ROS Damage Research Articles

Protective Effects of Antioxidant Supplements on Sperm Parameters, Sperm DNA Damage and Level of Seminal ROS in RPL Patients: A Clinical Trial Study

Background: Recurrent Pregnancy Loss (RPL) is an important medical problem comprising approximately 15% of all pregnancies. Sperm DNA damage and level of seminal ROS may have a role in RPL. Objectives: The main aim of the present study was to appraise the efficacy of antioxidants for improving these parameters in RPL patients. Methods: In a clinical trial, 90 couples suffering from RPL were divided into two groups from April 2014 to December 2015 at the Yazd Research and Clinical Center for Infertility in Iran. Group I received supplemental vitamin E (400 µg) daily in combination with Se (200 µg) and the other group received vitamin E plus zinc for three months. Sperm parameters, sperm DNA damage, and seminal ROS were evaluated before and after treatment and compared between the two groups. Data were analyzed by SPSS and P values of ≤ 0.05 were considered statistically significant. Results: Our results revealed normal sperm parameters in RPL patients. Sperm parameters substantially improved in the two treated groups (P ≤ 0.05). The ROS level dramatically reduced in the two groups (P = 0.01). The evaluation of chromatin integrity with AB staining did not show any remarkable changes in group 1 (P = 0.3), but it showed a considerable rate reduction in group 2 (P = 0.04). Also, it showed significant decreases with TB, CMA3, and TUNEL assays in both groups (P = 0.001). However, we did not find any significant difference between the two groups. Conclusions: Supplemental vitamin E in combination with selenium or zinc may improve semen quality by decreasing the level of seminal ROS and sperm DNA damage in RPL patients. We advocate their use for improving the quality of the men’s sperm DNA.

Salen‑manganese complexes for controlling ROS damage: Neuroprotective effects, antioxidant activity and kinetic studies

A new manganese(III) complex [MnL1(DCA)(H2O)](H2O),1 [H2L1 is the chelating ligand N,N′-bis(2-hydroxy-3-methoxybenzylidene)-1,2-diaminopropane, and DCA is dicyanamide], has been prepared and characterized by different analytical and spectroscopic techniques. The tetragonally elongated octahedral geometry for the manganese coordination sphere was revealed by X-ray diffraction studies for 1. The antioxidant behavior of this complex and other manganese(III)-salen type complexes was tested through superoxide dismutase and catalase probes, and through the study of their neuroprotective effects in SH-SY5Y neuroblastoma cells. In this human neuronal model, these model complexes were found to improve cell survival in an oxidative stress model. During studies aimed to getting a better understanding of the kinetics of the processes involved in this antioxidant behavior, an important effect on the solvent in the kinetics of reaction of the complexes with H2O2 was revealed that suggests a change in the mechanism of reaction of the complexes. The kinetic data in methanol and buffered aqueous solutions correlate well with the results of the test of catalase activity, thus showing that the rate determining step in the catalytic cycle corresponds to the initial reaction of the complexes with H2O2.

ITRAQ-based quantitative proteomic analysis of MG63 in response to HIF-1α inducers

Non-healing fractures constitute a serious clinical problem. HIF-1α is a crucial regulator in response to hypoxia and is proven to be pivotal in bone growth; however, the mechanism still needs further research. In this study, iTRAQ was used to study the effects of two HIF-1α inducers on the expression of proteins in MG63 cells. A total of 841 proteins were significantly changed after treatment with HIF-1α inducers. Among these, 12 proteins were functionally involved in the HIF-1 and VEGF signaling pathways. We then studied the protein and gene expression of the twelve proteins by western blot and RT-PCR, respectively. The results confirmed that VEGF, TFRC, ERK1/2, iNOS, GLUT1, ALDOA, ENO1 and IP3R1 were markedly upregulated, while NF-κB, RCN1, PLCγ1 and CaMKII were significantly downregulated upon treatment with HIF-1α inducers. Meanwhile, the intracellular levels of Ca2+, NO and ROS were closely related and significantly changed. Up-regulation of HIF can maintain high levels of Ca2+ and NO while reducing ROS and protect cells from apoptosis induced by low serum. This study presents a new way to study the regulation of HIF on bone growth by investigating the Ca2+, NO and ROS levels. SignifcanceWe found that the regulation of Ca2+ and NO proteins are tightly associated with HIF pathway using iTRAQ method. Furthermore, the concentration of Ca2+, NO and ROS are closely related in low serum cultured cells. Up-regulation of HIF pathway can maintain high levels of Ca2+ and NO while reducing ROS damage.

Biological and Phytochemical Screening of Fumaria indica extract on Chemically Induced Hepatocellular Carcinoma with Reference to Biochemical Parameters

Liver disease or liver cancer is the sixth most common cancer and the third leading cause of cancer mortality in the world. Hepatitis viral infection, food additives, alcohol, fungal toxins (aflatoxins), toxic industrial chemicals, air and water pollutants are the major risk factors of liver cancer. Moreover, due to high tolerance of liver, HCC is seldom detected at an early stage and once detected treatment faces a poor prognosis in most cases.Fumaria indica possesses hepatoprotective activity as evidenced by the significant and dose dependent restoring the activities of entire liver cancer marker enzymes, diminution in tumor incidence, decrease in lipid peroxidation (LPO) and increase in the level of antioxidant enzymes (GSH, CAT, SOD, GPx and GST) through scavenging of free radicals, or by enhancing the activity of antioxidant, which then detoxify free radicals. These factors protect cells from ROS damage in NDEA and CCl4-induced hepatocarcinogenesis. Histopathological observations of liver tissues too correlated with the biochemical observations. Thus, present investigation suggested that the Fumaria indica would exert a chemoprotective effect by reversing the oxidant-antioxidant imbalance during hepatocarcinogenesis induced by NDEA and CCl4. Besides Fumaria indicais very much effective in preventing NDEA-induced multistage hepatocarcinogenesis possibly through antioxidant and antigenotoxic nature, which was confirmed by various liver injury and biochemical tumour markers enzymes. The hepatoprotective activity of a Fumaria indicaof 50 % ethanolic extract was studied using rats. The animals received a single intraperitoneal injection of N-nitrosodiethylamine 200mg/kg body wt followed by subcutaneous injection of CCl4 in a dose of 3 ml/kg body wt. Fumaria indica extract dose dependently and significantly the increase in serum hepatic enzyme levels after NDEAand CCl4 treatment compared to the toxin control group. The results of this study confirmed the antioxidant and hepatoprotective activity of the Fumaria indicaextract against carbon tetrachlorideand N-nitrosodiethylamine induced hepatotoxicity in rats. In addition to this, studies on molecular aspect of hepatoprotective therapy will give mechanistic information in hepatoprotective therapy and also critical balance should be there between the animal model and clinical research. The hepatoprotective properties of Fumaria indicashould provide useful information in the possible application in hepatic liver disease.

Abstract 709: DNA Damage-free iPS-cardiomyocyte Reduces Cardiac Fibrosis Through Downmodulation of Exosomal miR-101

With the growing field of myocardial repair through transplantation of cardiomyocytes (CM) derived from human induced pluripotent stem cells (iPSC) calls for a method for reducing fibrotic tissue development at the site of cell transplantation. We tested the hypothesis of whether transplanting CMs derived from DNA damage-free (DdF) iPSCs could reduce the recruitment of myofibroblasts and reduce fibrosis at the site of transplantation. Transcription factor p53 functions are DNA damage-dependent: induce apoptosis in DNA damaged cells while promoting cell cycle in normal cells. Utilizing this discriminating nature of p53 we selected iPSCs that are DdF and differentiated them into CMs. Control CMs (Ctrl-CM) and DdF-CMs were tested for transplantation induced fibrosis in a mouse model. The inflammatory markers such as accumulation of ROS, superoxide and DNA damage were significantly reduced in DdF cells. RNA sequencing revealed that, in DdF-CMs, the inflammatory and fibrosis signaling pathways are significantly different compared to Ctrl-CMs. Transplantation of both CMs into the myocardium of ischemia-induced mice resulted in fibrosis around the transplantation site. However, the fibrotic area around transplanted sites and total fibrosis were significantly reduced in mice hearts that received DdF-CMs as evidenced by collagen accumulation and Sirus red staining of fibrotic tissue. Importantly, the number of myofibroblasts at the site of DdF-CM transplantation was significantly reduced compared to Ctrl-CM sites. In an in vitro migration study, fibroblasts preferentially migrated towards the Ctrl-CMs. Human fibroblast treatment with DdF-CM exosomes (DdF-Exo) resulted in reduced expression of fibrotic markers than Ctrl-CM exosomes (Ctrl-Exo) treatment. miRNA analysis in exosomes showed that the anti-inflammatory miR-26a is upregulated and the inflammatory miRNA101 is downregulated in DdF-Exo. Overexpression of miR-101 mimic in DdF-Exo abrogated the anti-fibrotic potential property of DdF-Exo. Our results clearly demonstrate that DdF-CMs has anti-fibrotic potential. Transplanting DdF-CMs attracted reduced myofibroblast to the engraft site and Ctrl-CMs transplantation induce fibrosis through the expression of exosomal miR-101.

MiRNAs deregulation in serum of mice is associated with lung cancer related pathway deregulation induced by PM2.5

Ambient fine particulate matter (PM2.5) as an environmental pollution has been associated with the lung cancer. However, the mechanism of epigenetics such as miRNAs deregulation between PM2.5-exposure and lung cancer has not been elucidated clearly. Twenty C57BL/6 mice were divided randomly into 2 groups and exposed to the filtered air (FA) and the concentrated air (CA), respectively. The FA mice were exposed to filtered air in chambers with a high-efficient particulate air filter (HEPA-filter), and the CA mice were exposed to concentration ambient PM2.5. The total duration of exposure was performed 6 h per day from December 1st, 2017 to January 27th, 2018. The mice exposed 900.21 μg/m3 PM2.5 for 6 h per day in CA chamber, which was nearly equaled to 225.05 μg/m3 for 24-h calculatingly. After exposure, the serum miRNAs levels were detected by microarray. Genetic and pathological alterations in lung of mice with/without PM2.5 exposure were detected. 38 differential miRNAs in serum of mice were found after PM2.5 exposure for 8 weeks. Among of them, 13 miRNAs related with lung cancer were consistent in serum and lung of mice. The target genes of 13 deregulated miRNAs including CRK, NR2F2, VIM, RASSF1, CCND2, PRKCA, SIRT1, CDK6, MAP3K7, HIF1A, UBE2V2, ATG10, BAX, E2F1, RASSF5 and CTNNB1, could involve in the pathway of lung cancer developing. Compared with the FA group, the significantly increases of histopathological changes, ROS and DNA damage were observed in lung of mice in CA group. Our study suggested that miRNAs in serum could be identified as candidate biomarkers to predict the lung cancer development during early PM2.5 exposure.

Nanomedicine-based Curcumin Approach Improved ROS Damage in Best Dystrophy-specific Induced Pluripotent Stem Cells

Best dystrophy (BD), also termed best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and can cause central visual loss. Unfortunately, there is no clear definite therapy for BD or improving the visual function on this progressive disease. The human induced pluripotent stem cell (iPSC) system has been recently applied as an effective tool for genetic consultation and chemical drug screening. In this study, we developed patient-specific induced pluripotent stem cells (BD-iPSCs) from BD patient-derived dental pulp stromal cells and then differentiated BD-iPSCs into retinal pigment epithelial cells (BD-RPEs). BD-RPEs were used as an expandable platform for in vitro candidate drug screening. Compared with unaffected sibling-derived iPSC-derived RPE cells (Ctrl-RPEs), BD-RPEs exhibited typical RPE-specific markers with a lower expression of the tight junction protein ZO-1 and Bestrophin-1 (BEST1), as well as reduced phagocytic capabilities. Notably, among all candidate drugs, curcumin was the most effective for upregulating both the BEST1 and ZO-1 genes in BD-RPEs. Using the iPSC-based drug-screening platform, we further found that curcumin can significantly improve the mRNA expression levels of Best gene in BD-iPSC-derived RPEs. Importantly, we demonstrated that curcumin-loaded PLGA nanoparticles (Cur-NPs) were efficiently internalized by BD-RPEs. The Cur-NPs-based controlled release formulation further increased the expression of ZO-1 and Bestrophin-1, and promoted the function of phagocytosis and voltage-dependent calcium channels in BD-iPSC-derived RPEs. We further demonstrated that Cur-NPs enhanced the expression of antioxidant enzymes with a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Collectively, these data supported that Cur-NPs provide a potential cytoprotective effect by regulating the anti-oxidative abilities of degenerated RPEs. In addition, the application of patient-specific iPSCs provides an effective platform for drug screening and personalized medicine in incurable diseases.

Action Mechanism of Streptomyces diastatochromogenes KX852460 Against Rhizoctonia solani AG-3 Involving Basidiospores Suppression and Oxidative Damage

This research was aimed to study the effect of ethyl acetate extracts from Streptomyces diastatochromogenes KX852460 on action mechanism at various stages of life cycle of Rhizoctonia solani AG-3, such as sclerotia, mycelium, basidiospores and hyphae. Suppression of sclerotia and mycelium was determined by measuring the inhibition zones. While morphology of mycelium and hyphae formation were observed by scanning electron microscopy and transmission electron microscopy, respectively, the basidiospores formation and ROS generation were detected by confocal laser scanning microscopy. Extract from S. diastatochromogenes KX852460 had 96% inhibition effect against the mycelium, formation of the sclerotia decreased from 71.73 to 0% as the concentration of the treatment increased from 1 × 10−4 to 7 × 10−4 mM and 88.22–97% inhibition effect on disease lesion. Suppression of basidiospores was one of the significant approaches evidenced by visualization using confocal laser scanning microscopy. Scanning electron microscopy analysis showed changes in morphological attributes of R. solani AG-3 caused by ethyl acetate extract of bacteria. Transmission electron microscopic observation revealed suppression of hyphae. Generation of endogenous ROS and oxidative damage were evidenced by the confocal laser scanning microscopy. Ethyl acetate extracts from Streptomyces diastatochromogenes KX852460 exhibited potential antagonistic action against the R. solani AG-3 at various stages of its life cycle including sclerotia, mycelium, basidiospores and hyphae which could be utilized as biocontrol agent.

Biological and Phytochemical Screening of Tephrosia purpurea extract on Chemically Induced Hepatocellular Carcinoma with Reference to Biochemical Parameters

Tephrosia purpurea possesses hepatoprotective activity as evidenced by the significant and dose dependent restoring the activities of entire liver cancer marker enzymes, diminution in tumor incidence, decrease in lipid peroxidation (LPO) and increase in the level of antioxidant enzymes (GSH, CAT, SOD, GPx and GST) through scavenging of free radicals, or by enhancing the activity of antioxidant, which then detoxify free radicals. These factors protect cells from ROS damage in NDEA and CCl4-induced hepatocarcinogenesis. Histopathological observations of liver tissues too correlated with the biochemical observations. Thus, present investigation suggested that the Tephrosia purpurea would exert a chemoprotective effect by reversing the oxidant-antioxidant imbalance during hepatocarcinogenesis induced by NDEA and CCl4. Besides Tephrosia purpurea is very much effective in preventing NDEA-induced multistage hepatocarcinogenesis possibly through antioxidant and antigenotoxic nature, which was confirmed by various liver injury and biochemical tumour markers enzymes. The hepatoprotective activity of aTephrosia purpurea of 50 % ethanolic extract was studied using rats. The animals received a single intraperitoneal injection of N-nitrosodiethylamine 200mg/kg body wt followed by subcutaneous injection of CCl4 in a dose of 3 ml/kg body wt.Tephrosia purpureaextract dose dependently and significantly the increase in serum hepatic enzyme levels after NDEAand CCl4 treatment compared to the toxin control group. The results of this study confirmed the antioxidant and hepatoprotective activity of the Tephrosia purpurea extract against carbon tetrachlorideand N-nitrosodiethylamine induced hepatotoxicity in rats.

Inhibitory Effects of Scutellaria baicalensis Root Extract on Linoleic Acid Hydroperoxide-induced Lung Mitochondrial Lipid Peroxidation and Antioxidant Activities.

In this study, we evaluated the ability of Scutellaria baicalensis Georgi to protect lipid-peroxidation (LPO) in lung tissue after free radical-induced injury. We prepared S. baicalensis root (SBR) extracts using different solvents. The total flavonoid and total phenol contents of each extract were measured, and the ROS damage protection was evaluated by analyzing linoleic acid hydroperoxide (LHP)-induced LPO in rat lung mitochondria. Moreover, evaluating diphenylpicrylhydrazyl (DPPH), hydrogen peroxide, superoxide anion radical, and hydroxyl radical scavenging abilities and using metal chelating assays were used to determine in vitro antioxidant activity. The ethyl acetate (EtOAc) extract showed high ROS scavenging ability, and four compounds were subsequently isolated and purified from this extract: baicalin, baicalein, wogonin, and oroxylin A. Baicalein in rat lung mitochondria the most significant LHP-induced LPO inhibition was shown and extracted with EtOAc that contained the highest amount of baicalein. Thus, baicalein and the EtOAc extract of SBR may be efficient in conferring ROS damage protection and inhibiting LHP-induced LPO in rat lung mitochondria. Additional studies are warranted to investigate their use as antioxidant therapy for respiration infections, nutrition supplements, and lead compounds in pharmaceuticals.

Episodic Hypoxia Promotes Defence Against Cellular Stress.

Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O₂) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p<0.001), and re-oxygenation after episodic hypoxia by 1.26 ± 0.16 fold (p<0.01). Staurosporine-induced active caspase-3 was highest after sustained, and lowest after episodic hypoxia. In vitro episodic hypoxia mimics the largely HIF-independent transcriptome observed after repetitive CsA treatment in vivo. In vitro preconditioning with episodic hypoxia protects against stress-induced apoptosis. Despite of its long-term adverse effects, CsA derived episodic hypoxia induces a unique renal hypoxia response that provides adaptation to re-oxygenation mediated ROS damage.

Lactobacillus paracasei PS23 modulated the age‐related inflammation in Senescence Accelerated Mouse Prone 8 (SAMP8) mice

Sarcopenia is a common impairment in the elderly population responsible for poor outcomes later in life, which is enhanced by inflammation. Only a few strategies have been reported to reduce sarcopenia. We investigated whether Lactobacillus paracasei PS23 (LPPS23) decelerates age‐related muscle loss and its inflammatory response; LPPS23 has been reported to delay some age‐related disorders. Female senescence‐accelerated mouse prone 8 (SAMP8) mice were divided into three groups (n = 6 each): non‐aging (16‐week‐old), control (28‐week‐old), and PS23 (28‐week‐old) groups. The control and PS23 groups were given saline and LPPS23, respectively. We evaluated the effects of LPPS23 by analyzing body weight and composition, reactive oxygen species, and IL‐6. The positive anti‐ROS effect of LPPS23 in the aged SAMP8 mice demonstrated that LPPS23 may protect muscle mitochondria against ROS damage, which may be associated with a lower level of sarcopenia in aged SAMP8 mice. Similar to LPPS23, some probiotics has been reported to have anti‐ROS properties; more probiotics may be also proofed to use for modulating sarcopenia in the future.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of Myocardial ischemia reperfusion injury by Geniposide preconditioning in diabetic rats

Studies have shown that the NRF-2 /HO-1 pathway participates in myocardial ischemic reperfusion injury (MI/R) and that Geniposide (GEN) could protect the myocardial against MI/R. This study aims to examine the protective effects of GEN on MI/R in diabetic rats and further explore the possible mechanism of action. During MI/R in rats, NRF-2 /HO-1signals changed significantly including NRF-2 and HO-1up-regulation, resulting in heart dysfuction, histological damage and increasing oxidative stress and cell apoptosis. Treatment with GEN can significantly improve the general condition and heart function in diabetic rats with decreasing the expression of cTnI, CK-MB, blood glucose, MDA, ROS, cell apoptosis and pathological damage in MI/R. In addition, GEN precondition can also significantly increase the weight of rats and the activity of SOD, CAT and GPx with up-regulating the expression of NRF-2 and HO-1 in MI/R. This study implied that Geniposide has a protective effect on myocardial ischemia reperfusion injury in diabetic rats, and its mechanism is associated with activating NRF2/HO-1 signaling pathway to suppress oxidative stress.

Elucidation of the Effects of Si-Wu Tang on Menstrual Disorder Patterns through Activation of Aromatase and Antioxidation.

Si-Wu Tang (SWT), a traditional Chinese formula, is commonly used for treating female diseases, such as relief of menstrual discomfort and climacteric syndrome. The aim of this study was to explore the synergistic effects between each herb in SWT on menstrual disorder patterns. Estradiol regulation and antioxidative effects were indicators that ameliorated menstrual disorder patterns and the total polyphenol and polysaccharide contents were quality markers. According to relationships of bioactivity and phytochemical contents, we discuss the effects of each herb in SWT. In a testosterone-treated MCF-7 cell model, Rehmannia glutinosa and catalpol significantly increased the estradiol content and aromatase upregulation in cell culture. We suggest that catalpol is an aromatase promoter in SWT, and R. glutinosa is a major actor. In terms of the antioxidant activity, pentagalloylglucose, gallic acid, and ferulic acid had stronger antioxidative effects than other compounds. We suggest that the antioxidative ability depends on polyphenols, and Paeonia lactiflora is a major contributor. Based on the prescribing principle of traditional Chinese medicine (TCM) theory, we suggest that R. glutinosa in SWT act as an aromatase promoter in the role of sovereign for ameliorating menstrual disorder patterns. As P. lactiflora has the strongest antioxidant effects and can prevent ROS damage ovarian; therefore, P. lactiflora could help R. glutinosa work as a minister for menstrual disorder patterns and R. glutinosa and P. lactiflora are a herbal pair in SWT.

Ultrastructural Changes of Organelles in Root Cap Cells of Tobacco Under Salinity

Abstract We investigated how an abiotic factor, i.e. salinity, affects specific intracellular organelles of cells of the root cap. We focused on: 1) amyloplasts, which are specific plastids of the cap that perform important metabolic and sensory functions; and 2) mitochondria, which protect cells from ROS damage by changing their ultrastructure. In our work we studied the ultrastructural changes of these organelles in different areas of the root cap under NaCl and Na2SO4 impact. We showed that the amyloplasts (statoliths) and mitochondria of the columella and the peripheral zone of the cap change their structural organisation in the presence of NaCl. Under action of Na2SO4, in plastids of columella the number of starch grains decreases dramatically, the cells of the peripheral part of the cap do not contain the amyloplasts, and the proplastids that do not store or store a little starch grains, which is attributed to leucoplasts with typical lamellae characteristic for root cortex cells. Thus, Na2SO4 influence has the most significant effect on the plastids of the root cap, and plastids of the cells of the peripheral zone of the cap are subjected to the most significant changes under salinity stress.

Responses of the weed Bidens pilosa L. to exogenous application of the steroidal saponin protodioscin and plant growth regulators 24‐epibrassinolide, indol‐3‐acetic acid and abscisic acid

The exogenous application of plant hormones and their analogues has been exploited to improve crop performance in the field. Protodioscin is a saponin whose steroidal moiety has some similarities to plant steroidal hormones, brassinosteroids. To test the possibility that protodioscin acts as an agonist or antagonist of brassinosteroids or other plant growth regulators, we compared responses of the weed species Bidens pilosa L. to treatment with protodioscin, brassinosteroids, auxins (IAA) and abscisic acid (ABA). Seeds were germinated and grown in agar containing protodioscin, dioscin, brassinolides, IAA and ABA. Root apex respiratory activity was measured with an oxygen electrode. Malondialdehyde (MDA) and antioxidant enzymes activities were assessed. Protodioscin at 48-240μm inhibited growth of B.pilosa seedlings. The steroidal hormone 24-epibrassinolide (0.1-5μm) also inhibited growth of primary roots, but brassicasterol was inactive. IAA at higher concentrations (0.5-10.0μm) strongly inhibited primary root length and fresh weight of stems. ABA inhibited all parameters of seedling growth and also seed germination. Respiratory activity of primary roots (KCN-sensitive and KCN-insensitive) was activated by protodioscin. IAA and ABA reduced KCN-insensitive respiration. The content of MDA in primary roots increased only after protodioscin treatment. All assayed compounds increased APx and POD activity, with 24-epibrassinolide being most active. The activity of CAT was stimulated by protodioscin and 24-epibrassinolide. The results revealed that protodioscin was toxic to B.pilosa through a mechanism not related to plant growth regulator signalling. Protodioscin caused a disturbance in mitochondrial respiratory activity, which could be related to overproduction of ROS and consequent cell membrane damage.

Cytotoxicity and DNA Damage Caused from Diazinon Exposure by Inhibiting the PI3K-AKT Pathway in Porcine Ovarian Granulosa Cells.

Organophosphorus insecticide diazinon (DZN) is diffusely used in agriculture, home gardening, and crop peats. Much work so far has focused on the link between DZN exposure and the occurrence of neurological diseases, while little is known on the reproductive toxicological assessment on DZN exposure. This research aimed to investigate the underlying mechanisms of toxic hazards for DZN exposure on cultured porcine ovarian granulosa cells. We analyzed the oxidative stress, energy metabolism, DNA damage, apoptosis, and autophagy by using high-throughput RNA-seq, immunofluorescence, Western blotting, and real-time PCR. The combined data demonstrated that DZN exposure could cause excessive ROS and DNA damage, which induced apoptosis and autophagy by inhibiting the PI3K-AKT pathway. The down-regulated CYP19A1 protein and granulosa cell deaths increase the risk for developing premature ovarian failure and follicular atresia. In conclusion, DZN exposure has obvious reproductive toxicity by induction of granulosa cell death through pathways connected to DNA damage and oxidative stress by inhibiting the PI3K-AKT pathway.

Abstract 17087: Competent p53 Stabilized iPS-Cardiomyocyte Transplantation in Ischemic Myocardium of Mice Reduce Fibrosis Through Exosomal miRNA-26a

Introduction: With the growing field of myocardial repair through transplantation of cardiomyocytes (CM) derived from human induced pluripotent stem cells (iPSC) calls for a method for reducing fibrotic tissue development at the site of cell transplantation. We tested the hypothesis whether transplanting CMs derived from p53 stabilized (p53S) iPSCs could reduce the recruitment of fibroblast and reduce fibrosis at the site of transplantation. Methods: Transcription factor p53 functions are inflammation dependent: inducing apoptosis in inflammatory and transformed cells while promoting cell cycle in normal cells. Utilizing this discriminating nature of p53 we selected iPSCs that express less inflammatory and more anti-inflammatory markers and differentiated them into CM. Control CM (ctrl-CM) and p53S-CMs were tested for transplantation induced fibrosis in a mice model. Results and Discussion: The inflammatory markers such as accumulation of ROS, superoxide and DNA damage were significantly lesser in p53S cells. RNA sequencing revealed that, in p53S-CMs, the anti-inflammatory and anti-fibrosis signaling pathways are upregulated compared to ctrl-CMs. Transplantation of both CMs into the myocardium of ischemia induced mice resulted in cell engraftment and fibrosis around the transplantation site. However, fibrotic area was significantly reduced in p53S-CM transplanted sites evidenced by COL1 and COL4 accumulation and Sirus red staining of fibrotic tissue. In vitro treatment of human fibroblast with p53S-CM exosomes (p53S-Exo) resulted in reduced expression of fibrotic markers than ctrl-CM exosomes (Ctrl-Exo) treatment. miRNA analysis of exosomes showed that the anti-inflammatory miRNA26a is upregulated and the inflammatory miRNA101 is downregulated in p53S-Exo. Overexpression of miRNA26a mimic in ctrl-Exo abrogated the fibrosis inducing property of ctrl-Exo. Interestingly, in migration study, fibroblasts preferentially migrated towards the ctrl-CMs. Conclusion: Our results clearly shows that anti-inflammatory cells can be isolated from a population of heterogeneous cells by activating p53. Transplanting p53S-CMs attracted reduced fibroblast to the engraft site and oppose fibrosis through exosomal expression of miRNA26a.

SPARC Inhibits Metabolic Plasticity in Ovarian Cancer.

The tropism of ovarian cancer (OvCa) to the peritoneal cavity is implicated in widespread dissemination, suboptimal surgery, and poor prognosis. This tropism is influenced by stromal factors that are not only critical for the oncogenic and metastatic cascades, but also in the modulation of cancer cell metabolic plasticity to fulfill their high energy demands. In this respect, we investigated the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in metabolic plasticity of OvCa. We used a syngeneic model of OvCa in Sparc-deficient and proficient mice to gain comprehensive insight into the paracrine effect of stromal-SPARC in metabolic programming of OvCa in the peritoneal milieu. Metabolomic and transcriptomic profiling of micro-dissected syngeneic peritoneal tumors revealed that the absence of stromal-Sparc led to significant upregulation of the enzymes involved in glycolysis, TCA cycle, and mitochondrial electron transport chain (ETC), and their metabolic intermediates. Absence of stromal-Sparc increased reactive oxygen species and perturbed redox homeostasis. Recombinant SPARC exerted a dose-dependent inhibitory effect on glycolysis, mitochondrial respiration, ATP production and ROS generation. Comparative analysis with human tumors revealed that SPARC-regulated ETC-signature inversely correlated with SPARC transcripts. Targeting mitochondrial ETC by phenformin treatment of tumor-bearing Sparc-deficient and proficient mice mitigated the effect of SPARC-deficiency and significantly reduced tumor burden, ROS, and oxidative tissue damage in syngeneic tumors. In summary, our findings provide novel insights into the role of SPARC in regulating metabolic plasticity and bioenergetics in OvCa, and shines light on its potential therapeutic efficacy.

Main Allelochemicals from the Rhizosphere Soil of Saussurea lappa (Decne.) Sch. Bip. and Their Effects on Plants' Antioxidase Systems.

Allelochemicals are the media of allelopathy and form the chemical bases of plant-environment interactions. To determine true allelochemicals and their autotoxic effects, seven compounds were isolated and identified from in-situ sampled rhizosphere soil of cultivated Saussurea lappa. Of these; costunolide (2), dehydrocostus lactone (3) and scopoletin (4) showed significant inhibition on seedling growth in a concentration-dependent manner. Detection and observation demonstrated that the antioxidase system was found to be affected by these chemicals, resulting in the accumulation of ROS and membrane damage. To investigate their release ways, the compounds were traced back and volumes quantified in rhizosphere soil and plant tissues. This work made clear the chemical bases and their physiological effects on the plants. These chemicals were found to be the secondary metabolites of the plants and included in the rhizosphere soil. The findings identified a potential pathway of plant-plant interactions, which provided theoretical basis to overcoming replanting problems. This research was also useful for exploring ecological effects of allelochemicals in green agriculture.