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Polysaccharides are a class of biological macromolecules made up of sugar chains with multiple glycosidic bonds. To explore the extracted, purified, carboxymethylated and phosphorylated modified to obtained carboxymethylated polysaccharides (HSPS) and phosphorylated polysaccharides (HSPP), and the HSPS and HSPP were characterized and analyzed, and their antioxidant activity measured. Response surface design was used for the synthesis process of HSPS and HSPP, the structure characterization and antioxidant activities of HSPS and HSPP were evaluated. The results showed that the Mw and Mn of HSP were 14.66 kDa and 8.39 kDa, respectively. The monosaccharides were mainly galactose, glucose, xylose, and arabinose in a molar ratio of 37.68%: 24.43%: 13.63%: 9.18%. The optimal synthesis conditions for HSPS were as follows: reaction time of 2.01 h, reaction temperature of 44.61°C and the amount of monochloroacetic acid necessary was 1.18 g. The optimal synthesis conditions for HSPP were: reaction time of 4.28 h, reaction temperature of 80.78°C, and reaction pH of 8.98. HSPS and HSPP have scavenging effects on ABTS, super oxideanion and hydroxyl radicals. The HSPS and HSPP were characterized and analyzed, and their antioxidant activity measured.

Zearalenone (ZEN), one of the most prevalent non-steroidal oestrogenic mycotoxins, is primarily produced by Fusarium fungi. Due to its toxicity as an oestrogenic compound and wide distribution in feed and foods, the reproductive toxicology of ZEN exposure is of public concern. The aim of the present study was to investigate the effect of ZEN on Sertoli cells to identify apoptotic pathways induced by this compound. We found that ZEN reduced the viability and caused apoptosis in Sertoli cells in vitro. Notably, we observed that such effects were associated with a significant increase in reactive oxygen species (ROS) and the number of cells that showed positive staining for γH2AX and RAD51, enzymes essential for repairing DNA damage. There was a parallel decrease in the expression of occludin and connexin 43, proteins that are present in the testis-blood barrier and gap junctions of Sertoli cells, respectively. Overall, the present study confirms that ZEN exposure can have serious deleterious effects on mammalian Sertoli cells and offers novel insight about its molecular targets in these cells.

Polysaccharides are good chelating agents for metal ions, which are often used to synthesize polysaccharide metal ion complexes. With carboxymethyl pachymaran (CMP) as the substrate, carboxymethyl pachymaran iron (CMPF), carboxymethyl pachymaran selenium (CMPS), and carboxymethyl pachymaran zinc (CMPZ) were synthesized by response surface methodology, and their biological characteristics were studied. The results showed that the CMP was a β‐polysaccharide, and the degree of carboxymethylation was 0.6352. The polysaccharide metal ion complexes were characterized by physicochemical methods, scanning electron microscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. All the polysaccharides and complexes possessed antioxidant activity in vitro with scavenging activities to ABTS, superoxide anions, and ferrous ions. CMPF, CMPS, and CMPZ caused significant inhibition of A2780 cell proliferation, promoted the production of reactive oxygen species, and induced apoptosis in a human ovarian cancer cell line (A2780 cells). These results suggest that the CMP complex may be an effective candidate drug for cancer treatment in the field of functional food and pharmacology.

In this study, lotus root polysaccharide was taken as raw material. The preparation technology of its polysaccharide selenium was established by response surface design, and its antioxidant activity in vitro was studied. Selenium content was taken as the detection index, and the effects of reaction time, the ratio of sodium selenite to the polysaccharide, and the volume fraction of nitric acid on selenium content were investigated. Through the response surface method, the optimal synthesis conditions of lotus root polysaccharide selenium were obtained as follows: reaction time of 5.36h, the ratio of sodium selenite and lotus root polysaccharide is 0.78, the volume fraction of nitric acid 0.47%, the selenium content of lotus root polysaccharide selenium 0.1106%. Selenium, a polysaccharide from lotus root, had a scavenging effect on ABTS radical and superoxide ion, and the maximum scavenging rate was 52.17% and 85.23%, respectively. Aa shows that the synthesized lotus root polysaccharide selenium can be used as an antioxidant or functional food.

In this study, we extracted lotus root polysaccharide (LRP) and synthesized carboxymethylated lotus root polysaccharide (CM-LRP) using response surface methodology (RSM). The monosaccharide component of LRP was analyzed by pre-column derivatization high performance liquid chromatography (PCD-HPLC). The polysaccharide was characterized by ultraviolet (UV) spectroscopy scan, Fourier transform infrared spectroscopy (FTIR), circular dichroism spectrometer (CD), and scanning electron microscopy (SEM). It was found that the main monosaccharide component of LRP was glucose, accounting for more than 98% of the total polysaccharide component. RSM analyses revealed that the optimal conditions for CM-LRP synthesis were a reaction duration of 2.22 h, the chloroacetic acid dosage is 2.02 g, and a temperature of 46.87 °C. Under these conditions, the predicted degree of substitution (DS) was determined to be 0.5101. The superoxide anion, ferrous ion and hydroxyl radical antioxidant system were constructed to study the antioxidant activity of LRP and CMLRP. The activity of CMLRP to remove ferrous ions and hydroxyl radicals were significantly stronger than that of LRP, and it also showed a certain concentration-dependent. LRP has a better scavenging ability than CMLRP in scavenging superoxide anion free radicals. Our data revealed that CM-LRP is a promising natural antioxidant with potential value as a food supplement.

Acute gouty arthritis (AGA) is characterized by the accumulation of pro‑inflammatory cytokines, which are immunological responses to monosodium urate (MSU) crystals. It has been demonstrated that long non‑coding RNA (lncRNA)‑MM2P is a novel regulator of M2 polarization of macrophages. The aim of the present study was to investigate whether lncRNA‑MM2P regulates the MSU‑induced inflammatory process. In cell models of RAW 264.7 and THP‑1‑derived macrophages, decreased expression of lncRNA‑MM2P was observed in lipopolysaccharide‑ and MSU‑treated macrophages, which was accompanied with obvious inflammatory responses. Using small interfering RNA to knockdown lncRNA‑MM2P led to the upregulation of MSU‑mediated inflammatory responses, both in RAW 264.7 and THP‑1‑derived macrophages. In conclusion, lncRNA‑MM2P could be an important regulator of MSU‑induced inflammation, and therefore could be involved in the development of AGA.

A response surface method (RSM) was employed to optimize polysaccharide extraction from Panax japonicus (PJPS). Also, carboxymethyl substitution of PJPS (CM-PJPS) was studied using a response surface methodology. A three-variable Box–Behnken design (BBD) methodology was applied. RSM analyses revealed that conditions that maximized polysaccharide yield were as follows: a liquid-to-material ratio 22:1, an extraction temperature of 83.99 °C and an extraction duration of 2.32 h. Under optimum extraction conditions, the average absorbance value of PJPS was 0.6001, and the purity of PJPS was 98.2%. A quadratic regression model for CM-PJPS was obtained via BBD. The optimum extraction conditions for the process were determined to include a 1.38 h reaction time, 1.24 g monochloroacetic acid (MCA) and a reaction temperature of 52.85 °C. Under optimum extraction conditions, the average degree of CM-PJPS substitution was 0.9733. The structure of the PJPS was examined by means of Fourier transform infrared spectroscopy (FTIR). CM-PJPS exhibited the strongest anti-oxidant activity in vitro. In addition, CM-PJPS inhibited Skov3 and A2780 cancer cell proliferation. Our data revealed that CM-PJPS is a promising natural antioxidant with potential value as a food supplement and for treatment of cancer.

In this study, we extracted lotus root polysaccharide (LRP) and synthesised phosphorylated lotus root polysaccharide (PLP) using response surface methodology (RSM). RSM analyses revealed that the optimal conditions for PLP synthesis were a reaction duration of 7 h, temperature of 70 °C and pH of 11.38. Under these conditions, the predicted degree of substitution (DS%) was determined to be 9.96%. The structure of the LRP1 was examined by ultraviolet (UV) spectroscopy scan, Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) (1H and 13C). The monosaccharide composition of LRP1 was determined to be mannose (0.12%), ribose(0.18%), glucuronic acid(0.60%), galacturonic acid(0.09%), glucose(98.79%) and galactose(0.21%). The number average molecular weight (Mn) and the weight average molecular weight (Mw) of LRP1 were 10236 and 251783 g/mol. LRP1 exhibited high antioxidant activities in scavenging ABTS radicals, Superoxide anion radicals and Metal ion scavenging activity. PLP exhibited strong antioxidant activity in vitro. In addition, PLP inhibited Skov3 cancer cell proliferation and induced reactive oxygen species (ROS) production. Our data revealed that PLP is a promising natural antioxidant with potential value as a food supplement and for the treatment of cancer.

Di(2-ethylhexyl)phthalate (DEHP) has proven characteristics of an endocrine-disrupting compound (EDC), which can threaten the reproductive health of humans and other animals. In mammals, a series of chromosomal events occur during the meiotic stage of oocytes. External toxins may enter the body and cause infertility and other related diseases. Therefore, it is crucial to explore the influence of DEHP exposure on the molecular mechanism of germ cell meiosis. We used single-cell RNA sequencing (scRNA-seq) to analyse the ovaries of foetal mice at embryonic day 12.5 (E12.5) and E14.5 after maternal DEHP exposure. DEHP exposure further activated the pathways related to DNA repair in germ cells, increased the expression of genes related to DNA damage and changed the developmental trajectory of germ cells. DEHP exposure may affect the proliferation of pregranulosa (PG) cells. Moreover, DEHP exposure altered the signal transduction between PG cells and germ cells. We showed that DEHP affects meiosis by causing DNA damage in oocytes and disrupting the signal transduction between PG cells and germ cells. These results provide a strong theoretical basis for the prevention and treatment of DEHP-mediated female reproductive health problems.

Due to the lack of a Panax japonicus var. major reference genome, we assembled a reference transcriptome from P. japonicus C. A. Mey transcriptome sequencing data, and 203 283 unigenes were obtained. In this study, with the assistance from the Trinity, Bowtie2 and SAMtools softwares, 218 465 single nucleotide polymorphisms (SNPs) were identified by mapping the Illumina sequences to the reference transcriptome. The SNP forms included 126 262 transformations and 92 203 transversions. A large number of SNP loci were associated with triterpenoid saponin synthesis: 54 SNPs were associated with cytochrome P450, one with glycosyl transferase and 94 with the biosynthesis of the triterpenoid saponin backbone.

Although Walnut oil (WO) has been reported to enhance cognitive function, the underlying molecular mechanisms are not well understood. This study was designed to assess the effects of WO on spatial memory in rats through modulation of the expression of acid‐sensing ion channel genes, Asic2a and Asic4. To investigate the effect of WO on cognitive performance, we supplemented the diet of female rats with WO. The results showed that supplementation with WO at doses of 2.2 and 11 g kg−1 day−1 significantly improved learning and memory. In vitro treatment of rat hippocampal neuronal cells with appropriate doses of WO revealed a significant increase in the expression of Asic2a and Asic4 in a dose‐dependent manner at both the mRNA and protein levels. We conclude that WO intake might help to prevent cognitive decline, particularly in the elderly, and that ASIC genes in neurons can be the targets of compounds contained in the oil.

Codonopsis pilosula is a kind of traditional Chinese medicine used to treat weak spleens, stomach problems, anemia, and fatigue. Polysaccharide is one of main components of Codonopsis pilosula. In this study, response surface methodology (RSM) was used to optimize the extraction parameters of Codonopsis pilosula polysaccharides (CPP) by fermentation. The exaction temperature (°C), yeast liquid volume (2 mg/ml, ml), and time (h) were employed effects. Results indicated that the best extraction conditions were the following: extraction temperature 24.75°C, yeast liquid volume 2.96 ml (5.92 mg), and a fermentation time of 21.03 hr. After purification with DE52 and Sephadex G‐100, the molecular structure was determined by ultraviolet–visible (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) (1H and 13C). The monosaccharide composition of CPP1 was determined to be mannose (1.76%), glucose (97.38%), and arabinose (0.76%). CPP1 exhibited high antioxidant activities in scavenging ABTS radicals, ferreous ions, and superoxide ion radicals. Thus, CPP1 could be used as an antioxidant or functional food., Response surface methodology (RSM) was used to optimize the extraction parameters of Codonopsis pilosula polysaccharides (CPPs) by fermentation.

Codonopsis pilosula is a kind of traditional Chinese medicine used to treat weak spleens, stomach problems, anemia, and fatigue. Polysaccharide is one of main components of Codonopsis pilosula. In this study, response surface methodology (RSM) was used to optimize the extraction parameters of Codonopsis pilosula polysaccharides (CPP) by fermentation. The exaction temperature (°C), yeast liquid volume (2 mg/ml, ml), and time (h) were employed effects. Results indicated that the best extraction conditions were the following: extraction temperature 24.75°C, yeast liquid volume 2.96 ml (5.92 mg), and a fermentation time of 21.03 hr. After purification with DE52 and Sephadex G-100, the molecular structure was determined by ultraviolet-visible (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) (1H and 13C). The monosaccharide composition of CPP1 was determined to be mannose (1.76%), glucose (97.38%), and arabinose (0.76%). CPP1 exhibited high antioxidant activities in scavenging ABTS radicals, ferreous ions, and superoxide ion radicals. Thus, CPP1 could be used as an antioxidant or functional food.

In this study, the optimum synthetic process of the Pyracantha polysaccharide-iron (PPI) complex was studied via response surface methodology (RSM). Its antioxidant and anti-cancer activities were also investigated. It was demonstrated that the optimal conditions for the synthetic process of the complex were as follows: a pH of 8.9, a reaction temperature of 70 °C and a trisodium citrate:polysaccharide ratio of 1:2. PPI were analysis by UV, FTIR, SEM, CD, XRD, TGA and NMR. PPI was able to scavenge the metal ion, ABTS and free radicals of the superoxide anion, demonstrating its potential antioxidant activity. PPI was found to display cytotoxicity to Skov3 cells, as shown by its ability to induce apoptosis and alter gene expression in Skov3 cells. These findings show than PPI may represent a novel antioxidant and chemotherapeutic drug.

Xi-Feng Zhang,1 Sangiliyandi Gurunathan2 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea Abstract: Graphene has been shown much interest, both in academics and industry due to its extraordinary physical, chemical, and biological proprieties. It shows great promises in biotechnological and biomedical applications as an antibacterial and anticancer agent, nanocarrier, sensor, etc. However, many studies demonstrated the toxicity of graphene in several cell lines, which is an obstacle to its use in biomedical applications. In this study, to improve the biocompatibility of graphene, we used nicotinamide (NAM) as a reducing and stabilizing agent to catalyze the reduction of graphene oxide (GO) to reduced graphene oxide (rGO). The resulted smaller-sized GO (NAM-rGO) showed excellent biocompatibility with mouse embryonic fibroblast cells, evidenced by various cellular assays. Furthermore, NAM-rGO had no effect on mitochondrial membrane permeability and caspase-3 activity compared to GO. Reverse transcription polymerase chain reaction analysis allowed us to identify the molecular mechanisms responsible for NAM-rGO-induced biocompatibility. NAM-rGO significantly induced the expression of genes encoding tight junction proteins (TJPs) such as zona occludens-1 (Tjp1) and claudins (Cldn3) without any effect on the expression of cytoskeleton proteins. Furthermore, NAM-rGO enhances the expression of alkaline phosphatase (ALP) gene, and it does this in a time-dependent manner. Overall, our study depicted the molecular mechanisms underlying NAM-rGO biocompatibility depending on upregulation of TJPs and ALP. This potential quality of graphene could be used in diverse applications including tissue regeneration and tissue engineering. Keywords: biocompatibility, graphene oxide, nicotinamide, reduced graphene oxide, tight junction proteins, alkaline phosphatase

Zhe Han,1,* Qi Yan,1,* Wei Ge,2 Zhi-Guo Liu,1 Sangiliyandi Gurunathan,3 Massimo De Felici,4 Wei Shen,2 Xi-Feng Zhang1 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, People’s Republic of China; 3Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul, Republic of Korea; 4Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy *These authors contributed equally tothis work Background: Nanoscience and nanotechnology are developing rapidly, and the applications of nanoparticles (NPs) have been found in several fields. At present, NPs are widely used in traditional consumer and industrial products, however, the properties and safety of NPs are still unclear and there are concerns about their potential environmental and health effects. The aim of the present study was to investigate the potential toxicity of ZnO NPs on testicular cells using both in vitro and in vivo systems in a mouse experimental model. Methods: ZnO NPs with a crystalline size of 70 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, and atomic force microscopy. The cytotoxicity of the ZnO NPs was examined in vitro on Leydig cell and Sertoli cell lines, and in vivo on the testes of CD1 mice injected with single doses of ZnO NPs.Results: ZnO NPs were internalized by Leydig cells and Sertoli cells, and this resulted in cytotoxicity in a time- and dose-dependent manner through the induction of apoptosis. Apoptosis likely occurred as a consequence of DNA damage (detected as γ-H2AX and RAD51 foci) caused by increase in reactive oxygen species associated with loss of mitochondrial membrane potential. In addition, injection of ZnO NPs in male mice caused structural alterations in the seminiferous epithelium and sperm abnormalities.Conclusion: These results demonstrate that ZnO NPs have the potential to induce apoptosis in testicular cells likely through DNA damage caused by reactive oxygen species, with possible adverse consequences for spermatogenesis and therefore, male fertility. This suggests that evaluating the potential impacts of engineered NPs is essential prior to their mass production, to address both the environmental and human health concerns and also to develop sustainable and safer nanomaterials. Keywords: ZnO nanoparticle, Sertoli cells, Leydig cells, mice

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The present study aims to investigate the protective effects of ω-3 polyunsaturated fatty acids (ω-3PUFAs) against high-fat diet induced male mouse reproductive dysfunction and to explore circadian regulation mechanisms. Male C57BL/6 mice were randomly divided into three groups and fed a normal chow diet (control group, CON), a high-fat diet (HFD group) or a HFD supplemented with fish oil (FO group) for 12 weeks. After 12 weeks of feeding, the body weight and the ratio of perinephric and epididymal fat weight to body weight were significantly higher in the HFD group compared with the CON group. The supplement of fish oil rich in ω-3PUFAs only slightly reduced the HFD-induced obesity but remarkably ameliorated HFD-induced dyslipidemia, sexual hormones disorder, testicle lesions and germ cell apoptosis. Fish oil supplementation restored the expression of steroid synthesis associated genes in HFD fed mouse and flattened the HFD-induced oscillations in circadian genes' expression. Fish oil supplementation prevented HFD-induced male mouse reproductive dysfunction and modified the rhythmic expression of testosterone synthesis related genes.

Zearalenone (ZEA), a metabolite of Fusarium fungi, is commonly found on moldy grains. Because it can competitively combine to estrogen receptor to disrupt estrogenic signaling, it has been reported to have serious adverse effects on animal reproduction systems. In order to explore the genotoxic effects of ZEA exposure on ovarian somatic cells, porcine granulosa cells were exposed to 10 μM and 30 μM ZEA for 24 or 72 h in vitro. The results showed that ZEA exposure for 24 h remarkably reduced the proliferation of porcine granulosa cells in a dose-dependent manner as determined by MTT analysis and flow cytometry. Furthermore, exposure to ZEA for 72 h induced apoptosis, and RNA sequence analysis also revealed that the expression of apoptosis related genes were altered. RT-qPCR, immunofluorescence and western blot analysis further confirmed the expression of DNA damage and repair related genes (γ-H2AX, BRCA1, RAD51 and PRKDC) were increased in ZEA exposed granulosa cells. When the estrogen antagonist, tamoxifen, was added with ZEA in the culture medium, the DNA damage and repairment by ZEA returned to normal level. Collectively, these results illustrate that ZEA disrupts genome stability and inhibits growth of porcine granulosa cells via the estrogen receptors which may promote granulosa cell apoptosis when the DNA repair system is not enough to rescue this serious damage.

Ding-Ping Bai,1,* Xi-Feng Zhang,2,* Guo-Liang Zhang,3,4 Yi-Fan Huang,1 Sangiliyandi Gurunathan5 1Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, China; 2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China; 3Dong-E-E-Jiao Co., Ltd., Shandong, China; 4National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Shandong, China; 5Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea *These authors contributed equally to this work Background: Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3). Methods: ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. Results: Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H2AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H2AX, p53, and LC3 and downregulation of Bcl-2. Conclusion: The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades. Keywords: zinc oxide nanoparticles, human ovarian cancer cells SKOV3, mitochondrial membrane potential, apoptosis, DNA fragmentation, autophagy

Xi-Feng Zhang,1,2 Feng-Hua Huang,1 Guo-Liang Zhang,3 Ding-Ping Bai,4 De Felici Massimo,5 Yi-Fan Huang,4 Sangiliyandi Gurunathan6 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China; 2Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, China; 3National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Dong-E-E-Jiao Co., Ltd, DongE, Shandong, China; 4Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, China; 5Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata’, Rome, Italy; 6Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea Background: Recently, there has been much interest in the field of nanomedicine to improve prevention, diagnosis, and treatment. Combination therapy seems to be most effective when two different molecules that work by different mechanisms are combined at low dose, thereby decreasing the possibility of drug resistance and occurrence of unbearable side effects. Based on this consideration, the study was designed to investigate the combination effect of reduced graphene oxide-silver nanoparticles (rGO-AgNPs) and trichostatin A (TSA) in human ovarian cancer cells (SKOV3). Methods: The rGO-AgNPs were synthesized using a biomolecule called lycopene, and the resultant product was characterized by various analytical techniques. The combination effect of rGO-Ag and TSA was investigated in SKOV3 cells using various cellular assays such as cell viability, cytotoxicity, and immunofluorescence analysis. Results: AgNPs were uniformly distributed on the surface of graphene sheet with an average size between 10 and 50 nm. rGO-Ag and TSA were found to inhibit cell viability in a dose-dependent manner. The combination of rGO-Ag and TSA at low concentration showed a significant effect on cell viability, and increased cytotoxicity by increasing the level of malondialdehyde and decreasing the level of glutathione, and also causing mitochondrial dysfunction. Furthermore, the combination of rGO-Ag and TSA had a more pronounced effect on DNA fragmentation and double-strand breaks, and eventually induced apoptosis. Conclusion: This study is the first to report that the combination of rGO-Ag and TSA can cause potential cytotoxicity and also induce significantly greater cell death compared to either rGO-Ag alone or TSA alone in SKOV3 cells by various mechanisms including reactive oxygen species generation, mitochondrial dysfunction, and DNA damage. Therefore, this combination chemotherapy could be possibly used in advanced cancers that are not suitable for radiation therapy or surgical treatment and facilitate overcoming tumor resistance and disease progression. Keywords: graphene, trichostatin, cytotoxicity, reactive oxygen species, apoptosis, DNA fragmentation, double-strand DNA breaks

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

Nanoscience and nanotechnology shows immense interest in various areas of research and applications, including biotechnology, biomedical sciences, nanomedicine, and veterinary medicine. Studies and application of nanotechnology was explored very extensively in the human medical field and also studies undertaken in rodents extensively, still either studies or applications in veterinary medicine is not up to the level when compared to applications to human beings. The application in veterinary medicine and animal production is still relatively innovative. Recently, in the era of health care technologies, Veterinary Medicine also entered into a new phase and incredible transformations. Nanotechnology has tremendous and potential influence not only the way we live, but also on the way that we practice veterinary medicine and increase the safety of domestic animals, production, and income to the farmers through use of nanomaterials. The current status and advancements of nanotechnology is being used to enhance the animal growth promotion, and production. To achieve these, nanoparticles are used as alternative antimicrobial agents to overcome the usage alarming rate of antibiotics, detection of pathogenic bacteria, and also nanoparticles being used as drug delivery agents as new drug and vaccine candidates with improved characteristics and performance, diagnostic, therapeutic, feed additive, nutrient delivery, biocidal agents, reproductive aids, and finally to increase the quality of food using various kinds of functionalized nanoparticles, such as liposomes, polymeric nanoparticles, dendrimers, micellar nanoparticles, and metal nanoparticles. It seems that nanotechnology is ideal for veterinary applications in terms of cost and the availability of resources. The main focus of this review is describes some of the important current and future principal aspects of involvement of nanotechnology in Veterinary Medicine. However, we are not intended to cover the entire scenario of Veterinary Medicine, despite this review is to provide a glimpse at potential important targets of nanotechnology in the field of Veterinary Medicine. Considering the strong potential of the interaction between the nanotechnology and Veterinary Medicine, the aim of this review is to provide a concise description of the advances of nanotechnology in Veterinary Medicine, in terms of their potential application of various kinds of nanoparticles, secondly we discussed role of nanomaterials in animal health and production, and finally we discussed conclusion and future perspectives of nanotechnology in veterinary medicine.

A retrospective study of consecutive patients. The purpose of this study was to compare the clinical effect of biomimetic mineralized collagen (MC) modified polymethylmethacrylate (PMMA) bone cement and traditional PMMA bone cement for the treatment of osteoporotic vertebral compression fractures (OVCF). New fracture on adjacent level is the major postoperative complication of percutaneous vertebroplasty (PVP). The clinical incidence was 12.4% to 27.7%. The increased stiffness of the treated vertebral body caused by filling bone cement is considered as one of the main reasons. A total of 30 patients treated with traditional PMMA bone cement from June 2013 to March 2016 were selected as the traditional group, while 50 patients treated with MC modified PMMA bone cement from July 2014 to March 2016 were selected as the modified group. The 2 groups were compared by injection time of the bone cements, postoperative pain relief effects, vertebral height restoration, CT value changes of the treated vertebral bodies, and postoperative complications in the clinical observations. The surgeries were successfully completed in both groups. In the treatment of OVCF, the MC modified bone cement was able to achieve the same pain relief and vertebral height restoration effects compared to traditional bone cement during the follow-ups, although the injection time of the cement was prolonged in the operation. MC modified bone cement significantly reduced the incidence of postoperative adjacent vertebral fracture from 13.3% to 2%, and significantly increased bone density of the treated vertebral bodies. The MC modified PMMA bone cement showed good clinical outcomes and better mechanical properties than the traditional bone cements.

The spatial and temporal specific activation and inhibition of numerous genes are required for successful oogenesis which is precisely regulated by germ cell-related transcription factors, and appropriate epigenetic modifications, including DNA methylation, histone modification and other mechanisms that closely regulate the functional exertion of these transcription factors. In this study, we characterized the correlation between the expression and epigenetic dynamics of Lhx8, a germ cell specific transcription factor during mouse oogenesis. Immunohistochemistry, quantitative PCR and western blots were performed to localize and quantify the expressional characteristics of Lhx8 in oocytes of 13.5 dpc (day post coitum), 17.5 dpc, 0 dpp (day post partum), 3 dpp, 7 dpp and 14 dpp. The results showed that LHX8 protein was located in the nucleus of oocytes, and increasingly expressed during primordial follicle activation. Sequencing of bisulfite-converted genomic DNAs revealed that the methylation dynamics of Lhx8-3' was highly changeable but almost no change occurred in Lhx8-5'. ChIP-QPCR analysis showed that histone H3 acetylation of Lhx8 was also increased during primordial follicle assembly and activation. In conclusion, Lhx8 expression is related with the activation of primordial follicles, which is highly correlated with the demethylation of Lhx8-3' untranslated region and the high acetylation of histone H3.

Xi-Feng Zhang,1 Sangiliyandi Gurunathan2 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul, South Korea Abstract: Ovarian cancer is one of the most important malignancies, and the origin, detection, and pathogenesis of epithelial ovarian cancer remain elusive. Although many cancer drugs have been developed to dramatically reduce the size of tumors, most cancers eventually relapse, posing a critical problem to overcome. Hence, it is necessary to identify possible alternative therapeutic approaches to reduce the mortality rate of this devastating disease. To identify alternative approaches, we first synthesized silver nanoparticles (AgNPs) using a novel bacterium called Bacillus clausii. The synthesized AgNPs were homogenous and spherical in shape, with an average size of 16–20nm, which are known to cause cytotoxicity in various types of human cancer cells, whereas salinomycin (Sal) is able to kill cancer stem cells. Therefore, we selected both Sal and AgNPs to study their combined effect on apoptosis and autophagy in ovarian cancer cells. The cells treated with either Sal or AgNPs showed a dose-dependent effect with inhibitory concentration (IC)-50 values of 6.0µM and 8µg/mL for Sal and AgNPs, respectively. To determine the combination effect, we measured the IC25 values of both Sal and AgNPs (3.0µM and 4µg/mL), which showed a more dramatic inhibitory effect on cell viability and cell morphology than either Sal or AgNPs alone. The combination of Sal and AgNPs had more pronounced effect on cytotoxicity and expression of apoptotic genes and also significantly induced the accumulation of autophagolysosomes, which was associated with mitochondrial dysfunction and loss of cell viability. Our data show a strong synergistic interaction between Sal and AgNPs in tested cancer cells. The combination treatment increased the therapeutic potential and demonstrated the relevant targeted therapy for the treatment of ovarian cancer. Furthermore, we provide, for the first time, a mode of action for Sal and AgNPs in ovarian cancer cells: enhanced apoptosis and autophagy. Keywords: apoptosis, autophagy, cell viability, caspase activity, ovarian cancer, salinomycin, silver nanoparticles

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

Due to their unique physical, chemical, and optical properties, gold nanoparticles (AuNPs) have recently attracted much interest in the field of nanomedicine, especially in the areas of cancer diagnosis and photothermal therapy. Because of the enormous potential of these nanoparticles, various physical, chemical, and biological methods have been adopted for their synthesis. Synthetic antioxidants are dangerous to human health. Thus, the search for effective, nontoxic natural compounds with effective antioxidative properties is essential. Although AuNPs have been studied for use in various biological applications, exploration of AuNPs as antioxidants capable of inhibiting oxidative stress induced by heat and cold stress is still warranted. Therefore, one goal of our study was to produce biocompatible AuNPs using biological methods that are simple, nontoxic, biocompatible, and environmentally friendly. Next, we aimed to assess the antioxidative effect of AuNPs against oxidative stress induced by cold and heat in Escherichia coli, which is a suitable model for stress responses involving AuNPs. The response of aerobically grown E. coli cells to cold and heat stress was found to be similar to the oxidative stress response. Upon exposure to cold and heat stress, the viability and metabolic activity of E. coli was significantly reduced compared to the control. In addition, levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and leakage of proteins and sugars were significantly elevated, and the levels of lactate dehydrogenase activity (LDH) and adenosine triphosphate (ATP) significantly lowered compared to in the control. Concomitantly, AuNPs ameliorated cold and heat-induced oxidative stress responses by increasing the expression of antioxidants, including glutathione (GSH), glutathione S-transferase (GST), super oxide dismutase (SOD), and catalase (CAT). These consistent physiology and biochemical data suggest that AuNPs can ameliorate cold and heat stress-induced oxidative damage in E. coli. Our results indicate that AuNPs may be effective antioxidants. However, further studies are needed to confirm the role of AuNPs as antioxidative agents, as well as their mechanism of action.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 μg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.

Xi-Feng Zhang,* Yun-Jung Choi,* Jae Woong Han, Eunsu Kim, Jung Hyun Park, Sangiliyandi Gurunathan, Jin-Hoi Kim Department of Animal Biotechnology, Konkuk University, Seoul, South Korea *These authors contributed equally tothis work Background: Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs). Methods: Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results: AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs. Conclusion: Our results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required. Keywords: Sertoli cells, Leydig cells, apoptosis, oxidative stress, tight junction proteins, apoptosis

Oxidative stress (OS), as a signal of aberrant intracellular mechanisms, plays key roles in maintaining homeostasis for organisms. The occurrence of OS due to the disorder of normal cellular redox balance indicates the overproduction of reactive oxygen species (ROS) and/or deficiency of antioxidants. Once the balance is broken down, repression of oxidative stress is one of the most effective ways to alleviate it. Ongoing studies provide remarkable evidence that oxidative stress is involved in reproductive toxicity induced by various stimuli, such as environmental toxicants and food toxicity. Zearalenone (ZEA), as a toxic compound existing in contaminated food products, is found to induce mycotoxicosis that has a significant impact on the reproduction of domestic animals, especially pigs. However, there is no information about how ROS and oxidative stress is involved in the influence of ZEA on porcine granulosa cells, or whether the stress can be rescued by curcumin. In this study, ZEA-induced effect on porcine granulosa cells was investigated at low concentrations (15 μM, 30 μM and 60 μM). In vitro ROS levels, the mRNA level and activity of superoxide dismutase, glutathione peroxidase and catalase were obtained. The results showed that in comparison with negative control, ZEA increased oxidative stress with higher ROS levels, reduced the expression and activity of antioxidative enzymes, increased the intensity of fluorogenic probes 2’, 7’-Dichlorodihydrofluorescin diacetate and dihydroethidium in flow cytometry assay and fluorescence microscopy. Meanwhile, the activity of glutathione (GSH) did not change obviously following 60 μM ZEA treatment. Furthermore, the underlying protective mechanisms of curcumin on the ZEA-treated porcine granulosa cells were investigated. The data revealed that curcumin pre-treatment significantly suppressed ZEA-induced oxidative stress. Collectively, porcine granulosa cells were sensitive to ZEA, which may induce oxidative stress. The findings from this study clearly demonstrate that curcumin is effective to reduce the dysregulation of cellular redox balance on porcine granulosa cells in vitro and should be further investigated for its protective role against ZEA in animals.