Differential expression and localisation of heat shock protein 70 (HSP70) and glutathione peroxidase 5 (GPX5) in the testis and epididymis of Sonid Bactrian camels.

CONTEXT: Heat shock protein 70 (HSP70) and glutathione peroxidase 5 (GPX5) are biomarkers of oxidative stress and stress in temperate, tropical environments, which are crucial for male reproduction. Their expression and distribution patterns in the testis and epididymis of Bactrian camels are still unknown. AIMS: This study aims to investigate the HSP70 and GPX5 expression and localisation in 3- and 6-year-old Bactrian camel testis and epididymis. METHODS: Reverse transcription quantitative polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry were used to detect HSP70 in the testis and epididymis (caput, corpus and cauda) and GPX5 in the epididymis at two developmental stages (3-year-old puberty group and 6-year-old adult group). KEY RESULTS: HSP70 was upregulated in the testis. Immunohistochemistry results indicated the HSP70 protein was mainly detected in spermatids and Leydig cells of testicular tissue. In the epididymis, HSP70 was located at the luminal spermatozoa, the epithelium lining the epididymal and the epididymal interstitium. GPX5 expression was significantly higher in the caput epididymis than in the corpus and cauda epididymis. GPX5 protein was observed in the epithelium lining the epididymal, interstitium and luminal spermatozoa in the epididymis by immunohistochemistry. CONCLUSIONS: Bactrian camel HSP70 and GPX5 exhibited spatiotemporal expression specificity. IMPLICATIONS: HSP70 and GPX5 may be essential for germ cell development and reproductive success after sexual maturation in Sonid Bactrian camels.

Hormone measurements and histomorphological observations in male Bactrian camels.

The aim of this study was to investigate the effect of age on the hypothalamic-pituitary-gonadal (HPG) axis hormones and to determine the morphological changes of the testis. The Bactrian camels were divided into two groups based on their ages. The results showed that the testicular weight was significantly heavier in adult male camels than in pubertal male camels (P < 0.05). There were also significant differences between testicular length, testicular width, and testicular volume (P < 0.05). In the testes of both pubertal and adult male camels, Sertoli cells, spermatogonia, spermatocytes, round spermatids, and elongated spermatids were observed. Adult male camels had more Sertoli cells (P < 0.01) and elongated spermatids (P < 0.05). The concentrations of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were higher in the plasma and testes of adult camels than in pubertal camels (P < 0.05). E2 concentrations were lower in adult camels than in pubertal camels (P < 0.05). The testosterone levels in testicular tissue were higher than in blood plasma in both adult and pubertal stage (P < 0.05). In conclusion, these findings provide supportive knowledge and show the significant differences in terms of testicular volume, testicular hormone concentrations, and testicular morphology between different developmental stages in Bactrian camels.

Whole transcriptome sequencing reveals core genes related to spermatogenesis in Bactrian camels.

Bactrian camels survive and reproduce better in extreme climatic conditions than other domestic animals can. However, the reproductive efficiency of camels under their natural pastoral conditions is low. Several factors affect mammalian reproductive performance, including testicular development, semen quality, libido, and mating ability. Testis is a main reproductive organ of the male and is responsible for producing spermatozoa and hormones. However, our understanding of the expression patterns of the genes in camel testis is minimal. Thus, we performed total RNA-sequencing to investigate the gene expression pattern. As a result, 1,538 differential expressed mRNAs (DEmRNAs), 702 differential expressed long non-coding RNAs (DElncRNAs), and 61 differential expressed microRNAs (DEmiRNAs) were identified between pubertal and adult Bactrian camel testes. Then the genomic features, length distribution, and other characteristics of the lncRNAs and mRNAs in the Bactrian camel testis were investigated. Target genes of the DEmiRNAs and DEmRNAs were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Genes, such as AMHR2, FGF1, ACTL7A, GATA4, WNT4, ID2, LAMA1, IGF1, INHBB, and TLR2, were mainly involved in the TGF-ß, PI3K-AKT, Wnt, GnRH, and Hippo signaling pathways which relate to spermatogenesis. Some of the DEmiRNAs were predicted to be associated with numerous DElncRNAs and DEmRNAs through competing endogenous RNA (ceRNA) regulatory network. At last, the candidate genes were validated by RT-qPCR, dual fluorescent reporter gene, and a fluorescence in situ hybridization (FISH) assay. This research provides high-throughput RNA sequencing data of the testes of Bactrian camels across different developmental stages. It lays the foundation for further investigations on lncRNAs, miRNAs, and mRNAs that involved in Bactrian camel spermatogenesis.

Bactrian camel breeding has a long history and has played an extremely important role in desert and semi-desert management and grassland culture, economy, and ecological development. As a precious livestock resource, the Bactrian camel has developed into an important part of China's grassland livestock industry. However, due to their biological characteristics, camels have lower fertility than other livestock. Fertility is one of the most important factors affecting camel productivity. Maintaining a high level of fertility is essential to improve their performance and genetic improvement. Fertility is mainly related to testicular development and regulation of gene expression during spermatogenesis. Therefore, the study of genes related to testicular development and spermatogenesis and the elucidation of their molecular mechanisms are important for improving and protecting male fertility and preventing male reproductive disorders. This study provided a theoretical foundation for further research into the molecular mechanisms of testis development and spermatogenesis in Bactrian camels by constructing the lncRNA-miRNA-mRNA regulatory interactions network.

Luteolin inhibits FcεRΙ- and MRGPRX2-mediated mast cell activation by regulating calcium signaling pathways.

Luteolin is a flavonoid found in many fruits, vegetables, and herbs. The antiinflammatory effects of luteolin have been reported. In this study, the effect of luteolin on allergic diseases and the underlying molecular mechanism were investigated. We found that luteolin inhibits Fc epsilon RΙ (FcεRΙ)- and Mas-related G protein-coupled receptor X2 (MRGPRX2)-mediated mast cells (MCs) activation, including degranulation and release of cytokines in vitro. Moreover, luteolin reduces the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner. The concentrations of histamine, TNF-α, MCP-1, IL-8, and IL-13 in mice serum are also decreased by luteolin administration. Our study reveals that luteolin can inhibit FcεRΙ- and MRGPRX2-mediated allergic responses in vivo and in vitro, and our results discover luteolin inhibited mast cells mediated anaphylactic reaction by inhibiting the phosphorylation level of PLCγ.

A rapid UHPLC-MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study.

A simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed and validated for the determination of ARQ531, a Bruton's tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C18 column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5-1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from -13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1-10 mg/kg and good oral bioavailability (>50%).

Granuloma formation in the liver is relatively delayed, although sustained, in BCG­infected mice co­infected with Plasmodium.

The purpose of the present study was to examine the effects of Plasmodium on the process of granuloma formation in Bacille Calmette­Guerin (BCG)­infected mice. Female six­week­old BALB/c mice were co­infected with BCG and Plasmodium. The liver index, pathological alterations and quantity of granulomas in the mice were observed when the mice were co­injected with BCG and Plasmodium. The expression of inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry and reverse transcription­polymerase chain reaction (RT­PCR) analysis. In addition, the expression of interleukin (IL)­10 in liver tissues was observed by RT­PCR. Following co­infection with BCG and Plasmodium, the swelling of the liver had been slowly restored to normal, and the time required to allow granulomas to subside had prolonged. In addition, the expression of iNOS increased, while the expression of IL­10 gradually decreased in Plasmodium­infected mice. It was concluded that the use of Plasmodium relatively delayed granuloma formation in livers of BCG­infected mice. In addition, iNOS and IL­10 are involved in this pathogenesis.

Comparison of naturally aging and D-galactose induced aging model in beagle dogs.

Animal models have been used to study aging for decades. In numerous aging studies, beagles are the most commonly used breed of dog. However, few studies have compared between naturally aging models and experimentally induced aging models in beagle dogs. In the present study, a D-galactose induced aging model was compared with a naturally aging model, and young adult dogs were considered as the young control group. The level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum, and brain tissue were measured. Histopathological comparisons of the liver, kidneys, heart, lungs and spleen were evaluated using hematoxylin and eosin (H&E) staining, in addition, the brain was evaluated by H&E staining, and Nissl staining. The expression levels of aging-associated factors in the hippocampus, including proliferating cell nuclear antigen (PCNA), P16 and P21 were also determined through reverse transcription quantitative-polymerase chain reaction, and western blot analysis. The results indicated that D-galactose induced aging significantly increased the MDA level, while the levels of SOD and GSH-Px were diminished when compared with the young control group, which was similar to the naturally aging group. Parallel histopathological features were observed in the D-galactose induced aging and naturally aging groups compared with the young control group. However, a reduced expression level of PCNA, and increased expression levels of P16 and P21 were observed in the naturally ageing and induced aging groups compared with the young control group. The results of the current study demonstrated that the beagle dogs in D-galactose induced aging model exhibited significant similarities with the naturally aging model, providing evidence to support that the D-galactose induced aging model may be applied to aging studies.

Aqueous Extract of Terminalia chebula Induces Apoptosis in Lung Cancer Cells Via a Mechanism Involving Mitochondria-mediated Pathways

The current study was designed to evaluate the aqueous extract of Terminalia chebula activity, and the main pathway was detected on lung cancer by extracts of T. chebula. Aqueous extract of T. chebula was separated using a zeolite, and five fractions of T. chebula extract were obtained and analyzed by ultraviolet (UV) and infrared (IR) spectroscopy. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods against human lung cancer (A549) and mouse lung cancer cell line LLC. T. chebula acts by regulating the Bcl-2 family protein-mediated mitochondrial pathway detected by western blot. Fraction 4 of the T. chebula extract showed much function and was thus studied further. Fraction 4 increased the activation of caspase-3, induced PARP cleavage, and promoted cytochrome c release into the cytoplasm. These data suggest that T. chebula acts by regulating the Bcl-2 family protein-mediated mitochondrial pathway and provide evidence that T. chebula deserves further investigation as a natural agent for treating and preventing cancer.