The enigmatic interplay of immune cells and abnormal spermatozoa through Mendelian randomization.

PURPOSE: Abnormal spermatozoa significantly impact reproductive health, affecting fertility rates, potentially prolonging conception time, and increasing the risk of miscarriages. This study employs Mendelian randomization to explore their potential link with immune cells, aiming to reveal their potential causal association and wider implications for reproductive health. METHODS: We conducted forward and reverse Mendelian randomization analyses to explore the potential causal connection between 731 immune cell signatures and abnormal spermatozoa. Using publicly available genetic data, we investigated various immune signatures such as median fluorescence intensities (MFI), relative cell (RC), absolute cell (AC), and morphological parameters (MP). Robustness was ensured through comprehensive sensitivity analyses assessing consistency, heterogeneity, and potential horizontal pleiotropy. The MR study produced a statistically significant p-value of .0000684, Bonferroni-corrected for the 731 exposures. RESULTS: The Mendelian randomization analysis revealed strong indications of a reciprocal relationship between immune cell pathways and sperm integrity. When examining immune cell exposure, a potential causal link with abnormal sperm was observed in 35 different types of immune cells. Conversely, the reverse Mendelian randomization results indicated that abnormal sperm might causally affect 39 types of immune cells. These outcomes suggest a potential mutual influence between alterations in immune cell functionality and the quality of spermatozoa. CONCLUSION: This study highlights the close link between immune responses and sperm development, suggesting implications for reproductive health and immune therapies. Further research may offer crucial insights into male fertility and immune disorders.

N -N-Butyl Haloperidol Iodide Mitigates Myocardial Ischemia/Reperfusion Injury Through Activation of SIRT1-Nrf2 Signaling Loop.

ABSTRACT: N -n-butyl haloperidol iodide (F 2 ), a derivative of haloperidol developed by our group, exhibits potent antioxidative properties and confers protection against cardiac ischemia/reperfusion (I/R) injury. The protective mechanisms by which F 2 ameliorates I/R injury remain obscure. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor transactivating many antioxidative genes, also attenuates I/R-induced myocardial damage. The present study investigated whether the cardioprotective effect of F 2 depends on Nrf2 using a mouse heart I/R model. F 2 (0.1, 0.2 or 0.4 mg/kg) or vehicle was intravenously injected to mice 5 minutes before reperfusion. Systemic administration of 0.4 mg/kg F 2 led to a significant reduction in I/R injury, which was accompanied by enhanced activation of Nrf2 signaling. The cardioprotection conferred by F 2 was largely abrogated in Nrf2-deficient mice. Importantly, we found F 2 -induced activation of Nrf2 is silent information regulator of transcription 1 (SIRT1)-dependent, as pharmacologically inhibiting SIRT1 by the specific inhibitor EX527 blocked Nrf2 activation. Moreover, F 2 -upregulated expression of SIRT1 was also Nrf2-dependent, as Nrf2 deficiency inhibited SIRT1 upregulation. These results indicate that SIRT1-Nrf2 signaling loop activation is indispensable for the protective effect of F 2 against myocardial I/R injury and may provide new insights for the treatment of ischemic heart disease.

N-n-butyl haloperidol iodide mediates cardioprotection via regulating AMPK/FoxO1 signalling.

Derangement of redox condition largely contributes to cardiac ischemia/reperfusion (I/R) injury. FoxO1 is a transcription factor which transcripts a series of antioxidants to antagonize I/R-induced oxidative myocardial damage. N-n-butyl haloperidol iodide (F2 ) is a derivative derived from haloperidol structural modification with potent capacity of inhibiting oxidative stress. This investigation intends to validate whether cardio-protection of F2 is dependent on FoxO1 using an in vivo mouse I/R model and if so, to further elucidate the molecular regulating mechanism. This study initially revealed that F2 preconditioning led to a profound reduction in I/R injury, which was accompanied by attenuated oxidative stress and upregulation of antioxidants (SOD2 and catalase), nuclear FoxO1 and phosphorylation of AMPK. Furthermore, inactivation of FoxO1 with AS1842856 abolished the cardio-protective effect of F2 . Importantly, we identified F2 -mediated nuclear accumulation of FoxO1 is dependent on AMPK, as blockage of AMPK with compound C induced nuclear exit of FoxO1. Collectively, our data uncover that F2 pretreatment exerts significant protection against post ischemic myocardial injury by its regulation of AMPK/FoxO1 pathway, which may provide a new avenue for treating ischemic disease.

Development and validation of a nomogram to predict lung metastasis in patients with testicular germ cell tumors.

Background: Lung metastatic tumor (LM) is one of testicular germ cell tumors' most common metastatic sites. Our study aimed to develop a nomogram for predicting the risk of LM among patients with testicular germ cell tumors (TGCTs). Methods: Clinicopathological information of 4078 patients with TGCT between 2010 and 2015 was obtained from SEER. Univariate and multivariate logistic regression analyses were performed to identify risk factors for LM, and a nomogram was developed based on these factors. Calibration curves, area under the receiver operating curve (AUC), and decision curve analysis (DCA) were used to evaluate the accuracy and discrimination of the model. Results: Study participants included 4078 people with TGCTs, including 305 people with LM. They were randomly divided into two groups (training cohort = 2854 and validation cohort = 1224) at a ratio of 7:3. The following variables were incorporated in the nomogram: marital status, tumor histological type, T stage, brain metastasis, liver metastasis, lactate dehydrogenase (LDH), and chemotherapy. Besides, the AUC of it was 0.922 in the training cohort, while was 0.930 in the validation cohort. Training and validation cohort calibrations showed that the nomogram had excellent predictive abilities. DCA suggested it was more clinically relevant than the traditional TN staging. Conclusion: We have established a nomogram to predict the risk of LM in patients with TGCTs. Doctors and patients can use this nomogram to monitor and identify lung metastasis of tumors through active monitoring and follow-up.

Prognostic and tumor microenvironmental feature of clear cell renal cell carcinoma revealed by m6A and lactylation modification-related genes.

Background: Both lactylation and m6A modification have important implications for the development of clear cell renal cell carcinoma (ccRCC), and we aimed to use crosstalk genes of both to reveal the prognostic and immunological features of ccRCC. Methods: Our first step was to look for lactylation-related genes that differed between normal and tumor tissues, and then by correlation analysis, we found the genes associated with M6A. Following that, ccRCC subtypes will be identified and risk models will be constructed to compare the prognosis and tumor microenvironment among different subgroups. A nomogram was constructed to predict the prognosis of ccRCC, and in vitro, experiments were conducted to validate the expression and function of key genes. Results: We screened 100 crosstalk genes and identified 2 ccRCC subtypes. A total of 11 prognostic genes were screened for building a risk model. we observed higher immune scores, elevated tumor mutational burden, and microsatellite instability scores in the high-risk group. Therefore, individuals classified as high-risk would derive greater benefits from immunotherapy. The nomogram's ability to predict overall survival with a 1-year AUC of 0.863 demonstrates its significant practical utility. In addition, HIBCH was identified as a potential therapeutic target and its expression and function were verified by in vitro experiments. Conclusion: In addition to developing a precise prognostic nomogram for patients with ccRCC, our study also discovered the potential of HIBCH as a biomarker for the disease.

Based on disulfidptosis, revealing the prognostic and immunological characteristics of renal cell carcinoma with tumor thrombus of vena cava and identifying potential therapeutic target AJAP1.

BACKGROUND: Patients with clear cell renal cell carcinoma (ccRCC) with venous tumor thrombus have a poor prognosis, high surgical risk, and lack of targeted therapeutic agents. METHODS: Genes with consistent differential expression trends in tumor tissues and VTT groups were first screened, and then differential genes associated with disulfidptosis were found by correlation analysis. Subsequently, identifying ccRCC subtypes and constructing risk models to compare the differences in prognosis and the tumor microenvironment in different subgroups. Finally, constructing a nomogram to predict the prognosis of ccRCC and validate key gene expression levels in cells and tissues. RESULTS: We screened 35 differential genes related to disulfidptosis and identified 4 ccRCC subtypes. Risk models were constructed based on the 13 genes, and the high-risk group had a higher abundance of immune cell infiltration, tumor mutational load, and microsatellite instability scores, predicting high sensitivity to immunotherapy. The 1-year AUC = 0.869 for predicting OS by nomogram has a high application value. The expression level of the key gene AJAP1 was low in both tumor cell lines and cancer tissues. CONCLUSIONS: Our study not only constructed an accurate prognostic nomogram for ccRCC patients but also identified an AJAP1 biomarker as a potential biomarker for the disease.

Effect of polymorphisms in drug metabolism and transportation on plasma concentration of atorvastatin and its metabolites in patients with chronic kidney disease.

Dyslipidemia due to renal insufficiency is a common complication in patients with chronic kidney diseases (CKD), and a major risk factor for the development of cardiovascular events. Atorvastatin (AT) is mainly used in the treatment of dyslipidemia in patients with CKD. However, response to the atorvastatin varies inter-individually in clinical applications. We examined the association between polymorphisms in genes involved in drug metabolism and transport, and plasma concentrations of atorvastatin and its metabolites (2-hydroxy atorvastatin (2-AT), 2-hydroxy atorvastatin lactone (2-ATL), 4-hydroxy atorvastatin (4-AT), 4-hydroxy atorvastatin lactone (4-ATL), atorvastatin lactone (ATL)) in kidney diseases patients. Genotypes were determined using TaqMan real time PCR in 212 CKD patients, treated with 20 mg of atorvastatin daily for 6 weeks. The steady state plasma concentrations of atorvastatin and its metabolites were quantified using ultraperformance liquid chromatography in combination with triple quadrupole mass spectrometry (UPLC-MS/MS). Univariate and multivariate analyses showed the variant in ABCC4 (rs3742106) was associated with decreased concentrations of AT and its metabolites (2-AT+2-ATL: ß = -0.162, p = 0.028 in the dominant model; AT+2-AT+4-AT: ß = -0.212, p = 0.028 in the genotype model), while patients carrying the variant allele ABCC4-rs868853 (ß = 0.177, p = 0.011) or NR1I2-rs6785049 (ß = 0.123, p = 0.044) had higher concentrations of 2-AT+2-ATL in plasma compared with homozygous wildtype carriers. Luciferase activity was enhanced in HepG2 cells harboring a construct expressing the rs3742106-T allele or the rs868853-G allele (p < 0.05 for each) compared with a construct expressing the rs3742106G or the rs868853-A allele. These findings suggest that two functional polymorphisms in the ABCC4 gene may affect transcriptional activity, thereby directly or indirectly affecting release of AT and its metabolites from hepatocytes into the circulation.

A nomogram model for determining optimal patients for local therapy in metastatic prostate cancer: a SEER database-based study.

BACKGROUND: Numerous studies have shown that local therapy can improve long-term survival in patients with metastatic prostate cancer. However, it is unclear which patients are the potential beneficiaries. METHODS: We obtained information on prostate cancer patients from the Surveillance, Epidemiology, and End Results database and divided eligible patients into the local treatment group and non-local treatment group. Propensity score matching (PSM) was used to reduce the influence of confounding factors. In the matched local treatment (LT) group, if the median overall survival time (OS) was longer than the Nonlocal treatment (NLT) group, it was defined as a benefit group, otherwise, it was a non-benefit group. Then, univariate and multivariate logistic regression were used to screen out predictors associated with benefits, and a nomogram model was constructed based on these factors. The accuracy and clinical value of the models were assessed through calibration plots and decision curve analysis. RESULTS: The study enrolled 7255 eligible patients, and after PSM, each component included 1923 patients. After matching, the median OS was still higher in the LT group than in the NLT group [42 (95% confidence interval: 39-45) months vs 40 (95% confidence interval: 38-42) months, p = 0.03]. The independent predictors associated with benefit were age, PSA, Gleason score, T stage, N stage, and M stage. The nomogram model has high accuracy and clinical application value in both the training set (C-index = 0.725) and the validation set (C-index = 0.664). CONCLUSIONS: The nomogram model we constructed can help clinicians identify patients with potential benefits from LT and formulate a reasonable treatment plan.

Resveratrol, novel application by preconditioning to attenuate myocardial ischemia/reperfusion injury in mice through regulate AMPK pathway and autophagy level.

Myocardial ischemia/reperfusion injury (MI/RI) is the main cause of deaths in the worldwide, leading to severe cardiac dysfunction. Resveratrol (RSV) is a polyphenol plant-derived compound. Our study aimed to elucidate the underlying molecular mechanism of preconditioning RSV in protecting against MI/RI. Mice were ligated and re-perfused by the left anterior descending branch with or without RSV (30 mg/kg·ip) for 7 days. Firstly, we found that RSV pretreatment significantly alleviated myocardial infarct size, improved cardiac function and decreased oxidative stress. Furthermore, RSV activated p-AMPK and SIRT1, ameliorated inflammation including the level of TNF-α and IL-1ß, and promoting autophagy level. Moreover, neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown the expression of AMPK, SIRT1 or FOXO1 were used to uncover the underlying molecular mechanism for the cardio-protection of RSV. In NRVMs, RSV increased cellular viability, decreased LDH release and reduced oxidative stress. Importantly, Compound C(CpC) and EX527 reversed the effect of RSV against MI/RI in vivo and in vitro and counteracted the autophagy level induced by RSV. Together, our study indicated that RSV could alleviate oxidative stress in cardiomyocytes through activating AMPK/SIRT1-FOXO1 signallingpathway and enhanced autophagy level, thus presenting high potential protection on MI/RI.

Geniposide suppresses NLRP3 inflammasome-mediated pyroptosis via the AMPK signaling pathway to mitigate myocardial ischemia/reperfusion injury.

BACKGROUND: NLRP3 inflammasome activation and pyroptosis play a significant role in myocardial ischemia reperfusion injury (MI/RI). Geniposide was reported to show potential therapeutic use for MI/RI with its anti-inflammatory and anti-oxidative properties. However, research on the specific mechanism of geniposide has not been reported. METHODS: The MIRI model of animal was created in male C57BL/6J mice and the hypoxia reoxygenation (H/R) model was established for the in vitro experiments. Neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown of TXNIP or NLRP3 were used. Geniposide was administered to mice before vascular ligation. HE staining, 2,3,5-triphenyltetrazolium chloride (TTC) staining, echocardiography, oxidative stress and myocardial enzyme detection were used to evaluate the cardioprotective effect of geniposide. Meanwhile, pharmacological approaches of agonist and inhibitor were used to observe potential pathway for geniposide cardioprotective in vitro and in vivo. Moreover, ELISA kits were adopted to detect the levels of inflammatory factors, such as IL-1ß and IL-18. The gene and protein expression of NLRP3 and pyroptosis-related factors in heart tissue were performed by RT-PCR, western blotting and immunofluorescence in vivo and in vitro, respectively. RESULTS: Our results indicate that geniposide can reduce the area of myocardial infarction, improve heart function, and inhibit the inflammatory response in mice after MI/RI. In addition, RT-PCR and western blotting shown geniposide promoting AMPK phosphorylation to activate myocardium energy metabolism and reducing the levels of genes and proteins expression of NLRP3, ASC, N-GSDMD and cleaved caspase-1, IL-1ß, IL-18. Meanwhile, geniposide improved NRVMs energy metabolism, which decreased ROS levels and the protein expression of TXNIP and thus suppressed the expression of NLRP3. AMPK antagonist or agonist and siRNA downregulation of TXNIP or NLRP3 were also verify the effect of geniposide against H/R injury. Further research found that geniposide promoted the translocation of TXNIP and reduce the binding of TXNIP and NLRP3. CONCLUSIONS: In our study, geniposide can significantly inhibit NLRP3 inflammasome activation via the AMPK signaling pathway and inhibit pyroptosis of cardiomyocytes in myocardial tissues.

LncRNA HOTAIR-mediated MTHFR methylation inhibits 5-fluorouracil sensitivity in esophageal cancer cells.

BACKGROUND: Esophageal cancer (EC) represents one of the most aggressive digestive neoplasms globally, with marked geographical variations in morbidity and mortality. Chemoprevention is a promising approach for cancer therapy, while acquired chemoresistance is a major obstacle impeding the success of 5-fluorouracil (5-FU)-based chemotherapy in EC, with the mechanisms underlying resistance not well-understood. In the present study, we focus on exploring the role of long non-coding RNA (lncRNA) HOTAIR in EC progression and sensitivity of EC cells to 5-FU. METHODS: Paired cancerous and pre-cancerous tissues surgically resected from EC patients were collected in this study. Promoter methylation of the MTHFR was assessed by methylation-specific PCR. RIP and ChIP assays were adopted to examine the interaction of DNA methyltransferases (DNMTs) with lncRNA HOTAIR and MTHFR, respectively. EC cells resistant to 5-FU were induced by step-wise continuous increasing concentrations of 5-FU. The sensitivity of EC cells to 5-FU in vivo was evaluated in nude mice treated with xenografts of EC cells followed by injection with 5-FU (i.p.). RESULTS: We found reciprocal expression patterns of lncRNA HOTAIR and MTHFR in EC tissues and human EC cells. Interference with lncRNA HOTAIR enhanced 5-FU-induced apoptosis, exhibited anti-proliferative activity, and reduced promoter methylation of the MTHFR in EC cells. Besides, overexpression of MTHFR attenuated the acquired chemoresistance induced by overexpression of lncRNA HOTAIR in EC cells. At last, enhanced chemosensitivity was observed in vivo once nude mice xenografted with lncRNA HOTAIR-depleted EC cells. CONCLUSION: Together, our study proposes that pharmacologic targeting of lncRNA HOTAIR sensitizes EC cells to 5-FU-based chemotherapy by attenuating the promoter hypermethylation of the MTHFR in EC.

High expression of forkhead box M1 (FOXM1) is a poor prognostic biomarker in lung adenocarcinoma.

BACKGROUND: Forkhead box M1 (FOXM1) is closely related to the formation and development of cancer. Because of differences in cellular origin, lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC) usually exhibit different signatures. Therefore, it is essential to investigate the abnormalities of FOXM1 in the two subtypes separately. METHODS: Through the Oncomine and TCGA databases, we investigated the expression of FOXM1 mRNA, its prognostic value and possible mechanisms leading to its dysregulation. Furthermore, networks involving FOXM1 and its significantly altered neighboring genes were identified using the cBioPortal database. GO and KEGG enrichment analyses were performed using DAVID. RESULTS: Expression of FOXM1 mRNA was higher in lung tumor tissues than in normal tissues, and higher in SCC tissues than in ADC tissues. FOXM1 mRNA expression was correlated with N stage, TNM stage, age, sex and smoking history in ADC, but only correlated with N stage, age and sex in SCC. Survival analysis indicated that high expression of FOXM1 mRNA resulted to poor overall survival (OS) for ADC patients, but not for SCC patients. Cox regression analysis confirmed that FOXM1 mRNA expression was an independent prognostic indicator for ADC patients, and regression analysis identified a moderately positive correlation between FOXM1 mRNA levels and copy number alterations (CNAs), but a weakly negative association with DNA methylation. FOXM1 was mainly involved in cell cycle regulation, G2/M transition, G1/S transition and p53, PI3K-Akt and TGF-beta signaling pathway. CONCLUSIONS: High expression of FOXM1 mRNA might be an independent biomarker of poor OS in ADC patients.

Ultra-performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan metabolites in human plasma and its application to clinical study.

A sensitive, rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to assay tryptophan (TRP) and its nine metabolites, including kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3-indolepropionic acid (IPA) and 3-indoleacetic acid (IAA) in human plasma. Tryptophan-d5 (TRP-d5) and carbamazepine (CAR) were applied to the method quantification, where TRP-d5 was the corresponding internal standard (IS) for TRP and KYN, and CAR was the corresponding IS for the other analytes. Plasma samples were processed by deproteinisation with acetonitrile, followed by separation on an Acquity UPLC HSS T3 column by using gradient elution with 0.1% (v/v) formic acid in water and acetonitrile and detection by electrospray ionisation tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) within a total run time of 5 min. The calibration ranges were 3-600 ng/mL for 3-HK, 1.5-300 ng/mL for 5-HT, 25-5000 ng/mL for KYN, 1-200 ng/mL for XA, 100-20,000 ng/mL for TRP, 5-1000 ng/mL for KYNA, 2-400 ng/mL for 3-HAA, 2.5-500 ng/mL for 5-HIAA and 10-2000 ng/mL for IAA and IPA. All intra- and inter-day analytical variations were acceptable. Matrix effect and recovery evaluation proved that matrix effect can be negligible, and sample preparation approach was effective. The newly developed method can simultaneously determine a panel of TRP metabolites and was successfully applied in the clinical study characterising TRP metabolism in healthy volunteers.

N-n-Butyl Haloperidol Iodide Ameliorates Oxidative Stress in Mitochondria Induced by Hypoxia/Reoxygenation through the Mitochondrial c-Jun N-Terminal Kinase/Sab/Src/Reactive Oxygen Species Pathway in H9c2 Cells.

Both c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS) play important roles in myocardial ischemia/reperfusion (I/R) injury. Our previous studies suggest that N-n-butyl haloperidol iodide (F2) exerts cardioprotection by reducing ROS production and JNK activation caused by I/R. In this study, we hypothesized that there is a JNK/Sab/Src/ROS pathway in the mitochondria in H9c2 cells following hypoxia/reoxygenation (H/R) that induces oxidative stress in the mitochondria and that F2 exerts mitochondrial protective effects during H/R injury by modulating this pathway. The results showed that H/R induced higher-level ROS in the cytoplasm on the one hand and JNK activation and translocation to the mitochondria by colocalization with Sab on the other. Moreover, H/R resulted in mitochondrial Src dephosphorylation, and subsequently, oxidative stress evidenced by the increase in ROS generation and oxidized cardiolipin in the mitochondrial membranes and by the decrease in mitochondrial superoxide dismutase activity and membrane potential. Furthermore, treatment with a JNK inhibitor or Sab small interfering RNA inhibited the mitochondrial translocation of p-JNK, decreased colocalization of p-JNK and Sab on the mitochondria, and reduced Src dephosphorylation and mitochondrial oxidative stress during H/R. In addition, Src dephosphorylation by inhibitor PP2 increased mitochondrial ROS production. F2, like inhibitors of the JNK/Sab/Src/ROS pathway, downregulated the H/R-induced mitochondrial translocation of p-JNK and the colocalization of p-JNK and Sab on the mitochondria, increased Src phosphorylation, and alleviated the above-mentioned mitochondrial oxidative stress. In conclusion, F2 could ameliorate H/R-associated oxidative stress in mitochondria in H9c2 cells through the mitochondrial JNK/Sab/Src/ROS pathway.

Angiotensin II confers resistance to apoptosis in cardiac myofibroblasts through the AT1/ERK1/2/RSK1 pathway.

Myofibroblast apoptosis is essential for normal resolution of wound repair, including cardiac infarction repair. Impaired cardiac myofibroblast (CMF) apoptosis is associated with excessive extracellular matrix (ECM) deposition, which could be responsible for pathological cardiac fibrosis. Conventionally, angiotensin II (Ang II), a soluble peptide, is implicated in fibrogenesis because it induces cardiac fibroblast (CFb) proliferation, differentiation, and collagen synthesis. However, the role of Ang II in regulation of CMF survival and apoptosis has not been fully clarified. In this report, we cultured neonatal rat CFbs, which transform into CMFs after passage 3 (6-8 days), and investigated the effects of Ang II on CMFs challenged by TNF-α combined with cycloheximide and the underlying mechanisms. Here, we show that Ang II rapidly activates MAPKs but not AKT in CMFs and confers apoptosis resistance, as evidenced by the inhibition of caspase-3 cleavage, early apoptotic cells and late apoptotic cells. This inhibitory effect of Ang II was reversed by blockade of AT1 or inactivation of ERK1/2 or RSK1 but not AT2, indicating that activation of the prosurvival AT1/ERK1/2/RSK1 signaling pathway mediates apoptosis resistance. TGF-ß, a latent fibrotic factor, was found to have no relation to Ang II-induced apoptosis resistance in our study. Furthermore, Ang II-mediated apoptosis resistance, which was conferred by activation of the AT1/ERK1/2/RSK1 signaling pathway, was also confirmed in human adult ventricular cardiac myofibroblasts. Collectively, our findings suggest a novel profibrotic mechanism of Ang II in which it promotes myofibroblast resistance to apoptosis in addition to classical mechanisms, providing a potential novel therapeutic approach by targeting prosurvival signaling pathways. © 2018 IUBMB Life, 71(1):261-276, 2019.

N-n-Butyl Haloperidol Iodide, a Derivative of the Anti-psychotic Haloperidol, Antagonizes Hypoxia/Reoxygenation Injury by Inhibiting an Egr-1/ROS Positive Feedback Loop in H9c2 Cells.

Early growth response-1 (Egr-1), a transcription factor which often underlies the molecular basis of myocardial ischemia/reperfusion (I/R) injury, and oxidative stress, is key to myocardial I/R injury. Silent information regulator of transcription 1(SIRT1) not only interacts with and is inhibited by Egr-1, but also downregulates reactive oxygen species (ROS) via the Forkhead box O1(FOXO1)/manganese superoxide dismutase (Mn-SOD) signaling pathway. N-n-butyl haloperidol iodide (F2), a new patented compound, protects the myocardium against myocardial I/R injury in various animal I/R models in vivo and various heart-derived cell hypoxia/reoxygenation (H/R) models in vitro. In addition, F2 can regulate the abnormal ROS/Egr-1 signaling pathway in cardiac microvascular endothelial cells (CMECs) and H9c2 cells after H/R. We studied whether there is an inverse Egr-1/ROS signaling pathway in H9c2 cells and whether the SIRT1/FOXO1/Mn-SOD signaling pathway mediates this. We verified a ROS/Egr-1 signaling loop in H9c2 cells during H/R and that F2 protects against myocardial H/R injury by affecting SIRT1-related signaling pathways. Knockdown of Egr-1, by siRNA interference, reduced ROS generation, and alleviated oxidative stress injury induced by H/R, as shown by upregulated mitochondrial membrane potential, increased glutathione peroxidase (GSH-px) and total SOD anti-oxidative enzyme activity, and downregulated MDA. Decreases in FOXO1 protein expression and Mn-SOD activity occurred after H/R, but could be blocked by Egr-1 siRNA. F2 treatment attenuated H/R-induced Egr-1 expression, ROS generation and other forms of oxidative stress injury such as MDA, and prevented H/R-induced decreases in FOXO1 and Mn-SOD activity. Nuclear co-localization between Egr-1 and SIRT1 was increased by H/R and decreased by either Egr-1 siRNA or F2. Therefore, our results suggest that Egr-1 inhibits the SIRT1/FOXO1/Mn-SOD antioxidant signaling pathway to increase ROS and perpetuate I/R injury. F2 inhibits induction of Egr-1 by H/R, thereby activating SIRT1/FOXO1/Mn-SOD antioxidant signaling and decreasing H/R-induced ROS, demonstrating an important mechanism by which F2 protects against myocardial H/R injury.

Purification, partial characterization and bioactivity of sulfated polysaccharides from Grateloupia livida.

Purification, preliminary characterization and bioactivity of polysaccharides from Grateloupia livida (GL) were investigated. Three water-soluble sulfated polysaccharide fractions (GLP-1, GLP-2 and GLP-3) were isolated and purified from the edible and medicinal red seaweed, Grateloupia livida (Harv.) Yamada by DEAE Sepharose CL-6B and Sephadex G-100 column chromatography, and chemical characterization was performed by HPGPC, GC-MS, FT-IR and SEM. In addition, anticoagulant activities were determined by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using normal human plasma in vitro. The antioxidant activities against DPPH and ABTS+ radicals were evaluated and compared. The molecular weights of GLP-1, GLP-2 and GLP-3 were 39.5, 60.4 and 3.36kDa, respectively. Monosaccharide analysis revealed that three polysaccharide fractions were homopolysaccharides and comprised of galactose only. Anticoagulant assays indicated that crude GLP, and purified GLP-1 and GLP-2 effectively prolonged APTT and TT, but not PT. All polysaccharide fractions exhibited significant in vitro antioxidant activities in a dose-dependent manner. GLP-2 showed consistently better anticoagulant and antioxidant activities compared with GLP, GLP-1 and GLP-3. These results demonstrate that sulfated polysaccharides isolated from Grateloupia livida can serve as readily available alternative natural sources of anticoagulant and antioxidant agents.

N-n-butyl haloperidol iodide ameliorates hypoxia/reoxygenation injury through modulating the LKB1/AMPK/ROS pathway in cardiac microvascular endothelial cells.

Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs.

N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F2), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F2. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F2, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F2 blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury.

Characterization and bioactivity of novel calcium antagonists - N-methoxy-benzyl haloperidol quaternary ammonium salt.

BACKGROUND AND PURPOSE: Calcium antagonists play an important role in clinical practice. However, most of them have serious side effects. We have synthesized a series of novel calcium antagonists, quaternary ammonium salt derivatives of haloperidol with N-p-methoxybenzyl (X1), N-m-methoxybenzyl (X2) and N-o-methoxybenzyl (X3) groups. The objective of this study was to investigate the bioactivity of these novel calcium antagonists, especially the vasodilation activity and cardiac side-effects. The possible working mechanisms of these haloperidol derivatives were also explored. EXPERIMENTAL APPROACH: Novel calcium antagonists were synthesized by amination. Compounds were screened for their activity of vasodilation on isolated thoracic aortic ring of rats. Their cardiac side effects were explored. The patch-clamp, confocal laser microscopy and the computer-fitting molecular docking experiments were employed to investigate the possible working mechanisms of these calcium antagonists. RESULTS: The novel calcium antagonists, X1, X2 and X3 showed stronger vasodilation effect and less cardiac side effect than that of classical calcium antagonists. They blocked L-type calcium channels with an potent effect order of X1 > X2 > X3. Consistently, X1, X2 and X3 interacted with different regions of Ca2+-CaM-CaV1.2 with an affinity order of X1 > X2 > X3. CONCLUSIONS: The new halopedidol derivatives X1, X2 and X3 are novel calcium antagonists with stronger vasodilation effect and less cardiac side effect. They could have wide clinical application.