Acute kidney injury (AKI) has a high morbidity and mortality rate but can only be treated with supportive therapy in most cases. The diagnosis of AKI is mainly based on serum creatinine level and urine volume, which cannot detect kidney injury sensitive and timely. Therefore, new diagnostic and therapeutic molecules of AKI are being actively explored. Extracellular vesicles (EVs), secreted by almost all cells, can originate from different parts of the kidney and mediate intercellular communication between various cell types of nephrons. At present, numerous successful EV-based biomarker discoveries and treatments for AKI have been made, such as the confirmed diagnostic role of urine-derived EVs in AKI and the established therapeutic role of mesenchymal stem cell-derived EVs in AKI have been confirmed; however, these related studies lack a full discussion. In this review, we summarize the latest progression in the profound understanding of the functional role of EVs in AKI caused by various etiologies in recent years and provide new insights into EVs as viable biomarkers and therapeutic molecules for AKI patients. Furthermore, the current challenges and prospects of this research area are briefly discussed, presenting a comprehensive overview of the growing foregrounds of EVs in AKI.

Background:Chronic kidney disease (CKD) is a global health issue, with renal fibrosis being a common pathway in CKD development. Histone modification plays crucial roles in transcriptional regulation, but their pathological functions and mechanisms in CKD are not well understood.Methods:We utilized chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-seq) and RNA-seq to evaluate the states and functions of H3 lysine 27 acetylation (H3K27ac) and H3 lysine 4 trimethylation (H3K4me3) in kidney of CKD mice. We identified epigenetic factors regulating H3K27ac through motif analysis. Expression of activating transcription factor 3 (ATF3) in CKD mouse models and patients' kidneys was validated via immunofluorescence staining or Western blot. We further generated the Atf3 deficient (Atf3-/-) mice to explore its effect in kidney function and fibrosis. ChIP-seq of H3K27ac from Atf3-/- CKD mice was employed to validate ATF3's regulatory effects. We explored how ATF3 maintains the state of H3K27ac by integrating the data sources from multiple databases.Results:The states of H3K27ac and H3K4me3 were changed during CKD, and positively correlated with differential gene expression. ATF3 was highly expressed in kidney of both patients and mice with CKD, and co-localized with H3K27ac in genome, epigenetically regulating H3K27ac state. Atf3 deficient in CKD mice significantly ameliorated kidney dysfunction and fibrotic phenotype, and reduced H3K27ac levels at the ATF3 binding sites. Mechanically, ATF3 may facilitate H3K27ac maintenance by corrodinating histone acetyltransferase (HATs)-associated regulatory machinery during CKD.Conclusion:ATF3 promotes kidney injury and fibrosis in CKD by maintaining the state of H3k27ac via coordinating HATs regulatory machinery.

Thromboangiitis obliterans (TAO) is characterized by progressive inflammatory vasculopathy featuring thrombotic occlusion. Aberrant thrombosis induces endothelial damage through pathological clotting, while iron may act as a pro-oxidant cofactor. However, the function and mechanism of iron in TAO pathogenesis and endothelial damage remain to be elucidated. In the current study, the iron status and key lipid peroxidation markers (MDA, 4HNE, and ACSL4) were evaluated in patients with TAO and the sodium laurate-induced rat model. The CCK-8 assay, immunofluorescence, western blot, qPCR, and transmission electron microscopy were employed to detect iron overload and ferroptosis in vascular endothelial cells. In addition, bioinformatics analysis, luciferase reporter gene assay, qPCR, and western blot were used to confirm the miR-32-5p/Neurofibromin-2 (NF2) pathway in vitro. The therapeutic feasibility was validated by deferoxamine and Ferrostatin-1 treatment in vivo. The results showed iron overload and increased TFR1 expression in the vessel lesions of patients with TAO, as well as significant increases in MDA, 4HNE, and ACSL4. Serum from patients with TAO increased intracellular iron and lipid peroxidation and decreased the viability of HUVECs in vitro. Mechanism studies indicated that exosomal miR-32-5p increased in patients with TAO and could target and decrease the expression of NF2, which then decreased the phosphorylation of YAP at Ser109 and Ser217 sites. Then the NF2-targeted genes TFR1 and ACSL4 were upregulated. Finally, deferoxamine and Ferrostatin-1 treatment relieved the disease score, inflammation, and ferroptosis in vivo. This study newly demonstrates that iron overload and ferroptosis are key risk factors in patients with TAO and that the exosomal miR-32-5p/NF2 pathway may play an important role in TAO pathogenesis.

Chronic kidney disease (CKD) is a global public health problem, involving about 10% of the global population. Unfortunately, there are currently no effective drugs. Kidney fibrosis is the main pathology of CKD, where integrins play crucial roles in renal fibrogenesis. Recently, Bexotegrast (PLN-74809) as a dual integrin αvβ1/αvβ6 inhibitor could reduce the degree of lung fibrosis in patients with idiopathic pulmonary fibrosis. However, the role of PLN-74809 remains unclear in fibrotic kidney disease. Here, we have revealed that PLN-74809 administration dose-dependently delayed the progression of renal fibrosis in both adenine diet- and unilateral ureteral obstruction (UUO)-induced mice. Mechanistically, PLN-74809 targeted integrin αvβ1/αvβ6 to inhibit FAK/Src/Akt/β-catenin cascade in fibrotic kidneys. In summary, our results for the first time highlighted the αvβ1/αvβ6 inhibitor PLN-74809 exerted potential therapeutic against kidney fibrosis.

BackgroundRenal ischemia reperfusion injury (IRI) is one of the pivotal event of acute kidney injury (AKI), and they are unavoidable in the process of kidney transplantation, which eventually leads to the loss of renal allograft. Ferroptosis is a newly identified programmed cell death. Recent studies have suggested that ferroptosis may participate in the pathophysiological process of renal IRI. Therefore, we aimed to determine biomarkers associated with ferroptosis during renal IRI and their impact on renal allografts.MethodsWe conducted a comprehensive bioinformatics analysis and established an IRI-AKI animal model to illustrate the critical role of ferroptosis-related hub genes (FRHGs) in IRI-AKI and their potential impact on kidney transplantation.ResultsIn this study, we identified 60 ferroptosis-related genes (FRGs) in renal IRI based on the GSE148420 dataset and FerrDb database. And then we performed functional annotation analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Protein-protein interaction (PPI) network was constructed by online tool String. EZH2, CDKN1A, PPARA, EGR1, ATF3, and CD44 were identited as six ferroptosis-related hubgenes (FRHGs) using four methods, including MMC, Degree, DMNC, and EPC. FRHGs expression level were verified by the validation sets GSE58438 and GSE126805. Protein expression level of FRHGs verified by Proteomics and Western blot. Cibersort was utilized to analyze immune cell infiltration during renal IRI as well as the correlation between FRHGs and immune cells. The GSE21374 dataset was used for renal allografts survival analysis. Finally, We induced the IRI-AKI animal model and illustrated the importance of FRGHs CD44 in ferroptosis and the accumulation of macrophages.ConclusionWe identified 6 FRHGs. We found that FRHGs not only exhibited significant correlation with immune cells but also directly influenced the survival of transplanted kidneys in the human population. Among six FRHGs, only CD44 was overexpressed at both the gene and protein levels. Anti-CD44 exerts a protective effect by inhibiting ferroptosis and the accumulation of M1 macrophages during renal IRI. This study provided new insights into the pathogenesis of renal IRI and provided new evidence for its treatment.

Abstract Purpose: The critical role played by miRNAs in sepsis-induced acute peritonitis has been repeatedly validated. This research focuses on elucidating the influence of endothelial progenitor cells (EPC)-derived exosomal miRNA-31 (exo-miR-31) on septic peritonitis (SP) and the role of E-selectin (SELE) in this process. Methods: Differences in serum miRNA-31 and soluble SELE (sSELE) expression between SP cases and healthy controls (HCs) were identified. Additionally, we built a mouse colon ascendens stent peritonitis (CASP) model and an in vitro sepsis model via lipopolysaccharide (LPS) stimulation of mouse venous endothelial cells to verify the specific mechanism of EPC-derived exo-miR-31 on SP in vitro and in vivo. Results: As indicated by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) results, SP cases showed markedly reduced serum miR-31 levels and evidently elevated sSELE expression than HCs, with the expression of both genes altered more significantly as the condition worsened. Correlation analysis suggested a strong association between serum miRNA-31 and sSELE levels and sepsis severity. miR-31 in the peritoneal lavage fluid (PLF) of CASP model mice was down-regulated, while sSELE and inflammatory factors (IFs) TNF-α, IL-6 and IL-10 were notably increased. Gimsa staining revealed the massive recruitment and aggregation of nucleated cells in the abdominal cavity of CASP mice, and HE staining showed extensive neutrophilic infiltration in lung, liver and colon tissues, with obvious inflammatory tissue destruction. Besides, large numbers of bacteria were detected in the peripheral blood and PLF of CASP mice, with many CASP mice dying within 48 hours, as indicated by the bacterial smears and culture tests. EPC-derived exo-miR-31 was effective in elevating PLF miR-31 expression in CASP mice while suppressing sSELE, TNF-α, IL-6 and IL-10 levels. In EPCs-ExosmiR-31 group, significant reductions in nucleated cell counts in the mouse PLF, neutrophil infiltration in lung, liver and colon tissues, and peripheral blood and PLF bacterial loads were observed, as well as an obvious extension in survival. In addition, in vitro experimental results of cell sepsis showed that miR-31 could obviously block the inhibitory effect of LPS stimulation on the proliferation level of mouse venous endothelial cells and inhibit the level of apoptosis. SELE, Caspase-3 and Bax protein levels were markedly lower in LPS+miR-31 group as compared to LPS and LPS-miR-NC groups, while Bcl-2 was increased (P<0.05). Dual luciferase reporter gene assay identified the role of miR-31 as the direct target of SELE. Conclusions: EPC-derived exo-miR-31 can effectively alleviate SP, possibly through targeted inhibition of SELE, providing a potential new direction for future prevention and treatment of SP.

Purpose Older patient population with acetabular fractures is increasing rapidly, requiring enhanced recovery. Acute total hip arthroplasty (THA) is a good option for these patients, and it is becoming increasing popular. However, acute THA has different indications in different studies. Therefore, a systematic review is needed to assess and comprehend the indications for acute THA in older patients. Methods A systematic literature review was conducted to identify a retrospective series or prospective studies in older patients (>60 years) with acetabular fractures. The search timeline was from database construction till December 2021; PubMed, Embase, and Cochrane Library databases were searched. Two trained professional reviewers independently read the full text of documents that met the inclusion criteria and extracted information on the specific methods used and indication information based on the research design. Results In total, there were 601 patients with acetabular fractures aged >60 years from 33 studies were obtained. Twenty-eight studies reported that THA was a feasible treatment option for acetabular fractures in geriatric patients with good outcome. The primary indications were dome impaction, irreducible articular comminution, femoral head injury, and pre-existing osteoarthritis or avascular necrosis. The most common patterns were anterior column and posterior hemitransverse, posterior wall, both columns, and T-type. Conclusion Acute THA is an effective treatment strategy for older patients with acetabular fractures and should be considered when the abovementioned indications are observed on preoperative images. (PROSPERO: CRD42022329555).

Ischemia/reperfusion (I/R) injury causes excessive oxidative events and initiates destructive inflammatory responses, and it is an important promoter to the pathology of various pathema states. Ferroptosis is an iron-dependent type of nonapoptotic cell death accompanied by the accumulation of membrane lipid peroxide and consumption of polyunsaturated fatty acid, and it plays a key role in I/R injury diseases. Moreover, the excessive production of inflammatory cytokines contributes to the development of acute kidney injury. Here, we reported neutrophil membrane-coated copper-based nanoparticles (N-Cu5.4O@DFO NPs) for I/R kidney injury treatment. The highly biocompatible and stable N-Cu5.4O@DFO NPs showed excellent antioxidant and iron ion scavenging abilities in vitro. Our finding showed that the N-Cu5.4O@DFO NPs strategy could significantly accumulate in the inflammatory kidney, reduce oxidative damage events and inflammatory response, and finally achieve synergistic therapy against renal I/R injury. This work promotes the development of nanoantioxidant agents with multiple antioxidant properties for the therapy of other I/R injury diseases.

Abstract Inflammation is a pathological feature of kidney injury and its progression correlates with the development of kidney fibrosis which can lead to kidney function impairment. This project investigated the regulatory function of WNT1-inducible signaling pathway protein 1 (WISP1) in kidney inflammation. Administration of recombinant WISP1 protein to healthy mice induced kidney inflammation (macrophage accrual and production of tumor necrosis factor α (TNF-α), CCL2 and IL-6), which could be prevented by inhibition of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Furthermore, inhibition of WISP1, by gene knockdown or neutralising antibody, could inhibit cultured macrophages producing inflammatory cytokines following stimulation with lipopolysaccharides (LPSs) and kidney fibroblasts proliferating in response to TNFα, which both involved NF-κB signaling. Kidney expression of WISP1 was found to be increased in mouse models of progressive kidney inflammation-unilateral ureter obstruction (UUO) and streptozotocin (STZ)-induced diabetic nephropathy (DN). Treatment of UUO mice with WISP1 antibody reduced the kidney inflammation in these mice. Therefore, pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting inflammation in kidney disease.

Formononetin (FMN) has been reported as a prospective antiosteoporotic medication. However, the antiosteoporotic properties of FMN are still unclear in a mouse model with diabetes-induced osteoporosis. An osteoporotic or osteopenic mouse model with type I diabetes mellitus (T1DM) was established using streptozotocin (40 mg/kg) injection for 5 consecutive days. After 12 weeks with FMN intragastric administration (0.5, 5, 20 mg/kg), the antiosteoporotic activity of FMN was evaluated in T1DM mice. FMN supplementation effectively improves Ca excretion and trabecular bone degeneration and impedes osteoclast differentiation and function to attenuate hyperglycemia-induced bone deterioration. In addition, FMN inhibited activating protein 1 (AP-1) and osteoclast-specific gene expression, Nfatc1, Ctsk, and TRAP. The administration of FMN has a beneficial effect to attenuate hyperglycemia-induced bone deteriorations, including osteoclastogenesis, trabecular bone, and Ca loss. Our study provided a prospective medication for the treatment of T1DM-related osteopenia or osteoporosis with FMN.

The polydopamine nanoparticles (PDA NPs) as a self-polymerized form of dopamine have occurred with growing interest in biomedical applications in late years. Its natural-inspired feature as a conjugated polymer endows excellent inactivating capability for radical species to PDA-based nanoparticles that provide a theoretical foundation for applications in preventing inflammation-mediated acute kidney injury (AKI) from ROS. Here, we develop a polydopamine wrapped manganese ferrite nanoparticles (PDA@MF NPs) strategy for acute kidney injury therapy by synergistically scavenging ROS and producing O2, which further regulates macrophages amounts by decreasing M1-type and increasing M2-type. Water-soluble PDA@MF NPs were prepared in one step after the oxidative and self-polymerized process of the dopamine monomer. Here, the biodegradable PDA NPs were applied to scavenge ROS. MF NPs undertake continuous O2 production in an H2O2-based hypoxic environment. Based on this system, we aim to relieve the hypoxia, pathological symptoms, and inflammation via scavenging ROS during the O2 production process, and effective polarization to M2-type macrophages. PDA@MF NPs in this study were verified could significantly attenuate oxidative stress in vivo, reduce inflammatory events in renal, and improve renal function, which might be a potential treatment to inhibit oxidative damages and inflammatory events in renal AKI disease

Oxidative stress is a critical event in neuronal damage following seizures. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been shown to be promising nanotherapeutic agents in neurological disorders. However, the mechanism underlying MSC-EVs therapeutic efficacy for oxidative stress-induced neuronal damage remains poorly understood. Methods: We investigated the antioxidant and restoration activities of MSC-EVs on hippocampal neurons in response to H2O2 stimulation in vitro and seizures in vivo. We also explored the potential underlying mechanism by injecting adeno-associated virus (AAV)-nuclear factor erythroid-derived 2, like 2 (Nrf2), a key antioxidant mediator, in animal models. Results: MSC-EVs were enriched in antioxidant miRNAs and exhibited remarkable antioxidant activity evident by increased ferric ion-reducing antioxidant ability, catalase, superoxide dismutase, and glutathione peroxidase activities and decreased reactive oxygen species (ROS) generation, DNA/lipid/protein oxidation, and stress-associated molecular patterns in cultured cells and mouse models. Notably, EV administration exerted restorative effects on the hippocampal neuronal structure and associated functional impairments, including dendritic spine alterations, electrophysiological disturbances, calcium transients, mitochondrial changes, and cognitive decline after oxidative stress in vitro or in vivo. Mechanistically, we found that the Nrf2 signaling pathway was involved in the restorative effect of EV therapy against oxidative neuronal damage, while AAV-Nrf2 injection attenuated the antioxidant activity of MSC-EVs on the seizure-induced hippocampal injury. Conclusions: We have shown that MSC-EVs facilitate the reconstruction of hippocampal neurons associated with the Nrf2 defense system in response to oxidative insults. Our study highlights the clinical value of EV-therapy in neurological disorders such as seizures.

Early brain injury (EBI) is a major cause of adverse outcomes following subarachnoid hemorrhage (SAH). There is evidence that mesenchymal stem cells (MSCs) - derived exosomes are involved in the repair of SAH. Exosomes were extracted from human umbilical cord mesenchymal stem cells (hubMSCs) and identified. OxyHb treated PC12 cells were transfected with exosomes alone or together with miR-26b-5p inhibitor. Hub-MSCs derived exosomes promote cell proliferation, inhibit apoptosis and reduce inflammatory mediator expression. Transfection of miR-26b-5p inhibitor abolished the promoting effect of exosomes on the proliferation of PC12 cells, as well as the inhibitory effect on cell apoptosis. In addition, methionine adenosyltransferase II alpha (MAT2A) was one target gene of miR-26b-5p. OxyHb treated PC12 cells were transfected with exosomes alone or together with pcDNA-MAT2A and observed that the promoting effect of exosomes on PC12 cell proliferation was abolished by pcDNA-MAT2A, which was the same as the effect of miR-26b-5p inhibitor. OxyHb treated PC cells incubated with exosomes were transfected with miR-26b-5p inhibitor alone or together with si-MAT2A, respectively, and it was observed that exosomes decreased the phosphorylation levels of p38 MAPK and STAT3 proteins, inhibited cell apoptosis and inflammatory mediator expression, and miR-26b-5p inhibitor abrogated the effects of exosomes, while transfection of si-MAT2A reversed the effects of miR-26b-5p inhibitor. Moreover, injection of miR-26b-5p inhibitor resulted in increased MAT2A and pathway protein expression, increased inflammatory mediators, and aggravated neurological symptoms in the brain tissues of SAH rats.

Traumatic brain injury (TBI) causes substantial mortality and long-term disability worldwide. TGFβ1 is a unique molecular and functional signature in microglia, but the role of TGFβ1 in TBI is not clear. The purpose of this study was to investigate the role of TGFβ1 in TBI. The weight dropping device was used to establish TBI model of rats. Hematoxylin eosin staining and Bielschowsky silver staining were used to assess tissue loss. Beam walking and muscle strength tests were used to assess neurological deficits. Immunohistochemical staining was used to assess axonal injures. Western blotting was used to detect expression of related proteins. RT-PCR was used to detect expression of cytokines. Immunofluorescence staining was used to assess the microglia/macrophages activation. We observed obvious axonal injury and microglia/macrophages activation in the peri-lesion cortex. The expression of inflammatory cytokines was markedly high after TBI. The expression of TGFβ1 and TGFβRI were significantly reduced after TBI. TGFβ1 promoted the functional recovery and alleviated axonal injury 1 day after TBI. TGFβ1 promoted microglia/macrophages polarizing to alternative activation and alleviated neuroinflammation. These effects of TGFβ1 could be inhibited by LY2109761, the inhibitor of TGFRI/II. These results suggested that TGFβ1 played a protective role in axonal injury and could be a potential therapeutic target in early stages following TBI.

Fibrosis is a pathological feature of chronic kidney disease and its progression correlates with declining renal function. Kidney fibrosis is driven by multiple profibrotic factors. This project examined the regulatory function of WNT1-inducible-signaling pathway protein 1 (WISP1) in the development of kidney fibrosis. Induction of WISP1 by transforming growth factor beta 1 (TGF-β1), and the role of WISP1 in TGF-β1/Smad signaling and fibrotic responses, was examined in multiple kidney cells. Kidney expression of WISP1 was examined in mouse models of unilateral ureter obstruction (UUO) and streptozotocin-induced diabetic nephropathy. WISP1 antibody was administered to UUO mice during the induction of kidney injury and the impact on kidney fibrosis was examined. WISP1 expression was upregulated in both mouse models. TGF-β1-induced expression of WISP1 and profibrotic genes in cultured kidney cells via TGF-βR1. Recombinant WISP1-induced expression of TGF-βR1 in kidney cells. Suppression of WISP1 by shRNA or neutralizing antibody reduced TGF-β1-mediated activation of Smad3, fibrotic gene expression, and fibroblast proliferation. Treatment with WISP1 antibody inhibited the development of kidney fibrosis in UUO mice. WISP1 mediates the profibrotic effects of TGF-β1 in kidney cells and in kidney disease. Pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting kidney fibrosis.

Tubular epithelial cells (TECs) represent the primary site of renal ischemia/reperfusion injury (RIRI). However, whether the damage of TECs could drive the initiation of inflammation was unclear. Here we investigated the role of the TECs and macrophages during RIRI. Increased expression of inflammation response and activated M1 macrophage were determined in the mice model of RIRI. Moreover, we demonstrated global miRNA expression profiling of renal exosomes, and miR-374b-5p was most upregulated in these exosomes in vivo. Inhibition of miR-374b-5p in the mice upon RIR operation would alleviate the kidney injury via decreasing the production of proinflammatory cytokines and suppressing the macrophage activation. Similar results were also identified in the hypoxia-induced cell model where exosomal miR-374b-5p was dramatically upregulated. Uptake of exosomes derived from the hypoxic TECs by macrophages would trigger M1 polarization via transferring miR-374b-5p. Besides, we confirmed that miR-374b-5p could directly bind to Socs1 using a dual-luciferase reporter assay. Notably, when we injected the miR-374b-5p-enriched exosomes into mice, a high-level inflammatory response and M1 macrophage activation were performed. Our studies demonstrated that exosomal miR-374b-5p played an essential role in the communication between injured TECs and macrophages, resulting in the M1 macrophage activation during RIRI. The blockage of the release of such exosomes may serve as a new therapeutic strategy for RIRI.

In Gram-negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.

Tissue plasminogen activator is usually used for the treatment of acute ischemic stroke, but the role of endogenous tissue plasminogen activator in traumatic brain injury has been rarely reported. A rat model of traumatic brain injury was established by weight-drop method. The tissue plasminogen activator inhibitor neuroserpin (5 μL, 0.25 mg/mL) was injected into the lateral ventricle. Neurological function was assessed by neurological severity score. Neuronal and axonal injuries were assessed by hematoxylin-eosin staining and Bielschowsky silver staining. Protein level of endogenous tissue plasminogen activator was analyzed by western blot assay. Apoptotic marker cleaved caspase-3, neuronal marker neurofilament light chain, astrocyte marker glial fibrillary acidic protein and microglial marker Iba-1 were analyzed by immunohistochemical staining. Apoptotic cell types were detected by immunofluorescence double labeling. Apoptotic cells in the damaged cortex were detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining. Degenerating neurons in the damaged cortex were detected by Fluoro-Jade B staining. Expression of tissue plasminogen activator was increased at 6 hours, and peaked at 3 days after traumatic brain injury. Neuronal apoptosis and axonal injury were detected after traumatic brain injury. Moreover, neuroserpin enhanced neuronal apoptosis, neuronal injury and axonal injury, and activated microglia and astrocytes. Neuroserpin further deteriorated neurobehavioral function in rats with traumatic brain injury. Our findings confirm that inhibition of endogenous tissue plasminogen activator aggravates neuronal apoptosis and axonal injury after traumatic brain injury, and activates microglia and astrocytes. This study was approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province of China in June 2015.

Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat's kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3'UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

We previously found that high-mobility group box protein 1 (HMGB1) promoted cell proliferation, migration, invasion, and autophagy in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), but little is known about its regulatory mechanism. The aim of this study was to investigate the regulatory mechanism of HMGB1 at the posttranscription level. Real-time qPCR, CCK-8 cell proliferation assay, transwell cell migration assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were used in this study. The targeting relationship between miRNA and mRNA was presented by the luciferase reporter assay. MiR-449a was downregulated in RA synovial tissue and inhibited RA-FLS proliferation, migration, and IL-6 production. MiR-449a directly targeted HMGB1 and inhibited its expression. Yin Yang 1(YY1) negatively regulated miR-449a expression and formed a mutual inhibition loop in RA-FLS. MiR-449a inhibited TNFα-mediated HMGB1 and YY1 overexpression and IL-6 production. Our results reveal the regulatory mechanism of HMGB1 in RA and demonstrate that miR-449a is a crucial molecule in RA pathogenesis and a suitable candidate for miRNA replacement therapies in RA.