E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis.

Intestinal ischemia-reperfusion (II/R) injury is an urgent clinical disease with high incidence and mortality, and impaired intestinal barrier function caused by excessive apoptosis of intestinal cells is an important cause of its serious consequences. Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase that is recently reported to suppress the inflammatory response and apoptosis. However, the biological function and regulation of TRIM65 in II/R injury are totally unknown. We found that TRIM65 was significantly decreased in hypoxia-reoxygenation (H/R) induced intestinal epithelial cells and II/R-induced intestine tissue. TRIM65 knockout mice markedly aggravated intestinal apoptosis and II/R injury. To explore the molecular mechanism of TRIM65 in exacerbating II/R-induced intestinal apoptosis and damage, thymocyte selection-associated high mobility group box factor 4 (TOX4) was screened out as a novel substrate of TRIM65 using the yeast two-hybrid system. TRIM65 binds directly to the N-terminal of TOX4 through its coiled-coil and SPRY structural domains. Immunofluorescence confocal microscopy showed that they can co-localize both in the cytoplasm and nucleus. Furthermore, TRIM65 mediated the K48 ubiquitination and degradation of TOX4 depending on its E3 ubiquitin ligase activity. In addition, TRIM65 inhibits H/R-induced intestinal epithelial apoptosis via TOX4. In summary, our results indicated that TRIM65 promotes ubiquitination and degradation of TOX4 to inhibit apoptosis in II/R. These findings provide a promising target for the clinical treatment of II/R injury.

Lidocaine induces neurotoxicity in spinal cord neurons in Goto-Kakizaki rats via AMPK-mediated mitophagy.

OBJECTIVE: Lidocaine has been reported to induce neurotoxicity, which is further enhanced by high glucose levels. This study is aimed to explore the underlying mechanisms of lidocaine neurotoxicity in spinal cord neurons of diabetes. METHODS: Take thirty specific pathogen-free (SPF) healthy Sprague-Dawley (SD) rats and thirty Goto-Kakizaki (GK) rats, aged 12 weeks, weighing 180-200 g. The spinal cord neurons of rats were isolated and cultured in vitro. Cell Counting Kit-8 was used to detect cell proliferation to determine the appropriate concentration and duration of lidocaine. Mitochondrial function was assessed using ATP content, cellular oxygen consumption rate, mitochondrial membrane potential, ROS production, and mitochondrial ultrastructure. Western blot was applied to detect the expression of autophagy- and mitophagy-related molecules PINK1, p-AMPK, LC-3II/LC3-I ratio and mTORC1. Immunofluorescent staining was used to detect the expression of PINK1 and LC3. RESULTS: Lidocaine decreased cell viability of spinal cord neurons in concentration- and time-dependent manners. And lidocaine treatment aggravated mitochondrial dysfunction in GK rats. Furthermore, mitophagy was activated in diabetes, and lidocaine exposure up-regulated mitophagy. AMPK activator MK8722 aggravated mitochondrial damage, increased the expression of PINK1, p-AMPK, LC-3II/LC3-I ratio, and decreased the expression of mTORC1, while AMPK inhibitor Compound C and autophagy inhibitor Bafilomycin A1 reduced mitochondrial damage and decreased the expression of PINK1, p-AMPK, LC-3II/LC3-I ratio, and increased the expression of mTORC1. CONCLUSIONS: Lidocaine induced neurotoxicity of spinal cord neurons in GK rats via AMPK-mediated mitophagy.

Stellate ganglion block alleviates postoperative cognitive dysfunction via inhibiting TLR4/NF-κB signaling pathway.

Postoperative cognitive dysfunction (POCD) is common in aged patients after major surgery and is associated with increased risk of long-term morbidity and mortality. However, the underlying mechanism remains largely unknown and the clinical management of POCD is still controversial. Stellate ganglion block (SGB) is a clinical treatment for nerve injuries and circulatory issues. Recent evidence has identified the benefits of SGB in promoting learning and memory. We thus hypothesize that SGB could be effective in improving cognitive function after surgery. In present study, we established POCD model in aged rats via partial liver resection surgery. We found that the development of POCD was associated with the activation of toll-like receptor 4/nuclear factor kapa-B (TLR4/NF-κB) signaling pathway in the microglia in dorsal hippocampus, which induced the production of pro-inflammatory mediators (TNF-α, IL-1ß, IL-6) and promoted neuroinflammation. More importantly, we showed evidence that preoperative treatment with SGB could inhibit microglial activation, suppress TLR4/NF-κB-mediated neuroinflammation and effectively attenuate cognitive decline after the surgery. Our study suggested that SGB may serve as a novel treatment to prevent POCD in elderly patients. As SGB is safe procedure widely used in clinic, our findings can be easily translated into clinical practice and benefit more patients.

Dexmedetomidine reduces enteric glial cell injury induced by intestinal ischaemia-reperfusion injury through mitochondrial localization of TERT.

This study was performed to uncover the effects of dexmedetomidine on oxidative stress injury induced by mitochondrial localization of telomerase reverse transcriptase (TERT) in enteric glial cells (EGCs) following intestinal ischaemia-reperfusion injury (IRI) in rat models. Following establishment of intestinal IRI models by superior mesenteric artery occlusion in Wistar rats, the expression and distribution patterns of TERT were detected. The IRI rats were subsequently treated with low or high doses of dexmedetomidine, followed by detection of ROS, MDA and GSH levels. Calcein cobalt and rhodamine 123 staining were also carried out to detect mitochondrial permeability transition pore (MPTP) and the mitochondrial membrane potential (MMP), respectively. Moreover, oxidative injury of mtDNA was determined, in addition to analyses of EGC viability and apoptosis. Intestinal tissues and mitochondria of EGCs were badly damaged in the intestinal IRI group. In addition, there was a reduction in mitochondrial localization of TERT, oxidative stress, whilst apoptosis of EGCs was increased and proliferation was decreased. On the other hand, administration of dexmedetomidine was associated with promotion of mitochondrial localization of TERT, whilst oxidative stress, MPTP and mtDNA in EGCs, and EGC apoptosis were all inhibited, and the MMP and EGC viability were both increased. A positive correlation was observed between different doses of dexmedetomidine and protective effects. Collectively, our findings highlighted the antioxidative effects of dexmedetomidine on EGCs following intestinal IRI, as dexmedetomidine alleviated mitochondrial damage by enhancing the mitochondrial localization of TERT.

JAK2/STAT3 inhibition attenuates intestinal ischemia-reperfusion injury via promoting autophagy: in vitro and in vivo study.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) causes severe injury to the intestine, leading to systemic inflammation and multiple organ failure. Autophagy is a stress-response mechanism that can protect against I/R injury by removing damaged organelles and toxic protein aggregates. Recent evidence has identified JAK-STAT signaling pathway as a new regulator of autophagy process, however, their regulatory relationship in intestinal I/R remains unknown. METHODS AND RESULTS: We systematically analyzed intestinal transcriptome data and found that JAK-STAT pathway was largely activated in response to I/R with most significant upregulation observed for JAK2 and STAT3. ChIP-Seq and luciferase assays in an in vitro oxygen-glucose deprivation and reoxygenation model revealed that activated JAK2/STAT3 signaling directly inhibited the transcription of autophagy regulator Beclin-1, leading to the suppression of autophagy and the activation of intestinal cell death. These findings were further confirmed in an in vivo mouse model, in which, intestinal I/R injury was associated with the activation of JAK2/STAT3 pathway and the deactivation of Beclin-1-mediated autophagy, while inhibiting JAK2/STAT3 with AG490 reactivated autophagy and improved survival after intestinal I/R injury. CONCLUSIONS: JAK2/STAT3 signaling suppresses autophagy process during intestinal I/R, while inhibiting JAK-STAT can be protective against intestinal I/R injury by activating autophagy. These findings expand our knowledge on intestinal I/R injury and provide therapeutic targets for clinical treatment.

Dexmedetomidine inhibits mitochondria damage and apoptosis of enteric glial cells in experimental intestinal ischemia/reperfusion injury via SIRT3-dependent PINK1/HDAC3/p53 pathway.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury commonly occurs during perioperative periods, resulting in high morbidity and mortality on a global scale. Dexmedetomidine (Dex) is a selective α2-agonist that is frequently applied during perioperative periods for its analgesia effect; however, its ability to provide protection against intestinal I/R injury and underlying molecular mechanisms remain unclear. METHODS: To fill this gap, the protection of Dex against I/R injury was examined in a rat model of intestinal I/R injury and in an inflammation cell model, which was induced by tumor necrosis factor-alpha (TNF-α) plus interferon-gamma (IFN-γ) stimulation. RESULTS: Our data demonstrated that Dex had protective effects against intestinal I/R injury in rats. Dex was also found to promote mitophagy and inhibit apoptosis of enteric glial cells (EGCs) in the inflammation cell model. PINK1 downregulated p53 expression by promoting the phosphorylation of HDAC3. Further studies revealed that Dex provided protection against experimentally induced intestinal I/R injury in rats, while enhancing mitophagy, and suppressing apoptosis of EGCs through SIRT3-mediated PINK1/HDAC3/p53 pathway in the inflammation cell model. CONCLUSION: Hence, these findings provide evidence supporting the protective effect of Dex against intestinal I/R injury and its underlying mechanism involving the SIRT3/PINK1/HDAC3/p53 axis.

The Emerging Roles of Tripartite Motif Proteins (TRIMs) in Acute Lung Injury.

Acute lung injury (ALI) is an inflammatory disorder of the lung that causes high mortality and lacks any pharmacological intervention. Ubiquitination plays a critical role in the pathogenesis of ALI as it regulates the alveolocapillary barrier and the inflammatory response. Tripartite motif (TRIM) proteins are one of the subfamilies of the RING-type E3 ubiquitin ligases, which contains more than 80 distinct members in humans involved in a broad range of biological processes including antivirus innate immunity, development, and tumorigenesis. Recently, some studies have shown that several members of TRIM family proteins play important regulatory roles in inflammation and ALI. Herein, we integrate emerging evidence regarding the roles of TRIMs in ALI. Articles were selected from the searches of PubMed database that had the terms "acute lung injury," "ubiquitin ligases," "tripartite motif protein," "inflammation," and "ubiquitination" using both MeSH terms and keywords. Better understanding of these mechanisms may ultimately lead to novel therapeutic approaches by targeting TRIMs for ALI treatment.

The α2AR/Caveolin-1/p38MAPK/NF-κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia-reperfusion.

Intestinal ischaemia-reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2-noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet-to-dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 expression. Correlation of Caveolin-1 (Cav-1) protein expression with p38, p-p38, p-p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal-1 and α2A -AR. Specifically, Dex promoted Cav-1 expression via α2A -AR activation and mitigated intestinal I/R-induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R-induced lung injury was also closely related to α2A -AR/p38 mitogen-activated protein kinases/nuclear factor-kappaB (MAPK/NF-κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav-1 to inhibit the activation of p38 and NF-κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.

Dexmedetomidine protects intestinal ischemia-reperfusion injury via inhibiting p38 MAPK cascades.

Intestinal ischemia-reperfusion (I/R) is a life-threatening condition associated with high morbidity and mortality. Dexmedetomidine (DEX), an agonist of α2-adrenoceptor with sedation and analgesia effect, has recently been identified with protective function against I/R injury in multiple organs. However, the mechanism underlying the beneficial effect of DEX on intestine after I/R injury remained poorly understood. In the present study, using in both in vitro and in vivo models, we found that intestinal I/R injury was associated with the activation of p38 MAPK cascade, while DEX was capable of deactivating p38 MAPK and thus protect intestinal cells from apoptosis by inhibiting p38 MAPK-mediated mitochondrial depolarization and cytochrome c (Cyto C) release. Moreover, through inhibiting p38 MAPK activity, the downstream production of pro-inflammatory cytokines-regulated by NF-κB was also suppressed by DEX treatment, leading to the resolution of I/R-induced inflammation in intestine. In general, our study provided evidence that DEX protected intestine from I/R injury by inhibiting p38 MAPK-mediated mitochondrial apoptosis and inflammatory response.

The Role of Janus Kinase/Signal Transducer and Activator of Transcription Signalling on Preventing Intestinal Ischemia/Reperfusion Injury with Dexmedetomidine.

Dexmedetomidine (Dex) works as a crucial agent for the treatment of intestinal ischemia/reperfusion (I/R), but its mechanism remains unclear. Recent articles demonstrated the pivotal role of Janus kinase/signal transducer and activator of transcription (JAK2/STAT3) signalling in I/R. Therefore, it is reasonable to explore the associated mechanism of JAK2/STAT3 signalling in Dex treatment. The study purpose was to evaluate the JAK2/STAT3 signalling regulatory mechanisms of Dex in preventing I/R. Anaesthetized rats were subjected to superior mesenteric artery occlusion consisting of 1 h of ischemia and 2 h of reperfusion while served as controls. Animals received subcutaneous administration of 50 µg/kg Dex, JAK1 and JAK2 inhibitor, Ruxolitinib, selective JAK2 inhibitor, 10 mg/kg AG490 or STAT inhibitor and 0.4 mg/kg rapamycin; or Dex-treatment in the presence of α2-adrenoceptor antagonists Atip or Dex-treatment alone after I/R. Injury was scored histologically, apoptosis was detected via the apoptotic mediators caspase-3 and Bcl-2/Bax and the degree of activation of the JAK/STAT pathway was evaluated. Dex inhibited I/R injury by decreasing apoptosis significantly with rescue of cleaved caspase-3 and the Bcl-2/Bax ratio. Furthermore, phosphorylation of JAK2, STAT1 and STAT3 was affected, suggesting the involvement of activated JAK/STAT in response to Dex. Meanwhile, the JAK2 or STAT inhibitors AG490 and rapamycin, but not Ruxolitinib, exhibited a similar but even greater JAK2 and STAT3 regulatory effect, thus leading to a greater benefit. JAK2/STAT3 activation is crucial to the diminishing effect of Dex on mesenteric I/R injury; however, the efficacy and timing of Dex administration should be considered in clinical practice.

MiR-520b promotes the progression of non-small cell lung cancer through activating Hedgehog pathway.

Although the non-small cell lung cancer (NSCLC) is one of the most malignant tumours worldwide, the mechanisms controlling NSCLC tumourigenesis remain unclear. Here, we find that the expression of miR-520b is up-regulated in NSCLC samples. Further studies have revealed that miR-520b promotes the proliferation and metastasis of NSCLC cells. In addition, miR-520b activates Hedgehog (Hh) pathway. Inhibitor of Hh pathway could relieve the oncogenic effect of miR-520b upon NSCLC cells. Mechanistically, we demonstrate that miR-520b directly targets SPOP 3'-UTR and decreases SPOP expression, culminating in GLI2/3 stabilization and Hh pathway hyperactivation. Collectively, our findings unveil that miR-520b promotes NSCLC tumourigenesis through SPOP-GLI2/3 axis and provide miR-520b as a potential diagnostic biomarker and therapeutic target for NSCLC.

KDM4B promotes DNA damage response via STAT3 signaling and is a target of CREB in colorectal cancer cells.

Resistance to radiotherapy is a major limitation for the successful treatment of colorectal cancer (CRC). Recently, accumulating evidence supports a critical role of epigenetic regulation in tumor cell survival upon irradiation. Lysine Demethylase 4B (KDM4B) is a histone demethylase involved in the oncogenesis of multiple human cancers but the underlying mechanisms have not been fully elucidated. Here we show that KDM4B is overexpressed in human colorectal cancer (CRC) tumors and cell lines. In CRC cells, KDM4B silencing induces spontaneous double-strand breaks (DSBs) formation and potently sensitizes tumor cells to irradiation. A putative mechanism involved suppression of Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, which is essential for efficient repair of damaged DNA. Overexpression of STAT3 in KMD4B knockdown cells largely attenuates DNA damage triggered by KDM4B silencing and increases cell survival upon irradiation. Moreover, we find evidence that transcription factor CAMP Responsive Element Binding Protein (CREB) is a key regulator of KMD4B expression by directly binding to a conserved region in KMD4B promoter. Together, our findings illustrate the significance of CREB-KDM4B-STAT3 signaling cascade in DNA damage response, and highlight that KDM4B may potentially be a novel oncotarget for CRC radiotherapy.

Dexmedetomidine promotes the recovery of neurogenesis in aged mouse with postoperative cognitive dysfunction.

Recently, growing evidence has demonstrated Dexmedetomidine (Dex) a promising intervene preventing postoperative cognitive decline (POCD) following surgery, which is associated with neuroinflammation leading to neuronal apoptosis and deregulated neurogenesis. Previous studies suggested the anti-inflammation and anti-neuroapoptosis action of Dex. Therefore we hypothesize the promoting neurogenesis of Dex linked to stimulating BDNF and subsequent p-MPAK production in a rat model of POCD. In the present study, the POCD animal model was established by performing an exploratory laparotomy under isoflurane anaesthesia in old rats, utilizing which Dex response is confirmed by behavioural tests. Inflammatory biomarkers as IL-1ß and TNF-α, mature neuron percentage measured by doublecortin staining (DCX), promoting factors as brain derived growth factor (BDNF), phosphorylated cAMP response element binding protein (CREB) and proteins of kinase A (PKA), MAPK production as p-P38-MAPK protein express were measured. Herein, we showed that surgery reduced DCX-positive neurons and expression of BDNF representing neurogenesis profoundly. As expected, Dex rescued the associated cognitive impairment and inflammatory changes, as well as up-regulated expression of BDNF, PKA, p-CREB/CREB and following p-P38-MAPK regulation. Our results confirmed the protective Dex response and indicated the proneurogenesis role of it as well, suggesting the mechanism of beneficial effects of Dex to prevent POCD.

Evaluation of dexmedetomidine in combination with sufentanil or butorphanol for postoperative analgesia in patients undergoing laparoscopic resection of gastrointestinal tumors: A quasi-experimental trial.

The aim of this study was to evaluate the efficacy of dexmedetomidine in combination with sufentanil or butorphanol for postoperative analgesia in patients undergoing laparoscopic resection of a gastrointestinal tumor.This quasi-experimental trial was conducted in Nanchang, China, from January 2014 to December 2015. Eighty patients (age 27-70 years, American Society of Anesthesiologists physical status I-II) undergoing laparoscopic resection of a gastrointestinal tumor were randomized into 4 groups and offered intravenous patient-controlled analgesia for pain control after surgery. The patients received sufentanil 2.0 µg/kg in combination with dexmedetomidine 1.5 µg/kg (group S1) or 2.0 µg/kg (group S2), or butorphanol 0.15 mg/kg in combination with dexmedetomidine 1.5 0 µg/kg (group N1) or 2.0 µg/kg (group N2). Oxygen saturation, mean arterial pressure (MAP), heart rate, visual analog scale score, and Ramsay sedation score were recorded at enrollment (T0), at extubation (T1), and 4 (T2), 8 (T3), 12 (T4), 24 (T5), and 48 (T6) hours thereafter. Side effects and satisfaction scores were evaluated after surgery.MAP increased in all groups at T1 but not significantly so when compared with T0. Heart rate decreased significantly in group S2 when compared with the other groups at T1-T5 (P < 0.05). MAP decreased significantly in group S2 when compared with group S1 at T4-T6 (P < 0.05). MAP increased significantly in group N1 when compared with group N2 at T4-T5 (P < 0.05). There was a statistically significant decrease in mean visual analog scale score in group S2 when compared with group S1 at T2 (P < 0.05) and group N2 at T1-T2 (P < 0.05). Two patients in group S1 had vomiting. There were no reports of drowsiness, respiratory depression, or other complications. The satisfaction score was higher in group S2 than in the other groups.Dexmedetomidine in combination with sufentanil or butorphanol can be used safely and effectively for postoperative analgesia in patients undergoing laparoscopic resection of a gastrointestinal tumor. The combination of dexmedetomidine 2.0 µg/kg and sufentanil is particularly beneficial in these patients.

The preventive effects of dexmedetomidine against intestinal ischemia-reperfusion injury in Wistar rats.

OBJECTIVES: Intestinal ischemia-reperfusion is a major problem, which may lead to multiorgan failure and death. The aim of this study was to evaluate the protective effects of dexmedetomidine on cell proliferation, antioxidant system, cell death, and structural integrity in intestinal injury induced by ischemia-reperfusion in rats. MATERIALS AND METHODS: Animals were randomized into three groups: group A, sham-operated or control; group B, intestinal ischemia/reperfusion (IR); and group C, intestinal IR pretreated with 50 µg of dexmedetomidine. Intestine tissue was collected from all rats 30 min after desufflation, and fresh frozen for histological and biochemical evaluation. RESULTS: The intestinal tissue of group B rats showed a significant decrease in the antioxidant enzyme activities. However, these enzyme activities were improved by the administration of dexmedetomidine. Inhibiting the protein expression of MCP7, PAR2, P-JAK, P-STAT1, and P-STAT3 proved the protective effect of dexmedetomidine. The immunohistochemical staining revealed its protective effect by maintaining the normal structural integrity, less caspase-3 immuno reactivity, and increased cell proliferation count in the intestinal tissues. CONCLUSIONS: Intraperitoneal injection of dexmedetomidine significantly protected intestine IR injury in rats by inhibiting the inflammatory response, intestinal epithelial apoptosis, and maintaining structural integrity of intestinal cells.

Comparison between nitroglycerin and remifentanil in acute hypervolemic hemodilution combined with controlled hypotension during intracranial aneurysm surgery.

Allogenetic transfusion has long been considered to be a relatively safe and extremely effective blood transfusion treatment. However, acute hypervolemic hemodilution (AHH) combined with the remifentanil-induced controlled hypotension (CH) have rarely been examined. Herein, 40 intracranial aneurysm surgery patients were randomly divided into nitroglycerin group (A group, n=20) and remifentanil group (B group, n=20). During intracranial aneurysm surgery, MAP, HR, Hb, and Hct were recorded. SjvO2, PjvO2, SaO2, PaO2 were measured, and CaO2, Da-jvO2, CjvO2, CERO2, VADL were calculated. In addition, The venous blood samples were collected for determining PT, TT, APTT, FBG, VIII, VWF and electrolytes. The results show that HR in nitroglycerin group dramatically accelerated and HR in remifentanil group slowed at 30 minutes after hypotension and 5 minutes after aneurysm occlusion (P<0.01) after hypotension. Compared with A group, the SjvO2 and CjvO2 of B group increased significantly and the Da-jvO2 and CERO2 decreased significantly at T3, T4. In addition, There were no significant differences between after AHH and before AHH in two groups (P>0.05) on TT, PT, APTT, FIB, VIII, VWF, Na(+), Cl(-), K(+), Ca(2+). These results suggest that AHH combined with remifentanil-based CH significantly lowered cerebral metabolic rate of oxygen and had effects on blood coagulation without clinical hemorrhagic signs increased and had important clinical significance for blood conservation.