IP3R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin.

INTRODUCTION: There is a high morbidity and mortality rate in mechanical trauma (MT)-induced hepatic injury. Currently, the molecular mechanisms underlying liver MT are largely unclear. Exploring the underlying mechanisms and developing safe and effective medicines to alleviate MT-induced hepatic injury is an urgent requirement. The aim of this study was to reveal the role of mitochondria-associated ER membranes (MAMs) in post-traumatic liver injury, and ascertain whether melatonin protects against MT-induced hepatic injury by regulating MAMs. METHODS: Hepatic mechanical injury was established in Sprague-Dawley rats and primary hepatocytes. A variety of experimental methods were employed to assess the effects of melatonin on hepatic injury, apoptosis, MAMs formation, mitochondrial function and signaling pathways. RESULTS: Significant increase of IP3R1 expression and MAMs formation were observed in MT-induced hepatic injury. Melatonin treatment at the dose of 30 mg/kg inhibited IP3R1-mediated MAMs and attenuated MT-induced liver injury in vivo. In vitro, primary hepatocytes cultured in 20% trauma serum (TS) for 12 h showed upregulated IP3R1 expression, increased MAMs formation and cell injury, which were suppressed by melatonin (100 µmol/L) treatment. Consequently, melatonin suppressed mitochondrial calcium overload, increased mitochondrial membrane potential and improved mitochondrial function under traumatic condition. Melatonin's inhibitory effects on MAMs formation and mitochondrial calcium overload were blunted when IP3R1 was overexpressed. Mechanistically, melatonin bound to its receptor (MR) and increased the expression of phosphorylated ERK1/2, which interacted with FoxO1 and inhibited the activation of FoxO1 that bound to the IP3R1 promoter to inhibit MAMs formation. CONCLUSION: Melatonin prevents the formation of MAMs via the MR-ERK1/2-FoxO1-IP3R1 pathway, thereby alleviating the development of MT-induced liver injury. Melatonin-modulated MAMs may be a promising therapeutic therapy for traumatic hepatic injury.

Adipsin inhibits Irak2 mitochondrial translocation and improves fatty acid ß-oxidation to alleviate diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) causes the myocardium to rely on fatty acid ß-oxidation for energy. The accumulation of intracellular lipids and fatty acids in the myocardium usually results in lipotoxicity, which impairs myocardial function. Adipsin may play an important protective role in the pathogenesis of DCM. The aim of this study is to investigate the regulatory effect of Adipsin on DCM lipotoxicity and its molecular mechanism. METHODS: A high-fat diet (HFD)-induced type 2 diabetes mellitus model was constructed in mice with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), glutathione-S-transferase (GST) pull-down technique, Co-immunoprecipitation (Co-IP) and immunofluorescence colocalization analyses were used to investigate the molecules which can directly interact with Adipsin. The immunocolloidal gold method was also used to detect the interaction between Adipsin and its downstream modulator. RESULTS: The expression of Adipsin was significantly downregulated in the HFD-induced DCM model (P < 0.05). Adipose tissue-specific overexpression of Adipsin significantly improved cardiac function and alleviated cardiac remodeling in DCM (P < 0.05). Adipsin overexpression also alleviated mitochondrial oxidative phosphorylation function in diabetic stress (P < 0.05). LC-MS/MS analysis, GST pull-down technique and Co-IP studies revealed that interleukin-1 receptor-associated kinase-like 2 (Irak2) was a downstream regulator of Adipsin. Immunofluorescence analysis also revealed that Adipsin was co-localized with Irak2 in cardiomyocytes. Immunocolloidal gold electron microscopy and Western blotting analysis indicated that Adipsin inhibited the mitochondrial translocation of Irak2 in DCM, thus dampening the interaction between Irak2 and prohibitin (Phb)-optic atrophy protein 1 (Opa1) on mitochondria and improving the structural integrity and function of mitochondria (P < 0.05). Interestingly, in the presence of Irak2 knockdown, Adipsin overexpression did not further alleviate myocardial mitochondrial destruction and cardiac dysfunction, suggesting a downstream role of Irak2 in Adipsin-induced responses (P < 0.05). Consistent with these findings, overexpression of Adipsin after Irak2 knockdown did not further reduce the accumulation of lipids and their metabolites in the cardiac myocardium, nor did it enhance the oxidation capacity of cardiomyocytes expose to palmitate (PA) (P < 0.05). These results indicated that Irak2 may be a downstream regulator of Adipsin. CONCLUSIONS: Adipsin improves fatty acid ß-oxidation and alleviates mitochondrial injury in DCM. The mechanism is related to Irak2 interaction and inhibition of Irak2 mitochondrial translocation.

MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.

AIM: Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored. METHODS: Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms. RESULTS: Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts. CONCLUSION: Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

SEVERE BURN-INDUCED MITOCHONDRIAL RECRUITMENT OF CALPAIN CAUSES ABERRANT MITOCHONDRIAL DYNAMICS AND HEART DYSFUNCTION.

ABSTRACT: Mitochondrial damage is an important cause of heart dysfunction after severe burn injury. However, the pathophysiological process remains unclear. This study aims to examine the mitochondrial dynamics in the heart and the role of µ-calpain, a cysteine protease, in this scenario. Rats were subjected to severe burn injury treatment, and the calpain inhibitor MDL28170 was administered intravenously 1 h before or after burn injury. Rats in the burn group displayed weakened heart performance and decreased mean arterial pressure, which was accompanied by a diminishment of mitochondrial function. The animals also exhibited higher levels of calpain in mitochondria, as reflected by immunofluorescence staining and activity tests. In contrast, treatment with MDL28170 before any severe burn diminished these responses to a severe burn. Burn injury decreased the abundance of mitochondria and resulted in a lower percentage of small mitochondria and a higher percentage of large mitochondria. Furthermore, burn injury caused an increase in the fission protein DRP1 in the mitochondria and a decrease in the inner membrane fusion protein OPA1. Similarly, these alterations were also blocked by MDL28170. Of note, inhibition of calpain yielded the emergence of more elongated mitochondria along with membrane invagination in the middle of the longitude, which is an indicator of the fission process. Finally, MDL28170, administered 1 h after burn injury, preserved mitochondrial function and heart performance, and increased the survival rate. Overall, these results provided the first evidence that mitochondrial recruitment of calpain confers heart dysfunction after severe burn injury, which involves aberrant mitochondrial dynamics.

Fibroblast-specific activation of Rnd3 protects against cardiac remodeling in diabetic cardiomyopathy via suppression of Notch and TGF-ß signaling.

Rationale: Extracellular matrix (ECM) remodeling, a key pathological feature in diabetic cardiomyopathy (DCM), is triggered by oxidative stress, inflammation, and various metabolic disorders in the heart. Cardiac fibroblasts (CFs) are the primary source of ECM proteins and the ultimate effector cells in ECM remodeling. CFs are turned on and differentiated into myofibroblasts in response to profibrotic signaling. Rnd3 is a small Rho-GTPase involved in the regulation of cell-cycle distribution, cell migration, and cell morphogenesis. Emerging evidence suggests a link between Rnd3 expression and onset of cardiovascular diseases. However, the role of Rnd3 in DCM remains unknown. Methods: Flow cytometry was employed to separate different types of cardiac cells. Type 2 diabetes mellitus was established in Rnd3 fibroblast-specific knockout and transgenic mice. RNA sequencing and chromatin immunoprecipitation assay were used to discern signaling pathways involved. Results: Rnd3 expression was reduced in cardiac tissues of diabetic mice, with CFs being the primary cell type. Fibroblast-specific upregulation of Rnd3 in vivo was protective against DCM, whereas Rnd3 downregulation in fibroblasts accentuated cardiac oxidative stress, fibrosis, ventricular remodeling, and dysfunction. Moreover, in vitro Rnd3 overexpression significantly attenuated reactive oxygen species production, CF migration and proliferation under high levels of glucose (35 mmol/L) and palmitic acid (500 µmol/L) challenge. Furthermore, RNA sequencing indicated that Notch and TGF-ß signaling were significantly suppressed upon Rnd3 overexpression. Mechanistically, Rnd3 regulated Notch and TGF-ß signaling by interacting with NICD and ROCK1, respectively. Specifically, glucotoxicity and lipotoxicity control Rnd3 expression by regulating the binding of Nr1H2 and Rnd3 promoter. Conclusions: Our findings provide compelling evidence in that fibroblast-specific activation of Rnd3 protects against cardiac remodeling in DCM, indicating promises of targeting Rnd3 in the treatment of DCM.

Unravelling relationships between fluorescence spectra, molecular weight distribution and hydrophobicity fraction of dissolved organic matter in municipal wastewater.

The characteristics of dissolved organic matter (DOM) in the influent and secondary effluent from 6 municipal wastewater treatment plants (WWTPs) were investigated with a size exclusion chromatogram (SEC) coupled with multiple detectors to simultaneously detect ultraviolet absorbance, fluorescence, dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) as a function of molecular weight (MW). The SEC chromatograms showed that biopolymers (>6 kDa) and humic substances (0.5-6 kDa) comprised the significant fraction in the influent, while humic substances became the abundant proportion in the secondary effluent. Direct linkages between MW distribution and hydrophobicity of DOM in the secondary effluent were further explored via SEC analysis of XAD resin fractions. DON and DOC with different hydrophobicity exhibited significantly distinct MW distribution, indicating that it was improper to consider DOC as a surrogate for DON. Different from DOC, the order of averaged MW in terms of DON was hydrophobic neutral ≈ transphilic neutral > hydrophobic acid > transphilic acid > hydrophilic fraction. Fluorescence spectral properties exhibited a significant semi-quantitative correlation with MW and hydrophobicity of DOC, with Pearson's coefficients of -0.834 and 0.754 (p < 0.01) for biopolymer and humic substances. Meanwhile, regional fluorescence proportion was demonstrated to indicate the MW and hydrophobicity properties of DON at the semi-quantitative level. The fluorescence excitation-emission matrix (EEM) could be explored to provide a rapid estimation of MW distribution and hydrophobic/hydrophilic proportion of DOC and DON in WWTPs.

Exosomes derived from pericardial adipose tissues attenuate cardiac remodeling following myocardial infarction by Adipsin-regulated iron homeostasis.

As a vital adipokine, Adipsin is closely associated with cardiovascular risks. Nevertheless, its role in the onset and development of cardiovascular diseases remains elusive. This study was designed to examine the effect of Adipsin on survival, cardiac dysfunction and adverse remodeling in the face of myocardial infarction (MI) injury. In vitro experiments were conducted to evaluate the effects of Adipsin on cardiomyocyte function in the face of hypoxic challenge and the mechanisms involved. Our results showed that Adipsin dramatically altered expression of proteins associated with iron metabolism and ferroptosis. In vivo results demonstrated that Adipsin upregulated levels of Ferritin Heavy Chain (FTH) while downregulating that of Transferrin Receptor (TFRC) in peri-infarct regions 1 month following MI. Adipsin also relieved post-MI-associated lipid oxidative stress as evidenced by decreased expression of COX2 and increased GPX4 level. Co-immunoprecipitation and immunofluorescence imaging prove a direct interaction between Adipsin and IRP2. As expected, cardioprotection provided by Adipsin depends on the key molecule of IRP2. These findings revealed that Adipsin could be efficiently delivered to the heart by exosomes derived from pericardial adipose tissues. In addition, Adipsin interacted with IRP2 to protect cardiomyocytes against ferroptosis and maintain iron homeostasis. Therefore, Adipsin-overexpressed exosomes derived from pericardial adipose tissues may be a promising therapeutic strategy to prevent adverse cardiac remodeling following ischemic heart injury.

The Direct Anterior Approach versus the Posterolateral Approach on the Outcome of Total Hip Arthroplasty: A Retrospective Clinical Study.

OBJECTIVE: To compare the clinical results of the direct anterior approach (DAA) and posterolateral approach (PLA) in total hip arthroplasty (THA) patients. METHODS: From January 2017 to September 2019, 80 patients who received primary THA in our hospital were retrospectively selected based on the propensity score matching (PSM) method. Baseline characteristics of patients who underwent the DAA and PLA were collected. Moreover, the incision length, intraoperative blood loss, operative time, length of stay, and Harris hip score were compared between patients in the two groups. The CK level was used to assess muscle damage between patients in the DAA and PLA groups. The complications of these two approaches were also evaluated at patients' 12-month follow-up evaluation. RESULTS: There was no significant difference in baseline characteristics between patients in the two groups (p > 0.05). The patients in the DAA group had a shorter incision length (9.2 ± 0.2 vs 14.7 ± 0.5, respectively; p < 0.05) and shorter length of hospital stay (9.5 ± 0.7 vs 12.9 ± 0.8, respectively, p < 0.05) than patients in the PLA group. Moreover, the DAA was associated with a decrease in intraoperative blood loss compared with the PLA (109.1 ± 12.6 vs 305.1 ± 14.1 ml, respectively, p < 0.05). However, the operation time was longer in patients in the DAA group (130.7 ± 1.7) than in patients in the PLA group (112.6 ± 1.3 min, p < 0.05). The CK level of patients in the DAA group was lower than that of patients in the PLA group (p < 0.05). The CK level at 48 h post-surgery was negatively correlated with the Harris hip scores at 6 months after THA (r = -0.538, p = 0.000). Compared with patients in the PLA group, the muscle strength of patients in the DAA group was significantly higher than that of patients in the DAA group at 4 days (p < 0.05) and 7 days (p < 0.05) after THA. The Harris hip scores of patients in the DAA group and PLA group were 81.0 ± 0.8 vs 70.8 ± 0.7 at 6 weeks, 93.4 ± 0.9 vs 86.4 ± 0.6 at 3 months, and 96.8 ± 1.1 vs 93.4 ± 0.8 at 6 months, respectively, both p < 0.05. There was no significant difference in the incidence of complications between patients in the DAA and PLA groups (p > 0.05). CONCLUSION: DAA was superior to the PLA in improving hip function after THA. Compared with the PLA, the DAA could reduce muscle damage, which is negatively correlated with hip function. Further multi-institution studies are required with longer follow-up durations, and larger patient populations are needed to provide more definitive conclusions.

Opportunities and challenges of pain-related myocardial ischemia-reperfusion injury.

Pain is one of the most serious problems plaguing human health today. Pain is not an independent pathophysiological condition and is associated with a high impact on elevated disability and organ dysfunction. Several lines of evidence suggested the associations of pain with cardiovascular diseases, especially myocardial ischemia-reperfusion (I/R) injury, while the role of pain in I/R injury and related mechanisms are not yet comprehensively assessed. In this review, we attempted to explore the role of pain in myocardial I/R injury, and we concluded that acute pain protects myocardial ischemia-reperfusion injury and chronic pain aggravates cardiac ischemia-reperfusion injury. In addition, the construction of different pain models and animal models commonly used to study the role of pain in myocardial I/R injury were discussed in detail, and the potential mechanism of pain-related myocardial I/R injury was summarized. Finally, the future research direction was prospected. That is, the remote regulation of pain to cardiac function requires peripheral pain signals to be transmitted from the peripheral to the cardiac autonomic nervous system, which then affects autonomic innervation during cardiac ischemia-reperfusion injury and finally affects the cardiac function.

Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch.

Imbalanced mitochondrial dynamics including inhibited mitochondrial fusion is associated with cardiac dysfunction as well as tumorigenesis. This study sought to explore the effects of promoting mitochondrial fusion on doxorubicin(Dox)-induced cardiotoxicity and its antitumor efficacy, with a focus on the underlying metabolic mechanisms. Herein, the inhibition of Mfn2-mediated mitochondrial fusion was identified as a key phenotype in Dox-induced cardiotoxicity. Restoration of Mfn2-mediated mitochondrial fusion enhanced mitochondrial oxidative metabolism, reduced cellular injury/apoptosis and inhibited mitochondria-derived oxidative stress in the Dox-treated cardiomyocytes. Application of lentivirus expressing Drp1 (mitochondrial fusion inhibitor) or Rote/Anti A (mitochondrial complex I/III inhibitors) blunted the above protective effects of Mfn2. Cardiac-specific Mfn2 transgenic mice showed preserved mitochondrial fusion and attenuated myocardial injury upon Dox exposure in vivo. The suppression of Mfn2-mediated mitochondrial fusion was induced by Dox-elicited upregulation of FoxO1, which inhibited the transcription of Mfn2 by binding to its promoter sites. In the B16 melanoma, Mfn2 upregulation not only attenuated tumor growth alone but also further delayed tumor growth in the presence of Dox. Mechanistically, Mfn2 synergized with the inhibitory action of Dox on glycolysis metabolism in the tumor cells. One common feature in both cardiomyocytes and tumor cells was that Mfn2 increased the ratio of oxygen consumption rate to extracellular acidification rate, suggesting Mfn2 triggered a shift from aerobic glycolysis to mitochondrial oxidative metabolism. In conclusion, targeting Mfn2-mediated mitochondrial fusion may provide a dual therapeutic advantage in Dox-based chemotherapy by simultaneously defending against Dox-induced cardiotoxicity and boosting its antitumor potency via metabolic shift.

Impairment of µ-calpain activation by rhTNFR:Fc reduces severe burn-induced membrane disruption in the heart.

Stress cardiomyopathy is a major clinical complication after severe burn. Multiple upstream initiators have been identified; however, the downstream targets are not fully understood. This study assessed the role of the plasma membrane in this process and its relationship with the protease µ-calpain and tumor necrosis factor-alpha (TNF-α). Here, third-degree burn injury of approximately 40% of the total body surface area was established in rats. Plasma levels of LDH and cTnI and cardiac cell apoptosis increased at 0.5 h post burn, reached a peak at 6 h, and gradually declined at 24 h. This effect correlated well with not only the disruption of cytoskeletal proteins, including dystrophin and ankyrin-B, but also with the activation of µ-calpain, as indicated by the cleaved fragments of α-spectrin and membrane recruitment of the catalytic subunit CAPN1. More importantly, these alterations were diminished by blocking calpain activity with MDL28170. Burn injury markedly increased the cellular uptake of Evans blue, indicating membrane integrity disruption, and this effect was also reversed by MDL28170. Compared with those in the control group, cardiac cells in the burn plasma-treated group were more prone to damage, as indicated by a marked decrease in cell viability and increases in LDH release and apoptosis. Of note, these alterations were mitigated by CAPN1 siRNA. Moreover, after neutralizing TNF-α with rhTNFR:Fc, calpain activity was blocked, and heart function was improved. In conclusion, we identified µ-calpain as a trigger for severe burn-induced membrane disruption in the heart and provided evidence for the application of rhTNFR:Fc to inhibit calpain for cardioprotection.

Lockdown in Chinese university dormitories: Significant increase in negative feelings and time perception distortion.

The COVID-19 pandemic has led to a global crisis resulting in lockdowns worldwide. Studies found that these could amplify negative affects predictive of disturbances in time perception. However, all existing studies on this topic concentrated on the general population during a lockdown at home. There was no research on university students living in dormitories. This study fills a gap in the literature by investigating the affective states and awareness of the passage of time of Chinese university students during an exceptional dormitory lockdown. Using a snowball sampling approach, this web-based study recruited 225 volunteers (136 girls, 17-27 years old) during a dormitory lockdown. The online survey consisted of questions on demographic information, affective states (the Chinese version of the depression anxiety stress scales-21 (DASS-21) on depression, anxiety, and stress), and sense of the passage of time (the translated version of the assessment of passage of time over daily, weekly and yearly periods). The researchers shared the questionnaire link via the social media application WeChat. The study applied descriptive statistics and column charts to represent students' affective states and sense of the passage of time under extreme conditions and Pearson's correlation analysis to examine the impact of affective states on their sense of time. The study's results indicate that the dormitory lockdown caused a significant increase in all depression, anxiety, and stress categories of the DASS-21, as well as a distortion of the sense of time passing. However, the direction of the distortion differed from that in previous studies. Moreover, a Pearson correlation analysis showed that none of the three negative affects could be associated with students' sense of time. The findings of this study indicate an increased vulnerability of students due to the lockdown. Their emotions were strongly affected, requiring particular interventions to preserve their mental well-being. In addition, they showed a different distortion direction of time passage. Finally, they differed in the correlations between negative affect and the sense of passage of time compared to the general population in lockdown at home. This shows a need to investigate the impact of psychological states on students' routines and quality of life in emergencies. This study's scope should be expanded by incorporating additional factors pertaining to the passage of time and by integrating cross-cultural comparisons.

Paeonol promotes Opa1-mediated mitochondrial fusion via activating the CK2α-Stat3 pathway in diabetic cardiomyopathy.

Diabetes disrupts mitochondrial function and often results in diabetic cardiomyopathy (DCM). Paeonol is a bioactive compound that has been reported to have pharmacological potential for cardiac and mitochondrial protection. This study aims to explore the effects of paeonol on mitochondrial disorderes in DCM and the underlying mechanisms. We showed that paeonol promoted Opa1-mediated mitochondrial fusion, inhibited mitochondrial oxidative stress, and preserved mitochondrial respiratory capacity and cardiac performance in DCM in vivo and in vitro. Knockdown of Opa1 blunted the above protective effects of paeonol in both diabetic hearts and high glucose-treated cardiomyocytes. Mechanistically, inhibitor screening, siRNA knockdown and chromatin immunoprecipitation experiments showed that paeonol-promoted Opa1-mediated mitochondrial fusion required the activation of Stat3, which directly bound to the promoter of Opa1 to upregulate its transcriptional expression. Moreover, pharmmapper screening and molecular docking studies revealed that CK2α served as a direct target of paeonol that interacted with Jak2 and induced the phosphorylation and activation of Jak2-Stat3. Knockdown of CK2α blunted the promoting effect of paeonol on Jak2-Stat3 phosphorylation and Opa1-mediated mitochondrial fusion. Collectively, we have demonstrated for the first time that paeonol is a novel mitochondrial fusion promoter in protecting against hyperglycemia-induced mitochondrial oxidative injury and DCM at least partially via an Opa1-mediated mechanism, a process in which paeonol interacts with CK2α and restores its kinase activity that subsequently increasing Jak2-Stat3 phosphorylation and enhancing the transcriptional level of Opa1. These findings suggest that paeonol or the promotion of mitochondrial fusion might be a promising strategy for the treatment of DCM.

Activation of κ-opioid receptor inhibits inflammatory response induced by sodium palmitate in human umbilical vein endothelial cells.

OBJECTIVES: The current study aims to investigate the effect of κ-opioid receptor (κ-OR) activation on sodium palmitate (SP)-induced human umbilical vein endothelial cells (HUVECs) inflammatory response and elucidate the underlying mechanisms. METHODS: A hyperlipidemic cell model was established and treated with κ-OR agonist (U50,488H), and antagonist (norbinaltorphimine, nor-BNI), or inhibitors targeting PI3K, Akt or eNOS (LY294002, MK2206-2HCl or L-NAME, respectively). Furthermore, the expression levels of NLRP3, caspase-1, p-Akt, Akt, p-eNOS, and total eNOS were evaluated. Additionally, the production of reactive oxygen species, and levels of inflammatory factors, such as TNF-α, IL-1ß, IL-6, IL-1 and adhesion molecules, such as ICAM-1, VCAM-1, P-selectin, and E-selectin were determined. The adherence rates of the neutrophils and monocytes were assessed as well. RESULTS: The SP-induced hyperlipidemic cell model demonstrated increased expression of NLRP3 and caspase-1 proteins (P < 0.05) and elevated ROS levels (P < 0.01), and decreased phosphorylated-Akt and phosphorylated-eNOS expression (P < 0.05). In addition, SP significantly increased TNF-α, IL-1ß, IL-6, ICAM-1, VCAM-1, P-selectin, and E-selectin levels (P < 0.01), decreased IL-10 levels (P < 0.01), and increased the adhesion rates of monocytes and neutrophils (P < 0.01). The SP-induced inflammatory response in HUVECs was ameliorated by κ-OR agonist, U50,488H. However, the protective effect of U50,488H was abolished by κ-OR antagonist, nor-BNI, and inhibitors of PI3K, Akt and eNOS. CONCLUSION: Our findings suggest that κ-OR activation inhibits SP-induced inflammation by activating the PI3K/Akt/eNOS signaling pathway.

Characterization of fragment sizes, copy number aberrations and 4-mer end motifs in cell-free DNA of hepatocellular carcinoma for enhanced liquid biopsy-based cancer detection.

Circulating cell-free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole-genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (< 150 bp) with tumor fraction (TF), copy number variation (CNV) alterations and the change in the proportion of 4-mer end motifs in HCC and HBV samples. Our analyses identified five typical CNV markers (i.e. loss in chr1p, chr4q and chr8p, and gain in chr1q and chr8q) in cfDNA with a cumulatively positive rate of ˜ 95% in HCC samples. Size selection (< 150 bp) significantly enhanced TF and CNV signals in HCC samples. Additionally, three 4-mer end motifs (CCCA, CCTG and CCAG) were identified as preferred end motifs in HCC samples. We identified 139 end motifs significantly associated with fragment size that showed similar patterns of associations between patients with HCC and HBV, suggesting that end motifs might be inherently coupled with fragment size by a ubiquitous mechanism. Here we conclude that CNV markers, fragment size selection and end-motif pattern in cfDNA have potential for effective detection of patients with HCC.

κ-opioid receptor stimulation alleviates rat vascular smooth muscle cell calcification via PFKFB3-lactate signaling.

In the present study, the effects and mechanism of action of U50,488H (a selective κ-opioid receptor agonist) on calcification of rat vascular smooth muscle cells (VSMCs) induced by ß-glycerophosphate (ß-GP) were investigated. VSMCs were isolated and cultured in traditional FBS-based media. A calcification model was established in VSMCs under hyperphosphatemia and intracellular calcium contents. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and lactate were detected in cell culture supernatants before and after treatment. Alizarin red staining was used to detect the degree of calcification of VSMCs. Expression levels of key molecules of osteogenic markers, fructose-2,6-biphosphatase 3 (PFKFB3), and proline hydroxylase 2 (PHD2), were determined using western blotting. Further, vascular calcification was induced by vitamin D3 plus nicotine in rats and isolated thoracic aortas, calcium concentration was assessed in rat aortic rings in vitro. We demonstrated that U50,488H inhibited VSMC calcification in a concentration-dependent manner. Moreover, U50,488H significantly inhibited osteogenic differentiation and ALP activity in VSMCs pretreated with ß-GP. Further studies confirmed that PFKFB3 expression, LDH level, and lactate content significantly increased during calcification of VSMCs; U50,488H reversed these changes. PHD2 expression showed the opposite trend compared to PFKFB3 expression. nor-BNI or 3-PO abolished U50,488H protective effects. Besides, U50,488H inhibited VSMC calcification in rat aortic rings ex vivo. Collectively, our experiments show that κ-opioid receptor activation inhibits VSMC calcification by reducing PFKFB3 expression and lactate content, providing a potential drug target and strategy for the clinical treatment of vascular calcification.

Genetic ablation of fas-activated serine/threonine kinase ameliorates alcoholic liver disease through modulating HuR-SIRT1 mRNA complex stability.

Chronic alcoholism often causes liver injuries characterized by hepatic steatosis, inflammation as well as oxidative stress and finally leads to advanced cirrhosis and liver cancer. Fas-activated serine/threonine kinase (FASTK) and its homologs are gradually known as multifunctional proteins involved in various biological processes; however, the role of FASTK and its family members in alcoholic liver disease (ALD) is still unexplored. Here we found that, among FASTK family members, the expression of FASTK was specifically induced both in livers of mice received chronic ethanol ingestion and in ethanol-stimulated hepatocytes. Animal studies showed that genetic deletion of FASTK attenuated chronic ethanol ingestion-induced liver damage, steatosis, and inflammation. Moreover, FASTK deficiency was associated with improved oxidative/anti-oxidative system homeostasis and reduced reactive oxygen species (ROS) generation in livers upon chronic ethanol stimulation. Importantly, FASTK ablation preserved hepatic sirtuin-1 (SIRT1) expression/activity upon chronic ethanol ingestion and SIRT1 silencing via adenovirus-mediated small interfering RNA transfer diminished FASTK deletion-elicited beneficial effects on alcohol-associated hepatic steatosis, inflammation, and oxidative stress. Mechanistically, ethanol increased the phosphorylation of human antigen R (HuR, a RNA binding protein that stabilizes SIRT1 mRNA) and triggered the dissociation of HuR-SIRT1 mRNA complex, in turn promoting SIRT1 mRNA decay. Genetic deletion of FASTK diminished ethanol-induced HuR phosphorylation and HuR-SIRT1 mRNA complex dissociation, thereby enhancing SIRT1 mRNA stability. Collectively, these findings for the first time highlight a critical role of FASTK in the pathogenesis of ALD and implicate HuR-SIRT1 mRNA complex involves in this process.

SHANK3 Co-ordinately Regulates Autophagy and Apoptosis in Myocardial Infarction.

Cardiac remodeling and dysfunction are responsible for the high mortality after myocardial infarction (MI). We assessed the potential for Shank3 to alleviate the post-infarction cardiac dysfunction. The experimental MI mice model was constructed by left anterior descending coronary artery ligation. Shank3 knockout aggravated cardiac dysfunction after MI, while Shank3 overexpression alleviated it. The histological examination showed that the infarct size was significantly increased in the acute phase of MI in the Shank3 knockout group, and the cardiac dysfunction of the Shank3 knockout group was even more severe than the Shank3 overexpression group, revealed by echocardiography analyses. In vitro, cultured neonatal cardiomyocytes were subjected to simulated MI. Shank3 downregulation curbed LC3 expression and autophagosome-lysosome fusion. Furthermore, Shank3 downregulation increased cardiomyocyte apoptosis. In contrast, Shank3 upregulation induced autophagy, and inhibited apoptosis under hypoxia. In vivo, western blot analysis showed decreased levels of Atg7, Beclin1, LC3-II, and Bcl-2 as well as increased expression of p62, cleaved caspase-3, and cleaved caspase-9 in the Shank3 knockout group which suffered from MI. On the other hand, it also revealed that Shank3 overexpression induced autophagy and inhibited apoptosis after MI. Shank3 may serve as a new target for improving cardiac function after MI by inducing autophagy while inhibiting apoptosis.

CircTADA2A suppresses the progression of colorectal cancer via miR-374a-3p/KLF14 axis.

BACKGROUND: Colorectal cancer (CRC) is one of the causes of cancer-related death worldwide. The aim of our study was to disclose the expression pattern and underlying molecular mechanism of circular RNA TADA2A (circTADA2A) in CRC. METHODS: The levels of circTADA2A, transcriptional adaptor 2A (TADA2A), microRNA-374a-3p (miR-374a-3p) and Kruppel like factor 14 (KLF14) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Xenograft tumor assay was used to uncover the function of circTADA2A in vivo. The miRNA targets of circTADA2A were searched using circbank and starbase softwares, while DIANA TOOL was used to explore miR-374a-3p-mRNA interactions. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to validate the target relationship of circTADA2A/miR-374a-3p/KLF14 axis. Cell cycle and apoptosis were analyzed by flow cytometry. The glycolysis of CRC cells was determined by Seahorse XFe 96 Extracellular Flux Analyzer, Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II and ATP Colorimetric Assay kit. KLF14 protein level was measured by Western blot assay. RESULTS: CircTADA2A was abnormally down-regulated in CRC tissues and cell lines. CircTADA2A overexpression impeded CRC tumor growth in vivo. MiR-374a-3p was verified as a target of circTADA2A in CRC cells, and circTADA2A inhibited the malignant potential of CRC cells through targeting miR-374a-3p. MiR-374a-3p interacted with KLF14 messenger RNA (mRNA), and miR-374a-3p deteriorated CRC through down-regulating KLF14. CircTADA2A enhanced the abundance of KLF14 through targeting miR-374a-3p in CRC cells. CONCLUSION: CircTADA2A functioned as a tumor suppressor in CRC to inhibit the glycolysis and cell cycle and potentiate the apoptosis of CRC cells via miR-374a-3p/KLF14 axis.