CDKN2A, DLAT, DLD, FDX1, GLS, PDHA1 and PDHB exhibited differential expression between ccRCC and normal tissues (|log2(fold change)| > 2/3 and p < 0.05). Utilizing an iterative sure independence screening (SIS) method, we separately constructed the prognostic signature of CRGs for predicting the overall survival (OS) and progression-free survival (PFS) in ccRCC patients. The prognostic score of CRGs yielded an area under the curve (AUC) of 0.658 and 0.682 for the prediction of 5-year OS and PFS, respectively. In the Kaplan−Meier survival analysis of OS, a higher risk score of cuproptosis-related gene signature was significantly correlated with worse overall survival (HR = 2.72 (2.01–3.68), log-rank p = 1.76 × 10−7). Patients with a higher risk had a significantly shorter PFS (HR = 2.83 (2.08–3.85), log-rank p = 3.66 × 10−7). Two independent validation datasets (GSE40435 (N = 101), GSE53757 (N = 72)) were collected for meta-analysis, suggesting that CDKN2A (log2(fold change) = 1.46, 95%CI: 1.75–2.35) showed significantly higher expression in ccRCC tissues while DLAT (log2(fold change) = −0.54, 95%CI: −0.93–−0.15) and FDX1 (log2(fold change) = −1.01, 95%CI: −1.61–−0.42) were lowly expressed. The expression of CDKN2A and FDX1 in ccRCC was also significantly associated with immune infiltration levels and programmed cell death protein 1 (PD-1) expression . In conclusion, the cuproptosis-related gene signature could serve as a potential prognostic predictor for ccRCC patients and may offer novel insights into the cancer treatment.Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma, which is characterized by metabolic reprogramming. Cuproptosis, a novel form of cell death, is highly linked to mitochondrial metabolism and mediated by protein lipoylation. However, the clinical impacts of cuproptosis-related genes (CRGs) in ccRCC largely remain unclear. In the current study, we systematically evaluated the genetic alterations of cuproptosis-related genes in ccRCC. Our results revealed that  Renal cell carcinoma (RCC) is one of the most common cancer types in the urinary system affecting more than 430,000 individuals in 2020 worldwide . Clear cCopper is an indispensable trace element involved in various biological processes. Recent studies showed that the copper levels of cancer patients are significantly elevated both in serum and tumor tissues compared to healthy counterparts ,6,7. WhiIn the present study, we intended to comprehensively investigate the molecular alterations and clinical relevance of cuproptosis-related genes (CRGs) in ccRCC. Our analysis highlights the importance of CRGs in ccRCC development and lays a foundation for the therapeutic application of cuproptosis regulators in ccRCC.https://portal.gdc.cancer.gov//, accessed on 15 March 2022). A total of 524 ccRCC patients and 72 adjunct nontumor samples were involved in the present study. Gene expression was measured with STAR as raw read counts, which subsequently transformed into transcripts per million (TPM). All gene features  were annotated by the GENCODE project (v22) [We obtained data of cuproptosis-related genes and clinical information of ccRCC patients from the The Cancer Genome Atlas (TCGA) database (ct (v22) . Clinicact (v22) . Only ccSomatic mutation data of ccRCC from whole exome/genome sequencing (WXS/WGS) were downloaded from the GDC TCGA-ccRCC project on the UCSC Xena server . The MuTDigital focal-level copy number variation (CNV) values were calculated from tumor aliquots using a “masked copy number fragment” file by GISTIC2  at the i2(fold change) | > 2/3 and p < 0.05, we considered it as statistically significant.We investigated the differential expression levels of CRGs between tumor and normal samples. Under the criterion of |log. Box plots were adopted to compare the expression of CRGs in various datasets using the R package “ggplot2”.For validation, we collected 2 datasets  as the assessment of the heterogeneity between multiple datasets. If there was no obvious heterogeneity , a fixed effects model was chosen. Otherwise, we selected a random effects model. A forest plot was utilized to show fold change and related 95% CI of CRGs using the R package “forestplot”.After computing the logTo analyze the potential interactions of these genes, we performed a gene network analysis with the GENEMANIA website . FurtherTo quantify cuproptosis-related genes at the individual level, we developed a signature based on iterative sure independence screening (SIS) . We usedWe then calculated the risk score using the regression coefficients of the identified prognostic signature of CRGs for OS and PFS, respectively. Subsequently, we classified patients into the high-risk and low-risk groups according to the median value of risk scores. The Kaplan−Meier survival curve was plotted to compare the OS or PFS between high-risk and low-risk groups by the R package “ggsurvplot”. Utilizing the signature, we also computed the 1-year survival, 3-year survival and 5-year survival based on the nearest neighbor nstimation (NNE) method . Receive+ T cells, CD8+ T cells, B cells, neutrophils, dendritic cells and macrophages).A Tumor Immune Estimation Resource   was usedWe also examined three important immune checkpoints (ICKs), including PD-1, PD-L1 and TIM-3 since the expression level of immune checkpoint-related genes is related to the treatment response of immune checkpoint inhibitors .The Pearson correlation analysis was used to examine the association between TME or ICKs and CRGs.First, we performed a descriptive statistical analysis of ccRCC patients in TCGA. Continuous variables were described as mean ± standard deviation, and categorical variables were described by frequency and proportion. A Kruskal−Wallis rank sum test  was applp values were two-sided, and we considered a level of p value less than 0.05 to be statistically significant.We utilized the Benjamini−Hochberg method for multiple correction, and a FDR < 0.05 was the standard of statistical significance . All staCDKN2A, FDX1, DLD, DLAT, LIAS, GLS, LIPT1, MTF1, PDHA1 and PDHB) that function closely with cuproptosis [CDKN2A (log2(fold change) = 2.12, p = 1.50 × 10−155) showed significantly higher expression while DLAT (log2(fold change) = −0.73, p = 5.12 × 10−26), DLD (log2 (fold change) = −0.97, p = 5.99 × 10−47), FDX1 (log2 (fold change) = −1.07, p = 9.21 × 10−54), GLS(log2 (fold change) = −0.94, p = 2.85 × 10−22), PDHA1(log2 (fold change) = −1.14, p = 7.17 × 10−32) and PDHB(log2 (fold change) = −1.12, p = 2.59 × 10−46) showed lower expression in ccRCC tissues than normal tissues   B.PDHB and CDKN2A. In contrast, GLS had the highest CNV amplifications. It is also interesting to notice the rare changes of DLD in mutation and CNV frequency. The somatic mutation status of 11 out of 370 (2.97%) ccRCC samples is shown (DLD (1%) and MTF1 (1%) showed higher mutation frequencies than others  in the univariate Cox proportional hazard regression model after the adjustment of age, gender, race and pathologic stage (FDX1 (HR = 0.54 (0.41–0.71), p = 1.56 × 10−5), DLD (HR = 0.74 (0.60–0.91), p = 4.10 × 10−3), DLAT (HR = 0.66 (0.54–0.82), p = 1.68 × 10−4), PDHB (HR = 0.70 (0.51–0.96), p = 2.91 × 10−2), MTF1 (HR = 0.76 (0.61–0.94), p = 1.01 × 10−2) and CDKN2A (HR = 1.21 (1.01–1.45), p = 3.52 × 10−2) remained statistically significant , p = 3.52 × 10−2, FDX1, LIPT1, LIAS, DLD, DLAT, PDHA1, PDHB, MTF1 and GLS) was remarkably related to better survival in ccRCC, showing features of tumor suppressors , log-rank p = 1.76 × 10−7, FDX1, DLAT and CDKN2A) correlated with PFS, and Expr (Gene) was the expression signature of corresponding genes. The same analyses were conducted for the PFS outcome. Patients with a higher risk score had significantly shorter PFS (HR = 2.83 (2.08–3.85), log-rank p = 3.66 × 10−7, Then, we separately constructed the prognostic signature of CRGs for OS and PFS in ccRCC utilizing SIS. For OS outcomes of ccRCC patients, three genes were selected to construct the prognostic score using their regression coefficients: ScoreTo facilitate the clinical application of the prediction model, we integrated clinical information and gene features of patients from TCGA and performed the multivariable Cox regression model to develop the nomogram. Discrimination and calibration methods were applied in both OS and PFS outcomes . The c-iCDKN2A showed a significantly higher expression in ccRCC tissues (GSE40435: log2(fold change) = 0.23, p = 3.76 × 10−19; GSE53757: log2(fold change) = 2.05, p = 5.02 × 10−27, DLAT, FDX1 and LIAS were significantly downregulated in their expression levels in ccRCC tissues (CDKN2A (log2(fold change) = 1.46, 95%CI: 1.75–2.35), DLAT (log2(fold change) = −0.54, 95%CI: −0.9–−0.15) and FDX1 (log2(fold change) = −1.01, 95%CI: −1.61–−0.42)  and performed a meta-analysis to derive the summary effect estimates. GSE40435 enrolled 101 adjacent nontumor tissues and 101 ccRCC tissues while GSE53757 included 72 normal and ccRCC tissues. In GEO datasets,  tissues A,B. Due 1–−0.42) C–F were CDKN2A, DLAT, FDX1 and LIAS and immune infiltration in ccRCC. The expression level of CDKN2A was positively associated with the immune infiltration level of CD8+ T cells (p = 2.89 × 10−2) and negatively correlated with macrophages (p = 2.89 × 10−2) (DLAT expression level was positively correlated with the immune infiltration level of B cells (p = 1.40 × 10−6), macrophages (p = 2.93E × 10−13), neutrophils (p = 1.89 × 10−6) and dendritic cells (p = 1.02 × 10−4) (FDX1 expression level was positively associated with the abundance of B cells (p = 2.33 × 10−3) and macrophages (p = 1.73 × 10−2) (LIAS expression and the abundance of CD8+ T cells (p = 3.86 × 10−2), macrophages (p = 1.12 × 10−5) and neutrophils (p = 1.15 × 10−3).It is uncertain whether CRGs would influence immune cell recruitment in the tumor microenvironment and, therefore, affect the prognosis of ccRCC. Thus, we performed an analysis to examine the relationships between  × 10−2) A. The DL × 10−4) B. The FD × 10−2) C. FigureCDKN2A expression in ccRCC had a positive correlation with PDCD1 expression levels . DLAT expression was associated with the expression level of PDCD1 , CD274  and HAVCR2 . FDX1 expression has a significant association with the expression of PDCD1 , CD274  and HAVCR2  from Our results also showed that CDKN2A, DLAT, FDX1 and LIAS) varied in different pathologic stages and histological grades of ccRCC, with the exception of DLD, which did not show any statistical difference across histological grades . FDX1 encodes a small iron−sulfur protein and was involved in the reduction of Cu2+ to Cu1+. In addition, FDX1 played the role of upstream regulator in the process of protein lipoylation in the TCA cycle and key regulator of cuproptosis [DLAT) was one of the components of the pyruvate dehydrogenase (PDH) complex. The oligomerization of DLAT was due to the integration of copper and lipoylated proteins in the TCA cycle [LIAS) encoded components of the lipoic acid pathway and synthesized a potent antioxidant termed α-Lipoic acid (LA) in mitochondria [CDKN2A expression functioned in the cell cycle control and was strongly correlated with the origin of a variety of tumors [CDKN2A was shown to be absent in 76% of metastatic ccRCC samples, according to a meta-analysis [The prognostic score constructed in this study consisted of four cuproptosis-related genes  showed a robust prognostic effect, regrettably, no dataset with a sufficiently large sample size (n > 50) and clinical prognostic information was available for further validation, which is urgently warranted in future research. Second, although the prognostic score focusing on CRGs’ expression signature showed a good performance in the prediction of ccRCC survivorship, some other significant genes with predictive values were not considered in this study. Third, given that the prognostic signature was built and validated by exploiting data from public databases, further biological evidence is needed apart from the statistical evidence we offered.There were several limitations in our study. First, although the validation of the expression of screened prognostic genes in TCGA and GEO demonstrated, to some extent, that three genes (In summary, this study systematically analyzed the landscape of molecular alterations and interactive genes of cuproptosis in ccRCC. Our study demonstrated that these cuproptosis-related genes may play a crucial role in ccRCC outcomes. The prognostic risk score based on the expression signature of CRGs showed a good performance for the prediction of OS and PFS of ccRCC patients and was significantly associated with immune infiltration levels and PD1 expression. Our results would also provide novel insights in developing pharmacological and nonpharmacological therapeutic strategies related to cuproptosis for cancer prevention and treatment.


DMC levels were estimated after harvest at the blush/background transition zone for fruit categorization on 300 fruits from each location. Fruits were coded with an individual number and grouped in different DMC categories. The distribution of the estimated DMC levels obtained from two locations was different. The results indicate that DMC levels were strongly and positively correlated with the soluble solids content (SSC) of the fruit (r = 0.81). Sensory evaluations also demonstrated that apples in the lowest DMC category (12.5% ± 0.5 from Cawston) were considered the least sweet apples with the least overall flavour quality by panellists compared to the apples from the other DMC categories included in the sensory evaluations from the two locations. Panellists also perceived less-than-expected “fresh apple” and “tropical” flavours but more-than-expected “no flavour” and “bland” off flavour from the lowest-DMC-category apples. The non-destructive DMC measurements show a potential to be used to sort apples for SSC, sweetness and flavour; nevertheless, they were not related to firmness or textural attributes.This research was conducted to determine the compositional and textural characteristics and sensory profile of ‘Ambrosia’ apples with different dry matter content (DMC) as estimated using a Felix-750 Produce Quality Meter . Fruits were harvested from a commercial orchard in Cawston and an experimental field in Summerland Research and Development Centre (SuRDC) in British Columbia, Canada, when the average absorbance difference index/coefficient of fruit skin  Fruit quality perceptions of consumers are influenced by both intrinsic and extrinsic characteristics of the fruit, and intrinsic attributes of the fruit such as its texture, flavour and appearance play important roles in consumers’ purchase decision-making process ,2. The iNon-destructive tools have been developed for the fruit maturity and/or quality assessments during different stages of production, harvest and storage. The measurement of the chlorophyll content of fruit skin ,9 and thThe non-destructive methods have been explored and proposed to measure the fruit DMC as a fruit quality indicator ,14,15,16δAbsorbance at 670–720 nm, δA) of fruit skin dropped under 0.45 ± 0.10   in two levels for each location: 12.5% ± 0.5 and 14.5% ± 0.5 for Cawston and 14.5% ± 0.5 to 16.5% ± 0.5 for SuRDC), Storage duration × Location interaction, DMC × Storage duration interaction [Location]; and random effects including judge, Judge × Location interaction, Judge × DMC[Location] interaction, and residual. The sensory dataset included 176 samples.Differences among judges are expected in sensory evaluations; however, a significant interaction effect of the judge with other factors can be concerning ,22 whichIn reporting the results of the ANOVA tests, significant main effects were reported only if the two-way interaction effects were not significant. In addition, chi-square tests were utilised to explore the independence of two categorical variables . Correlation coefficients were also explored to study relationships between the variables of interest.The distribution of DMCs from two locations are presented in p < 0.0001). Slightly higher correlation coefficients were observed between the DMC of the apples randomly selected for the instrumental tests and their mass (r(196) = 0.35, p < 0.0001).The correlation coefficient between DMC (%) and the fruit mass of individual apples was significant (r(538) = 0.16, p < 0.0001) and the intensity of the red over colour and DMC (r(539) = 0.24, p < 0.0001) were both significant. In addition, they were moderately correlated with each other (DMC (r(539) = 0.47, p < 0.0001). Furthermore, neither the red over colouration area nor intensity was correlated with the fruit mass.The correlation coefficients between the red over colouration area and DMC (r(539) = 0.30, The results of the ANCOVA test indicate that fruit mass was a significant covariate in the analysis, and differences in the DMC levels of the ground colour categories from two locations were significant. Apples with yellow background colour had significantly higher DMC levels  compared to the ones with green  or yellow-green  ground colours.p < 0.0001), but they also had larger red over colouration area  = 7.16, p < 0.0001) and more intense red over colouration  = 5.23, p < 0.0001). However, their fruit mass was not significantly different from those of Cawston samples  = −1.42, p = 0.16).Not only did SuRDC apples have greater DMC levels than those of Cawston apples  = 29.35, 2  = 85.62, p < 0.0001). Cawston samples had more-than-expected samples with either green or yellow ground colour but fewer-than-expected samples with yellow-green ground colour. The opposite was true about the SuRDC samples.Location and ground colour variables were not independent  = 13.47, p = 0.0003). The δA was higher at SuRDC (0.40 ± 0.01) than at Cawston (0.34 ± 0.01). The δA dropped from 0.43 ± 0.01 at two-week storage to 0.31 ± 0.01 after ten-week storage  = 41.32, p < 0.0001). The δA differed significantly among the DMC[Location × Storage duration] categories  = 4.20, p < 0.0001). At Cawston, the fruits in the higher DMC categories had less chlorophyll in the fruit skin, as indicated by lower δA on both test dates (Significant differences were observed in the st dates A.p = 0.02). Fruit mass was 206.58 ± 3.16 g at two-week storage, and decreased to 195.81 ± 3.17 g at ten-week storage. There was no significant difference rendered by locations or interaction. Fruits of higher DMC categories tended to have higher fruit mass; however, the difference was not significant at p = 0.05 (The effect of storage duration on fruit mass was significant (F (1180) = 5.8, p = 0.05 B.p = 0.005) and the interaction effect of Location × Storage duration  = 13.37, p = 0.0004). A higher firmness was found in the apples from SuRDC × two-week storage (18.20 ± 0.17 lbs) compared to fruits from the other three groups of Cawston × ten-week storage (17.39 ± 0.21 lbs), Cawston × two-week (17.24 ± 0.17 lbs) and SuRDC × ten-week storage (16.95 ± 0.21 lbs). In the flesh firmness, significant differences were observed in the main effect of storage duration  = 8.16, p < 0.0001). Storage duration and interaction effects were not significant. Among the DMC[Location × Storage duration] categories, SSC increased in both locations and on both test dates by increasing DMC  = 13.26, p < 0.0001; p < 0.0001; n = 47) (SuRDC apples had greater SSC levels (14.43% ± 0.07%) compared to the ones from Cawston (12.61% ± 0.07%)  = 311.07,  n = 47) A.−1 of malic acid) than in Cawston apples (3.50 ± 0.05 g L−1)  = 10.24, p = 0.03), and marginally higher in two-week stored apples (3.78 ± 0.04 g L−1) than in ten-week stored apples (3.44 ± 0.05 g L−1)  = 24.21, p < 0.0001). Significant difference was also found in the Location × Storage duration interaction, showing higher TA concentrations in two-week stored SuRDC apples (3.82 ± 0.06 g L−1), two-week Cawston apples (3.75 ± 0.06 g L−1) and ten-week SuRDC apples (3.63 ± 0.08 g L−1) compared to TA in ten-week Cawston apples (3.25 ± 0.08 g L−1)  = 5.08, p = 0.03). Among the DMC[Location × Storage duration] categories, the top two DMC categories from SuRDC and the top DMC category from Cawston had the highest level of TA compared to the next two DMC levels from SuRDC and the second DMC category from Cawston, which had higher TA than the last two DMC categories from Cawston  = 13.27, p < 0.0001) (Titratable acidity was significantly higher in SuRDC apples (3.72 ± 0.05 g L 0.0001) B.p = 0.003), and was significantly lower in 10-week stored apples (3.88 ± 0.01) than in two-week stored apples (4.07 ± 0.03)  = 25.62, p < 0.001). Significant difference was found in the Location × Storage duration interaction, showing higher pH in two-week stored SuRDC apples (4.19 ± 0.04), compared to two-week stored Cawston apples (3.95 ± 0.04), ten-week stored Cawston apples (3.89 ± 0.02) and ten-week stored SuRDC apples (3.88 ± 0.02)  = 11.11, p = 0.02). Significant difference was also found between the DMC levels of ten-week stored Cawston apples  = 5.15, p = 0.0498).The initial pH of the juice  was signΨfruit (−1.80 ± 0.02 MPa) than ten-week stored apples (−1.92 ± 0.03 MPa)  = 9.55, p = 0.004). Cawston fruits had higher Ψfruit (−1.77 ± 0.03 MPa) than SuRDC fruits (−1.95 ± 0.03 MPa)  = 22.86, p < 0.0001). The highest Ψfruit was observed in Cawston fruits with the lowest DMC levels; the lowest Ψfruit was in SuRDC fruits in the top DMC categories  = 2.97, p = 0.007; Ψfruit and SSC  (Two-week stored apples had higher  n = 47) B.p = 0.0004), and fruits were considered softer after storage in general . No significant differences were observed in the flesh hardness evaluations of apples from different DMC[Location] levels  = 13.40, ] levels .The model explained 25.34% of the variation in the juiciness data, and no significant differences were detected in the juiciness evaluations of the fruit from different DMC[Location] categories .The model explained 61.45% of the variation in the overall texture quality data, and no significant differences were detected in the overall texture quality evaluations of the fruit from different DMC[Location] categories .p = 0.005). Greater sweetness ratings were reported for the higher DMC categories  = 5.18, p = 0.015, The model explained 65.22% of the variation in the sweetness data. The evaluation of sweetness was impacted by both location and DMC[Location] factors. Apples from SuRDC  were considered to be sweeter than their Cawston  counterparts  = 12.66, The model explained 73.21% of the variation in the tartness data, and no significant differences were detected in the tartness evaluations of the fruit from different DMC[Location] categories .p = 0.04). This can be explained by better quality evaluations reported for the higher DMC categories  = 7.11, p = 0.005, The model explained 56.26% of the variation in the overall flavour quality data. The evaluations of overall flavour quality were impacted by both location and DMC[Location] factors. Apples from SuRDC  were considered to be better in flavour quality than their Cawston  counterparts  = 5.30, p = 0.03) and ”fresh apple”  = 17.08, p = 0.0007) flavours for the apples from the Cawston 12.5% ± 0.5 DMC category, while more-than-expected panellists reported “tropical” flavour for SuRDC 14.5% ± 0.5 apples. In addition, more-than-expected panellists reported ”no fruity flavour” for the apples from the Cawston 12.5% ± 0.5 DMC category  = 9.59, p = 0.02).Chi-square tests of independence were performed to examine the relationships between the DMC[Location] and the fruit flavour binomial CATA choices. Fewer-than-expected panellists reported “tropical”  = 9.042, p < 0.0001) while fewer-than-expected panellists reported ”no undesirable flavour” for the apples from those apples  = 18.49, p = 0.0003).Chi-square tests of independence were used to explore the relationships between the DMC[Location] and the off flavour binomial CATA choices. More than expected panellists reported “bland” flavour for the apples from the Cawston 12.5% ± 0.5 DMC category  = 30.77, δA in those apples [Apple mass was positively correlated with DMC level in the current study and similar results were reported by Saei et al. . Apples e apples . ‘Ambrose apples ,27. Devee apples  reportedThe results indicate that the textural attributes of ‘Ambrosia’ apples explored in this study were not influenced by the DMC of the fruit. This was confirmed by both instrumental and sensory data . In contThe soluble solids content was lower in fruits with lower DMC levels, especially in fruits from Cawston, as they were lower in DMC compared to those from SuRDC in general. The sensory results also indicate that the sweetness perception of panellists was lower in the lowest DMC category from Cawston  compared to other three categories. McGlone et al.  also repThe tartness perception of panellists was not significantly influenced by the DMC levels of the fruit. Nevertheless, TA compositional measurements demonstrated increases in the gram malic acid required per 100 L of the apple juice in fruits with higher DMC levels. Hampson et al.  also repThe sensory evaluations and textural measurements both indicated that apples were softer after a ten-week cold storage. Their mass shrank slightly and their TA levels dropped. Costa et al.  reportedΨfruit in this study  = 9.05, p = 0.013; data not graphed) and lower average daily minimum temperature in August (16.19 °C versus 15.68 °C)  . In addiThe application of DMC as a sorting factor demonstrated a potential for the selection of apples with higher SSC, sweetness and flavour but fruit firmness and textural attributes were not influenced by their DMC levels. As a result, DMC can be considered alongside indicators related to other intrinsic quality attributes to improve the precision of the sorting process. Fruit mass and near-infrared spectroscopic measurement of DMC can be used together to non-destructively characterize location-specific effects of preharvest growing conditions on fruit growth potential. Future studies will benefit from a diversity of apple varieties with different SSC and firmness profiles cultivated under different environmental and horticultural conditions.


To form these complexes, three thiosemicarbazone ligands (TSCs) were synthesized; L = 5-nitro-2-carboxyaldehyde-thiophen-N-methyl-thiosemicarbazone, (L1); 2-acetyl-5-bromo-thiophen-N-methyl-thiosemicarbazone, (L2) and 2-acetyl-5-bromo-thiophen-N,N-dimethyl-thiosemicarbazone, (L3). The isolated compounds were analyzed using spectroscopic techniques such as elemental analysis, conductance measurements, FT-IR, 1H NMR spectroscopy, MALDI-TOF mass spectrometry, and single-crystal XRD. Our results demonstrated that the synthesized thiosemicarbazone ligands (TSCs) are bound to the metal ion as a bidentate ligand that coordinates through the thiocarbonyl sulfur and azomethine nitrogen atoms in all complexes . The X-ray crystal structures of L1 and L2 revealed that both compounds are crystallized in the triclinic crystal system with space group P-1. The biological potency of newly synthesized TSC ligands  and their corresponding ruthenium complexes  were investigated on human primary ovarian (A2780) and human metastatic ovarian (OVCAR-3) cell lines. To get detailed information respecting antitumor properties, cytotoxicity, DNA/BSA binding affinity, cellular uptake, DNA binding competition, and trans-epithelial resistance measurement assays were performed. Our results demonstrate that newly synthesized ruthenium(II) complexes possess potential biological activity. Moreover, we observe that the ruthenium complexes reported here show anticancer activity on primary (A2780) and metastatic (OVCAR-3) ovarian cancer cells.In this study, we describe the synthesis, characterization and antiproliferative activity of three organo-ruthenium(II) half-sandwich complexes [RuCl(η Cancer is a complex disease that is often difficult to diagnose and has the ability to spread to other organs easily, which significantly lowers the patient’s survival rate. Considered as one of the most deadly diseases in the world, it has been reported that one in three people develop cancer during their lifetime . OvarianIn the last decades, attention on Ru(II)-arene complexes has significantly increased in the cancer field. These complexes have versatile pharmacological features. The arene ligands can be used to increase the stability of Ru(II) complexes. These ligands can also be utilized to provide a lipophilic/hydrophilic balance to the compounds. Moreover, different co-ligands are known to influence the activity of Ru(II)-based compounds. Small variations in the structure of the ligands can greatly change the biological activities. As a result, various ligands have been considered to be used in the studies that use Ru(II)-arene complexes .N, S chelating ligands, therefore metals can be coordinated with TSC ligands. Ru(II) complexes that have TSC ligands have been proposed to have potent anticancer activity -arene complexes that include different variations of bioactive ligands. In this way, the creation of a synergistic effect between the ligand and the metal center may increase the biological activity of the complex. TSCs have been of interest due to their different biological properties . Moreoveactivity .N-methyl-thiosemicarbazone, (L1); 2-acetyl-5-bromo-thiophen-N-methyl-thiosemicarbazone, (L2) and 2-acetyl-5-bromo-thiophen-N,N-dimethyl-thiosemicarbazone, (L3)] and their Ru (p-cym) complexes, [RuCl(η6-p-cym) ]Cl . Our results indicate that these newly synthesized Ru(II) complexes may have promising biological activity.In the current study, we synthesized new TSC derivatives,  was synthesized following literature procedures . 1,8,9-trihydroxyanthracene was used as a matrix for the mass analyses. Analyzes were recorded using a Bruker MALDI–TOF spectrometer. Conductance measurements were recorded on a Systronics automatic precision bridge 304 conductivity meter.LECOCHNS-O-9320 and Varian 1000 FT spectrophotometer were used for the elemental and FT-IR analysis, respectively. HP Digital FT NMR (400 MHz) was used for The synthesis of all ligands was carried out by following the given literature procedure . The ald7H8N4S2O2: Calc. %C: 34.42; H: 3.30; N: 22.93; O: 13.10; S: 26.25. Found: %C: 34.82; H: 3.17; N: 22.66; O: 13.87; S: 26.48. FTIR (KBr pellet), ʋ/cm−1: 3339 (s), 3142 (s), 3105 (s) (ν(N (1)H + N(2)H)sym), 1564 (s), 1537 (s) ν(C = N), 1037 (s) ν(N-N), 908 (s) ν(C = S), 731 (s), 694 (s) (thiophen ring stretchings). 1H NMR  δ 11.81 , 8.51 H), 8.18 H), 8.05 ; 7.49 , 2.99 . MALDI-TOF MS: m/z = 244 [M+]. Crystals were get by ethanol solution of L1.Orange powder, yield: 90%. C8H10N3S2Br: Calc. %C: 32.88; H, 3.45; N, 14.38; S, 21.95. Found: %C: 32.23; H, 3.12; N, 14.22; S, 21.44. FTIR (KBr pellet), ʋ/cm−1: 3370 (s), 3175 (m), 3062 (m) (ν(N (1)H + N(2)H)sym), 1547 (s), 1496 (s) ν(C = N), 1045 (s) ν(N–N), 974 (s) ν(C = S), 798 (s) and 694 (s) (thiophen ring stretchings). 1H NMR  δ 10.30 H), 8.06 H), 7.29 ; 7.18 , 3.01 , 2.26 . MALDI-TOF MS: m/z = 292 [M+]. Crystals were gained by slow evaporation of ethanol solution of L2.Yellow powder, yield: 85%. C9H12N3S2Br: Calc. %C: 38 78; H: 4, 07; N: 16,96; S: 20.91. Found %; C: 38, 22; H: 4, 86; N: 16, 75; S: 20.25. FTIR (KBr pellet), ʋ/cm−1: 3351 (s), 3236 (m), 3165 (m) (ν(N(2)H)sym), 1515 (s), 1488 (s) ν(C = N), 1064 (s) ν(N–N), 975 (s) ν(C = S), 789 (s), 705 (s) (thiophen ring stretchings). 1H NMR  δ 9.65 H), 7.34 ; 7.20 , 3.22 2), 2.31 . MALDI-TOF MS: m/z = 306 [M+].Light brown powder, yield: 84%. C6-p-cym)}2 (μ-Cl)2]  was dissolved in 10 ml of dry DCM and the solution was added to the TSC ligand. The reaction was continued for 24 h under nitrogen with stirring at ambient temperature. After the reaction was terminated, the volume of solution was halved using a rotary evaporator. Diethyl ether was added until a solid precipitate was observed. The product was obtained by filtration and dried in vacuo.RuCl(η6-p-cym) ]Cl, I.+; ɅM (Ω−1. m2. M−1) 75.RuCl(η6-p-cym) ]Cl,II.+; ɅM (Ω−1. m2. M−1) 72.RuCl(η6-p-cym) ]Cl,III.+; ɅM (Ω−1. m2. M−1) 78.Orange powder, yield: 66%. RuCα radiation (λ = 0.71073 Å) from an enhanced X-ray source. CrysAlisPro program  and the human metastatic ovarian cancer cell line (OVCAR-3) were obtained from the American Type Culture Collection (ATCC). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/ml). The cells were cultivated at 37°C in a humidified 5% CO50 values were defined as the drug concentration which limits cell growth at a 50% ratio and IC50 values were calculated by using cell survival diagrams.The antiproliferative effect of the newly synthesized TSC ligands, Ru(II) complexes, and chemotherapeutic drugs  as references were determined by the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Stock solutions of TSC ligands and Ru(II) complexes were freshly prepared in dimethyl sulfoxide (DMSO) at 5 mM concentration. Briefly, cells reaching approximately 80% confluency were placed into 96-well plates  and waited for nearly 16–18 h. Varying concentrations of TSC ligands  and Ru(II) complexes  were applied to the cells for 24 h. Then, the culture medium was taken and MTT (5 mg/ml) solution was put (100 μL/well) to each well. The plates were incubated for 4 h at 37°C. Then, the MTT solution in each well was carefully discarded and Dimethyl sulfoxide (DMSO) was placed in the wells. The absorbance was obtained by using a microplate reader (Thermo Multskan Go) at 540 nm. The experiments were performed in three biological replicas. IC4 cells in each well, then cells were kept in an incubator for overnight to adhere to the surface. Ru(II) complexes were applied to the cells at a certain concentration (20 µM) for 24 h. Cells were then washed with 1X phosphate salt buffer (PBS) and permeabilized with 4% paraformaldehyde (PFA) that contains 0.1% Triton-X-100. The cells were kept in an incubator at 25°C for 15 min. After incubation, the cells with increased pore permeability were applied in a 1: 2000 ratio of 4 6, 6-diamidino-2- phenylindole dihydrochloride (DAPI) (5 mg/ml) and were kept in the dark for 5 min at room temperature. After washing again with 1X PBS, coverslips were carefully covered on slides, and images were taken with a fluorescence microscope.Effective uptake of Ru(II) complexes into the cell were evaluated by fluorescence microscopy. Briefly, cells reaching approximately 70% confluency were counted with trypan blue and placed in 6-well plates with sterile coverslips containing 5 × 10DNA binding assay was performed in 1 cm quartz cuvettes. The temperature was set to 25°C. The absorption data were obtained using a UV-Vis spectrophotometer . Calf Thymus DNA (CT-DNA) concentration was acquired at 260 nm. CT-DNA purity was measured at 260/280 nm. Experiments were performed in 5 mM Tris-HCl buffer including 50 mM NaCl (pH 7.2). 10 µM concentration of TSC ligands and Ru(II) complexes were standardized with 0.07–1.4 mM CT-DNA concentrations at 37°C for 1 h, and the spectrum was recorded between 200–800 nm.The spectra of (BSA) in the presence and absence of ruthenium (II) complexes were carried out using the Spectrofluorophotometer  with excitation at 260 nm. The binding assay was performed in 50 mM phosphate buffer (pH 7.2). BSA emission changes were recorded between 250 and 450 nm by titrating the constant concentration of BSA (1 µM) with increasing Ru complex concentrations (0–50 µM).To determine the binding modes of Ru(II) complexes on DNA, two different competitive binding studies were performed using DAPI and methyl green. In the DAPI competitive binding study, Ru(II) complexes (100 µM) diluted in 10 mM pH 7.4 phosphate buffer were incubated with CT-DNA (20 µM) and DAPI (15 µM- 5 mg/ml) at 23°C for 15 min. The samples were scanned at the excitation wavelength of 338 nm and the emission wavelength of 461 nm in the spectrophotometer (Thermo Varioskan). In the methyl green competitive binding study, 2% methyl green, CT-DNA (50 µM), and Ru(II) complexes (100 µM) dissolved in 7.5 mM MgSO4 containing 50 mM pH 7.5 Tris-HCl solution were kept in a water bath at 37°C for 24 h. The samples were scanned on the spectrophotometer at an excitation wavelength of 630 nm and emission wavelength of 677 nm. The absorption plot was drawn by comparing the absorbances in solutions containing Ru(II) complexes compared to the control group without Ru(II) complexes.Trans-Epithelial Resistance of newly synthesized Ru(II) complexes was determined by using the Trans-epithelial resistance (TED) measurement device . Briefly, the cells were placed in transwells with a diameter of 0.4 µm pore at a concentration of 7,500 cells/250 µl medium. Approximately 1000 µl of growth medium was placed outside the transwells. The growth medium of the cells was changed every 3 days. Transepithelial resistance was measured before changing the medium. When the cells reached sufficient confluency after about 14 days, Ru(II) complexes were applied at IC50 concentrations, and differences in trans-epithelial resistance were observed by taking measurements at 24, 48, 72, 96, and 120 h.N-methyl-thiosemicarbazone, (L1); 2-acetyl-5-bromo-thiophen-N-methyl-thiosemicarbazone, (L2) and 2-acetyl-5-bromo-thiophen-N, N-dimethyl-thiosemicarbazone, (L3) were synthesized according to described procedures (6-p-cym) Cl]Cl were isolated in good yields.New ligands L = 5-nitro-2-carboxyaldehydethiophen-ocedures . Then ha1H NMR spectroscopy, MALDI-TOF spectrometry, and L1 and L2 structures were confirmed by single-crystal X-ray crystallography. In all cases, the metal binds to a chloride ion, a η6-p-cym ring, and a N, S bidentate TSC chelating ligand R(2)C(2) = N(3)N(2)(H)C(1) = (S)N(1)R(3)R(4)] commonly coordinate to metal as either of two tautomeric forms, a neutral thione form or the anionic thiol form. The existence of a band corresponds to the hydrazine N(2)–H group, which proposed the coordination of a TSC to the metal center in a neutral thione form, not an anionic thiol form (ν(C = N) stretching frequencies of the complexes  showed changes compared with free ligands L1, L2, and L3 which support complexation formation. The absorptions due to azomethine C=N of the free ligands at 1564, 1537 cm−1 (L1); 1547, 1496 cm−1 (L2), and 1515, 1488 cm−1 (L3) were observed. Coordination of the TSCs to Ru(II) through imine nitrogen is expected to change the electron density in the azomethine and thus alter ν(C = N) band frequency (ν(C= N) absorption frequencies were seen at 1576 and 1544 cm−1 (I); 1584 and 1564 cm−1 (II) and 1572 and 1535 cm−1 (III) in the FT-IR spectra of the complexes. The FTIR spectra of L1, L2, and L3 exhibited characteristic strong absorption bands attributed to thiocarbonyl (C = S) stretching, at 908, 974, and 975 cm−1 consecutively. They all were shifted to lower frequencies 876, 875, and 900 cm−1 for the complexes . These shifts proved that the ligand coordinated as a neutral, bidentate through thiocarbonyl sulfur and imine nitrogen atoms in . The ν (N-N) bands of TSCs were seen at 1037 cm−1, (L1); 1045 cm−1, (L2), and 1064 cm−1, (L3). The decrease in the frequency of these bands 1033 cm−1 (I), 1034 cm−1 (II), and 1032 cm−1 (III) confirms the coordination via the imine nitrogen as shown in the literature terature .1H NMR spectra. Two sets of signals of the p-cymene ring and the TSC protons of the coordinated ligand are seen definitely in the 1H NMR spectra. In the spectra of  all indications were that the TSCs remained in the neutral form due to the presence of the N(2)H protons. The NH absorptions in the spectra of the complexes were essentially altered from that of the free ligands. Absorptions of the free ligands N(2)H protons were observed at 8.51, (L1); 10.30, (L2), and 9.65, (L3) ppm whereas upon coordination the signals appeared at 8.11, (I); 7.84, (II), and 7.77, (III) ppm with slight highfield shifts. An upfield shift compared to the free TSCs upon coordination to Ru(II) ion, confirming coordination of the metal ion to the imine nitrogen atom as the signal became more shielded in each case. This was also observed for similar arene Ru(II) TSC complexes (3C = N) in L2 and L3 spectra were observed at around 2.26 and 2.31 ppm respectively, these absorptions moved slightly towards the upper field at 2.48 ppm for both of the complexes II and III.The L-to-M bonding is further supported by omplexes . In the omplexes . The sin2 protons in L3 are at 3.22 ppm were present at 2.07 ppm in the NMR spectrum of (III) with gradual upfield shifts.The N(1)H singlet signals in the complexes, 7.62 ppm, (I) and 7.61 ppm, (II) attributed to the TSC were slightly changed to the higher field from the free ligands, 8.18 ppm, (L1) and 8.06 ppm, (L2). The N(1) (Me)H group generated doublets at 2.99 ppm, (L1) and 3.01 ppm, (L2) with a slight change in complexes at 3.07 ppm (I) and 3.03 ppm (II). Singlet resonances of NMep-cym protons resonated at frequencies typically observed for this group ; 7.04–7.10 ppm, (II); 7.05–7.10 ppm, (III) for the complexes. The isopropyl methines of p-cym ring were emerged as multiplets at 2.79–2.81 ppm, in (I); 2,87–2.79 ppm, in (II), and 2.77–2.80 ppm, in (III), consecutively. The singlet signals owing to the methyl group on the p-cym ring at 2.24 ppm (I), 2.23 ppm (II), and 2.23 ppm (III) in sequence. The isopropyl methyls appeared as doublets at 1.18 ppm (I), 1.15 ppm (II), and 1.16 ppm (III).The is group . Multipl1H NMR spectra of  are actually a direct combination of the signals from the ligands plus those from the p-cym moiety. In the 1H NMR spectra of  all indications are that the ligands remain neutral form.All the protons resonated in commonly expected regions. The +, (I); [M-Cl-Me]+, (II); [M-Cl-Me]+, (III). The chloride ion loss confirmed all the complexes have chloride ions as a counter ion.The MALDI-TOF mass spectra of the TSCs and the complexes proved the suggested structures. Exact molecular ion peaks were observed for the ligands at 244 m/z (L1), 292 m/z (L2), and 306 m/z (L3). However, according to the mass analysis of the complexes the peaks at 515 m/z (I), 547 m/z (II), and 562 m/z (III) were equal to the total molecular weight of [M-Cl]–3 M in DMSO) in the range of 72–78 ɅM (Ω−1. m2. M−1) at 38°C suggesting 1:1 electrolytic behavior  at O1 and−0.236 (3) Å at C1, respectively. However, the 5-nitro thiophene ring in L1 [5-bromo thiophene ring in L2] and thiosemicarbazone fragment of the molecule were twisted concerning for to each other making a dihedral angle of 4.24 (6)° [9.085 (1)°].N4–O1 and N4-O2 bond lengths are 1.221 (3) and 1.236 (3) Å in compound L1, which are in agreement with bond distances reported for other compounds of 5-nitrothiophene . The Br1In the crystal packing of compounds L1 and L2, intermolecular interactions construct centrosymmetric dimeric motifs but give different supramolecular architecture. An S (5) ring motif is generated due to cyclic intramolecular N1–H1···N3 hydrogen bonds which support the molecular conformation of both compounds .In the crystal structure of L1, C6 atom act as hydrogen bond donor to O2 atom of an inversion-related molecule, producing an ractions . Furtherb-axis direction by π‒π interaction   complexes on human A2780 (primary) and human OVCAR-3 (metastatic) ovarian cancer cell lines. Carboplatin, Oxaliplatin, and Paclitaxel were used as comparative reference substances, and were investigated under identical conditions. Both cell lines were treated with different concentrations of TSC ligands  and Ru(II) complexes  for 24 h. The IC50 rate for Ru(II) complexes and reference substances against A2780 and OVCAR-3 cell lines were shown in The MTT assay procedure was applied to evaluate the 50 values, it was noticed that the highest cytotoxic activity on A2780 tumor cells was shown by complex II, and the highest cytotoxic activity on OVCAR-3 tumor cells was shown by complex III. This study showed that Ru(II) complexes  were more cytotoxic on the OVCAR-3 cell line than chemotherapeutic agents used as reference substances  in the recent study. The situation was slightly different considering the cytotoxicity experiments on the A2780 cell line. I and III complexes were more cytotoxic on the A2780 cell line than Paclitaxel and Carboplatin. However, Oxaliplatin showed the highest cytotoxic effect on A2780 cells than all Ru(II) complexes. When the IC50 values of the newly synthesized ruthenium (II) complexes obtained from the cytotoxicity experiments on both cell lines were examined, it was observed that the IC50 values of the Ru complexes on the A2780 cell line were lower. This showed that ruthenium complexes were more active in the primary ovarian cancer cell line. MTT results on ovarian cancer cell lines of ruthenium (II) complexes are in accordance with the literature studies  complexes I, II, and III treated with different concentrations of calf thymus DNA (CT DNA) were indicated in 0-I)/I) = nlog [Q] + logKb)  complexes that interact with BSA; [Q] denotes the complex concentration, and n shows the number of the binding site and Kb shows the binding constant. The binding constant Kb was determined by plotting according to log[(Io-I)/I] corresponding to log[Q]. As seen in 5, 7.67 × 105, and 2.03 × 104 for complex I, II, and III, respectively. The binding affinity of Ru(II) complexes to BSA followed the sequence of II > I > III. Taken together, these results showed that the binding of the Ru(II) complexes with BSA may be due to the presence of static quenching  is widely used in protein binding studies. BSA binding studies give information about pharmacokinetics and the biological distribution of molecules in the body. The binding of organo ruthenium complexes with BSA was evaluated using fluorescence spectra. The spectral emission was recorded at 280 nm excitation wavelength. The emission was monitored between 250 and 450 nm wavelength. Experiments were carried out by titrating BSA solution (1 μM) in varying Ru(II) complex concentrations (0–50 μM) in phosphate buffer (pH 7.2). + logKb) . I0 showuenching .Two different competitive binding studies were studied using DAPI and methyl green to find where the Ru(II) complexes bind on CT-DNA. In both cases, the competition of a second molecule for DNA binding resulted in loss the of absorbance . In thesOne of the most important functions of tight junctions between cells is to limit the spread of ions and hydrophilic non-ionic molecules depending on their load and size . Trans-eCancer cells have more transfer receptors than healthy cells to meet the increased iron need. Since Ru(II) complexes behave similarly to iron metal, they are more effectively taken up by cancer cells . TherefoToday, it is known that chemotherapeutic drugs, which are still widely used in cancer treatment, are not fully effective in all types of cancer, they cause drug resistance, and there have many important side effects. These adverse effects have led researchers to focus on metal-based anticancer drugs that show less toxicity and high selectivity to cancer cells. At this point, studies have rapidly increased in the field of metal-based pharmaceuticals in the treatment of cancer cells. Ruthenium complexes, metalopharmaceutical compounds, have shown attractive properties including high ligand exchange rates, higher cytotoxic effects, and high selectivity. Within the scope of this study, TSC ligands  and arene Ru(II) complexes  were synthesized and characterized structurally, and their biological potency was also investigated. It was seen that newly synthesized Ru(II) complexes were found to be taken into cancer cells effectively within 24 h, and had a strong cytotoxic effect on ovarian cancer cell lines. Considering the results of this study, it is thought that the newly synthesized organo Ru(II) complexes  may be potential molecules for effective treatment for the patients who have been diagnosed with ovarian cancer.


VSe2@MWCNT was mixed homogenously using sonication and fabricated film electrodes without a binder and substrate via vacuum filter. The hybrid electrode exhibited high-rate capability and stable cycling performance with a discharge capacity of 469.1 mAhg−1 after 200 cycles. Furthermore, VSe2@MWCNT exhibited coulombic efficiency of ~99.7%, indicating good cycle stability. Additionally, VSe2@MWCNT showed a predominant 85.5% of capacitive contribution at a scan rate of 1 mVs−1 in sodiation/desodiation process. These results showed that VSe2@MWCNT is a suitable anode material for flexible SIBs.As the market for electric vehicles and portable electronic devices continues to grow rapidly, sodium-ion batteries (SIBs) have emerged as energy storage systems to replace lithium-ion batteries (LIBs). However, sodium-ion is heavier and larger than lithium-ion, resulting in volume expansion and slower ion transfer. It is necessary to find suitable anode materials with high capacity and stability. In addition, wearable electronics are starting to be commercialized, requiring a binder-free electrode used in flexible batteries. In this work, we synthesized nano flake-like VSe Th. Th2@MWCtion (2) :(2)DNa=Rcy range . The DNao 2 Ag−1 d. The VS.01 Ag−1 e. VSe2@Mrocesses ; (2) VSeium-ions ; (3) nonuctivity .2@MWCNT, CV tests were conducted at various scan rates at 0.2 to 2 mVs−1 (−1) had a correlation, as shown in Equations (3) and (4). In Equation (4), the value of b (0.5 < b < 1) could be calculated based on the slope of the log (peak current)-log (scan rate) plot [To study sodium-ion storage and quantitative kinetics of VSe 2 mVs−1 a. With ate) plot .(3)i V=b close to 0.5 was a diffusion-controlled reaction (Faradaic process). A value close to 1 indicated a capacitive-controlled reaction (non-Faradaic process) [b value was 0.61 at an oxidation peak of 2.4 V and 0.63 at a reduction peak of 1.3 V  . The b vof 1.3 V b. Electrh k2ν1/2 .(5)i V=−1 for VSe2@MWCNT . These results indicate that a capacitive behavior-controlled process is better than a diffusion-controlled process. As previously reported, high capacitive contribution enables better cycle performance and plays a pivotal role in good stability and a long lifespan [2 and the high electrical conductivity of MWCNT can improve the diffusion kinetics of sodium ions.The obtained proportion of capacitive contribution (brown region) was 85.5% at a scan rate of 1 mVse2@MWCNT c. Figurelifespan ,62,63. T2 was synthesized with a colloidal method. It was hybridized with MWCNT through vacuum filtration. Such flake-like nanomaterials increased the surface area and made it easier to encounter electrolytes, thus facilitating sodium-ions kinetics. By using MWCNT with excellent flexibility and electrical conductivity, VSe2 problems could be solved. Electrodes were prepared without a binder and substrate by hybridization of MWCNT with VSe2. Therefore, using Raman spectroscopy, non-covalent functionalized CNT and VSe2 composite showed increased sp2 carbon structure crystallinity. The hybrid anode exhibited a high level of coulombic efficiency of 99.7 % and a discharge capacity of 469.1 mAhg−1, even after 200 cycles. The VSe2@MWCNT electrode measured various current densities and showed specific capacities of 319.6, 274.3, 239.2, 204.6, 186.9, 164.8 and 140.8 mAhg−1 at 0.05, 0.1 0.2, 0.5, 0.8, 1.0 and 2.0 Ag−1. We believe that such binder-free VSe2@MWCNT composite films can be easily prepared through the strategy described in this work and be successfully applied as new anode materials in SIBs.In summary, a flake-like VSe


Berberis lyceum was extensively used for the treatment of several human diseases.Traditionally, This study was undertaken to determine in vivo effects of Berberis lyceum root bark against doxorubicin-induced cardiotoxicity and cisplatin-induced neurotoxicity in Sprague Dawley rats.A single dose of doxorubicin (20 mg/ kg i. p) and cisplatin (4mg/kg i.p) was used to induce cardiotoxicity and neurotoxicity, respectively. Berberis lyceum methanolic extract was given orally (200 and 400 mg/ kg) to toxicity-induced rats. The cardiac biomarkers i.e. serum aspartate aminotransferase, alanine transaminase, lactate dehydrogenase, creatine kinase and creatine kinase MB were analyzed in blood collected from cardiotoxic rats. The tissue oxidative stress markers included protein, glutathione s-transferase specific activity, catalase activity, total glutathione, and malondialdehyde levels were measured in cardiac and brain homogenate of the respective groups.Berberis lyceum methanolic extract has the potential to reduce the doxorubicin-induced cardiotoxicity and cisplatin-induced neurotoxicity significantly (*p<0.05) by reducing the serum markers and oxidative stress parameters. Histopathological analysis exhibited a marked improvement in the morphology of cardiac and brain tissues.It is concluded that methanolic extract of Berberis lyceum root bark has the potential to protect and reverse anticancer drugs induced cardiotoxicity and neurotoxicity. Cancer is a neoplastic disorder that is a chief cause of death in many developed and emerging countries. The global economic burden of cancer is substantially raised and due to this disease, 7.6 million 13%) deaths are reported annually. For many years, medicinal plants have been used to cure or prevent a diverse range of disorders. There is a sufficient number of scientific literature is available that verified the anticancer potential of medicinal plants due to the presence of various bioactive chemicals.3% deaths2Doxorubicin (DOX) is a highly effective drug used to treat cancers like leukemia11Cisplatin is a platinum-based chemical and widely used to cure various malignancies. It exerts its antineoplastic effects by the formation of DNA adducts, by induction of oxidative stress, and by triggering apoptosis in tumor cells. Cisplatin being an inert molecule requires activation before interacting with nucleophilic target sites. A non-enzymatic water-based reaction activates cisplatin into a charged molecule.14Berberis lyceum Royle belongs to the family Berberidaceae and it is a common medicinal plant found in Pakistan. Berberis lyceum fruit is known as “Kashmal” while the root is called “Darhald”.20The study aimed to evaluate the protective effects of Berberis lyceum root bark against doxorubicin-induced cardiotoxicity and cisplatin-induced neurotoxicity in Sprague Dawley rats because previous no such study was found through an extensive literature survey.Doxorubicin (Brand name: Adrim) intravenous infusion was purchased from Atco Pharma Laboratories (Pvt). Ltd. Anthrone reagent was obtained from Sigma life sciences, Germany. Folin and Ciocalteu's phenol reagent and Triton X acquired from Unichem chemicals, Ireland. Vitamin E was procured from Merck Pvt. Ltd. For aspartate transaminase (AST) and alanine transaminase (ALT) analysis, kits by Singapore Biosciences (SBio) PTE Ltd. were used. The kits by Randox Laboratories Ltd. were used to estimate lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase myocardial band (CKMB). All chemicals were analytical grade.Berberis lyceum dried root bark was powdered. 250 g of powder was soaked in 800 mL of methanol and macerated for 6 days with intermittent stirring. This mixture was filtered and residues were again soaked in methanol for 3 days. The filtrate was pooled and dried by a rotary evaporator at 45°C. A total of 25 g of Berberis lyceum methanol extract (MEBL) was obtained. The extract was stored at 4 – 8°C.Qualitative phytochemical screening of Berberis lyceum methanolic extract was performed to detect the presence of various chemical compounds such as glycosides, saponins, tannins, alkaloids, flavonoids, triterpenoids, carbohydrates, proteins, fats and, fixed oils.The quantitative analysis of the phytochemicals (primary and secondary metabolites) was also performed according to the standard procedures. Primary metabolites estimation was included total protein 26Sprague Dawley rats were procured from the University of Health Sciences (UHS) Lahore. The animals were handled as per guidelines of an Animal Ethics Committee of University College of Pharmacy (PUCP), University of the Punjab Lahore, Pakistan. The committee reviewed and approved the experiment protocol (issued voucher no: AEC/PUCP/1051). The rats were acclimatized and maintained under controlled conditions; temperature 25°C ± 2 and 60% relative humidity with a 12/12 hour dark and light cycle. Animals were provided a standard diet and water ad libitum.For the in vivo study, animals were randomly selected from the animal house. The extract was dissolved in distilled water (DW) and vortexed to get a homogenous mixture. Animals were grouped as the following scheme having 6 rats in each group:GROUP 1 (Control): Rats were given water as a vehicle for 21 days.GROUP 2 (DOX treated): Animals were given vehicle for 21 days and on the 19th day, a single dose of doxorubicin 20 mg/ kg body weight (BW) was administered intraperitoneally.GROUP 3 (MEBL-200): Animals were administered Berberis lyceum methanol extract 200 mg/ kg BW through oral gavage for 21 days.GROUP 4 (MEBL-400): Animals were administered 400 mg/ kg BW of Berberis lyceum methanol extract through oral gavage for 21 days.GROUP 5 (DOX+MEBL-200): Rats were administered 200 mg/kg BW of Berberis lyceum methanol extract through oral gavage for 21 days. On the 19th day, a single injection of doxorubicin 20 mg/ kg was co-administered intraperitoneally.GROUP 6 (DOX+MEBL-400): Animals were administered 400 mg/ kg BW of Berberis lyceum methanol extract through oral gavage for 21 days. On the 19th day, a single injection of doxorubicin 20 mg/ kg BW was co-administered intraperitoneally.GROUP 7 (DOX+Vitamin E): Group was administered vitamin E 100 mg/ kg BW through oral gavage for 21 days. On the 19th day, a single injection of doxorubicin 20 mg/ kg BW was co-administered intraperitoneallyGROUP 8 (Cisplatin treated): Animals were given vehicle for 21 days and on the 19th day, a single dose of cisplatin 4 mg/ kg BW was administered intraperitoneally.GROUP 9 (Cisplatin+MEBL-200): Rats were administered 200 mg/kg BW of Berberis lyceum methanol extract through oral gavage for 21 days. On the 19th day, a single injection of cisplatin 4mg/ kg BW was co-administered intraperitoneally.GROUP 10 (Cisplatin+MEBL-400): Animals were administered 400 mg/ kg BW of Berberis lyceum methanol extract through oral gavage for 21 days. On the 19th day, a single injection of cisplatin 4 mg/ kg BW was co-administered intraperitoneally.Blood samples from each rat were taken after 24 hours of the last dose of doxorubicin and cisplatin. All the rats were sacrificed and the heart from cardiotoxic and brain from neurotoxic group rats was removed. A small section of the heart and brain from each group were preserved in 10% formalin for histopathological examination. All the tissue samples were stored at -80°C for further analysis.The blood samples from rats were taken by cardiac puncture and collected in the serum gel separating tube. Then these samples were allowed to stand at 25°C for 30 minutes and subsequently centrifuged at 3000 rpm for about 15 minutes. The clear serum was collected from the gel top while cell debris settled down in the gel. Serum was transferred to labeled tubes and refrigerated at -80°C for future experimentation.Preparation of heart homogenate and brain homogenate Pooled hearts were finely chopped in phosphate buffer through scissors. Heart tissues were then homogenized with the help of a homogenizer then centrifuged at 12,500 × g at 4°C for 20 minutes. The final post mitochondrial supernatant (PMS) was carefully transferred to Eppendorf 's tubes and stored at -80°C for further analysis.The serum biochemical markers in cardiotoxic groups included aspartate transaminase (AST), creatine kinase (CK), alanine transaminase (ALT), lactate dehydrogenase (LDH) , creatine kinase MB (CK-MB) were measured according to the procedure specified within the kits. The Lowry method was applied for the estimation of total protein in PMS.21The GST-specific activity was estimated by Habig's method with slight modification. For GST-specific activity, the reaction mixture contained post mitochondrial supernatant of brain and heart, 100 mM phosphate buffer (pH 6.5), 30 mM GSH, and 30 mM CDNB. Change in the absorbance was measured at 340 nm on a spectrophotometer for 5 minutes. GST activity expressed in µmol/minute/mg protein.27Total GSH content was determined by Sedlak and Lindsay method with slight modification. Heart and brain tissues were homogenized in 67 mM phosphate buffer (pH 7.4) separately. 25% trichloroacetic acid added to each homogenate and centrifuged at 4,200 rpm for 40 minutes. Afterward, supernatants of each sample were separated. 200 mM Tris HCl buffer containing 0.2 M EDTA (pH 7.5), 10 mM DTNB, and methanol added to each supernatant. Then, the reaction mixture was incubated for 30 minutes at 37°C. The absorbance of the final yellow solution was recorded on a spectrophotometer at wavelength 412 nm.28Catalase activity measured according to Sinha prescribed method with minor modification. The tissue homogenate (heart/ brain) was vortexed with 0.01 M phosphate buffer (pH 7) and freshly prepared 0.2 M H2O2 Dichromate/acetic acid added to the reaction mixture. Then, this reaction mixture heated for 10 minutes till permanent green color appeared. The absorbance was taken at 570 nm at room temperature, and catalase activity was measured directly from the standard curve.29Malondialdehyde (MDA) test was carried out by using thiobarbituric (TBA) assay followed by Ohkawa method with a slight alteration in the experimental steps. Heart and brain tissue samples (1mg) were homogenized in 1.2% KCl separately. To homogenate, 20% acetic acid, 8% sodium lauryl sulfate, 20.8% TBA, and distilled water were added. The reaction mixture was incubated for 1 hour at 98°C. After incubation, butanol: pyridine (15:1) was added to the mixture and centrifuged for 30 min . The absorbance of the resultant supernatant was recorded at 532nm. The concentration of MDA (mM/g tissue) was directly calculated from the MDA standard curve.3010% formalin solution was used to fix each heart and brain tissue. The specimens were handled as per standard procedure and embedded in paraffin wax. The blocks were sectioned and stained using the hematoxylin-eosin (H & E) method and examined by light microscopy.31Graph Pad Prism version 7.01 software was used for the statistical computation of results. Analysis was done by using unpaired t-test, one-way ANOVA, and by Dunnett's test. All data expressed in mean standard deviation (Mean ± SD) and *P <0.05 was considered statistically significant.The effect of Berberis lyceum on doxorubicin-induced cardiotoxicity and cisplatin-induced neurotoxicity in rats was estimated by measuring serum markers, tissue markers, and by histopathology of the heart and brain tissues. The general appearance of all groups of animals was observed and noted throughout the study. In the doxorubicin treated group, there was drastic weight loss and fur had a pink tinge. Rats had soft watery feces and red exudates also appeared on the sides of the eyes and nose. These observations were significantly less in concurrent administration therapy (doxorubicin and Berberis lyceum treated group).Berberis lyceum qualitative and quantitative phytochemical screening  revealedThe study outcomes showed that serum AST level in the doxorubicin treated group was significantly (*p < 0.05) increased (240.66 ± 8.32 U/L) than the control group (148.37 ± 11.5 U/L). The methanolic extract groups (MEBL-200 and MEBL-400) demonstrated an insignificant change in the AST level against the control. The concurrent administration of the doxorubicin and Berberis lyceum methanolic extract significantly decreased the AST level as compared to the doxorubicin treated group. For group 5 and group 6, a decrease in the AST level: 166.03 ± 8.58 U/L and 150.21 ± 4.49 U/L was reckoned, respectively. The co-administration of the doxorubicin and vitamin E also showed a significant drop (152.47 ± 8.01 U/L) in the AST level .This study has exhibited that the ALT level in the doxorubicin treated group was significantly (*p < 0.05) increased (77.07 ± 2.8 U/L) than the control group (40.6 ± 4.83 U/L). The methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in ALT level as compared to the control. The co-administration of doxorubicin and Berberis lyceum methanolic extract significantly reduced the ALT level in comparison with the doxorubicin-treated group. For group 5 and group 6, a decrease in the ALT level: 50.77 ± 1.93 U/L and 43.7 ± 2.20 U/L was observed, respectively. The concurrent administration of the doxorubicin and vitamin E displayed a significant reduction (42.97 ± 2.54 U/L) in the ALT level .The present study results revealed that serum CK level in the doxorubicin treated group was significantly (#p < 0.05) increased (497.7 ± 8.91 U/L) than the control group (156.23 ± 5.26 U/L). The methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in serum CK level in comparison to the control. The concurrent administration of the doxorubicin and Berberis lyceum methanolic extract significantly decreased CK level against the doxorubicin-treated group. For group 5 and group 6, a decrease in the CK level: 223 ± 20.25 U/L and 206.2 ± 13.86 U/L was noticed, respectively. The co-administration of the doxorubicin and vitamin E also showed a significant decrease (213.47 ±16.3 U/L) in serum CK level .The current investigation demonstrated that serum CK-MB level in the doxorubicin treated group was significantly (*p < 0.05) increased (206.16 ± 1.48 U/L) than the control group (65.93 ± 2.86 U/L). The methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in serum CK-MB level as compared to the control. The concomitant administration of the doxorubicin and Berberis lyceum methanolic extract significantly decreased the CK- MB level against the doxorubicin-treated group. For group 5 and group 6, a decrease in the CK-MB level: 140.73 ± 1.92 U/L and 116.1 ± 1.56 U/L was seen, respectively. The co-administration of the doxorubicin and vitamin E manifested a significant reduction (115.93 ±2.89 U/L) in serum CK-MB level as compared to the doxorubicin-treated group .This research outcome showed that serum LDH level in the doxorubicin-treated group was significantly (*p < 0.05) increased (837.43 ±8.44 U/L) than the control group (281.97 ± 23.2 U/L). The methanolic extract groups (MEBL-200 and MEBL-400) illustrated an insignificant change in serum LDH level contrasted with the control. The co-administration of doxorubicin and Berberis lyceum methanolic extract significantly decreased the LDH level as compared to the doxorubicin-treated group. For group 5 and group 6, a decrease in the LDH level: 588.5 ± 14.16 U/L and 395.27±14.70 U/L was observed, respectively. The concomitant use of the doxorubicin and vitamin E also exhibited a significant drop-off (381.07±14.69 U/L) in serum LDH level .The current study results showed that the protein content in the doxorubicin-treated group was significantly (# p < 0.05) decreased (39.13 ± 0.24 mg/mL) than the control group (68.07 ± 0.74 mg/mL). Berberis lyceum methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in protein content against the control. The parallel administration of the doxorubicin and Berberis lyceum methanolic extract significantly increased protein content over the doxorubicin-treated group. For group 5 and group 6, the respective increase in protein content was calculated as 68.43 ± 0.69 and 60.13 ± 0.25. The co-administration of the doxorubicin and vitamin E also showed a significant increase (60.80 ± 0.33 mg/mL) in protein content .The protein content in the cisplatin-treated group was significantly (*p < 0.05) decreased (28.9 ± 1 mg/mL) than the control group (68.07 ± 0.74 mg/mL). Berberis lyceum methanolic extract groups (MEB-200 and MEBL-400) have indicated an insignificant change in protein content as compared to the control.he simultaneous administration of the cisplatin and Berberis lyceum methanolic extract significantly increased protein content in contrast with the cisplatin-treated group. For group 5 and group 6, the respective increase in protein content 43.7 ± 1.4 mg/mL and 44.9 ± 1.2 mg/mL was noticed. The concurrent administration of the cisplatin and vitamin E also demonstrated a significant increase (47.2 ± 0.3 mg/mL) in protein content .This study showed that the GST specific activity in the doxorubicin-treated group was significantly (*p < 0.05) decreased (0.027 ± 0.006 µmol/min/mg) than the control group (0.068 ± 0.003 µmol/min/mg). The methanolic extract groups (MEBL-200 and MEBL-400) revealed an insignificant change in GST activity as compared to the control. The synchronous administration of the doxorubicin and Berberis lyceum methanolic extract significantly (*p < 0.05) enhanced the GST activity as compared to the doxorubicin-treated group. For group 5 and group 6, the increase in the GST activity was found to be 0.052 ± 0.001 µmol/min/mg and 0.056 ± 0.002 µmol/min/mg, respectively. The co-administration of the doxorubicin and vitamin E also exhibited a significant increase (0.057 ± 0.002 µmol/min/mg) in the GST activity as shown in The GST specific activity in the cisplatin-treated group was significantly (*p < 0.05) decreased (0.011 ± 0.01 µmol/min/mg) than the control group (0.058 ± 0.01 µmol/min/mg). The methanolic extract treated groups (MEBL-200 and MEBL-400) showed an insignificant change in the GST activity as compared to the control. The concurrent administration of the cisplatin and Berberis lyceum methanolic extract significantly enhanced GST activity as compared to the cisplatin-treated group. For group 5 and group 6, an increase in the GST activity 0.043 ± 0.01 µmol/min/mg and 0.049 ± 0.01 µmol/min/mg was estimated, respectively. The parallel administration of the cisplatin and vitamin E exhibited a marked increase (0.055 ± 0.01 µmol/min/mg) in the GST activity .The current study outcomes have demonstrated that the tGSH level in the doxorubicin-treated group was significantly (*p < 0.05) decreased (0.54 ± 0.03 mmol/mg) than thcontrol group (2.25 ± 0.053 mmol/mg). Berberis lyceum methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in the tGSH level against the control. The concurrent administration of the doxorubicin with different doses of Berberis lyceum methanolic extract treated groups has significantly increased the tGSH level over the doxorubicin-treated group. For group 5 and group 6, the respective increase of 1.79 ± 0.003 mmol/mg and 1.98 ± 0.03 mmol/mg was seen and the results are shown in The tGSH level in the cisplatin-treated group was significantly (*p < 0.05) decreased (0.48 ± 0.06 mmol/mg) than control group (1.25 ± 0.04 mmol/mg). Berberis lyceum methanolic extract groups (MEBL-200 and MEBL-400) have indicated an insignificant change in the tGSH level as compared to control. The co-administration of the cisplatin with different doses of Berberis lyceum methanolic extract treated groups significantly increased the tGSH level against the cisplatin-treated group. For group 5 and group 6, the respective increase 0.98 ± 0.1 mmol/mg and 1.06 ± 0.05 mmol/mg tissue was estimated .The present investigation illustrated that the CAT activity in the doxorubicin-treated group was significantly (*p < 0.05) decreased (0.287 ± 0.13 µmol/min/mg) than the control group (0.470 ± 0.12 µmol/min/mg). Berberis lyceum methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in the CAT activity as compared to the control. The parallel administration of the doxorubicin and Berberis lyceum methanolic extract significantly (*p < 0.05) decreased the CAT activity in comparison to the doxorubicin-treated group. For groups 5 and 6, the respective decrease in CAT activity was 0.3691 ± 0.085 µmol/min/mg and 0.484 ± 0.160 µmol/min/mg. The concurrent administration of the doxorubicin with vitamin E also exhibited a significant decrease (0.447 ± 0.068 µmol/min/mg) in the CAT activity when compared with the doxorubicin-treated group .The CAT activity in the cisplatin-treated group was significantly (*p < 0.05) decreased (0.32 ± 0.02 µmol/min/mg) than the control group (0.66 ± 0.02 µmol/min/mg). Berberis lyceum methanolic extract groups (MEBL-200 and MEBL-400) demonstrated an insignificant change in CAT activity as compared to the control. The co-administration of the cisplatin and Berberis lyceum methanolic extract treated groups significantly decreased the CAT activity than the cisplatin-treated group. For group 5 and group 6, the respective decrease was 0.59 ± 0.01 µmol/min/mg and 0.61 ± 0.02 µmol/min/mg. The concomitant administration of the cisplatin with vitamin E produced a significant decrease (0.64 ± 0.03 µmol/min/mg) in the CAT activity as compared to the cisplatin-treated group .The study results manifested that the MDA level in the doxorubicin-treated group was significantly (*p < 0.05) increased (38.01 ± 0.2 µmol/g) than the control group (14.2 ± 0.3 µmol/g). The methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in the MDA level as compared to the control. The concurrent administration of the doxorubicin with Berberis lyceum methanolic extract significantly decreased the MDA level in comparison to the doxorubicin treated group. For groups 5 and 6, the MDA level dropped to 22.3 µmol/g ± 0.46 and 18.76 ± 0.3 µmol/g, respectively. The synchronous administration of the doxorubicin with vitamin E also exhibited a significant decrease (18.4 ± 0.34 µmol/g) in the MDA level as compared to the doxorubicin-treated group .The MDA level in cisplatin-treated group was significantly (*p < 0.05) increased (183.9 ± 3.3 µmol/g) than control group (88.4 ± 2.9 µmol/g). The methanolic extract groups (MEBL-200 and MEBL-400) showed an insignificant change in the MDA level against the control. The co-administration of the cisplatin with Berberis lyceum methanolic extract significantly decreased the MDA level as compared to the cisplatin-treated group. For group 5 and group 6, the respective decrease was 93.4 ± 0.6 and 89 ± 2.6 and the results are presented in The histopathological examination of the control  heart tissue showed the normal architecture of myocardiocytes like well-arranged fibers and nucleus with no vacuolization . In the Doxorubicin-induced cardiotoxicity and cisplatin-induced neurotoxicity are foremost challenges for the current chemotherapy practices and need to be addressed on an urgent basis. Doxorubicin is one of the widely prescribed broad-spectrum anti-cancer drugs. Doxorubicin causes severe cardiotoxicity that limits its clinical use.35Globally, medicinal plants are used to treat numerous acute and chronic illnesses because these are considered safe, effective, easily accessible, and inexpensive source of therapy.39In the present investigation, it is proved that the administration of Berberis lyceum root bark methanolic extract can improve or reverse the doxorubicin-induced and cisplatin-induced changes. Earlier studies have confirmed that the release of ALT, AST, LDH, CK, and CK-MB will increase from myocardial tissues with doxorubicin use. These enzymes are valuable tools to assess doxorubicin-induced myocardial toxicity. This study found that the tissue levels of ALT, AST, LDH, CK, and CK-MB were increased in doxorubicin-treated rats, and pre-treatment with Berberis lyceum root bark methanolic extract significantly decreased the levels of these enzymes.41The histopathological examination of the heart and brain tissues revealed that doxorubicin and cisplatin both caused the damage of tissue morphology and resulted in cellular death. The histopathological evaluation of methanolic extract treated showed marked improvement in both organ tissues with moderate to mild degeneration. The results have demonstrated that the response of methanolic extract against anticancer drugs induced toxicity is dose-dependent as evident by improving enzymes level and tissue histopathology, i.e. mild protection is attained at a dose of 200 mg/kg while a significant defense against toxicity is achieved at an effective dose of 400 mg/kg.Berberis lyceum exhibited significant cardio and neuroprotection against anticancer drugs. The protective activity can be related to the plant antioxidant potential and its membrane-stabilizing effect by reducing lipid peroxidation. It is suggested that further research on Berberis lyceum Royle should be carried out to develop new and effective therapeutic agents to treat toxicity induced by anticancer drugs.


Traxoprodil is a selective N-methyl-d-aspartate receptor subunit 2B (NR2B) receptor inhibitor with rapid and long-lasting antidepressant effects. However, the appropriate dosage, duration of administration, and underlying mechanism of traxoprodil's antidepressant effects remain unclear. The purpose of this study is to compare the antidepressant effects of traxoprodil in different doses and different durations of administration and to explore whether traxoprodil exerts antidepressant effects via the brain-derived neurotrophic factor/extracellular signal-regulated kinase/cAMP-response element binding protein (BDNF/ERK/CREB) and protein kinase B/Forkhead box O/building information modelling (AKT/FOXO/Bim) signaling pathway. Mice were randomly divided into control group, chronic unpredictable mild stress (CUMS) + vehicle group, CUMS + traxoprodil  groups, and CUMS + fluoxetine (5 mg/kg) group, followed by a forced swimming test, tail suspension test, and sucrose preference test. Western blotting and immunohistochemistry were used to measure the protein expression of BDNF, p-ERK1/2, p-CREB, NR2B, AKT, FOXO1, FOXO3a, and Bim. Compared with the control group, CUMS treatment increased immobility time; decreased sucrose preference; reduced expression of BDNF, p-ERK1/2, and p-CREB; and increased expression of AKT, FOXO, and Bim in the hippocampus. These alterations were ameliorated by administration of 20 mg/kg or 40 mg/kg of traxoprodil after 7 or 14 days of administration and with 10 mg/kg of traxoprodil or 5 mg/kg of fluoxetine after 21 days of administration. At the 7-day and 14-day timepoints, traxoprodil displayed dose-dependent antidepressant effects, with 20 and 40 mg/kg doses of traxoprodil producing rapid and strong antidepressant effects. However, at 21 days of administration, 10 and 20 mg/kg doses of traxoprodil exerted more pronounced antidepressant effects. The mechanism of traxoprodil's antidepressant effects may be closely related to the BDNF/ERK/CREB and AKT/FOXO/Bim signaling pathway. Depression is a common mood disorder characterized by low mood, anhedonia, anxiety, and sleep disorders, and it is accompanied by changes in neurotransmitters in the brain . It is rGlutamate, as the major excitatory neurotransmitter in the central nervous system (CNS), plays an important role in learning, memory, and synaptic plasticity in physiological states . MultiplThe neurotrophic hypothesis holds that brain-derived neurotrophic factor (BDNF) regulates the function of neurons by activating tyrosine kinase receptor B (TrkB) and mitogen-activated protein kinase (MAPK) signaling pathways , 16. TheTraxoprodil is a selective NR2B receptor inhibitor. Administration of a subactive dose of traxoprodil with subactive doses of desipramine, paroxetine, and other traditional antidepressants at subtherapeutic doses led to an antidepressant-like effect in mice . A randoMale CD1 mice (5-6 weeks) were obtained from Liaoning Changsheng Biotechnology Co., Ltd.  and kept under standard laboratory conditions (temperature 25 ± 2°C and humidity 45-55%) with a 12 h light/dark cycle (light on: 7 : 00-19 : 00). Mice were provided with food and water freely except during behavioral tests or during a water and food deprivation stressor. All the experimental procedures were done in accordance with the Guide for The Care and Use of Laboratory Animals, National Institutes of Health, and were approved by the Experimental Animal Welfare and Ethics Committee of China Medical University (IACUC Issue No. CMU2019167). All efforts were taken to minimize animal suffering and reduce the number of animals used.n = 6), CUMS + vehicle group (n = 6), CUMS + traxoprodil (10 mg/kg (n = 6), 20 mg/kg (n = 6), and 40 mg/kg (n = 6)) groups, and a CUMS + fluoxetine (5 mg/kg) group (n = 6). The CUMS procedure was based on the methods as described previously. In brief, all mice except the control group were exposed to different stressors: (1) illumination overnight, (2) cage tilted 45° for 12 h, (3) wet bedding for 12 h, (4) no padding for 12 h, (5) restraint for 4 h, and (6) water and food deprivation for 24 h. The CUMS procedure lasted for 3 weeks, and the stressors were applied daily in a random order to add the unpredictable characteristic of the experiment [Mice were randomly divided into a control group  doses were dissolved in 0.9% saline containing 1% Tween-80, and the fluoxetine hydrochloride  doses were dissolved in 0.9% saline. Mice were administered traxoprodil , fluoxetine hydrochloride , or 0.9% saline for 7, 14, and 21 days, respectively. All drug solutions used in this study were administered at a volume of 10 ml/kg and prepared just before use.After treatment, the FST was performed on mice in a separate, quiet environment. The mice were placed individually in a glass cylinder (height 25 cm and diameter 16 cm) containing 20 cm of water (23-25°C) and adapted for 10 min the day before the test. All animals were forced to swim for 6 min, and their immobility time was recorded during the last 4 min of the test. A mouse was judged to be immobile when it stopped struggling and floated in the water or made only necessary movements to keep its head above the water.The TST was performed the day after the FST. In short, the mice were attached individually with adhesive tape 1.5 cm from the tip of the tail and suspended 15 cm above a table for 6 min. After 2 min of acclimatization, the immobility time of each mouse in the last 4 min was observed and recorded. Immobility time was defined as the duration in which a mouse did not struggle, bend, or twist.w/v, 100 ml) for 24 h. On the second day, one bottle of 1% sucrose solution was replaced with pure water for 24 h, and the position of the sucrose solution bottle and pure water bottle was changed at 12 h to avoid drinking position preference. On the third day, the mice were deprived of food and water for 24 h. Next, mice were given free access to a preweighed bottle of 1% sucrose solution (100 ml) and a preweighed bottle of pure water (100 ml). After 12 hours, the consumption of sucrose solution and pure water was recorded, and sucrose preference (%) was calculated by the following formula: Here SP(%) = sucrose solution consumption (g)/(sucrose solution consumption (g) + pure water consumption (g)) × 100%.The SPT lasted for 4 days. The first 3 days were devoted to training the mice, and testing occurred on the 4th day. On the first day, mice were housed individually and given two bottles of 1% sucrose solution . The PVDF membranes were washed and incubated at room temperature with goat anti-rabbit or goat anti-mouse IgG HRP-conjugated antibody  for 2 hours. The protein bands were visualized by enhanced ECL kit  in a gel image analyzing system. The quantification of protein bands was performed using ImageJ software and normalized against the level of β-actin protein.After the mice were decapitated, the hippocampus was dissected and immediately homogenized in RIPA buffer  containing PMSF  for 30 min on ice and then centrifugated at 14000 rpm for 25 min at 4°C. Total protein supernatant was collected, and the concentration was determined via BCA protein assay kit . Equal amounts of protein were loaded per well on 12% SDS-PAGE gels and then transferred onto PVDF membranes. After blocking in 5% skim milk or BSA for 1-2 hours at room temperature, the membranes were incubated at 4°C overnight with anti-BDNF antibody , anti-p-ERK1/2 antibody , anti-ERK1/2 antibody , anti-p-CREB antibody , anti-CREB antibody , anti-NR2B antibody , anti-AKT antibody , anti-FOXO1 antibody , anti-FOXO3a antibody , anti-Bim , or anti-μm using a paraffin slicer. Slices were boiled in a 10 mM citrate buffer (pH 6.0) for 10 min to expose antigens and then cooled to room temperature. The endogenous peroxides of sections were quenched with 0.3% H2O2 for 10 min. Slices were blocked with 5% normal goat serum for 10 min and then incubated with anti-BDNF antibody , anti-p-ERK1/2 antibody , anti-p-CREB antibody , anti-AKT antibody , or anti-FOXO3a antibody  overnight at 4°C. The resulting slides were washed and incubated with biotinylated secondary antibody  for 2 h at room temperature. Then, sections were stained with 3,3-diaminobenzidine  for 1-2 min, counterstained with hematoxylin for 20 s, decolorized, and sealed. Finally, the hippocampal subfields (CA1 and hilus) were observed under a 400x light microscope  and analyzed using ImageJ software.The brains of mice were fixed with 4% paraformaldehyde and embedded in paraffin and then cut into sections with thickness of 4 P < 0.05 was considered statistically significant.All data were expressed as mean ± SEM, analyzed, and plotted using GraphPad Prism 8.0 . Significant differences were compared using one-way analysis of variance (ANOVA). P < 0.001, respectively) and the TST  in the CUMS model groups were significantly increased at 7, 14, and 21 days of treatment compared to controls. Treatment with 20 or 40 mg/kg of traxoprodil significantly decreased the immobility times in the FST (P < 0.001 and 40 mg/kg: P < 0.001) and TST (P < 0.05 and 40 mg/kg: P < 0.001) in CUMS mice after 7 days of administration, but the 10 mg/kg traxoprodil group and 5 mg/kg fluoxetine group did not demonstrate such changes. After 14 days of administration, traxoprodil given at doses of 20 or 40 mg/kg significantly reduced the immobility times of the FST (P < 0.001 and 40 mg/kg: P < 0.001) and TST (P < 0.01 and 40 mg/kg: P < 0.001). The immobility time of CUMS mice in the 10 mg/kg traxoprodil group and fluoxetine group was slightly decreased at the 14-day timepoint, but the difference was not statistically significant. Furthermore, our results indicated that the immobility time during the FST  and TST  was reduced by the administration of traxoprodil at 10, 20, and 40 mg/kg or fluoxetine after 21-day treatment, but the immobility time in the 40 mg/kg traxoprodil group and 5 mg/kg fluoxetine group was longer than that in the 10 and 20 mg/kg traxoprodil groups at the same timepoint.After applications of the chronic unpredictable mild stressor protocol, the immobility times of the FST . After 7 days of administration, 20 (P < 0.001) and 40 (P < 0.001) mg/kg of traxoprodil significantly increased sucrose consumption in CUMS mice, but 5 mg/kg of fluoxetine and 10 mg/kg of traxoprodil did not (P < 0.01) and 40 (P < 0.01) mg/kg traxoprodil groups (P < 0.001) and 20 (P < 0.001) mg/kg traxoprodil treatment led to significantly increased sucrose preference after 21 days, as did 5 mg/kg fluoxetine (P < 0.01), but 40 mg/kg of traxoprodil did not  = 6.032), p-ERK1/2  = 11.27), and p-CREB  = 10.98) in the CUMS model group were significantly decreased compared with the control group, using β-actin, ERK1/2, and CREB as inner controls, respectively. The expression of BDNF (P < 0.01 and 40 mg/kg: P < 0.01) and p-ERK1/2 (P < 0.01 and 40 mg/kg: P < 0.01) in the hippocampus was significantly increased in the 20 and 40 mg/kg traxoprodil groups. The expression level of p-CREB in the hippocampus of CUMS mice was significantly increased in the 40 mg/kg traxoprodil group (P < 0.01), but the increase in the 20 mg/kg traxoprodil group did not reach significance. Neither 10 mg/kg traxoprodil nor 5 mg/kg fluoxetine influenced the expression of BDNF, p-ERK1/2, or p-CREB in the hippocampus of CUMS mice after 7 days of treatment.After 7 days of administration, western blot analysis showed that the protein expression levels of BDNF  = 6.567), p-ERK1/2  = 7.192), and p-CREB  = 4.303) in the hippocampus compared with the control group. Traxoprodil at 20 and 40 mg/kg significantly increased the hippocampal protein levels of BDNF (P < 0.05 and 40 mg/kg: P < 0.01), p-ERK1/2 (P < 0.01 and 40 mg/kg: P < 0.001), and p-CREB (P < 0.05 and 40 mg/kg: P < 0.05), while 10 mg/kg of traxoprodil only induced a significant increase in p-ERK1/2 (P < 0.05) expression. BDNF and p-CREB levels in the hippocampus were slightly increased in the 10 mg/kg traxoprodil group and 5 mg/kg fluoxetine group, but the differences did not reach significance.At 14 days of administration, CUMS treatment significantly reduced the expression of BDNF  = 11.07), p-ERK1/2  = 6.128), and p-CREB  = 7.746) compared to the control group. Nevertheless, 21-day treatment with fluoxetine and 10 and 20 mg/kg doses of traxoprodil markedly upregulated the expression of BDNF , p-ERK1/2 , and p-CREB , but treatment with 40 mg/kg of traxoprodil did not.After 21 days of administration, CUMS mice displayed significant reductions in hippocampal BDNF  = 11.60), AKT  = 5.811), FOXO1  = 13.22), FOXO3a  = 19.83), and Bim  = 9.878) in the CUMS group compared with the control group. The 20 and 40 mg/kg traxoprodil groups reduced the protein levels of NR2B (P < 0.001 and 40 mg/kg: P < 0.01), AKT (P < 0.01 and 40 mg/kg: P < 0.05), FOXO1 (P < 0.001 and 40 mg/kg: P < 0.01), FOXO3a (P < 0.001 and 40 mg/kg: P < 0.001), and Bim (P < 0.001 and 40 mg/kg: P < 0.05) in CUMS mice. However, there was no significant difference in the 10 mg/kg traxoprodil and 5 mg/kg fluoxetine groups compared with the CUMS group.To determine whether the AKT/FOXO pathway is involved in the effects of CUMS on depression-like behaviors, western blotting was used to examine the expression of related proteins in the hippocampus. After 7 days of administration, our results revealed a significant increase in the protein levels of NR2B  = 9.373), AKT  = 8.178), FOXO1  = 17.36), FOXO3a  = 11.25), and Bim  = 15.82) protein levels, whereas traxoprodil at 20 and 40 mg/kg significantly reduced the protein levels of NR2B (P < 0.001 and 40 mg/kg: P < 0.001), AKT (P < 0.01 and 40 mg/kg: P < 0.01), FOXO1 (P < 0.05 and 40 mg/kg: P < 0.001), FOXO3a (P < 0.05 and 40 mg/kg: P < 0.01), and Bim (P < 0.01 and 40 mg/kg: P < 0.01) in CUMS mice. There was no significant difference in levels of NR2B, AKT, FOXO1, FOXO3a, or Bim in the 10 mg/kg traxoprodil and 5 mg/kg fluoxetine groups.After 14 days of administration, CUMS increased NR2B  = 29.30), AKT  = 14.26), FOXO1  = 11.14), FOXO3a  = 18.44), and Bim  = 13.88) compared to the control group. However, the 5 mg/kg fluoxetine group and 10 and 20 mg/kg traxoprodil groups decreased the expression of NR2B , AKT , FOXO1 , and FOXO3a . Although 40 mg/kg traxoprodil significantly reduced FOXO1 (P < 0.001) protein level, it did not significantly alter levels of any other protein compared with the CUMS group. Levels of Bim were only significantly decreased in the 20 mg/kg traxoprodil group (P < 0.05).After 21 days of administration, CUMS mice displayed significant increases in hippocampal NR2B  = 40.74 and DG: P < 0.05, F = 15.68), p-ERK1/2  = 18.53and DG: P < 0.001, F = 35.84), and p-CREB  = 17.62 and DG: P < 0.001, F = 37.37) in the CUMS group compared to the control group. However, these changes in expression of BDNF , p-ERK1/2 , and p-CREB  were reversed with the administration of traxoprodil at 20 and 40 mg/kg doses for 7 days. No significant differences were found in the 5 mg/kg fluoxetine and 10 mg/kg traxoprodil groups.Immunohistochemistry showed that in the hippocampal CA1 and DG regions, there was lower expression of BDNF  = 35.61 and DG: P < 0.001, F = 19.19), p-ERK1/2  = 34.90 and DG: P < 0.001, F = 19.05), and p-CREB  = 21.84 and DG: P < 0.001, F = 15.06) in the hippocampal CA1 and DG regions were significantly reduced in the CUMS mice. Treatment with 20 and 40 mg/kg of traxoprodil significantly increased the levels of BDNF , p-ERK1/2 , and p-CREB  in the CA1 and DG regions of the hippocampus in CUMS mice. The protein levels in the 10 mg/kg traxoprodil group and 5 mg/kg fluoxetine group were slightly changed, but no significant differences were found.After 14 days of administration, compared with the control group, the protein levels of BDNF  = 16.28 and DG: P < 0.001, F = 45.68), p-ERK1/2  = 16.5 and DG: P < 0.001, F = 30.14), and p-CREB  = 25.14 and DG: P < 0.01, F = 9.952) in the hippocampus compared with the control group. However, traxoprodil at 10 and 20 mg/kg doses as well as 5 mg/kg fluoxetine significantly elevated the BDNF , p-ERK1/2 , and p-CREB  protein levels in the hippocampus. However, in the 40 mg/kg traxoprodil group, no significant differences were observed compared to CUMS + vehicle mice.After 21 days of administration, CUMS treatment resulted in decreased expression of BDNF (CA1: P < 0.001 and DG: P < 0.001) expression in the CUMS administration compared to the control group was determined in hippocampal CA1 and DG regions. Traxoprodil at 20 and 40 mg/kg doses reversed this change after 7  and 14 days of treatment , but no significant differences were found in the 5 mg/kg fluoxetine and 10 mg/kg traxoprodil groups. After 21 days of administration, traxoprodil and fluoxetine treatments significantly decreased the levels of FOXO3a  in CUMS mouse hippocampus, except that treatment with 40 mg/kg of traxoprodil had no effect on FOXO3a level in the DG region of the hippocampus.Molecules of FOXO are important factors to regulation of several cellular functions. Excessive production of FOXO can lead to neuronal cell death through an apoptosis pathway. Current research has shown that the transcriptional activity of FOXO3a is mediated by the PI3K/AKT pathway and other signal pathways. As shown in P < 0.001 and DG: P < 0.001) in the CA3 and DG regions of the hippocampus was significantly increased in the CUMS group. After 7 days of administration, 20 and 40 mg/kg of traxoprodil decreased the levels of AKT in the hippocampus  and 14 , but no difference was found in the 5 mg/kg fluoxetine and 10 mg/kg traxoprodil groups. After 21 days of administration, traxoprodil at 10 and 20 mg/kg doses as well as 5 mg/kg fluoxetine significantly decreased the AKT  protein levels in the hippocampus. However, in the 40 mg/kg traxoprodil group, no significant differences were observed compared to CUMS + vehicle mice.As presented in Current antidepressants are primarily based on altering monoaminergic signaling mechanisms, but delayed onset and treatment resistance limit their clinical use in many patients . TraxoprIn this study, the traditional antidepressant fluoxetine was used as a positive control drug to evaluate the antidepressant effects of traxoprodil at different concentrations and days of administration. We found that traxoprodil at 10 mg/kg for 21 days significantly reduced immobility time during the FST and TST in mice, similar to the antidepressant effect of fluoxetine at 5 mg/kg. Administration of traxoprodil at 20 and 40 mg/kg for 7 or 14 days significantly improved depressive-like behaviors, indicating that traxoprodil produced rapid and robust antidepressant effects in the CUMS mice. However, we did not observe any significant difference in depressive behaviors of the 10 mg/kg traxoprodil or 5 mg/kg fluoxetine groups compared with the CUMS group at 7 and 14 days. Thus, these results indicate that doses of 20 and 40 mg/kg of traxoprodil elicit rapid and significant antidepressant effects in CUMS mice. Similar to fluoxetine, the administration of traxoprodil at 10 mg/kg showed antidepressant effects only after more than 2 weeks. Additionally, the adverse psychiatric reactions caused by excessive drug accumulation may lead to a decrease in the effect of 40 mg/kg of traxoprodil after 21 days of administration and thereby negatively affect its antidepressant efficacy.One of the main points of this study was to investigate whether traxoprodil exerts antidepressant effects through the BDNF/ERK/CREB signaling pathway. A large number of previous studies have shown that chronic stress can lead to depression and may be associated with impairment of structural plasticity and neural cellular resilience –39. BDNFOn the other hand, FOXO transcription factors are regulators that mediate different physiological functions by regulating the expression of target genes . FOXO suA limitation of this study is that the protein expression of BDNF/ERK/CREB and Akt/FOXO/Bim signaling pathway in the hippocampus was measured only by western blot and immunohistochemistry. Furthermore, assessment of phosphorylated FOXO transcription factors and apoptosis-related experiments was not performed in this study. Additionally, further detailed studies are required that utilize inhibitors to block the expression of members of the BDNF/ERK/CREB and Akt/FOXO/Bim signaling pathways to confirm the mechanism by which traxoprodil exerts its antidepressant effects.Overall, this study illustrated the antidepressant effects of traxoprodil at different concentrations after different durations of treatment. After 7 days and 14 days of treatment, the antidepressant effects of traxoprodil on the depressive mice increased with increasing dose, and both 20 and 40 mg/kg of traxoprodil produced rapid and strong antidepressant effects. However, after 21 days of administration, treatment with 10 and 20 mg/kg of traxoprodil exerted more pronounced antidepressant effects. The mechanism of antidepressant effects of traxoprodil may be closely related to the activation of BDNF/ERK/CREB and Akt/FOXO/Bim signaling pathway.