NADPH oxidase 2 mediates cardiac sympathetic denervation and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced cardiomyopathy.

Doxorubicin has been used extensively as a potent anticancer agent, but its clinical use is limited by its cardiotoxicity. However, the underlying mechanisms remain to be fully elucidated. In this study, we tested whether NADPH oxidase 2 (Nox2) mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced heart failure. Nox2 knockout (KO) and wild-type (WT) mice were randomly assigned to receive a single injection of doxorubicin (15 mg/kg, i.p.) or saline. WT doxorubicin mice exhibited the decreases in survival rate, left ventricular (LV) wall thickness and LV fractional shortening and the increase in the lung wet-to-dry weight ratio 1 week after the injections. These alterations were attenuated in Nox2 KO doxorubicin mice. In WT doxorubicin mice, myocardial oxidative stress was increased, myocardial noradrenergic nerve fibers were reduced, myocardial expression of PGP9.5, GAP43, tyrosine hydroxylase and norepinephrine transporter was decreased, and these changes were prevented in Nox2 KO doxorubicin mice. Myocyte autophagy was increased and myocyte size was decreased in WT doxorubicin mice, but not in Nox2 KO doxorubicin mice. Nox2 mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy-both of which contribute to cardiac atrophy and failure after doxorubicin treatment.

NADPH oxidase 2 inhibitor GSK2795039 prevents doxorubicin-induced cardiac atrophy by attenuating cardiac sympathetic nerve terminal abnormalities and myocyte autophagy.

Doxorubicin is widely used for the treatment of human cancer, but its clinical use is limited by a cumulative dose-dependent cardiotoxicity. However, the mechanism of doxorubicin-induced cardiac atrophy and failure remains to be fully understood. In this study, we tested whether the specific NADPH oxidase 2 (Nox2) inhibitor GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, leading to the amelioration of cardiac atrophy and dysfunction in chronic doxorubicin-induced cardiomyopathy. Mice were randomized to receive saline, doxorubicin (2.5 mg/kg, every other day, 6 times) or doxorubicin plus GSK2795039 (2.5 mg/kg, twice a day, 9 weeks). Left ventricular (LV) total wall thickness and LV ejection fraction were decreased in doxorubicin-treated mice compared with saline-treated mice and the decreases were prevented by the treatment of the specific Nox2 inhibitor GSK2795039. The ratio of total heart weight to tibia length and myocyte cross-sectional area were decreased in doxorubicin-treated mice, and the decreases were attenuated by the GSK2795039 treatment. In doxorubicin-treated mice, myocardial Nox2 and 4-hydroxynonenal levels were increased, myocardial expression of GAP43, tyrosine hydroxylase and norepinephrine transporter, markers of sympathetic nerve terminals, was decreased, and these changes were prevented by the GSK2795039 treatment. The ratio of LC3 II/I, a marker of autophagy, and Atg5, Atg12 and Atg12-Atg5 conjugate proteins were increased in doxorubicin-treated mice, and the increases were attenuated by the GSK2795039 treatment. These findings suggest that inhibition of Nox2 by GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, thereby ameliorating cardiac atrophy and dysfunction after chronic doxorubicin treatment.

GSK2795039 prevents RIP1-RIP3-MLKL-mediated cardiomyocyte necroptosis in doxorubicin-induced heart failure through inhibition of NADPH oxidase-derived oxidative stress.

Doxorubicin (DOX), which is widely used for the treatment of cancer, induces cardiomyopathy associated with NADPH oxidase-derived reactive oxygen species. GSK2795039 is a novel small molecular NADPH oxidase 2 (Nox2) inhibitor. In this study, we investigated whether GSK2795039 prevents receptor-interacting protein kinase 1 (RIP1)-RIP3-mixed lineage kinase domain-like protein (MLKL)-mediated cardiomyocyte necroptosis in DOX-induced heart failure through NADPH oxidase inhibition. Eight-week old mice were randomly divided into 4 groups: control, GSK2795039, DOX and DOX plus GSK2795039. H9C2 cardiomyocytes were treated with DOX and GSK2795039. In DOX-treated mice, the survival rate was reduced, left ventricular (LV) end-systolic dimension was increased and LV fractional shortening was decreased, and these alterations were attenuated by the GSK2795039 treatment. GSK2795039 inhibited not only myocardial NADPH oxidase subunit gp91phox (Nox2) protein, but also p22phox, p47phox and p67phox proteins and prevented oxidative stress 8-hydroxy-2'-deoxyguanosine levels in DOX-treated mice. RIP3 protein and phosphorylated RIP1 (p-RIP1), p-RIP3 and p-MLKL proteins, reflective of their respective kinase activities, markers of necroptosis, were markedly increased in DOX-treated mice, and the increases were prevented by GSK2795039. GSK2795039 prevented the increases in serum lactate dehydrogenase and myocardial fibrosis in DOX-treated mice. Similarly, in DOX-treated cardiomyocytes, GSK2795039 improved cell viability, attenuated apoptosis and necrosis and prevented the increases in p-RIP1, p-RIP3 and p-MLKL expression. In conclusion, GSK2795039 prevents RIP1-RIP3-MLKL-mediated cardiomyocyte necroptosis through inhibition of NADPH oxidase-derived oxidative stress, leading to the improvement of myocardial remodeling and function in DOX-induced heart failure. These findings suggest that GSK2795039 may have implications for the treatment of DOX-induced cardiomyopathy.

Enhanced oxidative stress mediates pathological autophagy and necroptosis in cardiac myocytes in pressure overload induced heart failure in rats.

In cardiac myocytes in vitro, hydrogen peroxide induces autophagic cell death and necroptosis. Oxidative stress, myocyte autophagy and necroptosis coexist in heart failure (HF). In this study, we tested the hypothesis that excessive oxidative stress mediates pathological autophagy and necroptosis in myocytes in pressure overload-induced HF. HF was produced by chronic pressure overload induced by abdominal aortic constriction (AAC) in rats. Rats with AAC or sham operation were randomised to orally receive an antioxidant N-acetylcysteine (NAC) or placebo for 4 weeks. Echocardiography was performed for the assessments of left ventricular (LV) structure and function. AAC rats exhibited decreased LV fractional shortening (FS) at 4 weeks after surgery. NAC treatment attenuated decreased LV FS in AAC rats. In AAC rats, myocardial level of 8-hydroxydeoxyguanosine assessed by immunohistochemical staining, indicative of oxidative stress, was increased, LC3 II protein, a marker of autophagy, Beclin1 protein and Atg4b, Atg5, Atg7 and Atg12 mRNA expression were markedly increased, RIP1, RIP3 and MLKL expression, indicative of necroptosis, was increased, and all of the alterations in AAC rats were prevented by the NAC treatment. NAC treatment also attenuated myocyte cross-sectional area and myocardial fibrosis in AAC rats. In conclusion, NAC treatment prevented the increases in oxidative stress, myocyte autophagy and necroptosis and the decrease in LV systolic function in pressure overload-induced HF. These findings suggest that enhanced oxidative stress mediates pathological autophagy and necroptosis in myocytes, leading to LV systolic dysfunction, and antioxidants may be of value to prevent HF through the inhibition of excessive autophagy and necroptosis.

The efficacy, biodistribution and safety of an inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis.

DNA vaccines, the third-generation vaccines, were extensively studied. The attenuated Salmonella choleraesuis (S. choleraesuis) was widely focused as a carrier to deliver DNA vaccines in the chromosome-plasmid balanced-lethal system. The efficacy of inhibin DNA vaccine delivered by attenuated S. choleraesuis was proved in mice and cows in our previous studies. In this study, the efficacy of inhibin DNA vaccine was confirmed in rhesus monkeys. To further study the biodistribution and safety, the mice were immunized under laboratory conditions. The results of the rhesus monkeys showed the plasma IgA and IgG titres against inhibin were elevated, and the oestradiol (E2 ) and progesterone (P4 ) levels were increased with immunizing inhibin DNA vaccine. The biodistribution and safety assessment displayed the body weight, pathological change and haematology indexes where there is no significant difference between vaccinated mice and control. And the genomics analysis showed there was no integration of the inhibin gene into the mouse genome 2 months after immunization. This study indicated the inhibin DNA vaccine delivered by attenuated S. choleraesuis was safe. And this vaccine was a potential means to improve their reproductive traits in primates and other animals.

The role of Melatonin receptor MTNR1A in the action of Melatonin on bovine granulosa cells.

Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin ß and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin ß abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.

Preventive effects of ß-cryptoxanthin against cadmium-induced oxidative stress in the rat testis.

ß-cryptoxanthin (CRY), a major carotenoid of potential interest for health, is obtained naturally from orange vegetables and fruits. A few research studies have reported that CRY could decrease oxidative stress and germ cell apoptosis. The purpose of this study was to examine the effects of CRY on acute cadmium chloride (CdCl 2 )-induced oxidative damage in rat testes. For this study, 24 rats were divided into four groups, one of which serves as a control group that received intraperitoneal (i.p.) injections of corn oil and physiological saline. The other rats were i.p. injected with CRY (10 µg kg-1 ) every 8 h, beginning 8 h before CdCl 2 (2.0 mg kg-1 ) treatment. The pathological and TUNEL findings revealed that CRY ameliorated the Cd-induced testicular histological changes and germ cell apoptosis in the rats. Furthermore, the Cd-induced decrease in the testicular testosterone (T) level was attenuated after CRY administration (P < 0.05). The administration of CRY significantly reversed the Cd-induced increases in the lipid peroxide (LPO) and malondialdehyde (MDA) levels (P < 0.01). The testicular antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were decreased by treatment with Cd alone but were restored by CRY co-treatment. These results demonstrated that the application of CRY can enhance the tolerance of rats to Cd-induced oxidative damage and suggest that it has promised as a pharmacological agent to protect against Cd-induced testicular toxicity.

Anti-inflammatory potential of ß-cryptoxanthin against LPS-induced inflammation in mouse Sertoli cells.

ß-cryptoxanthin (CX), a major carotenoid pigment, can inhibit inflammatory gene expression in mice with nonalcoholic steatohepatitis. In the present study, we examined the anti-inflammatory effects of CX on lipopolysaccharide (LPS)-induced inflammation in mouse primary Sertoli cells and the possible molecular mechanisms behind its effects. The results showed that CX significantly inhibited LPS-induced decreases in cell viability and in the percentage of apoptotic cells. Moreover, CX inhibited the LPS-induced up-regulation of tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in Sertoli cells. In addition, CX significantly limited the LPS-induced down-regulation of AR, HSF2, CREB, FSHR, INHBB and ABP in Sertoli cells. Western blot analysis showed that CX significantly suppressed NF-κB (p65) activation as well as MAPK phosphorylation. All the results suggested that CX suppressed inflammation, possibly associated with the NF-κB activation and MAPK of phosphorylation. Thus, CX may possess therapeutic potential against inflammation-related diseases.

Effects in cattle of genetic variation within the IGF1R gene on the superovulation performance and pregnancy rates after embryo transfer.

The insulin-like growth factor 1 receptor (IGF1R) is a membrane glycoprotein mediating most biological actions of IGF1 and IGF2, and has an important effect on ovulation, pre-implantation embryo development and pregnancy rate. The objectives of this study were to detect IGF1R gene polymorphisms of cattle and analyze the relationship with superovulation performance and pregnancy rates after embryo transfer (ET), as well as the hormone concentrations at the day of ET. One reported SNP of IGF1R G404T and a novel SNP of IGF1R G399A were analyzed in 170 Chinese Holstein donor cows and 118 Luxi recipients cattle. Statistical analysis revealed that the G404T mutation was associated (p=0.019) with increased ovulation rate and females with this mutation had enhanced performance in producing transferable embryos. For the polymorphic locus G399A, recipients with g.399 GG and g.399 GA genotypes had greater pregnancy rates after ET than that of g.399 AA genotype. Furthermore, the same tendency was observed that the genotype groups with greater pregnancy rates had greater progesterone and lesser estrogen concentrations, but these did not reach statistical significance. Results of the present study showed, for the first time, that the polymorphism in IGF1R is associated with superovulation traits, and indicated that the IGFIR gene can be used as a potential marker for donor selection.

Effects of PGR and ESRα genotypes on the pregnancy rates after embryo transfer in Luxi cattle.

Progesterone receptor (PGR) and estrogen receptor alpha (ESRα), which mediate the biological effects of the steroid hormones progesterone and estrogen, play a central role in the establishment and maintenance of pregnancy. The objectives of this study were to detect bovine PGR and ESRα genes polymorphisms and analyze their relationships with the pregnancy rates after embryo transfer and the hormone concentrations at the day of embryo transfer. One reported SNP of PGR G59752C and a novel SNP of ESRα G75935C were analyzed in 132 recipients of Luxi cattle. For the PGR gene, recipients with g.59752 GG and g.59752 GC genotypes had obviously higher pregnancy rates than g.59752 CC genotype. For the ESRα gene, recipients with g.75935 GC and g.75935 CC genotypes had obviously higher pregnancy rates than g.75935 GG genotype. Furthermore, the same tendency was observed for these two genes that the same genotype groups with high pregnancy rates had high progesterone concentration and low estrogen concentration at the day of embryo transfer. These results showed for the first time that PGR G59752C and ESRα G75935C polymorphisms had obvious effects on the pregnancy rates after embryo transfer, and indicated that PGR G59752C and ESRα G75935C polymorphisms could be potential markers for recipient selection of embryo transfer.

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats.

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n=18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 µg (T1, T2 and T3, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P<0.05) in T3 than in either T1 or T2 (0.341±0.123, 0.236±0.068, and 0.251±0.077, respectively, mean±SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P<0.05) in T3, and were higher (P<0.05) in T1 and T2 than in control groups. For antibody-positive rats, there was a correlation (P<0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T3 group (46.00±4.65) was higher (P<0.05) than in two control groups (29.25±3.72 and 27.92±3.48), and also higher (P<0.05) than in T1 and T2 (37.17±4.99 and 38.75±7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75±4.23 vs. 35.60±3.38, P<0.05). Moreover, litter size and number of placentas were increased (P<0.05) in the pcISI immunization groups, except for the T1 group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 µg yielded the best immune response.

Polymorphisms in luteinizing hormone receptor and hypothalamic gonadotropin-releasing hormone genes and their effects on sperm quality traits in Chinese Holstein bulls.

Genes of hypothalamic-pituitary-gonadal axis play a key role in male reproductive performance. This study evaluated the polymorphisms of luteinizing hormone receptor (LHR) and hypothalamic gonadotropin-releasing hormone (GnRH) genes and their effects on sperm quality traits including semen volume per ejaculate (VOL), sperm density (SD), fresh sperm motility (FSM), thawed sperm motility (TSM), acrosome integrity rate (AIR), and abnormal sperm rate (ASR) collected from 205 Chinese Hostein bulls. The study bulls consisted of 205 mature Chinese Holstein, 27 Simmental, 28 Charolais, and 14 German yellow cattle. One single nucleotide polymorphism (SNP) (A883G) in exon 2 of GnRH and two SNPs (A51703G and G51656T) in intron 9 of LHR were identified in 274 bulls. Analysis of variance in 205 Chinese Holstein bulls showed that age had significant effect on both SD and FSM (P < 0.01), and ASR (P < 0.05). With regards to genotype and its interaction with age, only the SNP of G51656T in LHR gene had significant effect on SD (P < 0.05, P < 0.01; respectively). The association result showed that bulls with AG genotype had higher FSM than bulls with AA and GG genotype in LHR at 51,703 locus (P < 0.10), and bulls with GG genotype had higher SD than bulls with TT genotype in LHR at G51656T locus (P < 0.10). Phenotypic correlation among the traits revealed that significant negative correlations were observed between ASR and AIR (r = -0.736, P < 0.01), ASR and AIR (r = -0.500, P < 0.01). There were moderate positive correlations between VOL and SD (r = 0.422, P < 0.01), as well as FSM (r = 0.411, P < 0.01). In conclusion, LHR may be a potential marker for sperm quality of SD and FSM.

Polymorphisms in follicle stimulation hormone receptor, inhibin alpha, inhibin bata A, and prolactin genes, and their association with sperm quality in Chinese Holstein bulls.

Candidate genes follicle stimulation hormone receptor (FSHR), inhibin alpha (INHA), inhibin bata A (INHBA) and prolactin (PRL) were investigated for their association with sperm quality traits of semen volume per ejaculate (VOL), sperm concentration (SCON), motility (MOT), sperm motility in frozen semen (FMOT), acrosome integrity rate (AIR) and abnormal sperm rate (ASR) in a total of 123 normal mature Holstein bulls. Three reported single nucleotide polymorphisms (SNPs) of FSHR A-234500T (rs43676359), IHNA A192G (rs41257116), and IHNBA C7639T (rs43408735), and 3 novel SNPs (G7550A, C7661T, and T8370C) in exons 4 and 5 of bovine PRL gene (NC 007324) were analyzed. Analysis of variance revealed that FSHR A-234500T and INHBA C7639T polymorphisms significantly associated with VOL (P<0.05) and SCON (P<0.05), and the polymorphism of INHBA C7639T also had significant effects on MOT (P<0.05). Furthermore, the INHA A192G polymorphism significantly associated with AIR (P<0.05). No significant differences were found between the polymorphisms of PRL gene and sperm quality traits.

Effects of immunization against a DNA vaccine encoding somatostatin gene (pGM-CSF/SS) by attenuated Salmonella typhimurium on growth, reproduction and lactation in female mice.

A novel somatostatin (SS) DNA vaccine (pGM-CSF/SS), delivered orally by attenuated Salmonella typhimurium (CSO22), was used to immunize female mice at 5, 7, and 11 wk of age; the objective was to investigate the humoral immune response and effects of this vaccine on growth, reproduction and lactation. The pGM-CSF/SS induced SS-specific antibodies, which peaked (3.69 ± 0.89; mean ± S.D) 4 wk after the first booster immunization. Compared with a saline-treated control group, body weight gain of a pGM-CSF/SS immunized group increased 30.3% (23.88 vs. 18.32 g, P < 0.05) during the growth period (from 2 wk after primary immunization to 4 wk after the first booster immunization). Immunized mice had higher plasma estradiol concentrations (84.10 ± 2.16 vs. 81.45 ± 2.12 pg/ml, P < 0.05) and a shorter estrous cycle (4.06 ± 0.75 vs. 5.33 ± 0.49 d, P < 0.05), but serum progesterone concentrations were not significantly affected. Since offspring produced by immunized mice gained weight faster (P < 0.05) in the first 2 wk of life (4.27 ± 0.62 and 7.81 ± 1.30 g in Weeks 1 and 2, respectively vs. 3.70 ± 0.23 and 7.14 ± 0.48 g), we inferred that pGM-CSF/SS could have a direct or indirect role in regulating lactation in mice. In conclusion, GM-CSF and CSO22 served as adjuvant and attenuated live vector, respectively, with efficient oral delivery of an SS DNA vaccine which successfully induced a humoral immune response and enhanced rate of weight gain. Furthermore, the DNA vaccine pGM-CSF/SS affected plasma estradiol concentrations and the estrous cycle, and seemed to enhance lactation performance of female mice.

Somatostatin can alter fertility genes expression, oocytes maturation, and embryo development in cattle.

This study was designed to investigate the effect of somatostatin on oocytes maturation and subsequent embryo development in cattle. Bovine granulosa cells separated from oocytes, cultured for 24 h and transfected with pEGFP-N1 vector with mouse SST gene (Experimental) and with out plasmid transfection (Control). RT-PCR and Real-Time PCR were used to estimate the expression of bovine receptors of androgen, estrogen beta, growth hormone, and follicular stimulating hormone. Culture media concentrations of hormones were measured by kits using radioimmunoassay. COCs aspirated from ovaries were co-cultured with granulosa cells layers (transfected or control) at 38.5 degrees C in CO(2) incubator for maturation. We found a significant (2.37X) increase in estrogen receptor beta expression in experimental group. There was a decrease in androgen receptor, growth hormone releasing hormone receptor, and follicular stimulating hormone receptor (P < 0.05). But, 96 h of post transfection, culture media concentration of estradiol-17beta was increased significantly (P < 0.05) and testosterone, growth hormone and follicular stimulating hormone showed opposite trend (P < 0.05) in experimental groups. Co-culture of somatostatin transfected granulosa cells with oocytes, reduced the maturation rate from 70% to 66% but had no effect on subsequent fertilization and embryo development.