Protective effect of luteoloside against Toxoplasma gondii-induced liver injury through inhibiting TLR4/NF-κB and P2X7R/NLRP3 and enhancing Nrf2/HO-1 signaling pathway.

Toxoplasma gondii (T. gondii) infection can cause liver injury by inducing inflammation and oxidative stress. The Chinese herbal extract luteoloside (Lut) has considerable anti-inflammatory and antioxidant properties, but its effects on the liver injury during T. gondii infection have not been reported. This study investigated the hepatoprotective effects of Lut by treating T. gondii-infected mice with 0-200 mg/kg doses of Lut and further examined the expression of key proteins in the inflammation and oxidative stress-related pathways in the liver to investigate the potential mechanism of the hepatoprotective effects of Lut. Results showed that Lut remarkably reduced serum ALT and AST levels, considerably decreased inflammatory factors TNF-α, IL-6, and IL-1ß, as well as oxidative products MDA, and greatly increased antioxidant enzymes SOD and GSH. The expression of key proteins TLR4, Myd88, TRAF6, p-NF-κB p65 in the TLR4/NF-κB pathway and P2X7R, NLRP3, caspase 1, IL-1ß, IL-18 in the P2X7R/NLRP3 pathway were significantly decreased in the liver. And the expression of key proteins Nrf2, HO-1, NQO-1, and GCLC in the Nrf2/HO-1 antioxidant-related pathway was significantly upregulated. In conclusion, Lut attenuated T. gondii-induced liver injury by inhibiting the inflammatory response and enhancing antioxidant capacity. The hepatoprotective mechanisms of Lut are involved in inhibiting TLR4/NF-κB and P2X7R/NLRP3 inflammatory signaling pathways, as well as enhancing the Nrf2/HO-1 antioxidant pathway. These findings not only provide some reference for further exploring the specific hepatoprotective mechanism of Lut during T. gondii infection, but also provide some theoretical basis for the future clinical application of Lut as a hepatoprotective drug in T. gondii infection.

Rationale and design for the study of recombinant human hepatocyte growth factor plasmid in the treatment of patients with chronic limb-threatening ischemia (HOPE CLTI): Randomized, placebo-controlled, double-blind, phase III clinical trials.

BACKGROUND: Although patients with CLTI have benefited from the rapid development of endovascular techniques, many patients are considered unsuitable for revascularization procedures. A previous phase II clinical trial has suggested that recombinant human hepatocyte growth factor plasmid (NL003) can salvage limbs during the treatment of patients with CLTI. However, the safety and efficacy of this drug need to be evaluated in a larger cohort. STUDY DESIGN: HOPE CLTI is a multicenter, randomized, double-blind, placebo-controlled phase III clinical study to evaluate the efficacy and safety of intramuscular injection of NL003 in CLTI patients. This study consisted of 22 trials: HOPE CLTI-1, which includes patients with rest pain (Rutherford stage 4), and HOPE CLTI-2, which includes patients with limb ulcers (Rutherford stage 5). In both trials, patients are randomized with a 2:1 ratio of intramuscular injection of NL003 to placebo. The primary endpoint of HOPE CLTI-1 is the complete pain relief rate. The primary endpoint of HOPE CLTI-2 is the complete ulcer healing rate. The safety endpoint was assessed based on adverse events after injection of NL003. Enrollment began in July 2019. The HOPE CLTI-1 trial aims to complete the randomization of at least 300 patients, while the HOPE CLTI-2 trial aims to enroll at least 240 patients. Both trials are organized such that patients will be followed for 6 months after the first intramuscular injection. CONCLUSIONS: HITOP CLTI, which is comprised of 2 multicenter, double-blind, placebo-controlled phase III clinical trials, aims to evaluate the efficacy and safety of the intramuscular administration of NL003 in patients with CLTI.

A first-in-human, randomized, double-blind, single- and multiple-dose, phase I study of recombinant human thymosin ß4 in healthy Chinese volunteers.

The study evaluated the safety, tolerability, pharmacokinetics (PK) and anti-drug antibody (ADA) of the recombinant human thymosin ß4 (NL005) for single and multiple intravenous injections in healthy subjects. Seven cohorts, with 54 healthy subjects, were given a single intravenous dose of NL005 or placebo and were observed for 28 days. The cohorts received ascending doses of either 0.05, 0.25, 0.5, 2.0, 5.0, 12.5 or 25.0 µg/kg in the single-dose trial. A total of 30 healthy subjects were randomly enrolled in the multiple-dose trial, and 3 cohorts (0.5, 2.0 and 5.0 µg/kg) were administered once human thymosin ß4 daily for 10 days and observed for 28 days. The adverse events were mild to moderate in intensity. There were no dose-limiting toxicities or serious adverse events. The plasma concentration, maximum peak concentration (Cmax ) and AUC of each dose group increased with the increase in the dose. The tendency of terminal clearance in each dose group was consistent, and there was no obvious accumulation after continuous administration. Thus, the drug can be concluded to be well tolerated and safe in healthy people and suitable for use in a clinical study for the treatment of acute myocardial infarction.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Hepatocyte Growth Factor in the Treatment of Critical Limb Ischemia.

NL003 is a plasmid engineered to simultaneously express two isoforms of hepatocyte growth factor. This phase II study was performed to assess the clinical safety and efficacy of intramuscular injection of NL003 in critical limb ischemia (CLI) patients for 6 months. Two hundred patients (Rutherford scale 4-5) were randomly assigned: placebo (n = 50), low-dose NL003 (n = 50), middle-dose NL003 (n = 50), or high-dose NL003 (n = 50). The drug was administered in the affected limb of 197 patients on days 0, 14, and 28. No significant differences in the incidence of adverse events (AEs) or serious AEs were found among the groups. At 6 months, pain severity was significantly reduced in all NL003 groups, but not in the placebo group (p < 0.05). The proportion of patients with complete ulcer healing in the high-dose group was significantly higher than that of the placebo group (p = 0.0095). There were no statistically significant differences in transcutaneous oxygen pressure (TcPO2), ankle-brachial index (ABI), or toe-brachial index (TBI) value among the four groups throughout the study period. These results provide the first effective evidence of significant improvements in total healing of ulcers in treated legs, complete pain relief without analgesics, and safety for NL003 in patients with Rutherford stage 4-5.

Identification and characterization of ERV transcripts in goat embryos.

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

Temporary sensory separation of lamb groups from ewes affects behaviors and serum levels of stress-related indicators of small-tailed Han lambs.

Under the current meat sheep breeding system, newborn lambs usually live with their mothers until weaning, and in daily management, they often need to be separated from their ewes for a short period due to dehorning, disease treatment, etc. Such short-term separation was considered to be a high-intensity stress for the lambs. This study aimed to explore the effects of 1 h sensory separations on behaviors and the concentration of stress-related indicators of small-tailed Han lambs. Lambs were assigned to four groups: auditory, visual, and tactile separation (AVT) group; visual and tactile separation (VT) group; tactile separation (T) group; and control (C) group. Then they were separated from their mothers for one hour on postnatal days 14, 21, 28, 35 and 42. Results showed the separated lambs (AVT, VT, and T groups) spent less time lying down relaxing and more time looking around, exploring, vocalizing, and attempting to escape (P < 0.05). Lambs separated by lack of tactile contact only exhibited the most escaping and moving behavior. Twin-born lambs showed less moving, escaping, and vocalizing than single-born lambs (P < 0.05). The separation also led to a rise in serum globulin levels and a decrease in tetraiodothyronine. In conclusion, this study showed that temporary 1 h ewe-lamb separations could affect behaviors and the serum levels of stress indicators of lambs. The behavioral responses were more obvious when lambs were separated by lack of tactile contact only, and in single-born lambs. It can conclude that indicated that when lambs need to be temporarily separated from ewes in daily management production, it would be better to let them stay together with their littermates, and make them avoid hearing or seeing the ewes, such management may partially reduce the separation stress, thereby improving the welfare and breeding efficiency of sheep.

Functions of intrinsic disorder in proteins involved in DNA demethylation during pre-implantation embryonic development.

DNA demethylation is involved in many biological processes during pre-implantation embryonic development in mammals. To date, the complicated mechanism of DNA demethylation is still not fully understood. Ten-eleven translocation family (TET3, TET1 and TET2), thymine DNA glycosylase (TDG) and DNA methyltransferase 1 (DNMT1) are considered the major protein enzymes of DNA demethylation in pre-implantation embryos. TET3, TET1, TET2, TDG, and DNMT1 contain abundant levels of intrinsically disordered protein regions (IDPRs), which contribute to increasing the functional diversity of proteins. Thus we tried to explore the complicated DNA demethylation in pre-implantation embryos from the intrinsic disorder perspective. These five biological macromolecules all have DNA demethylation-related functional domains. They can work together to fulfill DNA demethylation in pre-implantation embryos through complex protein-protein interaction networks. Intrinsic disorder analysis results showed these proteins were partial intrinsically disordered proteins. Many identifiable disorder-based DNA-binding sites, protein-binding sites and post-translational modification sites located in the intrinsically disordered regions, and DNA demethylation deficiency point mutations in the IDPRs could significantly change the local disorder propensity of these proteins. To the best of our knowledge, this work provides a new viewpoint for studying the mechanism of DNA methylation reprogramming during mammalian pre-implantation embryonic development.

Plasticizer Bis(2-ethylhexyl) Phthalate Causes Meiosis Defects and Decreases Fertilization Ability of Mouse Oocytes in Vivo.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride (PVC) plastics. Humans and animals are widely and continuously exposed to DEHP, especially with respect to diet, which is associated with reproductive diseases. Nevertheless, the effects and underlying mechanisms of DEHP exposure on oocytes in vivo remain ambiguous. In this study, we found that oral administration of DEHP (40 µg/kg body weight per day for 14 days) markedly reduced the maturation and fertilization of oocytes in vivo. In addition, DEHP caused oxidative stress, increased reactive oxygen species generation, promoted early apoptosis, and resulted in DNA damage in mouse oocytes. Moreover, DEHP exposure caused mitochondrial damage, reduced ATP content, down-regulated actin expression, and disturbed the spindle assembly and chromosome alignment in mouse oocytes. Furthermore, DEHP exposure remarkably impaired the localization and protein level of Juno, the sperm receptor on the membrane of oocytes. The levels of DNA methylation, H3K9me3, and H3K9ac were also altered in the DEHP-exposed mouse oocytes. Thus, our results indicated that DEHP exposure reduced the maturation and fertilization capabilities of mouse oocytes by affecting cytoskeletal dynamics, oxidative stress, early apoptosis, meiotic spindle morphology, mitochondria, ATP content, Juno expression, DNA damage, and epigenetic modifications in mouse oocytes.

Treating donor cells with 2-PCPA corrects aberrant histone H3K4 dimethylation and improves cloned goat embryo development.

Epigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p < 0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2µM compared with the control group (p < 0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p < 0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA. ABBREVIATIONS: SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase.

Protective Effects of Quercetin Against Cadmium Chloride-Induced Oxidative Injury in Goat Sperm and Zygotes.

Quercetin, a plant-derived flavonoid, is frequently used as an antioxidant for efficient anti-oxidative capacity. However, whether quercetin has protective effects on goat sperm and preimplantation embryos against Cd2+-induced oxidative injury is still unclear. So, we researched the influence of quercetin on goat sperm and zygotes respectively under the oxidative stress induced by Cd2+. In our study, quercetin decreased the malonaldehyde (MDA) and reactive oxygen species (ROS) levels caused by Cd2+ in goat sperm (p < 0.05), which facilitated sperm characteristics including motility, survival rates, membrane integrity, and mitochondria activity during storage in vitro and subsequent embryo development (p < 0.05). Moreover, in goat zygotes, quercetin decreased peroxidation products including ROS, MDA, and carbonyl through preserving or maintaining mitochondrial function, gene expression, and anti-oxidative products such as glutathione peroxidase, superoxide dismutase, and catalase, which ameliorated subsequent embryo development and embryo quality (p < 0.05). Taken together, these results suggest that quercetin protects both goat sperm and preimplantation embryos from Cd2+-induced oxidative stress.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency.

Effects of granulosa cells on steroidogenesis, proliferation and apoptosis of stromal cells and theca cells derived from the goat ovary.

The aim of this study was to investigate the effect of granulosa cells from small antral follicles on steroidogenesis, proliferation and apoptosis of goat ovarian stromal and theca cells in vitro. Using Transwell co-culture system, we evaluated androgen production, LH responsiveness, cell proliferation and apoptosis and some molecular expression regarding steroidogenic enzyme and apoptosis-related genes in stromal and theca cells. The results indicated that the co-culture with granulosa cells increased steroidogenesis, LH responsiveness and bcl-2 gene expression as well as decreased apoptotic bax and bad expressions in stromal and theca cells. Thus, granulosa cells had a capacity of promoting steroidogenesis in stromal cell and LH responsiveness in cortical stromal cells, maintaining steroidogenesis in theca cells, inhibiting apoptosis of cortical stromal cells and improving anti-apoptotic abilities of stromal and theca cells.

Anti-bacterial activity of recombinant human ß-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

The present study was conducted to determine whether recombinant human ß-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.