Protective effects of verbenalin and (+)-eudesmin against 6-hydroxydopamine-induced oxidative/nitrosative stress in SH-SY5Y cells.

BACKGROUND: The purpose of this research was to study whether verbenalin, an iridoid glucoside, and (+)-eudesmin, a furofuran lignan isolated from different plant families, can attenuate cell damage and death induced by 6-hydroxydopamine (6-OHDA) in human neuroblastoma SH-SY5Y cells. METHODS: SH-SY5Y cells were incubated with 6-OHDA (35 µM) for 1 day. Verbenalin and (+)-eudesmin were administrated with various concentrations (1, 2.5, 5, 10, 20, and 50 µM) one hour before the 6-OHDA treatment. After 1 day, cell viability and neuroprotective effect were investigated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Nitrosative stress was determined with measurements of nitric oxide (NO) and 3-nitrotyrosine (3-NT), a biomarker of peroxynitrite formation. RESULTS: We observed that 6-OHDA declined viability and augmented LDH leakage in SH-SY5Y cells. MTT analyses showed that pretreatment with verbenalin and (+)-eudesmin markedly prevented the toxicity due to 6-OHDA (P < 0.05). Verbenalin and (+)-eudesmin suppressed LDH release induced by 6-OHDA (P < 0.01). Although 6-OHDA treatment produced no marked effects on NO levels, (+)-eudesmin at high concentrations (10-50 µM) markedly attenuated NO levels (P < 0.01). There was a significant increase in 3-NT levels with 6-OHDA exposure in cells. Pretreatment with verbenalin, but not (+)-eudesmin, diminished 3-NT levels at low concentrations (1-20 µM) and prevented the cytotoxic effect of 6-OHDA (P < 0.01). CONCLUSION: These results indicated that verbenalin and (+)-eudesmin exert potent cytoprotective activities against cytotoxicity triggered by 6-OHDA in neuroblastoma cells. This is the first report demonstrating that verbenalin may act as a peroxynitrite scavenger.

Comparison of serum dynamic thiol/disulphide homeostasis and nitric oxide levels of total intravenous vs inhaled anaesthesia in endoscopic transsphenoidal pituitary surgery.

BACKGROUND: Transsphenoidal pituitary surgery (TPS) is traditionally performed under general anaesthesia. This study aimed to compare the effects of total intravenous anaesthesia (TIVA) or sevoflurane, an inhalation anaesthetic, on thiol-disulphide homeostasis in patients undergoing endoscopic endonasal TPS. METHODS: In this study, 84 patients scheduled for TPS were randomly categorised into two groups: propofol (n = 42, the TIVA group) or sevoflurane (n = 42, the SEVO group). Blood samples were taken before induction of general anaesthesia and at the 30 minutes of postoperation. Serum native thiol and total thiol levels were detected, and the number of dynamic disulphide bonds and related ratios were calculated from these values. Serum nitric oxide (NO) levels were measured using a chemiluminescence method. RESULTS: Although native thiol levels in TIVA postoperation group were markedly increased (P < .05), total thiol levels in SEVO postoperation group were significantly decreased (P < .01). Disulphide levels were declined in both groups (P < .05 for TIVA and P = .001 for SEVO groups). Disulphide/native thiol (P < .05 for both groups) and disulphide/total thiol ratios (P < .05 for TIVA and P < .01 for SEVO groups) were depressed in postoperation groups. We found a marked elevation in native thiol/total thiol ratio in both groups (P < .05 for TIVA and P < .01 for SEVO groups). There was significant augmentation in serum NO levels in the SEVO postoperation group (P < .05). CONCLUSION: These results are the first to show that both TIVA and sevoflurane showed similar antioxidant effect with reduced disulphide levels, but sevoflurane may offer more robust oxidative stress protection and augmented NO production than TIVA during TPS. However, the clinical effect is needed to further investigate.

Dynamic thiol/disulfide homeostasis in children with community-acquired pneumonia.

BACKGROUND: Alteration in thiol level under oxidative stress may contribute to community-acquired pneumonia (CAP). The goal of this study was to determine whether there are changes in thiol/disulfide homeostasis and nitric oxide (NO) in children with CAP. METHODS: In total, 130 participants were involved in the study. Of these, 65 had been diagnosed with CAP on admission, and the remaining 65 were healthy individuals. Serum total thiol and native thiol were measured in each participant using a novel automated spectrophotometric method. The amount of dynamic disulfide bonds and related ratios were calculated from these values. Serum NO was measured on chemiluminescence assay. RESULTS: Average native thiol, total thiol, and disulfide in the CAP group were significantly lower than in the healthy individuals (P < 0.0001, P < 0.0001, P = 0.0126, respectively). In addition, disulfide/native thiol (P = 0.0002), and disulfide/total thiol ratios (P = 0.0004) were significantly higher, whereas the native thiol/total thiol ratio (P = 0.0004) was lower in the CAP group. High serum NO was noted in the CAP group (P = 0.0003), but there was no marked correlation between thiol/disulfide and NO. CONCLUSION: The changes in endogenous thiol levels under oxidative stress may be associated with the pathogenesis of CAP in pediatric patients.

Determination of dynamic thiol/disulphide homeostasis in children with tetralogy of Fallot and ventricular septal defect.

Oxidative stress may contribute to the pathogenesis of congenital heart defects, but the role of dynamic thiol/disulphide homeostasis has not been evaluated. The objective of this study was to assess whether there are changes in thiol/disulphide homeostasis and nitric oxide levels in children with tetralogy of Fallot (TOF) and ventricular septal defect (VSD). A total of 47 children with congenital heart defects (24 TOF and 23 VSD) and 47 healthy age- and sex-matched controls were included in this study. Serum total thiol and native thiol levels were measured using a novel automatic spectrophotometric method. The amount of dynamic disulphide bonds and related ratios were calculated from these values. Serum nitric oxide levels were detected using a chemiluminescence assay. We found that the average native thiol, total thiol, and disulphide levels were decreased in patients with VSD when compared with healthy individuals (p < 0.001, p < 0.001, and p < 0.01, respectively). While native thiol levels were decreased (p < 0.01), disulphide levels were elevated in the TOF group (p < 0.05). We observed marked augmentation of disulphide/native thiol (p < 0.001) and disulphide/total thiol ratios (p < 0.01) in the TOF group. However, there was a significant decrease in native thiol/total thiol ratio in patients with TOF. No significant changes in these ratios were noted in the VSD group. We detected significant elevations in serum nitric oxide levels in children with TOF and VSD (p < 0.001 for all). These results are the first to demonstrate that thiol/disulphide homeostasis and nitric oxide are associated with TOF and VSD in children.

Impact of Radiation Therapy on Serum Humanin and MOTS-c Levels in Patients with Lung or Breast Cancer.

BACKGROUND: Lung and breast cancer are the most frequent causes of death from cancer globally. The objectives of this research were to evaluate the serum mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) and humanin levels in lung or breast cancer patients, and investigate the impacts of radiation therapy on the circulating levels of these peptides. METHODS: 35 lung cancer patients, 34 breast cancer patients, and healthy volunteers as a control group were recruited in this prospective observatory research. Lung cancer patients with stage IIIA/IIIB were treated with paclitaxel-based chemotherapy plus radiotherapy (2 Gy per day, 30 times, 60 Gy total dose). Breast cancer stage IIA/IIB patients were treated with postoperative locoregional radiation therapy (2 Gy per day, 25 times, 50 Gy total dose). The ELISA method was used to detect serum humanin and MOTS-c levels during, before, and after radiotherapy. RESULTS: We observed marked elevations in circulating MOTS-c, but not humanin levels in patients with lung cancer (P < 0.001). Radiation therapy led to a marked augmentation in MOTS-c levels in these patients (P < 0.001). On the other hand, there was a marked decline in humanin, but not MOTS-c, levels in breast cancer patients (P < 0.001). CONCLUSION: Our research has shown, for the first time, that increased MOTS-c and decreased humanin levels play a role in lung cancer and breast cancer, respectively. Additionally, radiotherapy modifies MOTS-c levels in patients with lung, but not breast cancer.

Evaluation of Serum Humanin and MOTS-c Peptide Levels in Patients with COVID-19 and Healthy Subjects.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) is a life-threatening and persistent pandemic with high rates of mortality and morbidity. Although a dysfunction in the mitochondria occurs in COVID-19 pathogenesis, the contribution of mitochondrial-derived peptides to its pathophysiology has not yet been completely elucidated. The goals of this research were to assess the circulating humanin and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) levels in COVID-19 patients and explore the effects of antiviral drug therapy on these peptide levels. METHODS: Thirty adult COVID-19 patients and 32 gender-matched healthy volunteers were enrolled in this study. Circulating humanin and MOTS-c levels were detected using the ELISA method during pretreatment (before drug therapy) and post-treatment (on the 7th day of drug therapy). RESULTS: We found that there was significant attenuation of the serum humanin levels in COVID-19 patients (P < 0.001). However, we detected a significant augmentation in serum MOTS-c levels when compared to controls (P < 0.01 for pre-treatment and P < 0.001 for post-treatment). Interestingly, antiviral drug therapy did not modify the serum MOTS-c and humanin levels. CONCLUSION: Our findings suggest that MOTS-c and humanin were involved in the COVID-19 pathogenesis. Our data may also imply that elevated MOTS-c could act as a compensatory mechanism to eliminate the effects of decreased humanin levels.

Expression Analysis of the Small GTP-Binding Protein Rac in Pterygium

Objectives: To determine the roles of small GTP-binding proteins Rac1, Rac2, and Rac3 expression in pterygial tissue and to compare these expressions with normal conjunctival tissue. Materials and Methods: Seventy-eight patients with primary pterygium were enrolled. Healthy conjunctival graft specimens obtained during pterygium surgery were used as control tissue. The real-time polymerase chain reaction method on the BioMark HD dynamic array system was utilized in genomic mRNA for the gene expression analysis. Protein expressions were analyzed using western blot and immunohistochemical methods. Results: RAC1, RAC2, and RAC3 gene expressions in pterygial tissues were not markedly elevated when compared to the control specimens (p>0.05). As a very low level of RAC1 gene expression was observed, further protein expression analysis was performed for the Rac2 and Rac3 proteins. Western blot and immunohistochemical analysis of Rac2 and Rac3 protein expression revealed no significant differences between pterygial and healthy tissues (p>0.05). Conclusion: This is the first study to identify the contribution of Rac proteins in pterygium. Our results indicate that the small GTP-binding protein Rac may not be involved in pterygium pathogenesis.

Effects of antiviral drug therapy on dynamic thiol/disulphide homeostasis and nitric oxide levels in COVID-19 patients.

The novel coronavirus disease 2019 (COVID-19) has led to a serious global pandemic. Although an oxidative stress imbalance occurs in COVID-19 patients, the contributions of thiol/disulphide homeostasis and nitric oxide (NO) generation to the pathogenesis of COVID-19 have been poorly identified. Therefore, the aim of this study was to evaluate the effects of antiviral drug therapy on the serum dynamics of thiol/disulphide homeostasis and NO levels in COVID-19 patients. A total of 50 adult patients with COVID-19 and 43 sex-matched healthy control subjects were enrolled in this prospective study. Venous blood samples were collected immediately on admission to the hospital within 24 h after the diagnosis (pre-treatment) and at the 15th day of drug therapy (post-treatment). Serum native thiol and total thiol levels were measured, and the amounts of dynamic disulphide bonds and related ratios were calculated. The average pre-treatment total and native thiol levels were significantly lower than the post-treatment values (P < 0.001 for all). We observed no significant changes in disulphide levels or disulphide/total thiol, disulphide/native thiol, or native thiol/total thiol ratios between pre- and post-treatments. There was also a significant increase in serum NO levels in the pre-treatment values when compared to control (P < 0.001) and post-treatment measurements (P < 0.01). Our results strongly suggest that thiol/disulphide homeostasis and nitrosative stress can contribute to the pathogenesis of COVID-19. This study was the first to show that antiviral drug therapy can prevent the depletion in serum thiol levels and decrease serum NO levels in COVID-19 patients.

Investigation of Dynamic Thiol/Disulfide Homeostasis and Nitrosative Stress in Patients with Wilson Disease.

BACKGROUND: Wilson disease (WD) is an autosomal recessive inherited disorder of copper (Cu2+) metabolism, resulting in Cu2+ accumulation and liver and central nervous system toxicity. Oxidative stress may have a role in the pathogenesis of Wilson disease, but the roles of thiol/disulfide homeostasis and nitrosative stress have not been examined. The purpose of this study was to evaluate whether there is a modification in thiol/disulfide homeostasis and nitrosative stress in patients with Wilson disease. METHODS: A total of 50 patients with Wilson disease (42 under drug treatment and 8 newly diagnosed patients with no drug treatment) and 50 healthy gender- and age-matched controls were enrolled for this study. Serum native thiol and total thiol levels were measured with a spectrophotometric method. The number of disulfide bonds and the related ratios were determined from these measurements. Serum nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels were analyzed using chemiluminescence and ELISA assays, respectively. RESULTS: The average native thiol levels of the patient group under drug treatment were found to be markedly higher than the levels of controls (P < .05). We detected no marked changes in total thiol and disulfide levels, and disulfide/total thiol, disulfide/native thiol, or native thiol/total thiol ratios between groups. We found significant elevations in NO levels in Wilson disease group before drug treatment, and the 3-NT levels in the Wilson disease groups prior to (P < .05) and under drug treatment (P < .01), when compared to controls. CONCLUSION: Our data are the first to show that nitrosative stress and thiol/disulfide homeostasis can contribute to the pathogenesis of Wilson disease.

Increased Expressions of ICAM-2 and ICAM-3 in Pterygium.

Purpose: Pterygium, one of the most common ocular surface diseases, is characterized by inflammatory infiltrates, proliferation, angiogenesis, fibrosis, and extracellular matrix breakdown. The objective of this study was to elucidate the levels of the intercellular adhesion molecule (ICAM)-2, and ICAM-3 gene and protein expressions in pterygium. Methods: A total of 59 patients with pterygium were included in this study. mRNA from pterygial and conjunctival autograft tissues were extracted, and real-time polymerase chain reaction on the BioMark HD dynamic array system was performed for the ICAM-2 and ICAM-3 gene expressions. ICAM-2 and ICAM-3 protein expressions using western blot and immunohistochemistry methods were also investigated in pterygial and conjunctival autograft tissues. Results: ICAM-2 and ICAM-3 gene expressions were markedly augmented in pterygial tissues (P = 0.0018 and P = 0.0023, respectively). Significant increases in protein expressions in pterygial tissues were also detected for ICAM-2 and ICAM-3 (P = 0.0116 and P = 0.0252, respectively). In the immunohistochemical studies, there was a marked increase in ICAM-3 (P = 0.0152), but not in ICAM-2 (P = 0.1041), protein expressions in pterygial tissues. Significant positive correlations between pterygia grading with ICAM-2 protein expression (P = 0.0398) and ICAM-3 immunohistochemical scores (P = 0.0138) were observed. Conclusion: These results demonstrate, for the first time, the expressions of ICAM-2 and ICAM-3 in the pterygium. These findings may help to understand the signal transduction mechanisms in the pterygium formation and provide a new therapy strategy for pterygium treatment.

Toward Novel Diagnostics for Primary Open-Angle Glaucoma? An Association Study of Polymorphic Variation in Ras Homolog Family Member (A, B, C, D) Genes RHOA, RHOB, RHOC, and RHOD.

The annual economic burden of visual disorders in the United States was estimated as $139 billion. The World Health Organization has listed glaucoma in the top 10 priority eye diseases. Primary open-angle glaucoma (POAG) is a common subtype, with a lack of clinical tools for early diagnosis. The Rho GTPases belong to the Ras superfamily of proteins; the RhoA immunostaining in the optic nerve head in human glaucoma is reportedly increased. We investigated the association of polymorphisms in the Ras Homolog Family Member A, B, C, and D genes (RHOA, RHOB, RHOC, and RHOD, respectively). In a total sample of 361 unrelated subjects (179 patients with POAG and 182 age- and sex-matched healthy controls), RHOA (rs6784820, rs974495), RHOB (rs62121967), RHOC (rs11102522), and RHOD (rs61891303, rs2282502) polymorphisms were characterized by the BioMark HD dynamic array system with real-time polymerarse chain reaction. Among these candidate genetic markers and considering the Bonferroni correction, RHOA rs974495 polymorphism was significantly associated with POAG (p = 0.0011), with the TT genotype increasing the disease risk 4.9 times (95% CI 1.630-15.023). The allele and haplotype distributions of the above RHO candidate polymorphisms did not diplay a significant association. This is the first study, to the best of our knowledge, to identify a significant genotypic association between POAG and RHOA gene rs974495 polymorphism. These observations warrant replication in independent samples in the pursuit of precision medicine for rapid and early glaucoma diagnosis, and molecular targets for innovation in therapeutics of this common eye disease.