Small Peptides from Periplaneta americana Inhibits Oxidative Stress-Induced KGN Cell Apoptosis by Regulating Mitochondrial Function Through Bcl2L13.

This study examined the protective effects of small peptides from Periplaneta americana against H2O2-induced mitochondrial injury in human ovarian granulosa cells. The ATP level and mitochondrial membrane potential as well as the quantity and ultrastructure of mitochondria in cells were detected. Mitochondrial DNA copy number and expression levels of Bcl2L13, LC3B, and p62 were tested. Targeted silencing of Bcl2L13 expression in KGN cells. The expression levels of Bcl2L13 and LC3B as well as interaction were evaluated. The ATP level, mtDNA-CN, and MMP of the H2O2 group were significantly lower than those of the normal control group (P < 0.05), accompanied by a reduction in mitochondrial mass and mitochondrial fluorescence intensity (P < 0.05). However, the ATP level, mtDNA, and MMP in KGN cells were increased after SPPA treatment (P < 0.05). Scanning electron microscopy shows that SPPA ameliorates H2O2-induced structural damage to mitochondria. Moreover, the expression levels of Bcl2L13 and p62 in the H2O2 group were downregulated significantly compared with those of the normal control group (P < 0.05), while LC3B was upregulated (P < 0.05). After SPPA treatment, the expression levels of Bcl2L13 and p62 were upregulated (P < 0.05), while LC3B was downregulated (P < 0.05). The Co-IP results indicated that Bcl2L13 and LC3B interacted, and this interaction was weakened after cell treatment with H2O2, and dissociation between Bcl2L13 and LC3B declined after SPPA treatment. SPPA inhibits KGN cell apoptosis induced by oxidative stress via inhibition of mitochondrial injury Bcl2L13-mediated mitochondrial autophagy might participate in the regulation process.

Signaling pathways of Periplaneta americana peptide resist H2O2-induced apoptosis in pig-ovary granulosa cells through FoxO1.

Granulosa cell apoptosis induced by oxidative stress is an important cause of follicular atresia. Our previous studies found that Periplaneta americana peptide (PAP) decreased H2O2-induced apoptosis of pig-ovary granulosa cells (PGCs) through FoxO1. The aim of this study is to investigate the signaling pathways involved in PAP resistance against H2O2-induced apoptosis of PGCs. PGCs obtained from the follicles of non-estrous Duroc × Landrace × Yorkshire gilts (5 months old, 50-55 kg) were treated with H2O2 and PAP, or together with inhibitors against PI3K and JNK, and then collected for ROS levels and SOD activities detection, TUNEL staining, qRT-PCR, western blotting, immunofluorescence or coimmunoprecipitation. Results showed that the increased ROS levels and decreased activities of SOD caused by H2O2 stimulation were reversed by PAP. Additionally, PAP downregulated the differential abundance of mRNA of Bax and FasL, thus inhibiting H2O2-induced apoptosis of PGCs. PAP significantly reduces p-JNK expression and increases the p-FoxO1/FoxO1 expression ratio, thereby decreasing caspase-3 expression and cell apoptosis in H2O2-induced PGCs. PAP promotes the combination of FoxO1 with the 14-3-3 protein, increases FoxO1 translocation to the cytoplasm, and decreases FoxO1 acetylation. Therefore, PAP regulates FoxO1 expression through the JNK/FoxO1 signaling pathway and effects the translocation of FoxO1 to the cytoplasm by the FoxO1 interaction with 14-3-3, enabling reversal of the H2O2-induced apoptosis of PGCs. Acetylation of FoxO1 is also involved in the antiapoptotic effect of PAP.

Mechanism of action and the uses betaine in pig production.

Betaine, the trimethyl derivative of glycine, is a good methyl group donor, and an important component in pig production. However, betaine has not been extensively studied in this field. Therefore, in this study, we reviewed the effects of betaine in pig production performance, meat quality and reproductive performance, as well as its mechanisms, to provide a theoretical basis for the optimal use and development of this compound.

Protective effect of small peptides from Periplaneta americana on cyclophosphamide-induced premature ovarian failure.

AIM: To investigate the protective effect of small peptides from Periplaneta americana (SPPA) on cyclophosphamide (CP)-induced premature ovarian failure (POF) in mice. Silent mating type information regulation 2 homolog 1 (SIRT1) /tumor-associated protein 53 (p53) signaling pathway plays an important role in delaying POF. Hematopoietic progenitor cell antigen (CD34) reflects ovarian aging from the side. However, whether SPPA inhibits POF in mice by influencing the SIRT1/p53 pathway and CD34 expression remains to be studied. METHODS: Forty female Kun Ming (KM) mice were divided into four groups: a control group (normal saline, n = 10), POF model group (160 mg/kg CP, n = 10), SPPA low-dosage group (160 mg/kg CP + 100 mg/kg SPPA, n = 10), and SPPA high-dosage group (160 mg/kg CP + 200 mg/kg SPPA, n = 10). CP administration route is intraperitoneal injection, and SPPA administration route is intragastric. Eyeball enucleation blood samples and the ovaries of mice were collected by midline laparatomy and oopherectomy, and the malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) concentrations were tested. Immunohistochemical tests for the expressions of SIRT1, p53, and CD34 were carried out. Finally, ovarian mRNA levels of SIRT1 and p53 were detected with real-time fluorescence quantification PCR (qRT-PCR). RESULTS: A mouse model of POF was generated using 160 mg/kg of CP. Compared with POF group, we found that plasma NO, MDA, and FSH decreased, while AMH and SOD increased in the SPPA low-dose group. Compared with the POF group, the SPPA low- and high-dosage groups achieved significant growth in the number of primordial, primary, and total number of healthy follicles at all levels, but sharp reductions in the number of atretic follicles. In addition, we found downregulated protein and mRNA expression of SIRT1, and upregulated that of p53 were observed in ovarian tissues of treated mice with POF, in immunohistochemistry experiments and qPCR experiments. In contrast, high protein and mRNA expression of SIRT1, and low that of p53 were observed in SPPA treatment groups. And the results of CD34 protein expression were consistent with that of SIRT1. CONCLUSION: In total, SPPA significantly inhibited POF caused by CP in mice via activation of the SIRT1/p53 signaling pathway in the mouse ovary.

Analysis of the resistance of small peptides from Periplaneta americana to hydrogen peroxide-induced apoptosis in human ovarian granular cells based on RNA-seq.

Apoptosis of ovarian granular cells is closely related with weakening fertility of women. Hence, resisting apoptosis of human ovarian granular cells is of important significance. According to previous studies, DAPI fluorescence staining experiment and Western Blot test of Caspase-3 demonstrate that small peptides from Periplaneta americana (SPPA) can improve hydrogen peroxide (H2O2) -induced apoptosis of human ovarian granular cells (KGN cells). However, the molecular mechanism of SPPA resistance against apoptosis of granular cells still remains unknown. In this study, key genes and signaling pathways for SPPA to resist H2O2-induced apoptosis of KGN cells were determined through transcriptome sequencing (RNA-seq). Experiments were divided into three groups, namely, the control group, H2O2 group and H2O2 + SPPA group. A total of 1196 differentially expressed genes (DEGs) were screened by comparing the control group and the H2O2 group, and 2805 DEGs were screened by comparing the H2O2 group and H2O2 + SPPA group. It is important to note that 87 overlapping genes were identified upregulating in H2O2 exposure, but downregulating in SPPA repair. Another 151 overlapping genes were identified downregulating in H2O2 exposure, but upregulating in SPPA repair. These 238 overlapping genes have significant enrichment in multiple KEGG pathways. Among them, 13 genes play significant roles in SPPA resistance process of cell apoptosis: EIF3D, RAN, UPF1 and EIF2B4 participate in RNA transport; ACTG1, SIPA1 and CTNND1 participate in Leukocyte transendothelial migration; S100A7, S100A9, RELA and IL17RE participate in IL-17 signaling pathway; BCL2L13, EIF2AK3 and RELA participate in Mitophapy-animal. Ten genes were selected for florescence quantitative PCR (qPCR) verification and the expression level was consistent with sequencing results. Finally, a control network of SPPA resistance against the H2O2-induced KGN cell apoptosis was built based on the target genes screened by the RNA-seq technology. This study provides a direction and some references to further understand the molecular mechanism of SPPA resistance against the H2O2-induced KGN cell apoptosis.

Periplaneta americana peptide decreases apoptosis of pig-ovary granulosa cells induced by H2 O2 through FoxO1.

Oxidative stress can induce apoptosis of granulosa cells and lead to follicular atresia, thereby reducing the number of pigs giving birth. The aim of this study was to investigate the protective effect of Periplaneta americana peptide (PAP) on the apoptosis of the granulosa cells of pig ovaries (PGCs) induced by hydrogen peroxide (H2 O2 ) via FoxO1. PGCs were treated with H2 O2 to establish a cell apoptosis model. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was detected using flow cytometry. The malondialdehyde (MDA) level and nitric oxide (NO) content were detected to reflect the oxidative stress. Western blotting, qRT-PCR and overexpression were undertaken to determine the expression of FoxO1 and caspase-3, and immunofluorescence was used to detect FoxO1 in the nucleus and cytoplasm. PGCs were treated with 100 µM H2 O2 for 6 hr, which resulted in oxidative damage and apoptosis and an apoptosis rate for PGCs of 32.95%. Next, PGCs were treated with 400 µg/ml PAP for 24 hr to repair the apoptosis induced by H2 O2 . PAP improved cell viability in H2 O2 -stimulated PGCs, the increased MDA level and NO content caused by H2 O2 stimulation were reversed and the apoptotic rate of PGCs was reduced. The qRT-PCR and Western blotting results indicated that PAP decreased the H2 O2 -induced apoptosis and the expression of FoxO1 and caspase-3 in PGCs. The effect of PAP was the same following FoxO1 overexpression. FoxO1 was expressed in the nucleus when stimulated by H2 O2 or overexpression; however, it migrated to the cytoplasm following PAP treatment. PAP decreased the apoptosis of PGCs induced by H2 O2 by regulating FoxO1 expression and nuclear translocation.

The protective effect of small peptides from Periplaneta americana on hydrogen peroxide-induced apoptosis of granular cells.

This study investigates the protective effect of small peptides from Periplaneta americana (SPPA) on hydrogen peroxide (H2O2)-induced apoptosis of ovarian granular cells. H2O2 was applied to human ovarian granular cells (KGN cell strains). Cell viability was tested by cell counting Kit-8 (CCK-8). Cell apoptosis was tested by flow cytometry, and a cell apoptosis model was established. The model cells were treated with SPPA, and the cell survival rate was monitored using the CCK-8 method. The oxidative stress state of cells was examined using SOD, ROS, MDA, and NO kits. The protein expression levels of SIRT1, p53, and the apoptosis-related gene Caspase3 were measured using Western Blot methodology. Relative to the control group, cell viability declined significantly after the H2O2 treatment only (P < 0.01), while the apoptosis rate increased significantly (P < 0.01). The activity of SOD was weakened significantly (P < 0.01), while the cell levels of ROS, MDA, and NO increased dramatically (P < 0.01). Cell viability dramatically recovered (P < 0.01), and the SOD activity is hugely increased (P < 0.01) after SPPA treatment. In contrast, contents of ROS, MDA, and NO decreased sharply (P < 0.01), and significant dose-response relationships are characterized. Moreover, the H2O2 treatment group showed significantly downregulated expression of SIRT1 (P < 0.01) but significantly upregulated expressions of p53 and Caspase3 (P < 0.01) compared to the control group. Following the SPPA treatment of apoptosis cells, expression of SIRT1 increased significantly, while expressions of p53 and Caspase3 declined significantly (P < 0.01). This study suggests that SPPA inhibits H2O2-induced human KGN cell apoptosis through antioxidation, and the SIRT1/p53 signal pathway mediates the antioxidation.

Sexually dimorphic effects of maternal dietary protein restriction on fetal growth and placental expression of 11ß-HSD2 in the pig.

Placental 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) inactivates glucocorticoids (GCs) to protect fetuses from over-exposure to maternal GCs, yet how maternal malnutrition affects placental 11ß-HSD2 expression is unknown. In this study, Meishan sows were fed standard-protein (SP) or low-protein (LP, 50% of SP) diets and fetuses/newborn piglets were weighed and the corresponding placenta and umbilical cord blood were collected on gestational day 70 and the day of parturition. Significant growth retardation was observed in female, but not male, fetuses (P < 0.05) and the newborns (P < 0.01) of the LP group, which was accompanied by sexually dimorphic expression of 11ß-HSD2 in placentas. Female fetuses in LP group showed significant decrease in placental 11ß-HSD2 protein content (P < 0.05) and enzyme activity (P < 0.05), whereas male fetuses demonstrated significantly enhanced placental 11ß-HSD2 activity (P < 0.05). Serum cortisol levels were significantly higher (P < 0.05) in male piglets compared to females, and the effects of maternal protein restriction on thyroid hormones (T3 and T4) in the umbilical cord blood were also sex dimorphic. Male piglets in LP group had significantly higher T3 (P < 0.01) and lower T4 (P < 0.01), whereas female piglets showed significantly lower T4 (P < 0.01) with no change in T3. As a result, male piglets in LP group exhibited significantly higher T3/T4 ratio compared to female counterparts. These results indicate that the effects of maternal protein restriction on placental 11ß-HSD2 expression are gender-dependent in the pig, and thyroid hormones may be involved in such effects.

Glucocorticoid receptor is involved in the breed-dependent transcriptional regulation of 3ß-hydroxysteroid dehydrogenase in the liver of preweaning piglets.

BACKGROUND: Hepatic 3ß-hydroxysteroid dehydrogenase (3ß-HSD) plays an important role in steroid inactivation and catabolism. Serum concentrations of steroid hormones differ significantly between breeds in pigs, however the molecular mechanism regulating hepatic 3ß-HSD expression in different breeds of pigs is poorly understood. In the present study, we used preweaning purebred male Large White (LW) and Erhualian (EHL) piglets as model to investigate the breed difference in the expression and regulation of 3ß-HSD gene in porcine liver. RESULTS: The hepatic expression of 3ß-HSD mRNA was significantly lower (P < 0.01) in EHL piglets compared to that in LW piglets. Significant breed differences were detected for the hepatic expression of transcription factors such as androgen receptor (AR), glucocorticoid receptor (GR), and CCAAT/enhancer binding protein ß (C/EBPß). The nucleoprotein contents of AR (P < 0.05), GR (P < 0.01) and phospho-Ser(211)GR (P < 0.01) were significantly higher in the liver of EHL piglets. Chromatin immunoprecipitation (ChIP) assay demonstrated significantly lower binding of GR, but not AR or C/EBPß, to 3ß-HSD gene promoter in EHL piglets (P < 0.05). GR was not detected to interact with C/EBPß or AR in the co-immunoprecipitation analysis. CONCLUSIONS: These results indicate that GR binding to 3ß-HSD promoter is involved in the breed-dependent 3ß-HSD expression in the liver of piglets.

Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions.

PURPOSE: The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms. METHODS: Gestational sows were fed control or betaine-supplemented (3 g/kg) diets throughout the pregnancy. Immediately after birth, male piglets were killed, and the hippocampus was dissected for analyses. The mRNA abundance was determined by reverse transcription real-time polymerase chain reaction. Protein content was measured by Western blot, and DNA methylation was detected by methylated DNA immunoprecipitation assay. RESULTS: Prenatal betaine supplementation did not alter the body weight or the hippocampus weight, but increased the hippocampal DNA content as well as the mRNA expression of proliferation-related genes. Prenatal betaine supplementation increased serum level of methionine (P < 0.05) and up-regulated (P < 0.05) the mRNA and protein expression of betaine-homocysteine methyltransferase, glycine N-methyltransferase and DNA methyltransferase 1 in the neonatal hippocampus. Hippocampal expression of insulin growth factor II (IGF2) and its receptors IGF1R and IGF2R were all significantly up-regulated (P < 0.05) in betaine-treated group, together with a significant activation (P < 0.01) of the downstream extracellular signal-regulated kinase 1/2. Moreover, the differentially methylated region (DMR) 1 and 2 on IGF2 locus was found to be hypermethylated (P < 0.05) in the hippocampus of betaine-treated piglets. CONCLUSIONS: These results indicate that maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression, causes hypermethylation of DMR on IGF2 gene, which was associated with augmented expression of IGF2 and cell proliferation/anti-apoptotic markers in the hippocampus of neonatal piglets.

Maternal Low-protein Diet Alters Ovarian Expression of Folliculogenic and Steroidogenic Genes and Their Regulatory MicroRNAs in Neonatal Piglets.

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets.

To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory element-binding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27α-hydroxylase (CYP27α1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7α-hydroxylase (CYP7α1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.

Betaine supplementation in maternal diet modulates the epigenetic regulation of hepatic gluconeogenic genes in neonatal piglets.

In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism.

Maternal protein restriction during gestation and lactation programs offspring ovarian steroidogenesis and folliculogenesis in the prepubertal gilts.

Maternal malnutrition may disrupt ovarian functions in adult offspring. Steroidogenesis and folliculogenesis in the offspring ovary appear to be the major targets of nutritional programming. Nevertheless, the mechanism by which maternal low-protein diet affects the offspring steroidogenesis and folliculogenesis, and the possible pathway linking these two processes remain unclear. In this study, Landrace×Yorkshire crossbred sows were fed either standard (SP) or low-protein (LP, 50% of the SP) diets throughout gestation and lactation. Female offspring were fed the same diet after weaning until 6 months of age. LP offspring had higher serum 17ß-estradiol level (P<0.01), which was accompanied by lower mRNA (P<0.05) but higher protein (P<0.05) expression of cytochrome P450 aromatase (CYP19A1) in the ovary. CYP19A1 protein up-regulation was associated with lower ovarian expression of drosha (P<0.05) and miRNAs targeting CYP19A1 (P<0.05). LP offspring had less graafian follicles with more apoptotic granulosa cells (P<0.05), as well as higher caspase 3 activity (P<0.05) and FasL expression (P<0.05) in the ovary. FasL gene up-regulation was associated with higher ERα protein expression (P<0.05) and binding to FasL gene promoter. These results suggest that a maternal LP diet in pregnancy and lactation elevated serum 17ß-estradiol level by activating CYP19A1 through miRNA-mediated mechanism, and induced granulosa apoptosis in graafian follicles through ER-activated Fas/FasL-caspase 3 pathway.