Dongcheng Feng

miR-34a-5p Inhibition Alleviates Intestinal Ischemia/Reperfusion-Induced Reactive Oxygen Species Accumulation and Apoptosis via Activation of SIRT1 Signaling.

AIMS Reactive oxygen species (ROS) generation and massive epithelial apoptosis are critical in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. We previously found that the Sirtuin 1 (SIRT1)-mediated antioxidant pathway was impaired in the intestine after I/R. Here, we investigate the potential role of SIRT1-targeting microRNAs (miRNAs) in regulating ROS accumulation and apoptosis in intestinal I/R, and the important role SIRT1 involved in. RESULTS C57BL/6 mice were subjected to intestinal I/R induced by occlusion of the superior mesenteric artery followed by reperfusion. Caco-2 cells were incubated under hypoxia/reoxygenation condition to mimic I/R in vivo. We find that SIRT1 is gradually repressed during the early reperfusion, and that this repression results in intestinal ROS accumulation and apoptosis. Using bioinformatics analysis and real-time PCR, we demonstrate that miR-34a-5p and miR-495-3p are significantly increased among the 41 putative miRNAs that can target SIRT1. Inhibition of miR-34a-5p, but not miR-495-3p, attenuates intestinal I/R injury, as demonstrated by repressing p66shc upregulation, manganese superoxide dismutase repression, and the caspase-3 activation in vitro and in vivo; it further alleviates systemic injury, as demonstrated by reducing inflammatory cytokine release, attenuating lung and liver lesions, and improving survival. Interestingly, SIRT1 plays an indispensable role in the protection afforded by miR-34a-5p inhibition. INNOVATION This study provides the first evidence of miRNAs in regulating oxidative stress and apoptosis in intestinal I/R. CONCLUSION miR-34a-5p knockdown attenuates intestinal I/R injury through promoting SIRT1-mediated suppression of epithelial ROS accumulation and apoptosis. This may represent a novel prophylactic approach to intestinal I/R injury. Antioxid. Redox Signal. 24, 961-973.

miR-381-3p knockdown improves intestinal epithelial proliferation and barrier function after intestinal ischemia/reperfusion injury by targeting nurr1

Impairment in gut barrier function induced by intestinal ischemia/reperfusion (I/R) injury is associated with high morbidity and mortality. Intestinal barrier function requires the tight coordination of epithelial migration, proliferation and differentiation. We previously observed that nuclear receptor-related protein 1 (nurr1)-mediated proliferative pathway was impaired in intestinal I/R injury. Here, we aimed to assess the effect of nurr1 on intestinal barrier function and to evaluate microRNA (miRNA)-nurr1-mediated restoration of intestinal barrier function in intestinal I/R injury. We induced an in vivo intestinal I/R injury mouse model by clamping and then releasing the superior mesenteric artery. We also performed an in vitro study in which we exposed Caco-2 and IEC-6 cells to hypoxia/reoxygenation (H/R) conditions to stimulate intestinal I/R injury. Our results demonstrated that nurr1 regulated intestinal epithelial development and barrier function after intestinal I/R injury. miR-381-3p, which directly suppressed nurr1 translation, was identified by microarray and bioinformatics analysis. miR-381-3p inhibition enhanced intestinal epithelial proliferation and barrier function in vitro and in vivo and also attenuated remote organ injury and improved survival. Importantly, nurr1 played an indispensable role in the protective effect of miR-381-3p inhibition. Collectively, these findings show that miR-381-3p inhibition mitigates intestinal I/R injury by enhancing nurr1-mediated intestinal epithelial proliferation and barrier function. This discovery may lead to the development of therapeutic interventions for intestinal I/R injury.

Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion

Autophagy is an essential cytoprotective response against pathologic stresses that selectively degrades damaged cellular components. Impaired autophagy contributes to organ injury in multiple diseases, including ischemia/reperfusion (I/R), but the exact mechanism by which impaired autophagy is regulated remains unclear. Several researchers have demonstrated that microRNAs (miRNAs) negatively regulate autophagy by targeting autophagy-related genes (ATGs). Therefore, the effect of ATG-related miRNAs on I/R remains a promising research avenue. In our study, we found that autophagy flux is impaired during intestinal I/R. A miRNA microarray analysis showed that miR-665-3p was highly expressed in the I/R group, which was confirmed by qRT-PCR. Then, we predicted and proved that miR-665-3p negatively regulates ATG4B expression in Caco-2 and IEC-6 cells. In ileum biopsy samples from patients with intestinal infarction, there was an inverse correlation between miR-665-3p and ATG4B expression, which supports the in vitro findings. Moreover, based on miR-665-3p regulation of autophagy in response to hypoxia/reoxygenation in vitro, gain-of-function and loss-of-function approaches were used to investigate the therapeutic potential of miR-665-3p. Additionally, we provide evidence that ATG4B is indispensable for protection upon inhibition of miR-665-3p. Finally, we observed that locked nucleic acid-modified inhibition of miR-665-3p in vivo alleviates I/R-induced systemic inflammation and apoptosis via recovery of autophagic flux. Our study highlights miR-665-3p as a novel small molecule that regulates autophagy by targeting ATG4B, suggesting that miR-665-3p inhibition may be a potential therapeutic approach against inflammation and apoptosis for the clinical treatment of intestinal I/R.

PKC|[zeta]| phosphorylates TRAF2 to protect against intestinal ischemia|[ndash]|reperfusion|[ndash]|induced injury

Intestinal ischemia–reperfusion (I/R) is a common clinical problem that occurs during various clinical pathological processes. Excessive apoptosis has an indispensable role in intestinal I/R injury. Tumor necrosis factor receptor-associated factor 2 (TRAF2) and PKCζ have an essential role in apoptosis. Here, we aimed to investigate the effects of PKCζ and TRAF2 and to explore the correlation between PKCζ and TRAF2 in intestinal I/R injury. Mice were subjected to intestinal I/R injury in vivo. In vitro experiments were conducted by treating Caco-2 cells with hypoxia/reoxygenation (H/R) stimulation to simulate intestinal I/R. Intestinal tissue samples and Caco-2 cells were examined using various approaches. Intestinal I/R induced the membrane translocation and phosphorylation of PKCζ. Pretreatment with the PKCζ activator phosphatidylcholine remarkably attenuated gut injury by suppressing apoptosis. H/R induced PKCζ to combine with TRAF2, which was phosphorylated by PKCζ at Ser55, but not at Ser11, under intestinal I/R or H/R conditions. In addition, TRAF2 Ser55 phosphorylation increased cell survival by inhibiting cell apoptosis in the H/R model. Mechanistically, TRAF2 Ser55 phosphorylation promoted NF-κB activation but suppressed c-Jun activation in Caco-2 cells under H/R conditions. The results of this study demonstrate that the PKCζ/TRAF2 pathway represents a novel protective mechanism against intestinal I/R injury. Therefore, the PKCζ/TRAF2 pathway is a novel target for potential treatments of intestinal I/R injury-related diseases.

Salvianolic acid A alleviates chronic ethanol-induced liver injury via promotion of β-catenin nuclear accumulation by restoring SIRT1 in rats

ABSTRACT In recent years, alcoholic liver disease (ALD) has emerged as a growing public health problem worldwide. &bgr;‐catenin plays an important role in the growth, development, regeneration and metabolic activity of the liver. Salvianolic acid A (SalA) is a water‐soluble component from the root extract of Salvia miltiorrhiza Bunge, and its effect on ALD has not yet been investigated. This study aimed to investigate the effect of SalA on chronic alcohol‐induced liver injury and to explore the role of SIRT1‐mediated &bgr;‐catenin deacetylation in such an effect. In this study, SalA treatment significantly alleviated the accumulation of lipid droplets and reduced the plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), alcohol and ammonia levels in rats. SalA enhanced ethanol and ammonia metabolism and maintained mitochondrial homeostasis. Moreover, SalA restored the activity of the major ethanol‐metabolizing enzymes and oxidative stress functions in the liver. Importantly, we found that SalA treatment effectively inhibited the ethanol‐mediated decrease in nuclear &bgr;‐catenin by upregulating SIRT1 in the liver. SIRT1 then deacetylated &bgr;‐catenin to promote its accumulation in the nucleus, thereby preventing alcohol‐induced liver injury. The results demonstrate that the SIRT1/&bgr;‐catenin pathway is a key therapeutic target in liver injury caused by chronic alcohol exposure and that SalA protects against alcohol‐induced liver injury via the SIRT1‐mediated deacetylation of &bgr;‐catenin. HighlightsSalA regulates the SIRT1/&bgr;‐catenin pathway.SIRT1/&bgr;‐catenin pathway is a key therapeutic target in ALD.SalA protects against ALD via SIRT1‐mediated deacetylation of &bgr;‐catenin.

Carnosic acid prevents COL1A2 transcription through the reduction of Smad3 acetylation via the AMPKα1/SIRT1 pathway

&NA; Carnosic acid (CA), a major bioactive component in rosemary extract, has many biological and pharmaceutical activities. Smad3 acetylation can regulate the transcription of type I &agr;2 collagen (COL1A2), which is the major component of the extracellular matrix (ECM). The aim of the current study was to evaluate whether CA inhibits COL1A2 transcription via the reduction of Smad3 acetylation against liver fibrosis. The results showed that CA treatment significantly suppressed COL1A2 transcription and markedly decreased the deposition of ECM induced by dimethylamine (DMN) in rats. Importantly, the suppression of COL1A2 transcription following CA treatment depended on the reduction of Smad3 acetylation via the activation of Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide+ (NAD+)‐dependent deacetylase. SIRT1 siRNA increased the acetylation of Smad3 and blocked CA‐down‐regulated Smad3 deacetylation. Notably, CA‐mediated AMP‐activated protein kinase‐&agr;1 (AMPK&agr;1) activation not only increased AMPK&agr;1 phosphorylation but also increased SIRT1 expression, thus leading to a significant reduction in Smad3 acetylation. Furthermore, CA‐mediated SIRT1 activation was inhibited by AMPK&agr;1 siRNA. Collectively, CA can inhibit the transcription of COL1A2 through SIRT1‐mediated Smad3 deacetylation, and the activation of SIRT1 by CA involves the AMPK&agr;1/SIRT1 pathway in liver fibrosis. HighlightsCA alleviates DMN‐induced liver fibrosis.CA suppresses the expression of COL1A2 at the level of transcription.The protective effect of CA is associated with the acetylation of Smad3.CA regulates the AMPK&agr;1/SIRT1 pathway.

Microarray Analysis of Differentially Expressed Profiles of Circular RNAs in a Mouse Model of Intestinal Ischemia/Reperfusion Injury with and Without Ischemic Postconditioning

Background/Aims: Ischemic postconditioning (iPoC) represents a promising strategy to mitigate ischemia/reperfusion (I/R) injury of the intestine, yet the mechanisms of this treatment remain to be elucidated. Circular RNAs (circRNAs), a novel class of endogenous non-coding RNAs, have recently been recognized as important regulators of gene expression and pathological processes. Here, we aimed to investigate the expression patterns of circRNAs after intestinal I/R with and without iPoC and, furthermore, to explore the potential mechanisms of iPoC in relation to the differentially expressed circRNAs. Methods: The global circRNA and mRNA expression profiles in mouse intestinal mucosa were initially screened by microarray (n = 3 per group) and quantitative real-time PCR was used to validate the expression pattern of circRNAs and mRNAs. Bioinformatics analysis including Gene ontology, KEGG pathway analysis, microRNA binding sites identification and circRNA-miRNA-mRNA network construction were utilized for in-depth mechanism exploration. Results: There were 4 up- and 58 downregulated circRNAs as well as 322 up- and 199 downregulated mRNAs in the intestinal I/R group compared with the sham group, whereas compared with I/R, iPoC treatment significantly upregulated 12 circRNAs and 129 mRNAs and downregulated 21 circRNAs and 174 mRNAs. The expression levels of a randomly selected set of 6 circRNAs and 5 mRNAs were successfully validated by qRT-PCR. Through a systematic comparison of the direction of circRNA expression changes in all groups, we identified two circRNAs, circRNA_012412 and circRNA_016863, that may be closely associated with the protective mechanisms of iPoC. Finally, four possible circRNA_012412/circRNA_016863-miRNA-mRNA pathways were predicted, which may play important roles in endogenous protective signaling in iPoC. Conclusions: This study was the first to comprehensively delineate the expression profiles of circRNAs in a mouse model of intestinal I/R and iPoC and provides novel clues for understanding the mechanisms of iPoC against intestinal I/R injury.

Inhibition of the ubiquitination of HSF1 by FBXW7 protects the intestine against ischemia–reperfusion injury

Epithelial apoptosis is an important factor in intestinal ischemia–reperfusion (I/R) injury. Heat shock factor 1 (HSF1) is a classical stress response factor that directly regulates the transcription of heat shock proteins (HSPs) under stress conditions. Although HSPs are involved in protecting the intestine against I/R, the mechanism whereby HSF1 is regulated in I/R is poorly understood. Here, we show that the ubiquitin ligase FBXW7 targets HSF1 for ubiquitination and degradation in intestinal I/R. In this study, we found that FBXW7 expression was upregulated at the transcriptional level in intestinal mucosae subjected to I/R. In Caco-2 and IEC-6 cells subjected to hypoxia/reoxygenation (H/R), a high FBXW7 level led to excessive HSF1 ubiquitination and degradation. FBXW7 knockdown attenuated HSF1 ubiquitination and downregulation and accelerated HSPB1 and HSP70 expression. In addition, FBXW7 deletion alleviated the apoptosis of intestinal epithelial cells, as evidenced by decreased activation of caspase-3 and caspase-9. The results suggest that FBXW7 suppression protects against intestinal I/R, at least partly through the HSF1/HSP pathway. These findings indicate that FBXW7 may be a potential therapeutic target for inhibiting intestinal mucosa apoptosis during intestinal I/R.

Carnosol-mediated Sirtuin 1 activation inhibits Enhancer of Zeste Homolog 2 to attenuate liver fibrosis

&NA; Quiescent hepatic stellate cell (HSC) activation and subsequent conversion into myofibroblasts is the central event in hepatic fibrosis pathogenesis. Epithelial−mesenchymal transition (EMT), another vital participant in liver fibrosis, has the potential to initiate HSC activation, which promotes abundant myofibroblast production. Previous studies suggest that Enhancer of Zeste Homolog 2 (EZH2) plays a significant role in myofibroblast transdifferentiation; however, the underlying mechanisms remain largely unaddressed. Carnosol (CS), a compound extracted from rosemary, displays multiple pharmacological activities. This study aimed to investigate the signaling mechanisms underlying EZH2 inhibition and the anti‐fibrotic effect of CS in liver fibrosis. We found that CS significantly inhibited CCl4‐ and TGF&bgr;1‐induced liver fibrosis and reduced both HSC activation and EMT. EZH2 knockdown also prevented these processes induced by TGF&bgr;1 in HSCs and AML‐12 cells. Interestingly, the protective effect of CS was positively associated with Sirtuin 1 (SIRT1) activation and accompanied by EZH2 inhibition. SIRT1 knockdown attenuated the EZH2 inhibition induced by CS and increased EZH2 acetylation, which enhanced its stability. Conversely, upon TGF&bgr;1 exposure, SIRT1 activation significantly reduced the level of EZH2 acetylation; however, EZH2 overexpression prevented the SIRT1 activation that primed myofibroblast inhibition, indicating that EZH2 is a target of SIRT1. Thus, SIRT1/EZH2 regulation could be used as a new therapeutic strategy for fibrogenesis. Together, this study provides evidence of activation of the SIRT1/EZH2 pathway by CS that inhibits myofibroblast generation, and thus, CS may represent an attractive candidate for anti‐fibrotic clinical therapy. Graphical abstract Figure. No caption available.