Zuogui Wan improves trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats by regulating orexin-A and orexin receptor.

OBJECTIVE: To investigate the protective effects of Zuogui Wan (ZGW) on bone loss induced by ovariectomy (OVX) and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model. METHODS: Fifty Sprague-Dawley female rats were randomly divided into sham-operated (sham) group and four OVX subgroups. Rats subjected to sham and OVX were treated with the vehicle (OVX, 1 mL/100 g weight, n = 10), 17ß-estradiol (E2, 50 µg*kg-1*d-1), and ZGW at the doses of 2.3 (ZGW-L) and 4.6 (ZGW-H) g/kg/day lyophilized powder daily for 3 months, respectively. The serum biochemical parameters of 17ß-estrogen (17ß-E2), tartrate-resistant acid phosphatase (TRACP-5b) and bone alkaline phosphatase (BALP) were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones. Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur. The gene or protein expression of orexin-A, orexin receptor 1 (OX1R), orexin receptor 2 (OX2R), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were assayed by either quantitative polymerase chain reaction or Western blot analysis. RESULTS: Compared with the OVX group, ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and17ß-E2 (P < 0.01). Meanwhile, ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number, and arranging the trabeculae structure properly. Compared with the OVX group, it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae, and OPG in the tibiae of ZGW groups (P < 0.01, < 0.05), while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae (P < 0.01). CONCLUSION: ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.

Genome-wide identification, classification and expression profile analysis of the HSF gene family in Hypericum perforatum.

Heat shock transcription factors (HSFs) are critical regulators of plant responses to various abiotic and biotic stresses, including high temperature stress. HSFs are involved in regulating the expression of heat shock proteins (HSPs) by binding with heat stress elements (HSEs) to defend against high-temperature stress. The H. perforatum genome was recently fully sequenced; this provides a valuable resource for genetic and functional analysis. In this study, 23 putative HpHSF genes were identified and divided into three groups (A, B, and C) based on phylogeny and structural features. Gene structure and conserved motif analyses were performed on HpHSFs members; the DNA-binding domain (DBD), hydrophobic heptad repeat (HR-A/B), and exon-intron boundaries exhibited specific phylogenetic relationships. In addition, the presence of various cis-acting elements in the promoter regions of HpHSFs underscored their regulatory function in abiotic stress responses. RT-qPCR analyses showed that most HpHSF genes were expressed in response to heat conditions, suggesting that HpHSFs play potential roles in the heat stress resistance pathway. Our findings are advantageous for the analysis and research of the function of HpHSFs in high temperature stress tolerance in H. perforatum.

Montelukast alleviates inflammation in experimental autoimmune encephalomyelitis by altering Th17 differentiation in a mouse model.

Montelukast is a leukotriene receptor antagonist that is known to prevent allergic rhinitis and asthma. Blocking the Cysteinyl leukotriene receptor (CysLTR1), one of the primary receptors of leukotrienes, has been demonstrated to be efficacious in ameliorating experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), through disrupting chemotaxis of infiltrating T cells. However, the role of CysLTR1 in the pathogenesis of MS is not well understood. Here, we show that MS patients had higher expression of CysLTR1 in the circulation and central nervous system (CNS). The majority of CD4+ T cells expressed CysLTR1 in MS lesions. Among T-cell subsets, Th17 cells had the highest expression of CysLTR1, and blocking CysLTR1 signalling abrogated their development in vitro. Inhibition of CysLTR1 by montelukast suppressed EAE development in both a prophylactic and therapeutic manner and inhibited myelin loss in EAE mice. Similarly, the in vivo results showed that montelukast inhibited Th17 response in EAE mice and that Th17 cells treated with montelukast had reduced encephalitogenic in adoptive EAE. Our findings strongly suggest that targeting Th17 response by inhibiting CysLTR1 signalling could be a promising therapeutic strategy for the treatment of MS and CNS inflammatory diseases.

Identification of core genes and prediction of miRNAs associated with osteoporosis using a bioinformatics approach.

Osteoporosis (OP) is an age-related disease, and osteoporotic fracture is one of the major causes of disability and mortality in elderly patients (>70 years old). As the pathogenesis and molecular mechanism of OP remain unclear, the identification of disease biomarkers is important for guiding research and providing therapeutic targets. In the present study, core genes and microRNAs (miRNAs) associated with OP were identified. Differentially expressed genes (DEGs) between human mesenchymal stem cell specimens from normal osseous tissues and OP tissues were detected using the GEO2R tool of the Gene Expression Omnibus database and Morpheus. Network topological parameters were determined using NetworkAnalyzer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery, and ClueGO. Cytoscape with the Search Tool for the Retrieval of Interacting Genes and Molecular Complex Detection plug-in was used to visualize protein-protein interactions (PPIs). Additionally, miRNA-gene regulatory modules were predicted using CyTargetLinker in order to guide future research. In total, 915 DEGs were identified, including 774 upregulated and 141 downregulated genes. Enriched GO terms and pathways were determined, including 'nervous system development', 'regulation of molecular function', 'glutamatergic synapse pathway' and 'pathways in cancer'. The node degrees of DEGs followed power-law distributions. A PPI network with 541 nodes and 1,431 edges was obtained. Overall, 3 important modules were identified from the PPI network. The following 10 genes were identified as core genes based on high degrees of connectivity: Albumin, PH domain leucine-rich repeat-containing protein phosphatase 2 (PHLPP2), DNA topoisomerase 2-α, kininogen 1 (KNG1), interleukin 2 (IL2), leucine-rich repeats and guanylate kinase domain containing, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit γ (PIK3CG), leptin, transferrin and RNA polymerase II subunit A (POLR2A). Additionally, 15 miRNA-target interactions were obtained using CyTargetLinker. Overall, 7 miRNAs co-regulated IL2, 3 regulated PHLPP2, 3 regulated KNG1, 1 regulated PIK3CG and 1 modulated POLR2A. These results indicate potential biomarkers in the pathogenesis of OP and therapeutic targets.

Effect of Zuoguiwan on osteoporosis in ovariectomized rats through RANKL/OPG pathway mediated by ß2AR.

ETHNOPHARMACOLOGICAL RELEVANCE: The deficiency of kidney Yin is the main pathogenesis of postmenopausal osteoporosis (PMOP) according to traditional Chinese medicine (TCM). Zuoguiwan (ZGW) is among the classical prescriptions in TCM and has been applied to various diseases that are due to deficiency of kidney Yin, including osteoporosis, fractures, menopausal syndromes. However, the underlying mechanism of ZGW in treating PMOP remains poorly understood. AIM OF THE STUDY: ZGW, a traditional Chinese prescription, has been used to nourish Yin and reinforce the kidney since ancient times. The investigation aimed to explore the mechanism of ZGW via the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway as mediated by the ß2-adrenergic receptor (ß2AR) in an osteoporosis rat model. MATERIALS AND METHODS: An osteoporosis model induced by ovariectomy was established in rats. A total of 40 female Sprague-Dawley rats were randomly assigned into bilateral ovariectomy group (OVX), sham operated group (Sham), 17ß-estradiol-treated positive group (E2, 25 µg/kg/d), ZGW low-dose group (ZGW-L, 2.3 g/kg/d lyophilized powder) and ZGW high-dose group (ZGW-H, 4.6 g/kg/d lyophilized powder). The serum markers of bone turnover were measured using enzyme-linked immunosorbent assay (ELISA). The morphological structure changes in bones were detected through H&E staining. Local bone mineral density (BMD) and trabecular bone microarchitecture of the right distal femur were measured and evaluated by using micro-CT. Furthermore, the mRNA and protein expressions levels of ß2AR, OPG and RANKL were measured by qPCR and Western blot analysis. RESULTS: Compared with the OVX group, ZGW groups showed significantly reduced levels of serum tartrate-resistant acid phosphatase 5b (TRACP-5b) and ß-cross-linked c-telopeptide of type I collagen (ß-CTX) (P ＜ 0.01), increased levels of serum bone-specific alkaline phosphatase (BALP) (P ＜ 0.01) and OPG (P ＜ 0.05), prevention of OVX-induced bone loss, and improved microarchitecture of the trabecular bone of distal femur. Moreover, ZGW mediated the osteoporosis syndrome by reducing the empty bone lacunae, promoting the ordered arrangement of trabeculae structure, and increasing the trabeculae structure thickness. Furthermore, in ZGW groups, the protein expression of OPG in the tibia was notably up-regulated (P ＜ 0.01), whereas the mRNA and protein expression of ß2AR in the hippocampus (P ＜ 0.01), and the protein expressions levels of ß2AR (P ＜ 0.01) and RANKL (P ＜ 0.05) in the tibia were down-regulated compared with OVX group. CONCLUSIONS: ZGW through its protective effects, stimulates bone formation and suppresses bone resorption. The underlying mechanism of ZGW in improving perimenopausal syndrome and increasing bone mass might be attributed to the regulation of RANKL/OPG, as mediated by ß2AR. Therefore, ZGW may be used as an alternative treatment for PMOP.

Ultrasound microbubble contrast agent-mediated suicide gene transfection in the treatment of hepatic cancer.

The aim of this study was to investigate the role of ultrasound microbubble contrast agent-mediated suicide gene transfection in the treatment of hepatic cancer. We intratumorally injected KDR-TK, AFP-TK and microbubble contrast agent into nude mice prior to ultrasound treatment and administration of prodrugs (GCV and 5-FC). The tumor volume, tumor inhibition rate, survival time and apoptosis of tumor cells was determined. The sizes of subcutaneous hepatic cancers in mice receiving treatment were comparable to those in the control group, and the survival time was similar between the two groups (P>0.05). However, the tumor inhibition rate and the number of apoptotic cells in the treatment group was markedly higher compared with that in the control group (P<0.05). Evident tumor necrosis was absent in both groups, except at the needle tract. Ultrasound therapy following injection of suicide genes and microbubble contrast agents is able to inhibit cancer growth in vivo. This may be attributed to the induction of cancer cell apoptosis.