The role of miRNA-144-3p/Oprk1/KOR in nicotine dependence and nicotine withdrawal in male rats.

INTRODUCTION: The kappa-opioid receptor (KOR) has been implicated in mediating the behavioral and biochemical effects associated with nicotine reward and withdrawal; however, its underlying mechanisms remain to be further explored. METHODS: Adult male Sprague-Dawley rats were used to establish a nicotine dependence and withdrawal model by injecting nicotine (3 mg/kg/day, s.c.) or vehicle for 14 days, followed by the termination of nicotine for 7 days. Body weight gain, pain behaviors, and withdrawal scores were assessed in succession. MicroRNA (miRNA) sequencing was performed, and quantitative real-time PCR was used to detect the expression of candidate miRNAs and Oprk1. Western blotting was performed to examine KOR protein expression of KOR. Luciferase assay was conducted to validate the relationship of certain miRNAs/Oprk1. RESULTS: The behavioral results showed that nicotine dependence and withdrawal induced behavioral changes. Biochemical analyses demonstrated that miR-144-3p expression decreased and Oprk1/KOR expression increased in the prefrontal cortex, nucleus accumben, and hippocampus. Further investigation suggested that miR-144-3p exerted an inhibitory effect on Oprk1 expression in PC12 cells. CONCLUSIONS: This study revealed that miR-144-3p/Oprk1/KOR might be a potential pathway underlying the adverse effects induced by nicotine dependence and withdrawal, and might provide a novel therapeutic target for smoking cessation. IMPLICATIONS: This study demonstrates an impact of nicotine dependence and nicotine withdrawal on behavioral outcomes and the expressions of miR-144-3p/Oprk1/KOR in male rats. These findings have important translational implications given the continued use of nicotine and the difficulty in smoking cessation worldwide, which can be applied to alleviated the adverse effects induced by nicotine dependence and withdrawal, thus assist smokers to quit smoking.

Mu-opioid receptor alleviated ferroptosis in hepatic ischemia-reperfusion injury via the HIF-1α/KCNQ1OT1 axis.

Ferroptosis is the ideal therapeutic target for hepatic ischemia and reperfusion injury (HIRI). The µ opioid receptor (MOR) is associated with ferroptosis in HIRI. We aimed to determine the ferroptosis-related therapeutic mechanism of MOR in HIRI. A model of HIRI was established in BALB/c mice. Primary hepatocytes isolated from mice were stimulated by hypoxia/reoxygenation (H/R). Changes in histopathology were determined by H&E staining. Alterations in ferroptosis were evaluated by malondialdehyde (MDA), iron, glutathione (GSH), ACSL4, GPX4, and mitochondrial morphology. ALT and AST were used to determine hepatic function. First, we found that hepatic ischemia/reperfusion (I/R) induced the destruction of hepatic tissue structure and dead hepatocytes and determined that ferroptosis occurred in vivo and in vitro. During HIRI, the expression levels of HIF-1α and KCNQ1OT1 were significantly upregulated. We demonstrated that sufentanil improved the damage in the liver and hepatocytes undergoing I/R. Importantly, sufentanil inhibited ferroptosis in HIRI. In addition, sufentanil downregulated the expression levels of HIF-1α and KCNQ1OT1 in HIRI. Increases in HIF-1α and KCNQ1OT1 reversed the role of sufentanil in ferroptosis and HIRI. Subsequently, we determined that HIF-1α could activate the transcription of KCNQ1OT1 by binding to its promoter. In addition, KCNQ1OT1 was demonstrated to enhance ACSL4 stability by interacting with SRSF1. Finally, we observed that KCNQ1OT1 downregulation protected hepatocytes from hepatic I/R and inhibited ferroptosis. KCNQ1OT1 upregulation aggravated ferroptosis and hepatic injury during I/R. However, decreases in ACSL4 and SRSF1 reversed the harmful role of KCNQ1OT1 upregulation in HIRI. MOR alleviated ferroptosis in HIRI via the HIF-1α/KCNQ1OT1 axis.

Silencing lncRNA KCNQ1OT1 reduced hepatic ischemia reperfusion injury-induced pyroptosis by regulating miR-142a-3p/HMGB1 axis.

BACKGROUND: Based on pre-existing evidence, KCNQ1OT1 has been pointed out to be closely related to myocardial and cerebral ischemia reperfusion injury diseases. Herein, the objective of our study is to probe into the potential function as well as the underlying mechanism of KCNQ1OT1 on hepatic ischemia reperfusion injury (HIRI). METHODS: Using C57BL/6 J mice and primary mouse hepatocytes were conducted to establish HIRI model in vivo and in vitro. Cell viability was examined using CCK-8 assay and EdU assay. Flow cytometric analysis was performed to evaluate the pyroptosis. Dual-luciferase reporter assay was employed to verify the interaction relationships. qRT-PCR and Western blot were adopted to analyze the mRNA and protein level. Histopathological alteration of liver tissue was evaluated by HE staining. Immunohistochemistry (IHC) was performed to measure NLRP3 and caspase 1. RESULTS: Our data revealed that KCNQ1OT1 expression was ascending in hepatic tissue of HIRI mouse. Moreover, deprivation of KCNQ1OT1 mitigated I/R-induced hepatic injury and pyroptosis in vivo. Further experiments demonstrated that silencing KCNQ1OT1 promoted proliferation and inhibited pyroptosis in hypoxia/reoxygenation (H/R)-induced primary mouse hepatocytes. Mechanistically, KCNQ1OT1 functioned as a competing endogenous RNA which sponged miR-142a-3p, therefore promoted HMGB1 expression to activate TLR4/NF-κB signaling pathway in HIRI. CONCLUSION: LncRNA KCNQ1OT1 elevated HMGB1 expression through binding to miR-142a-3p, thereby promoting pyroptosis in HIRI.

Octreotide ameliorates hepatic ischemia-reperfusion injury through SNHG12/TAF15-mediated Sirt1 stabilization and YAP1 transcription.

BACKGROUND: Hepatic ischemia-reperfusion (HIR) injury is a pathological condition initiated by interrupted hepatic blood supply and exaggerated after reperfusion, which is one of the most lethal risks in liver transplantation and other liver surgeries. We aimed to investigate the protective mechanism of octreotide (Oct) against HIR injury. METHODS: The function of Oct was evaluated in the in vivo mouse model of HIR injury. Histological examinations were performed to assess the pathological changes. Serum parameters including ALT and AST were measured to evaluate the liver damage. qRT-PCR and western blot analysis were employed to determine the levels of long non-coding RNA SNHG12 (SNHG12) and autophagy or apoptosis-related proteins. RNA pull-down and RIP assays were used to verify the interaction between SNHG12 and TAF15. The transcriptional regulation of TAF15 in YAP1 was validated by ChIP and luciferase reporter assays. RESULTS: In the in vivo HIR injury model, Oct efficiently alleviated HIR-caused hepatic damage by suppressing apoptosis and activating autophagy. However, silencing of SNHG12 abrogated the protective effects of Oct via inactivating autophagy. Further mechanism investigation revealed that SNHG12 promoted the stabilization of Sirt1 and increased YAP1 transcriptional activity via interacting with TAF15. Up-regulation of Sirt1 and YAP1 was essential for maintaining the protective effect of Oct against HIR injury through increasing autophagic flux and suppressing apoptosis. CONCLUSIONS: Oct-induced up-regulation of SNHG12 attenuated HIR injury via promoting Sirt1 stabilization and YAP1 transcription to activate autophagy and repress apoptosis.

Effect of Edaravone on MicroRNA Expression in Exosomes after Hepatic Ischemia-reperfusion Injury.

BACKGROUND AND OBJECTIVE: Hepatic ischemia-reperfusion injury (HIRI) results in serious complications after liver resection and transplantation. Edaravone (ED) has a protective effect on IRI. This study was designed to evaluate whether ED could protect the liver of rats from HIRI injury and explored its exosomal miRNA-related mechanism. METHODS: The sham group, hepatic ischemia/reperfusion (IR group), and hepatic ischemia/reperfusion + edaravone (ED group) models were established. We determined the protective effect of ED by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD); enzyme-linked immunosorbent assay for tumor necrosis factor- α (TNF-α) and interleukin-1ß (IL-1ß); hematoxylin-eosin staining and immunohistochemistry for histopathological changes. Exosomal miRNAs were subjected to second-generation sequencing to identify their differential expression. The results were analyzed using bioinformatics methods and validated using real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: HIRI rats showed higher levels of ALT, AST, oxidative stress, and inflammatory markers; ED attenuated these effects. The sequencing results showed 6 upregulated and 13 downregulated miRNAs in the IR vs. sham groups, 10 upregulated and 10 downregulated miRNAs in the ED vs. IR groups. PC-3p-190-42101 was screened as an overlapping differentially expressed miRNA, and RT-qPCR validation showed that its expression in HIRI rats was significantly decreased; ED prevented this downregulation. Moreover, the expression of PC-3P-190-42101 was significantly correlated with the level of inflammatory factors. CONCLUSION: These findings indicate that ED can regulate the level of inflammatory factors by affecting the expression of miRNA PC-3p-190-42101 in plasma exosomes to protect the liver from IRI.

Sufentanil postoperative analgesia reduce the increase of T helper 17 (Th17) cells and FoxP3+ regulatory T (Treg) cells in rat hepatocellular carcinoma surgical model: A randomised animal study.

BACKGROUND: Surgery-related pain and opioids might exacerbate immune defenses in immunocompromised cancer patients which might affect postoperativd overall survival. Sufentanil is a good postoperative pain control drug,the present study aimed to figure out whether it effect T cell immunity in rat hepatocellular carcinoma surgical model. METHODS: A rat hepatocellular carcinoma (HCC) models was established by N-nitrosodiethylamine. Forty-eight of them were randomly divided into 3 equal groups: surgery without postoperative analgesia (Group C), surgery with morphine postoperative analgesia (Group M), surgery with sufentanil postoperative analgesia (Group S). Each animal underwent a standard left hepatolobectomy, and intraperitoneally implanted with osmotic minipumps filled with sufentanil, morphine or normal saline according to the different group. The food and water consumptions, body weight changes, locomotor activity and mechanical pain threshold (MPT) were observed. The ratio of CD4+/CD8+, proportions of Th1, Th2, Th17 and Treg cells in blood were detected using flow cytometry. The liver function and the rats' survival situation of each group were observed. RESULTS: The food and water consumption, locomotor activity and MPT of group C declined than those of group S and M on d1, d2, d3 (P < 0.05). The CD4+/CD8+ ratio and the proportion of Th1 cells were significantly higher while the proportion of Th2, Th17 and Treg cells were significantly lower in group S and group M compared with group C. The rats of group S have higher CD4+/CD8+ ratio on d3, while lower proportion of Treg cells on d7 compared with group M. The plasma ALT and AST values in group C were significantly higher than that of group S and group M on both d3 and d7. There were not significant differences in mortality rate between 3 groups. CONCLUSIONS: Sufentanil and morphine postoperative analgesia in HCC rats accepted hepatectomy could relieve postoperative pain, promote the recovery of liver function after surgery, alleviate the immunosuppressive effect of pain. Furthermore, Compared to morphine, sufentanil might have a slighter effect on CD4+/CD8+ ratio and Treg frequencies. Therefore, sufentanil postoperative analgesia is better than morphine in HCC hepatectomy rats.

Noncovalently Caged Firefly Luciferins Enable Amplifiable Bioluminescence Sensing of Hyaluronidase-1 Activity in Vivo.

Hyaluronidase 1 (Hyal-1) is an important enzyme involved in intracellular hyaluronic acid (HA) catabolism for performing various physiological functions, and its aberrant level is closely associated with many malignant diseases. Bioluminescence imaging is advantageous for monitoring Hyal-1 activity in vivo, but it remains challenging to design an available probe for differentiating Hyal-1 from other isoforms by a traditional strategy that covalently masks the firefly luciferase substrate. Herein, we, for the first time, present a noncovalently caging approach to construct a Hyal-1-specific bioluminogenic nanosensor by entrapping d-luciferin (d-Luc) inside the cholesterylamine-modified HA (CHA) nanoassembly to inhibit the bioluminescence production. When encountered with intracellular Hyal-1, CHA could be fully dissembled to liberate multiple copies of the loaded d-Luc, thereby emitting light by the luciferase-catalyzed bioluminescence reaction. Because of its cascade signal amplification feature, d-Luc@CHA displayed a remarkable "turn-on" response (248-fold) to 5 µg/mL Hyal-1 with a detection limit of 0.07 ng/mL. Importantly, bioluminescence imaging results validated that d-Luc@CHA could be competent for dynamically visualizing endogenous Hyal-1 changes in living cells and animals and possessed the capability of discriminating between normal and cancer cells, thus offering a promising toolbox to evaluate Hyal-1 roles in biological processes as well as to diagnose Hyal-1-related diseases.

Octreotide Attenuates Acute Kidney Injury after Hepatic Ischemia and Reperfusion by Enhancing Autophagy.

Octreotide exerts a protective effect in hepatic ischemia-reperfusion (HIR) injury. However, whether octreotide preconditioning could also reduce acute kidney injury (AKI) after HIR is unknown. This study was designed to investigate the role of octreotide in AKI after HIR. Male Sprague-Dawley rats were pretreated with octreotide or octreotide combined with 3-methyladenine (autophagy inhibitor, 3MA). Plasma creatinine, inflammation markers (e.g., TNF-α and IL-6 etc.), apoptosis, autophagy and phosphorylation of protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase (Akt/mTOR/p70S6K) in the kidney were measured after 60 minutes of liver ischemia and 24 hours of reperfusion for each rat. Octreotide pretreatment significantly preserved renal function and reduced the severity of renal injury. Moreover, octreotide alleviated inflammation and apoptosis in the kidney after HIR. Additionally, octreotide induced autophagy and autophagy inhibition with 3MA markedly reversed the renoprotective, anti-inflammatory and anti-apoptotic effects of octreotide after HIR. Finally, octreotide abrogated the activation of phosphorylation of Akt, mTOR and p70S6K in the kidney after HIR. Our results indicate that octreotide reduced renal injury after HIR due to its induction of autophagy. The enhancement of autophagy may be potentially linked to the octreotide mediated Akt/mTOR/p70S6K pathway deactivation and reduction of kidney inflammation and apoptosis after HIR.