akt/mTOR signalling was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced cardioprotective effect in rats subjected to LPS possibly through inhibiting the iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway in vivo. Furthermore, the pharmacological effect of Sal associated with the ROS‐mediated PI3K/Akt/mTOR pathway was proved by the use of ROS scavenger, N‐acetyl‐l ‐cysteine, in LPS‐stimulated H9C2 cells. Our results indicated that Sal could be a potential therapeutic agent for the treatment of cardiovascular disease.     

Characteristics of circular RNA expression of pulmonary macrophages in mice with sepsis‐induced acute lung injury

Circular RNAs (circRNAs) make up a large class of non‐coding RNAs and play important roles in the pathology of a variety of diseases. However, their roles in pulmonary macrophage polarization after sepsisinduced lung injury is unknown. In this study, mice were divided into two groups: Sham control group and cecal ligation and puncture (CLP)‐induced ALI group. Macrophages were isolated from lung homogenates 24 hours after SCLP/CLP. We started with RNA‐seq of circRNA changes in macrophages and validated by RT‐PCR in the following experiments. A total of 4318 circRNAs were detected in the two groups. Of these, 11 and 126 circRNAs were found to be significantly upregulated and downregulated, respectively, compared to the control (p≤0.05, Fold Change ≥2). Differentially expressed circRNAs with a high foldchange (fold‐change >4, P<0.05) were selected for validation by qRT‐PCR, 10 of which were verified. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. The network of circRNA‐miRNA‐mRNA was illustrated by cytoscape software. Gene ontology analyses indicated the upregulated circRNAs were involved in the multiple biological functions such as regulation of mitochondrion distribution and Notch binding, while the down‐regulated circRNAs mainly involved in the biological process as histone H3K27 methylation. KEGG pathway analysis revealed TGF‐beta signaling pathway was related to the upregulated circRNAs. The present study provides a novel insight into the roles of circRNAs in pulmonary macrophage differentiation and polarization post septic lung injury.     

Resveratrol decreases CD45+CD206− subtype macrophages in LPS‐induced murine acute lung injury by SOCS3 signalling pathway

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are life‐threatening condition in critically ill patients. Resveratrol (Res), a natural polyphenol, has therapeutic effect in animal model with ALI; however, whether Res attenuates ALI through modulation of macrophage phenotypes in the animal model remains unknown. We in this study treated LPS‐induced murine ALI with 30 mg/kg Res and observed significantly reduced severity of ALI in the Res‐treated mice 48 hours after Res treatment. Neutrophil infiltrates were significantly reduced, accompanied with lower infiltration of CD45⁺Siglec F^? phenotype macrophages, but higher population of CD45⁺Siglec F⁺ and CD45⁺CD206⁺ alternatively activated macrophages (M2 cells) in the Res‐treated mice with ALI. In addition, the expression of IL‐1beta and CXCL15 cytokines was suppressed in the treated mice. However, Res treatment in mice with myeloid cell‐restricted SOCS3 deficiency did not significantly attenuate ALI severity and failed to increase population of both CD45⁺Siglec F⁺ and CD45⁺CD206⁺ M2 subtype macrophages in the murine ALI. Further studies in wild‐type macrophages revealed that Res treatment effectively reduced the expression of IL‐6 and CXCL15, and increased the expression of arginase‐1, SIRT1 and SOCS3. However, macrophages’ lack of SOCS3 expression were resistant to the Res‐induced suppression of IL‐6 and CXCL15 in vitro. Thus, we conclude that Res suppressed CD45⁺Siglec F^? and CD45⁺CD206^? M1 subtype macrophages through SOCS3 signalling in the LPS‐induced murine ALI.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells

MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D₃ (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBPβ and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.     

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS‐induced lung injury

Bone marrow‐derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline‐stimulated BMSCs on lipopolysaccharide (LPS)‐induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS‐induced injury were co‐cultured with BMSCs. LPS‐stimulated alveolar macrophages were co‐cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α‐ and β‐adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS‐injured lung cells or lung tissue. Adrenaline‐stimulated BMSCs decreased the inflammation of LPS‐stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS‐injured rats. Our data indicate that adrenaline‐stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation.     

Ursodeoxycholic acid protects against lung injury induced by fat embolism syndrome

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life‐threatening disease with a high mortality rate, which was a common complication of fat embolism syndrome (FES). Ursodeoxycholic acid (UDCA) has been reported to exert potent anti‐inflammatory effects under various conditions. In vivo, perinephric fat was injected via tail vein to establish a rat FES model, the anti‐inflammatory effects of UDCA on FES‐induced lung injury were investigated through histological examination, ELISA, qRT‐PCR, Western blot and immunofluorescence. In vitro, human lung microvascular endothelial cells (HPMECs) were employed to understand the protective effects of UDCA. The extent of ALI/ARDS was evaluated and validated by reduced PaO₂/FiO₂ ratios, increased lung wet/dry (W/D) ratios and impaired alveolar‐capillary barrier, up‐regulation of ALI‐related proteins in lung tissues (including myeloperoxidase [MPO], vascular cell adhesion molecule 1 [VCAM‐1], intercellular cell adhesion molecule‐1 [ICAM‐1]), elevated protein concentration and increased proinflammatory cytokines levels (TNF‐α and IL‐1β) in bronchoalveolar lavage fluid (BALF). Pre‐treatment with UDCA remarkably alleviated these pathologic and biochemical changes of FES‐induced ALI/ARDS; our data demonstrated that pre‐treatment with UDCA attenuated the pathologic and biochemical changes of FES‐induced ARDS, which provided a possible preventive therapy for lung injury caused by FES.     

Eriodictyol ameliorates lipopolysaccharide‐induced acute lung injury by suppressing the inflammatory COX‐2/NLRP3/NF‐κB pathway in mice

The purpose of this paper is to observe the protective action and its effective mechanism of eriodictyol on lipopolysaccharide (LPS)‐induced acute lung injury (ALI). In this study, our results indicated that eriodictyol could dramatically suppress the inflammatory mediators, including interleukin‐6 (IL‐6), IL‐1β, prostaglandin E2, and tumor necrosis factor‐α in bronchoalveolar lavage fluid of LPS‐challenged mice. Eriodictyol also alleviated the wet/dry ratio and improved pathological changes of the lung. In addition, eriodictyol significantly decreased myeloperoxidase activity and malondialdehyde content as well as increased superoxide dismutase activity. Moreover, eriodictyol inhibited the COX‐2/NLRP3/NF‐κB signaling pathway in the lung tissues of ALI mice. In conclusion, our observations validated that eriodictyol processed the protective effects on ALI mice, which was related to the regulation of the COX‐2/NLRP3/NF‐κB signaling pathway.     

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.     

MG53 attenuates nitrogen mustard‐induced acute lung injury

Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant‐induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM‐induced lung injury than the wild‐type mice. Treatment of wild‐type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM‐induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM‐induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant‐induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant‐induced lung injury.