Neutrophil-Driven M2-Like Macrophages Are Critical for Skin Fibrosis in a Systemic Sclerosis Model.

Systemic sclerosis (SSc) is a challenging autoimmune disease characterized by progressive fibrosis affecting the skin and internal organs. Despite the known infiltration of macrophages and neutrophils, their precise contributions to SSc pathogenesis remain elusive. In this study, we elucidated that CD206hiMHCIIlo M2-like macrophages constitute the predominant pathogenic immune cell population in the fibrotic skin of a bleomycin-induced SSc mouse model. These cells emerged as pivotal contributors to the profibrotic response by orchestrating the production of TGF-ß1 through a MerTK signaling-dependent manner. Notably, we observed that neutrophil infiltration was a prerequisite for accumulation of M2-like macrophages. Strategies such as neutrophil depletion or inhibition of CXCR1/2 were proven effective in reducing M2-like macrophages, subsequently mitigating SSc progression. Detailed investigations revealed that in fibrotic skin, neutrophil-released neutrophil extracellular traps were responsible for the differentiation of M2-like macrophages. Our findings illuminate the significant involvement of the neutrophil-macrophage-fibrosis axis in SSc pathogenesis, offering critical information for the development of potential therapeutic strategies.

Multispecies transcriptomics identifies SIKE as a MAPK repressor that prevents NASH progression.

Nonalcoholic fatty liver (NAFL) remains relatively benign, but high-risk to end-stage liver diseases become highly prevalent when it progresses into nonalcoholic steatohepatitis (NASH). Our current understanding of the development of NAFL to NASH remains insufficient. In this study, we revealed MAP kinase (MAPK) activation as the most notable molecular signature associated with NASH progression across multiple species. Furthermore, we identified suppressor of IKKε (SIKE) as a conserved and potent negative controller of MAPK activation. Hepatocyte-specific overexpression of Sike prevented NASH progression in diet- and toxin-induced mouse NASH models. Mechanistically, SIKE directly interacted with TGF-ß-activated kinase 1 (TAK1) and TAK1-binding protein 2 (TAB2) to interrupt their binding and subsequent TAK1-MAPK signaling activation. We found that indobufen markedly up-regulated SIKE expression and effectively improved NASH features in mice and macaques. These findings identify SIKE as a MAPK suppressor that prevents NASH progression and provide proof-of-concept evidence for targeting the SIKE-TAK1 axis as a potential NASH therapy.

Biodistribution-based Administration of cGMP-compliant Human Umbilical Cord Mesenchymal Stem Cells Affects the Therapeutic Effect of Wound Healing.

BACKGROUND: Although mesenchymal stem cells (MSCs) are used as therapeutic agents for skin injury therapy, few studies have reported the effects of dosing duration and delivery frequency on wound healing. In addition, before the clinical application of MSCs, it is important to assess whether their usage might influence tumor occurrence. METHODS: We described the metabolic patterns of subcutaneous injection of hUC-MSCs using fluorescence tracing and qPCR methods and applied them to the development of drug delivery strategies for promoting wound healing. RESULTS: (i) We developed cGMP-compliant hUC-MSC products with critical quality control points for wound healing; (ii) The products did not possess any tumorigenic or tumor-promoting/inhibiting ability in vivo; (iii) Fluorescence tracing and qPCR analyses showed that the subcutaneous application of hUC-MSCs did not result in safety-relevant biodistribution or ectopic migration; (iv) Reinjecting hUC-MSCs after significant consumption significantly improved reepithelialization and dermal regeneration. CONCLUSIONS: Our findings provided a reference for controlling the quality of MSC products used for wound healing and highlighted the importance of delivery time and frequency for designing in vivo therapeutic studies.

The Impact of Rayleigh Scattering in UWFBG Array-Based Φ-OTDR and Its Suppression Method.

Ultra-weak fiber Bragg grating (UWFBG) array-based phase-sensitive optical time-domain reflectometry (Φ-OTDR) utilizes the interference interaction between the reference light and the reflected light from the broadband gratings for sensing. It significantly improves the performance of the distributed acoustic sensing system (DAS) because the intensity of the reflected signal is much higher than that of the Rayleigh backscattering. This paper shows that Rayleigh backscattering (RBS) has become one of the primary noise sources in the UWFBG array-based Φ-OTDR system. We reveal the impact of the Rayleigh backscattering signal on the intensity of the reflective signal and the precision of the demodulated signal, and we suggest reducing the pulse duration to improve the demodulation accuracy. Experimental results demonstrate that using light with a 100 ns pulse duration can improve the measurement precision by three times compared with the use of a 300 ns pulse duration.

Network-based analysis identifies key regulatory transcription factors involved in skin aging.

Skin aging is a complex process involving intricate genetic and environmental factors. In this study, we performed a comprehensive analysis of the transcriptional regulatory landscape of skin aging in canines. Weighted Gene Co-expression Network Analysis (WGCNA) was employed to identify aging-related gene modules. We subsequently validated the expression changes of these module genes in single-cell RNA sequencing (scRNA-seq) data of human aging skin. Notably, basal cell (BC), spinous cell (SC), mitotic cell (MC), and fibroblast (FB) were identified as the cell types with the most significant gene expression changes during aging. By integrating GENIE3 and RcisTarget, we constructed gene regulation networks (GRNs) for aging-related modules and identified core transcription factors (TFs) by intersecting significantly enriched TFs within the GRNs with hub TFs from WGCNA analysis, revealing key regulators of skin aging. Furthermore, we demonstrated the conserved role of CTCF and RAD21 in skin aging using an H2O2-stimulated cell aging model in HaCaT cells. Our findings provide new insights into the transcriptional regulatory landscape of skin aging and unveil potential targets for future intervention strategies against age-related skin disorders in both canines and humans.

A high-throughput drug screening identifies luteolin as a therapeutic candidate for pathological cardiac hypertrophy and heart failure.

Background: Pathological cardiac hypertrophy is commonly resulted from sustained pressure overload and/or metabolic disorder and eventually leads to heart failure, lacking specific drugs in clinic. Here, we aimed to identify promising anti-hypertrophic drug(s) for heart failure and related metabolic disorders by using a luciferase reporter-based high-throughput screening. Methods: A screen of the FDA-approved compounds based on luciferase reporter was performed, with identified luteolin as a promising anti-hypertrophic drug. We systematically examined the therapeutic efficacy of luteolin on cardiac hypertrophy and heart failure in vitro and in vivo models. Transcriptome examination was performed to probe the molecular mechanisms of luteolin. Results: Among 2,570 compounds in the library, luteolin emerged as the most robust candidate against cardiomyocyte hypertrophy. Luteolin dose-dependently blocked phenylephrine-induced cardiomyocyte hypertrophy and showed extensive cardioprotective roles in cardiomyocytes as evidenced by transcriptomics. More importantly, gastric administration of luteolin effectively ameliorated pathological cardiac hypertrophy, fibrosis, metabolic disorder, and heart failure in mice. Cross analysis of large-scale transcriptomics and drug-target interacting investigations indicated that peroxisome proliferator activated receptor γ (PPARγ) was the direct target of luteolin in the setting of pathological cardiac hypertrophy and metabolic disorders. Luteolin can directly interact with PPARγ to inhibit its ubiquitination and subsequent proteasomal degradation. Furthermore, PPARγ inhibitor and PPARγ knockdown both prevented the protective effect of luteolin against phenylephrine-induced cardiomyocyte hypertrophy in vitro. Conclusion: Our data clearly supported that luteolin is a promising therapeutic compound for pathological cardiac hypertrophy and heart failure by directly targeting ubiquitin-proteasomal degradation of PPARγ and the related metabolic homeostasis.

Honokiol acts as an AMPK complex agonist therapeutic in non-alcoholic fatty liver disease and metabolic syndrome.

BACKGROUND: Non-alcoholic fatty liver (NAFLD) and its related metabolic syndrome have become major threats to human health, but there is still a need for effective and safe drugs to treat these conditions. Here we aimed to identify potential drug candidates for NAFLD and the underlying molecular mechanisms. METHODS: A drug repositioning strategy was used to screen an FDA-approved drug library with approximately 3000 compounds in an in vitro hepatocyte model of lipid accumulation, with honokiol identified as an effective anti-NAFLD candidate. We systematically examined the therapeutic effect of honokiol in NAFLD and metabolic syndrome in multiple in vitro and in vivo models. Transcriptomic examination and biotin-streptavidin binding assays were used to explore the underlying molecular mechanisms, confirmed by rescue experiments. RESULTS: Honokiol significantly inhibited metabolic syndrome and NAFLD progression as evidenced by improved hepatic steatosis, liver fibrosis, adipose inflammation, and insulin resistance. Mechanistically, the beneficial effects of honokiol were largely through AMPK activation. Rather than acting on the classical upstream regulators of AMPK, honokiol directly bound to the AMPKγ1 subunit to robustly activate AMPK signaling. Mutation of honokiol-binding sites of AMPKγ1 largely abolished the protective capacity of honokiol against NAFLD. CONCLUSION: These findings clearly demonstrate the beneficial effects of honokiol in multiple models and reveal a previously unappreciated signaling mechanism of honokiol in NAFLD and metabolic syndrome. This study also provides new insights into metabolic disease treatment by targeting AMPKγ1 subunit-mediated signaling activation.

Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in Allium wallichii.

Himalayan onion (Allium wallichii) is a perennial bulbous herb with high ornamental value and has long been used as traditional medicines in Nepal and China because of the anti-cancer and anti-microbial activities. Wild Allium wallichii features different flower colors, including purple, pink, deep purple and white. However, little is known about the molecular mechanisms of color formation during A. wallichii flower development stages due to the lack of optimal reference genes. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for quantifying expression levels of target genes. The accuracy of qRT-PCR analyses is largely dependent on the identification of stable reference genes for data normalization. The stability of reference gene expression may vary with plant species and environmental conditions. The aim of this study was to select stable reference genes for qRT-PCR analyses of target genes at flower development stages, in different flower colors and organs for Allium wallichii. The CDSs of eight potential reference genes (TUB2, ACT1, GAPC, EF1α, UBQ, UBC, SAND and CYP1) were cloned and their stability was evaluated by four programs (Delta Ct, geNorm, NormFinder and BestKeeper), and the results were further integrated into a comprehensive rank by RefFinder. The results showed that TUB2 and GAPC were the most stable two reference genes at different developmental stages of purple- and white-flower genotypes and across all samples. UBC and TUB2 expression was stable at different developmental stages of purple flowers. CYP1 and TUB2 were stably expressed at different developmental stages of white flowers. GAPC and SAND showed the highest rankings in different flower colors. TUB2 and EF1α performed the best in different tissues. ACT1 was the least stable gene in all tested samples. Moreover, DIHYDROFLAVONOL-4-REDUCTASE (DFR) gene that involved in anthocyanin synthesis was used to evaluate the effectiveness of the selected candidates. This study identified the first set of suitable reference genes for qRT-PCR analyses, which will lay the foundation for gene function study in A. wallichii.

Mesenchymal stem cells alleviate systemic sclerosis by inhibiting the recruitment of pathogenic macrophages.

Systemic sclerosis (SSc) is a recalcitrant autoimmune disease for which there is no cure. Mesenchymal stem cell (MSC)-based treatment has emerged as a promising therapeutic option for several autoimmune diseases. Previously, we found that the immunoregulatory potential of MSCs can be greatly enhanced by IFN-γ and TNF-α. Here, we found that IFN-γ- and TNF-α-pretreated MSCs significantly alleviated skin fibrosis in a bleomycin (BLM)-induced SSc model. Macrophages were found to be the predominant profibrotic immune cell population in the pathogenesis of SSc. The accumulation of macrophages was significantly decreased by MSC treatment. Importantly, MSCs primarily reduced the population of maturing macrophages with high CCR2 expression by inhibiting the generation of CCL2 from fibroblasts and macrophages. This finding may help to improve MSC-based clinical treatments for SSc patients.

Effects of treatment of non-alcoholic fatty liver disease on heart failure with preserved ejection fraction.

In the past few decades, non-alcoholic fatty liver disease (NAFLD) and heart failure with preserved ejection fraction (HFpEF) have become the most common chronic liver disease and the main form of heart failure (HF), respectively. NAFLD is closely associated with HFpEF by sharing common risk factors and/or by boosting systemic inflammation, releasing other secretory factors, and having an expansion of epicardial adipose tissue (EAT). Therefore, the treatments of NAFLD may also affect the development and prognosis of HFpEF. However, no specific drugs for NAFLD have been approved by the Food and Drug Administration (FDA) and some non-specific treatments for NAFLD are applied in the clinic. Currently, the treatments of NAFLD can be divided into non-pharmacological and pharmacological treatments. Non-pharmacological treatments mainly include dietary intervention, weight loss by exercise, caloric restriction, and bariatric surgery. Pharmacological treatments mainly include administering statins, thiazolidinediones, glucagon-like peptide-1 receptor agonists, sodium-glucose cotransporter 2 inhibitors, and metformin. This review will mainly focus on analyzing how these treatments may affect the development and prognosis of HFpEF.

A small molecule targeting ALOX12-ACC1 ameliorates nonalcoholic steatohepatitis in mice and macaques.

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease and has become a leading indication for liver transplantation in the United States. The development of effective therapies for NASH is a major unmet need. Here, we identified a small molecule, IMA-1, that can treat NASH by interrupting the arachidonate 12-lipoxygenase (ALOX12)­acetyl-CoA carboxylase 1 (ACC1) interaction. IMA-1 markedly blocked diet-induced NASH progression in both male mice and Cynomolgus macaque therapeutic models. The anti-NASH efficacy of IMA-1 was comparable to ACC inhibitor in both species. Protein docking simulations and following functional experiments suggested that the anti-NASH effects of IMA-1 were largely dependent on its direct binding to a pocket in ALOX12 proximal to its ACC1-interacting surface instead of inhibiting ALOX12 lipoxygenase activity. IMA-1 treatment did not elicit hyperlipidemia, a known side effect of direct inhibition of ACC enzymatic activity, in both mice and macaques. These findings provide proof of concept across multiple species for the use of small molecule­based therapies for NASH.

Genome-Wide Analysis of the Auxin/Indoleacetic Acid Gene Family and Response to Indole-3-Acetic Acid Stress in Tartary Buckwheat (Fagopyrum tataricum).

Auxin/indoleacetic acid (Aux/IAA) family genes respond to the hormone auxin, which have been implicated in the regulation of multiple biological processes. In this study, all 25 Aux/IAA family genes were identified in Tartary buckwheat (Fagopyrum tataricum) by a reiterative database search and manual annotation. Our study provided comprehensive information of Aux/IAA family genes in buckwheat, including gene structures, chromosome locations, phylogenetic relationships, and expression patterns. Aux/IAA family genes were nonuniformly distributed in the buckwheat chromosomes and divided into seven groups by phylogenetic analysis. Aux/IAA family genes maintained a certain correlation and a certain species-specificity through evolutionary analysis with Arabidopsis and other grain crops. In addition, all Aux/IAA genes showed a complex response pattern under treatment of indole-3-acetic acid (IAA). These results provide valuable reference information for dissecting function and molecular mechanism of Aux/IAA family genes in buckwheat.

Pharmacological inhibition of arachidonate 12-lipoxygenase ameliorates myocardial ischemia-reperfusion injury in multiple species.

Myocardial ischemia-reperfusion (MIR) injury is a major cause of adverse outcomes of revascularization after myocardial infarction. To identify the fundamental regulator of reperfusion injury, we performed metabolomics profiling in plasma of individuals before and after revascularization and identified a marked accumulation of arachidonate 12-lipoxygenase (ALOX12)-dependent 12-HETE following revascularization. The potent induction of 12-HETE proceeded by reperfusion was conserved in post-MIR in mice, pigs, and monkeys. While genetic inhibition of Alox12 protected mouse hearts from reperfusion injury and remodeling, Alox12 overexpression exacerbated MIR injury. Remarkably, pharmacological inhibition of ALOX12 significantly reduced cardiac injury in mice, pigs, and monkeys. Unexpectedly, ALOX12 promotes cardiomyocyte injury beyond its enzymatic activity and production of 12-HETE but also by its suppression of AMPK activity via a direct interaction with its upstream kinase TAK1. Taken together, our study demonstrates that ALOX12 is a novel AMPK upstream regulator in the post-MIR heart and that it represents a conserved therapeutic target for the treatment of myocardial reperfusion injury.

Milk Fat Globule-Epidermal Growth Factor-Factor 8 Improves Hepatic Steatosis and Inflammation.

BACKGROUND AND AIMS: Milk fat globule-epidermal growth factor-factor 8 (MFGE8) has been shown to be a critical extracellular molecule that mediates apoptotic signaling in the pathological process of nonalcoholic fatty liver disease (NAFLD). MFGE8 is abundantly expressed in hepatocytes, but its function in the pathogenesis of NAFLD has not been characterized. APPROACH AND RESULTS: In our current study, hepatic MFGE8 showed a protective role in the pathogenesis of NAFLD. Hepatic MFGE8 deletion largely exacerbated lipid accumulation and inflammatory responses in the liver in response to overnutrition. Mechanistically, intercellular MFGE8 was shown to directly bind to apoptosis signal-regulating kinase 1 (ASK1) and to inhibit its dimerization and phosphorylation under a normal diet. However, under metabolic challenges, decreased cytoplasmic MFGE8 facilitated the dimerization and phosphorylation of ASK1 and subsequent mitogen-activated protein kinase signaling in hepatocytes. CONCLUSIONS: Hepatic MFGE8 is an endogenous inhibitor that halts the progression of hepatic steatosis and inflammation. Metabolic challenge-induced loss of intracellular MFGE8 facilitates ASK1 dimerization and phosphorylation. Therefore, maintaining hepatic MFGE8 levels may serve as an alternative strategy for the treatment of NAFLD.

Prevalence and influencing factors of nonunion in patients with tibial fracture: systematic review and meta-analysis.

OBJECTIVE: The aim of this study is to assess the prevalence of nonunion in patients with tibia fracture and the association between influencing factors and tibia fracture nonunion. METHOD: A database searches of PubMed, the Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), Weipu database, and Wanfang database from inception until June 2019 was conducted. The pooled prevalence, odds ratio (OR), and 95% confidence intervals (CI) were calculated with Stata software. RESULTS: In this study, 111 studies involving 41,429 subjects were included. In the study of the relationship between influencing factors and tibia fracture nonunion, 15 factors significantly influenced the fracture union, including > 60 years old, male, tobacco smoker, body mass index > 40, diabetes, nonsteroidal anti-inflammatory drugs (NSAIDs) user, opioids user, fracture of middle and distal tibia, high-energy fracture, open fracture, Gustilo-Anderson grade IIIB or IIIC, Müller AO Classification of Fractures C, open reduction, fixation model, and infection. CONCLUSION: The prevalence of nonunion in patients with tibia fracture was 0.068 and 15 potential factors were associated with the prevalence. Closed reduction and minimally invasive percutaneous plate osteosynthesis (MIPPO) have the low risks of nonunion for the treatment of tibial fractures.

Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease worldwide and is strongly associated with the presence of oxidative stress. Disturbances in lipid metabolism lead to hepatic lipid accumulation, which affects different reactive oxygen species (ROS) generators, including mitochondria, endoplasmic reticulum, and NADPH oxidase. Mitochondrial function adapts to NAFLD mainly through the downregulation of the electron transport chain (ETC) and the preserved or enhanced capacity of mitochondrial fatty acid oxidation, which stimulates ROS overproduction within different ETC components upstream of cytochrome c oxidase. However, non-ETC sources of ROS, in particular, fatty acid ß-oxidation, appear to produce more ROS in hepatic metabolic diseases. Endoplasmic reticulum stress and NADPH oxidase alterations are also associated with NAFLD, but the degree of their contribution to oxidative stress in NAFLD remains unclear. Increased ROS generation induces changes in insulin sensitivity and in the expression and activity of key enzymes involved in lipid metabolism. Moreover, the interaction between redox signaling and innate immune signaling forms a complex network that regulates inflammatory responses. Based on the mechanistic view described above, this review summarizes the mechanisms that may account for the excessive production of ROS, the potential mechanistic roles of ROS that drive NAFLD progression, and therapeutic interventions that are related to oxidative stress.

Truncated TEAD-binding protein of TAZ inhibits glioma survival through the induction of apoptosis and repression of epithelial-mesenchymal transition.

Transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, promotes tumor progression by serving as a transcriptional coactivator with TEAD. Here, we introduced a new construct which can express the TEAD-binding domain of TAZ protein (TAZBD), and determined its antitumor effect in malignant glioma both in vitro and in vivo. We first observed that TAZ was upregulated in glioma tissues and related to malignant clinicopathologic characteristic, indicating the crucial role of TAZ during glioma progression. In U87 and U251 cells, TAZBD expression increased the proportion of apoptotic cells, and suppressed the colony formation and tumorigenicity. Further, TAZBD also decreased cell metastasis through the repression of epithelial-mesenchymal transition. The mechanistic study showed that TAZBD suppression of glioma cells was predominantly through blocking the TAZ-TEAD complex formation by competing with endogenous TAZ. Thus, the gene therapy of malignant glioma through blocking TAZ-TEAD complex by TAZBD may provide a new way for the targeted therapy of glioma.

Integrated Omics Reveals Tollip as an Regulator and Therapeutic Target for Hepatic Ischemia-Reperfusion Injury in Mice.

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.

An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) injury is a common clinical issue lacking effective therapy and validated pharmacological targets. Here, using integrative 'omics' analysis, we identified an arachidonate 12-lipoxygenase (ALOX12)-12-hydroxyeicosatetraenoic acid (12-HETE)-G-protein-coupled receptor 31 (GPR31) signaling axis as a key determinant of the hepatic IR process. We found that ALOX12 was markedly upregulated in hepatocytes during ischemia to promote 12-HETE accumulation and that 12-HETE then directly binds to GPR31, triggering an inflammatory response that exacerbates liver damage. Notably, blocking 12-HETE production inhibits IR-induced liver dysfunction, inflammation and cell death in mice and pigs. Furthermore, we established a nonhuman primate hepatic IR model that closely recapitulates clinical liver dysfunction following liver resection. Most strikingly, blocking 12-HETE accumulation effectively attenuated all pathologies of hepatic IR in this model. Collectively, this study has revealed previously uncharacterized metabolic reprogramming involving an ALOX12-12-HETE-GPR31 axis that functionally determines hepatic IR procession. We have also provided proof of concept that blocking 12-HETE production is a promising strategy for preventing and treating IR-induced liver damage.