The role of insulin-like growth factor-1 in bone remodeling: A review.

Insulin-like growth factor (IGF)-1 is a polypeptide hormone with vital biological functions in bone cells. The abnormal expression of IGF-1 has a serious effect on bone growth, particularly bone remodeling. Evidence from animal models and human disease suggested that both IGF-1 deficiency and excess cause changes in bone remodeling equilibrium, resulting in profound alterations in bone mass and development. Here, we first introduced the functions and mechanisms of the members of IGFs in bone. Subsequently, the critical role of IGF-1 in the process of bone remodeling were emphasized from the aspects of bone resorption and bone formation respectively. This review explains the mechanism of IGF-1 in maintaining bone mass and bone homeostasis to a certain extent and provides a theoretical basis for further research.

Porcine IGF-1R synonymous mutations in the extracellular domain affect proliferation and differentiation of skeletal muscle cells.

OBJECTIVE: Miniature pigs are considered ideal organ donors for xenotransplantation in humans, but the mechanism underlying their dwarfism remains to be elucidated. IGF-1R is a crucial factor in body size formation in mammals, including skeletal muscle formation and development. The extracellular domain (ECD) binds to the ligand, a phenomenon that results in the activation of downstream pathways. METHODS: In this study, the coding sequences of two IGF-1R ECD haplotypes of the large Landrace (LP) pig and the small Bama Xiang (BM) pig were cloned into pcDNA3.1 vectors to generate pcDNA3.1-LP and pcDNA3.1-BM. The two recombinant vectors were then transfected into skeletal muscle cells. RESULTS: IGF-1R transcript was found to be expressed at higher levels in the pcDNA3.1-LP group than in the pcDNA3.1-BM group. The IGF-1R ECD from LP promoted cell proliferation and CyclinD1 expression, and promoted the phosphorylation of protein kinase B (to yield p-AKT). Moreover, the IGF-1R ECD from LP increased cell differentiation and the expression of myogenic determination factor (MyoD). CONCLUSION: Our data indicated that the IGF-1R ECD haplotypes between pig breeds with different body sizes affect IGF-1R expression, in turn affecting the proliferation and differentiation of skeletal muscle cells by activating downstream signalling pathways.

Peptides Isolated from Amphibian Skin Secretions with Emphasis on Antimicrobial Peptides.

The skin of amphibians is a tissue with biological functions, such as defense, respiration, and excretion. In recent years, researchers have discovered a large number of peptides in the skin secretions of amphibians, including antimicrobial peptides, antioxidant peptides, bradykinins, insulin-releasing peptides, and other peptides. This review focuses on the origin, primary structure, secondary structure, length, and functions of peptides secreted from amphibians' skin. We hope that this review will provide further information and promote the further study of amphibian skin secretions, in order to provide reference for expanding the research and application of amphibian bioactive peptides.

Peptide-Calcium Chelate from Antler (Cervus elaphus) Bone Enhances Calcium Absorption in Intestinal Caco-2 Cells and D-gal-Induced Aging Mouse Model.

Antler bone calcium (AB-Ca) and bioactive peptides (ABPs) were extracted from antler bones (Cervus elaphus) to maximize their value. In this study, 0.14 g calcium was obtained from 1 g antler bone. The peptide-calcium chelate rate was 53.68 ± 1.80%, and the Gly, Pro, and Glu in ABPs were identified to donate most to the increased calcium affinity through the mass spectrometry. Fourier transform infrared spectroscopy showed that calcium predominantly interacted with amino nitrogen atoms and carboxyl oxygen atoms, thereby generating a peptide-calcium chelate. The peptide-calcium chelates were characterized using scanning electron microscopy. A Caco-2 cell monolayer model showed that ABPs significantly increased calcium transport. Furthermore, the D-gal-induced aging mouse model indicated that the ABPs + AB-Ca group showed higher Ca and PINP levels, lower P, ALP, and CTX-1content in serum, and considerably higher tibia index and tibia calcium content. Results showed that ABPs + AB-Ca increased bone formation and inhibited bone resorption, thereby providing calcium supplements for ameliorating senile osteoporosis (SOP).

MGF-19E peptide promoted proliferation, differentiation and mineralization of MC3T3-E1 cell and promoted bone defect healing.

The repair of segmental bone defects and bone fractures is a clinical challenge involving high risk and postsurgical morbidity. Bone injury and partial bone tumor resection via traditional bone grafting result in high complications. Growth factors have been proposed as alternatives to promote bone repair and formation and circumvent these limitations. In this study, we classified different lengths of mechano growth factor (MGF) E peptides in different species and analyzed their effects on MC3T3-E1 cell proliferation, cell cycle, alkaline phosphatase (ALP) activity, differentiation-related factor expression, and cell mineralization. A rabbit bone injury model was constructed, and the repair function of MGF E peptide was verified by injecting the candidate MGF E peptide. We analyzed 52 different MGF-E peptides and classified them into the following four categories: T-MGF-25E, M-MGF-25E, T-MGF-19E, and M-MGF-19E. These peptides were synthesized for further study. T-MGF-19E peptide obviously promoted cell proliferation by regulating cell cycle after MGF E peptide treatment at 72 h. T-MGF-25E and T-MGF-19E peptide significantly promoted the differentiation of osteoblasts on day 14, and M-MGF-25E peptide promoted cell differentiation on day 7. T-MGF-19E, T-MGF-25E, and M-MGF-19E significantly promoted osteoblast mineralization, with T-MGF19E showing the most significant effect. These results implied that T-MGF19E peptide could remarkably promote MC3T3-E1 cell proliferation, differentiation, and mineralization. The rabbit bone defect model showed that the low-dose T-MGF-19E peptide significantly promoted bone injury healing, suggesting its promoting effect on the healing of bone injury.

Identification of hub genes and potential molecular mechanisms of chickpea isoflavones on MCF-7 breast cancer cells by integrated bioinformatics analysis.

BACKGROUND: Chickpea isoflavones have been demonstrated to play an inhibitory role in breast cancer cells. In this study, we aimed to explore the mechanism of chickpea isoflavones inhibiting the formation and development of breast carcinoma through the integration of wet and dry experiments. METHODS: Chickpea isoflavones were added to the MCF-7 cells for 48 hours, and the subsequent morphological changes of cells were observed using an inverted microscope, while apoptosis was quantified by flow cytometry. The mRNA and LncRNA expression profiles were detected by RNA-sequencing (RNA-Seq) technology. The protein-protein interaction (PPI) network was constructed from the STRINGdb database. To identify the co-expressed long non-coding RNA and messenger RNA (lncRNA-mRNA) pairs, Pearson's correlation coefficients were calculated based on the expression value between every differentially expressed lncRNA and mRNA pair. The hub gene expression was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and survival analysis results were provided by The Human Protein Atlas website. RESULTS: Microscopic observation and flow cytometry results confirmed that chickpea isoflavones with a final concentration of 32.8 µg/mL could cause apoptosis of the MCF-7 cells. Transcriptome results showed that a total of 1,094 mRNAs and 378 lncRNAs were differentially expressed in isoflavone-treated cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that inhibition of cell proliferation was mainly due to the up-regulation of genes in the apoptosis signaling pathway and the down-regulation of genes in mRNA splicing pathway. The co-expressed genes of the top 10 down-regulated lncRNAs were mainly heterogeneous nuclear ribonucleoproteins (HNRNP) family genes, which interacted with apoptosis-related genes through ubiquitin C (UBC). The abnormal expression of 11 hub genes (degree >10) of PPI networks were beneficial to improve the overall survival time of breast cancer patients. CONCLUSIONS: Our results reveal a potential mechanism for chickpea isoflavones to inhibit MCF-7 breast cancer cell proliferation and provide a reference for the development of new anti-cancer drugs used in breast cancer.

miR-709 inhibits GHRP6 induced GH synthesis by targeting PRKCA in pituitary.

Pituitary growth hormone (GH) plays an essential role in processes of organism growth and metabolism. MicroRNA (miRNA) could also participate in diverse biological processes. However, the role of miRNA in the regulation of pituitary GH during the growth process remains unclear. In this study, we firstly confirmed that the second highly expressed pituitary miRNA (miR-709) significantly inhibited the GH synthesis and suppressed the viability of GH3 cells. The bioinformatics analysis and dual luciferase report system were used to ascertain the PRKCA is the direct target gene of miR-709, which is the coding gene of PKCα. Then the transcription and translation levels of Prkca were obvious reduced by the over-expression of miR-709 in GH3 cells, followed by the inhibition of the transcription factor (CREB1) of Gh1 gene and the ERK1/2 signaling pathway or the possible cross-talk signaling pathway (cAMP/PKA signaling pathway) detected by western blot, suggesting that ERK1/2 maybe an important factor involved in the GH3 cell viability mediated by PKCα. At last, GHRP6 increased PKCα and GH expression but reduced miR-709 expression in vitro and vivo assays, and this conclusion was further confirmed by the result of GHRP6 attenuated the inhibition of miR-709 on GH expression. These findings will provide new molecular mechanism on the regulation of pituitary GH.

Association of SNPs in GnRH gene with sperm quality traits of Chinese water buffalo.

Hypothalamic gonadotropin-releasing hormone (GnRH) controls the activity of hypothalamic-pituitary-gonadal axis and plays a key role in the reproductive performance of animals. In this study, five single nucleotide polymorphisms (SNPs), namely g.991T > C, g.1041T > C g.3424T > C, g.3462C > A and g.3463Inde A, were detected in the GnRH gene of 162 water buffaloes by Sanger sequencing. Each SNP was associated with more than two sperm quality traits of ejaculate volume, sperm concentration, post-thaw sperm motility and sperm abnormality. g.3424T > C and g.3462C > A were related to these four traits and had a remarkable effect on ejaculate volume. The three other SNPs were related to sperm concentration, post-thaw sperm motility and sperm abnormality. Moreover, six haplotypes (H1: TCCAI, H2: CTTC-, H3: TCCCI, H4: CTTA-, H5: CCTA- and H6: CTCC-) composed of five SNPs comprising seven different combined genotypes were generated by linkage disequilibrium analysis. Statistics followed by one-way ANOVA indicated that water buffaloes with the haplotype combination H1H1 had the highest genotypic frequency, and those with the H4H4 haplotype combination had the highest ejaculate volume. The sperm concentration of those with haplotype combination H1H5 was higher than that of the other genotypes. In summary, our study showed a remarkable association between the SNPs of GnRH and sperm quality traits of Chinese water buffalo.

A miR-511-binding site SNP in the 3'UTR of IGF-1 gene is associated with proliferation and apoptosis of PK-15 cells.

Insulin-like growth factor-1 (IGF-1) is a functional candidate gene for pig growth and development due to its crucial role in the growth axis of growth hormone-IGF-1. Considering that the 3' untranslated region (3'UTR) of gene may affect its expression, we analyzed the effect of a single-nucleotide polymorphism (SNP) (rs34142920, c.674C > T) on gene expression, cell proliferation, and apoptosis and the possible related molecular mechanisms in PK-15 cells. The SNP was found in the 3'UTR of IGF-1 in Bama Xiang pig in previous investigations. Results showed that the SNP was located at the target site binding to microRNA (miR-511). The 3'UTR of IGF-1 gene with C allele significantly downregulated the expression of IGF-1 gene compared with that of the gene with T allele by luciferase assay. miR-511 was transfected into porcine kidney cell line (PK-15 cells) to reveal its effects on cells and whether or not it targets IGF-1. The expression levels of IGF-1 at mRNA and protein levels were remarkably downregulated. miR-511 significantly inhibited cell proliferation and promoted cell apoptosis by downregulating the phosphorylation level of AKT and ERK1/2. This finding confirmed that miR-511 inhibits proliferation and promotes apoptosis by downregulating the IGF-1 in PK-15 cells.

Systematic review on the efficacy and safety of immune checkpoint inhibition in renal cell carcinoma.

To perform a systematic review of the relevant literature about clinical trials on efficacy and safety of immune checkpoint inhibition, whether it is used alone, in combination or with other targeted therapies in patients with advanced and metastatic renal cell carcinoma (RCC), two team members reviewed the abstracts and selected pertinent articles from the relevant databases. A narrative review of randomized controlled trials was performed and seven randomized controlled trials were identified in this systematic review. In treatment of RCC, nivolumab has superior efficacy and safety compared with second-line everolimus. Combination strategies, especially those combined with anti-VEGF agents presents better efficacy but worse outcomes in term of safety than monotherapy and conventional treatment and might guide treatment choice for patients with RCC.

Liposomes Incorporating Transferrin and Stearic Acid-modified Octa-arginine for siRNA Delivery.

Liposomes incorporating stearic acid-modified octa-arginine (StA-R8) and conjugated to transferrin (Tf) were synthesized and evaluated for delivery of small interfering RNA (siRNA) against survivin. Characteristics, cytotoxicity, cellular uptake and biological activity of liposomes complexed to survivin siRNA were investigated. Tf-conjugated liposomes were shown to have reduced cytotoxicity and improved delivery efficiency for siRNA into cancer cells compared to non-conjugated StA-R8 liposomes. These data suggest that Tf-conjugated StA-R8 liposomes are promising vehicles for siRNA delivery in cancer therapy.

Polypeptides from the Skin of Rana chensinensis Exert the Antioxidant and Antiapoptotic Activities on HaCaT Cells.

Studies have shown that frog skin secretes many types of peptides that are good for human skin. In this study, acid and enzymatic extracts of Rana skin peptides (acid/enzymatic Rana skin peptides, ARPs/ERPs) were obtained. The chemical and physical properties of the ARPs and ERPs were identified through UV scanning, HGLC, FRIT, and MS. MTS and flow cytometry were used to test the proproliferative and antiapoptotic effects of the ARPs and ERPs on human immortalized keratinocytes (HaCaT cells). To elucidate the antiapoptotic mechanisms, the mRNA and protein levels of EGF (epidermal growth factor, which enhances stimulation of cellular proliferation in both cells and epithelial tissues) and caspase-3 were evaluated using quantitative RT-PCR. The results indicated that the ARPs and ERPs were extracted from the Rana skin with yields of 0.65% and 0.52%, respectively. Treatment with ARPs (1.6 g/L) and ERPs (0.8 g/L) showed a 1.66-fold (p < 0.001) and 2.1-fold (p < 0.001) enhancement in the proliferation rates of HaCaT cells. The rate of apoptosis decreased by 2.6 fold (p < 0.01) and 3.4 fold (p < 0.01) under the UVB stimulation, respectively, at the same time, the up-regulation of EGF and down-regulation of caspase-3 were found. These results suggested that we can dig into the potential value of ARPs/ERPs in a new field.

Identification of Four SNPs in LHB Gene and Their Associations with Sperm Qualities of Chinese Buffaloes.

Luteinizing hormone beta polypeptide (LHB) gene has been considered important for sexual behavior and has associations with sperm quality. In this study, four SNPs (g.276 T>C, g.377A>C, g.401T>C, and g.412A>G) were detected in the LHB gene of 165 water buffaloes by direct sequencing and identification of overlap peaks, each of which was associated with at least one sperm quality trait of ejaculate volume, sperm concentration, post-thaw sperm motilities, and sperm abnormalities by chi-square analysis. Among them, g.276 T>C was associated with ejaculate volume (F = 2.857, p < 0.05), sperm concentration (F = 2.052, p < 0.05), and post-thaw sperm motilities (F = 3.480, p < 0.05); g.377A>C was related to ejaculate volume (F = 4.178, p < 0.05), g.401T>C had a marker effect on sperm abnormalities (F = 3.332, p < 0.05), g.412A>G was associated with sperm concentration (F = 3.579, p < 0.05), and sperm abnormalities (F = 3.408, p < 0.05). Furthermore, four haplotypes (H1: ACG, H2: CCG, H3: CTA, H4: CCA) were generated by linkage disequilibrium analysis, which composed seven genotypes. Among them, the buffaloes with combined genotype H2H2 had the higher ejaculate volume and the individuals with the combined haplotypes H1H4 had higher sperm concentration. In summary, our study showed that there was a significant association between SNPs of LHB gene and Chinese water buffalo sperm quality traits. To the best of our knowledge, this is the first report addressing the associations between the SNPs in the LHB gene and the sperm qualities of Chinese buffaloes.

Delivery of siRNA Using Cationic Liposomes Incorporating Stearic Acid-modified Octa-Arginine.

Cationic liposomes incorporating stearic acid-modified octa-arginine (StA-R8) were evaluated for survivin small interfering RNA (siRNA) delivery. StA-R8 was synthesized and incorporated into liposomes. The composition of liposomes was optimized. Physicochemical properties, cytotoxicity, cellular uptake and gene silencing activity of the liposomes complexed to survivin siRNA were investigated. The results showed that StA-R8-containing liposomes had reduced cytotoxicity and improved delivery efficiency of siRNA into cancer cells compared with StA-R8 by itself.

Missense mutations in the signal peptide of the porcine GH gene affect cellular synthesis and secretion.

CONTEXT: In previous investigations, we have demonstrated the mutations in the signal peptide of porcine GH gene were associated with the body size. METHODS: In this study, the fusion gene expression vectors which consisted of eight signal peptide mutants of GH gene and EGFP gene were constructed according to three missense mutations (p.Val9Ala, p.Gln22Arg and p.Asp25Gly), and they were transfected into the GH3 cell line. RESULTS: The inhibition levels of EGFP gene transcriptions with different signal peptide mutants were significantly different. Typically, the allelic variants carrying Val in codon nine showed higher protein synthesis (P < 0.05), and the allelic variants carrying neutral Gln in codon 22 and Gly in codon 25 showed higher secretion proportion (P < 0.05) compared with the other groups as assessed by western blotting. In silico RNA folding prediction indicated that the mutations gave rise to different RNA secondary structures, suggesting that they might affect translation and protein synthesis. CONCLUSION: We conclude that the missense mutations within the signal sequence influence the expression and the secretion of the protein. To the best of our knowledge, this is the first report addressing the functional consequences of the mutations in the signal peptide of porcine GH gene.

Ceftiofur attenuates lipopolysaccharide-induced acute lung injury.

Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Our laboratory has previously been reported that ceftiofur can modulate early cytokine responses and increase mouse survival in endotoxemia. In the present study, we investigated the effect of ceftiofur on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in vivo. Mice were pretreated with ceftiofur 1h before challenge with a dose of 0.5mg/kg LPS. Mice treated with LPS alone showed marked increased TNF-alpha, IL-6, and IL-8 levels in the bronchoalveolar lavage fluid (BALF). When pretreated with 30mg/kg of ceftiofur, the TNF-alpha, IL-6, and IL-8 levels were significantly decreased. In addition, the W/D ratio of the lung tissue and the number of total cells, neutrophils and macrophages in the BALF significantly decreased at 8h after pretreatment with ceftiofur. Furthermore, ceftiofur markedly attenuated the LPS-induced histological alteration. These studies indicate that ceftiofur significantly decreases the inflammation in a murine model of LPS-mediated ALI and may represent a novel prevention strategy for nonspecific inflammation in the lungs.

PLGA microsphere-mediated growth hormone release hormone expression induces intergenerational growth.

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3-21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.

Simultaneous expression of growth hormone releasing hormone (GHRH) and hepatitis B surface antigen/somatostatin (HBsAg/SS) fusion genes in a construct in the skeletal muscle enhances rabbit weight gain.

Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are synthesized and secreted by the hypothalamus, which can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. In this article, we describe the regulation of animal growth using plasmid DNA encoding both the GHRH gene and the SS gene fused with the hepatitis B surface antigen (HBsAg) gene. We constructed a series of expression plasmids to express the GHRH and HBsAg-SS fusion genes individually as well as collectively. The fusion gene and GHRH were successfully expressed in Chinese hamster ovary (CHO) cells, as proven by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting tests. Poly D, L-lactide-co-glycolic acid (PLGA) plasmid-encapsulating microspheres were prepared and injected intramuscularly into the leg skeletal muscles of rabbits. Weight gain/day and the levels of insulinlike growth factor-I (IGF-I), SS, and hepatitis B surface antibody (HBsAb) were monitored. During days 30 postinjection, increase in weight gain/day and IGF- I concentration and decrease in SS were observed in treatment groups. From days 15 to 30 postinjection, the weight gain/day significantly increased (P < 0.05) by 129.13%, 106.8%, and 72.82% relative to the control group in the co-expression GHRH and fusion gene (named P-G-HS), fusion gene (named P-HS), and GHRH (named P-G) groups, respectively. And most importantly, the P-G-HS group showed significant weight gain/day (P < 0.05) relative to the P-G and P-HS groups. A significant increase in the IGF-I concentration and decrease in the SS level relative to the control group were also observed. The results indicated that the combination of plasmid-mediated GHRH supplementation and positive immunization against SS led to more robust weight gain/day in rabbits.