Apigenin inhibits STAT3/CD36 signaling axis and reduces visceral obesity.

Visceral obesity is the excess deposition of visceral fat within the abdominal cavity that surrounds vital organs. Visceral obesity is directly associated with metabolic syndrome, breast cancer and endometrial cancer. In visceral obese subjects, signal transducer and activator of the transcription 3 (STAT3) in adipocytes is constitutively active. In this study, we aimed to screen for dietary herbal compounds that possess anti-visceral obesity effect. Apigenin is abundant in fruits and vegetables. Our data show that apigenin significantly reduces body weight and visceral adipose tissue (VAT), but not subcutaneous (SAT) and epididymal adipose tissues (EAT), of the high fat diet (HFD)-induced obese mice. Mechanistic studies show that HFD increases STAT3 phosphorylation in VAT, but not in SAT and EAT. Further studies suggest that apigenin binds to non-phosphorylated STAT3, reduces STAT3 phosphorylation and transcriptional activity in VAT, and consequently reduces the expression of STAT3 target gene cluster of differentiation 36 (CD36). The reduced CD36 expression in adipocytes reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) which is the critical nuclear factor in adipogenesis. Our data show that apigenin reduces CD36 and PPAR-γ expressions and inhibits adipocyte differentiation; overexpression of constitutive active STAT3 reverses the apigenin-inhibited adipogenesis. Taken together, our data suggest that apigenin inhibits adipogenesis via the STAT3/CD36 axis. Our study has delineated the mechanism of action underlying the anti-visceral obesity effect of apigenin, and provide scientific evidence to support the development of apigenin as anti-visceral obesity therapeutic agent.

Overexpression of Rho-Associated Coiled-Coil Containing Protein Kinase 2 Is Correlated with Clinical Progression and Poor Prognosis in Breast Cancer.

BACKGROUND Rho-associated coiled-coil containing protein kinases 2 (ROCK2) is one of the best characterized targets for the small GTPase Rho. It has been reported that ROCK2 is critical for cancer cell migration and invasion. The objective of this study was to investigate the association of ROCK2 expression with clinicopathological features and overall survival of breast cancer patients. MATERIAL AND METHODS The expression of ROCK2 in breast cancer and paired adjacent normal tissues was detected and compared by immunohistochemical staining of tissue array. ROCK2 mRNA expression and clinicopathological information was extracted from the TCGA breast cancer dataset. The association of ROCK2 expression with the clinicopathological characteristics of patients with breast cancer was evaluated using univariate and multivariate Cox proportional hazards models. Overall survival was analyzed using the Kaplan-Meier method. RESULTS Immunohistochemistry showed that ROCK2 expression was significantly higher in tumor tissues than in paired adjacent normal tissues [immunoreactivity score (IRS): tumor, 5.25±2.10, n=40 vs. adjacent normal 3.83±1.06, n=40, P<0.01]. The IRS was correlated to breast cancer staging. Similarly, the mRNA level of ROCK2 was correlated to tumor stage. Notably, ROCK2 mRNA expression (hazard ratio [HR] 1.665 and 95% confidence interval [CI] 1.115-2.488, P=0.013) were also associated with overall survival in a multivariate analysis. CONCLUSIONS Upregulation of ROCK2 was associated with the progression of breast cancer. High expression of ROCK2 may predict poor overall survival rates for breast cancer patients.

[Experimental study on preclinical quality control, urgent poison and irritation of Dendrobium aurantiacum eye drops, a class I new drug against diabetic cataract].

To establish a quality control method of Dendrobium aurantiacum eye drops, in order to evaluate acute toxicity, irritation and irritability and lay a foundation for its development and utilization in the future. The content of gigantol and SA in D. aurantiacum eye drops were determined by high-performance liquid chromatography (HPLC). The linear ranges of gigantol and SA were 0.040 8-1.530 0 g x L(-1) (r = 0.999 9) and 0.100 8-0.504 0 g x L(-1) (r = 0.999 9), with the average recoveries being 100.8%, 99.84%, and RSD being 1.4%, 1.8% (n = 9) respectively. The sample solution was stable at room temperature within 72 h. The acute toxicity test showed no toxic reaction of D. aurantiacum eye drops in mice. The irritating test for single-dose and multiple-dose administrations of D. aurantiacum eye drops and physiological saline in rabbit eyes and skin, as well as the allergic test in guinea pigs showed no eye irritation and skin irritation and irritability. These findings indicated that D. aurantiacum eye drops are safe and stable, with a good druggability.

Transcriptomic analysis of Bombyx mori corpora allata with comparison to prothoracic glands in the final instar larvae.

The corpus allatum (CA) is an endocrine organ of insects that synthesizes juvenile hormone (JH). Yet little is known regarding the global gene expression profile for the CA, although JH signaling pathway has been well-studied in insects. Here, we report the availability of the transcriptome resource of the isolated CA from the final (fifth) instar larvae of the silkworm, Bombyx mori when the JH titer is low. We also compare it with prothoracic gland (PG) that produces the precursor of 20-hydroxyecdysone (20E), to find some common features in the JH and 20E related genes between the two organs. A total of 17,262 genes were generated using a combination of genome-guided assembly and annotation, in which 10,878 unigenes were enriched in 58 Gene Ontology terms, representing almost all expressed genes in the CA of the 5th instar larvae of B. mori. Transcriptome analysis confirmed that gene for Torso, the receptor of prothoracicotropic hormone (PTTH), is present in the PG but not in the CA. Transcriptome comparison and quantitative real time-PCR indicated that 11 genes related to JH biosynthesis and regulation and six genes for 20E are expressed in both the CA and PG, suggesting that the two organs may cross talk with each other through these genes. The temporal expression profiles of the two genes for the multifunctional neurohormonal factor sericotropin precursor and the uncharacterized protein LOC114249572, the most abundant in the CA and PG transcriptomes respectively, suggested that they might play important roles in the JH and 20E biosynthesis. The present work provides new insights into the CA and PG.

Molecular cloning, expression pattern and phylogenetic analysis of the will die slowly gene from the Chinese oak silkworm, Antheraea pernyi.

The will die slowly (wds) gene coding for a WD-repeat protein with seven repeats has been characterized in Drosophila melanogaster. In this paper, the wds gene was isolated and characterized from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae). The obtained 1733 bp cDNA sequence contains an open reading frame of 1041 bp encoding a polypeptide of 346 amino acids, with 85% sequence identity to that from D. melanogaster. RT-PCR analysis showed that the wds gene was transcribed during four developmental stages and in all the tissues tested, consistent with the result observed in Bombyx mori based on EST resources and genome-wide microarray information. The mRNA expression level of the A. pernyi wds gene was not significantly down- or up- regulated under temperature stress compared to the control, indicating that it may be not involved in temperature stress tolerance. In search of database, the wds protein homologues were found in various kinds of eukaryotes, including fungi, plants, invertebrates and vertebrates, with 50-93% amino acid sequence identities between them, suggesting that they are highly conserved during the evolution of eukaryotes. Phylogenetic analysis based on the wds protein homologue sequences clearly separated the known fungi, plants, invertebrates and vertebrates, consistent with the topology tree on the classical systematics, suggesting the potential value of wds protein in eukaryotic phylogenetic inference. In vertebrates, two apparent types of the wds proteins were also defined by sequence alignment and phylogenetic analysis.

Metabolomics Reveals the Mechanisms for the Pulmonary Toxicity of Siegesbeckia orientalis L. and the Toxicity-Reducing Effect of Processing.

Siegesbeckia orientalis L. (SO) is a commonly used Chinese medicinal herb. It has long been used as a remedy in traditional Chinese medicine (TCM) for symptoms that resemble inflammatory joint disorders. However, it is slightly toxic. According to the TCM theory, processing can reduce the toxicity of the herbs. Here, we performed metabolomics to determine whether processing with rice wine reduces the toxicity of raw SO, and to explore the mechanisms underlying the raw SO-induced toxicity and the toxicity-reducing effect of processing. Our results showed that raw SO has long-term toxicity in rats. It significantly elevated the serum level of LDH and caused histopathological damages in the lung tissues. It is worth noting that the LDH level in the PSO group was lower than that in the raw SO group, and the damages in lung tissues were relatively mild in PSO-treated rats, suggesting that processing reduces the pulmonary toxicity of the raw. Moreover, a total of 32 significantly changed metabolites were identified. Based on the MetaboAnalyst pathway analysis, we found that two characteristic metabolic pathways including alanine, aspartate and glutamate metabolism and glycerophospholipid metabolism were only changed in the raw SO group, while histidine metabolism was only changed in the PSO group, which suggests that induction of oxidative stress contributes to raw SO-induced pulmonary toxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing. Our data shed new light on how raw SO induces pulmonary toxicity and how the toxicity can be reduced by processing. This study not only provides scientific justifications for the traditional processing theory of SO, but also helps to optimize the processing protocol and the clinical drug combination of SO.

Paeoniflorin regulates the hypothalamic-pituitary-adrenal axis negative feedback in a rat model of post-traumatic stress disorder.

OBJECTIVES: To investigate the effects of paeoniflorin (PEF) on the hypothalamic-pituitary-adrenal (HPA) axis feedback function of post-traumatic stress disorder (PTSD). MATERIALS AND METHODS: Single-prolonged stress (SPS) was used to establish a PTSD-like rat model. The contents of plasma corticosterone (CORT), adrenocorticotropin hormone (ACTH) and corticotropin-releasing hormone (CRH) were measured by ELISA. Glucocorticoid receptor (GR), mineralocorticoid receptor (MR), adrenocorticotropic hormone-releasing factor I receptor (CRF1R), and adrenocorticotropic hormone-releasing factor II receptor (CRF2R) in the hippocampus and amygdala were measured by RT-PCR and immunohistochemistry. RESULTS: The results showed that on day 8 after SPS, model rats showed enhanced HPA axis negative feedback lasting to day 29. On day 29, plasma CORT levels increased in model rats, while plasma CRH levels had no significant difference on days 8, 22, and 29. The expression of GR and MR of model rats significantly increased in the hippocampus, while the expression of GR, MR, and CRF1R significantly decreased in the amygdala. After 14 days of continuous administration of PEF, the enhanced negative feedback was inhibited, and the plasma CORT level significantly reduced after 21 days of administration. Moreover, PEF could significantly decrease the expression of GR and MR in the hippocampus, and increase the expression of GR, MR, and CRF1R significantly in the amygdala. CONCLUSION: PEF could regulate HPA axis dysfunction in a rat model of PTSD, which may be related to regulating expression of GR and MR in the hippocampus and amygdala and regulating expression of CRF1R in the amygdala.

A high level of chloroplast genome sequence variability in the Sawtooth Oak Quercus acutissima.

The Sawtooth Oak, Quercus acutissima Carruth., is an economically and ecologically important tree species in the family Fagaceae with a wide distribution in China. Here, we examined its intraspecific chloroplast (cp) genome variability using available and a newly sequenced genome. The new cp genome comes from a Q. acutissima individual collected from Shenyang (Northeast China; "Q. acutissima Shenyang" in the following), and then is compared with two recently published cp genomes from Tongchuan (Northwest China) and Nanjing (East China). The cp genome of Q. acutissima Shenyang exhibits a slightly larger genome size than the other two individuals, although each encodes 86 protein-coding genes, 40 tRNA genes and eight rRNA genes. We also found the length difference for the IR/SC boundary region among the three cp genomes. Sequence comparison revealed a high intraspecific genetic divergence: the three cp genomes differ by 332 sequence patterns including 77 single nucleotide polymorphisms, and 255 indels (each gap considered) scattering across 67 regions. Phylogenetic analyses based on the cp genome recovered the split between the subgenus Cerris and the subgenus Quercus, but revealed that three Q. acutissima individuals did not cluster together, indicating that even complete cp genome data fail to reproduce species boundaries in Asian members of section Cerris. Our results show that more complete plastomes covering remote ranges needs to be sequenced to provide a solid backbone for future population-scale in-depth studies and phylogenetic analysis of section Cerris.

Characterization of the Adult Head Transcriptome and Identification of Migration and Olfaction Genes in the Oriental Armyworm Mythimna separate.

The oriental armyworm Mythimna separate is an economically important insect with a wide distribution and strong migratory activity. However, knowledge about the molecular mechanisms regulating the physiological and behavioural responses of the oriental armyworm is scarce. In the present study, we took a transcriptomic approach to characterize the gene network in the adult head of M. separate. The sequencing and de novo assembly yielded 63,499 transcripts, which were further assembled into 46,459 unigenes with an N50 of 1,153 bp. In the head transcriptome data, unigenes involved in the 'signal transduction mechanism' are the most abundant. In total, 937 signal transduction unigenes were assigned to 22 signalling pathways. The circadian clock, melanin synthesis, and non-receptor protein of olfactory gene families were then identified, and phylogenetic analyses were performed with these M. separate genes, the model insect Bombyx mori and other insects. Furthermore, 1,372 simple sequence repeats of 2-6 bp in unit length were identified. The transcriptome data represent a comprehensive molecular resource for the adult head of M. separate, and these identified genes can be valid targets for further gene function research to address the molecular mechanisms regulating the migratory and olfaction genes of the oriental armyworm.

Antidepressant-Like Effects of Fractions Prepared from Danzhi-Xiaoyao-San Decoction in Rats with Chronic Unpredictable Mild Stress: Effects on Hypothalamic-Pituitary-Adrenal Axis, Arginine Vasopressin, and Neurotransmitters.

The aim of the present study was to investigate the antidepressant-like effects of two fractions, including petroleum ether soluble fraction (Fraction A, FA) and water-EtOH soluble fraction (Fraction B, FB) prepared from the Danzhi-xiaoyao-san (DZXYS) by using chronic unpredictable mild stress-induced depressive rat model. The results indicated that DZXYS could ameliorate the depression-like behavior in chronic stress model of rats. The inhibition of hyperactivity of HPA axis and the modulation of monoamine and amino acid neurotransmitters in the hippocampus may be the important mechanisms underlying the action of DZXYS antidepressant-like effect in chronically stressed rats.

[Effects of Lycium barbarum polysaccharide on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice].

OBJECTIVE: To study the effects of Lycium barbarum polysaccharide (LBP) on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice and the mechanisms for intervention of tumor immune escape by LBP. METHODS: H22-bearing mice were given LBP orally for two weeks. T-lymphocyte subsets and the phenotypes of dendritic cells in tumor-infiltrating lymphocytes (TIL) were detected by flow cytometry (FCM). RESULTS: LBP could significantly increase the numbers of CD4(+) and CD8(+) T cells in TIL as compared with those in model control group (P<0.05). In model control group, the number of dendritic cells in tumor microenvironment decreased markedly, while in LBP-treated group, the increased number of dendritic cells and B7-1 expression were observed, but there were no significant differences between these two groups. CONCLUSION: LBP has anti-tumor effect probably by increasing the numbers of CD4(+) and CD8(+) T cells in TIL to relieve the immunosuppression and enhance the anti-tumor function of the immune system. But whether LBP can recover the phenocyte and function of dendritic cells in H22-bearing mice should be further studied.

Identification and Characterization of a Novel Microvitellogenin from the Chinese Oak Silkworm Antheraea pernyi.

Microvitellogenin (mVg) is a relatively small vitellogenic protein only characterized in the eggs of the lepidopteran insects Manduca sexta and Bombyx mori. In the present study, we report a novel mVg (ApmVg) isolated from the Chinese oak silkworm Antheraea pernyi. The obtained ApmVg cDNA sequence contains an open reading frame of 783 bp encoding a protein of 260 amino acids with a predicted molecular weight of 29.96 kDa. This gene does not contain introns. Structural analysis revealed that this protein shares putative conserved domains with the lepidopteran low-molecular weight lipoprotein, which belongs to the lipoprotein_11 superfamily. The protein sequence of ApmVg exhibits 48% sequence identity with mVg from M. sexta and 40-47% sequence identity with the 30K lipoproteins from B. mori. Phylogenetic analysis suggests that ApmVg is a novel member of the lepidopteran low-molecular weight lipoproteins. Transcriptional analysis indicated that ApmVg mRNA is mainly expressed in the fat body (both female and male) during post-diapause development of the pupal stage, and it was also detected in ovaries and spermaries in smaller amounts. RT-PCR and Western blot analyses revealed that ApmVg is synthesized by the fat body and secreted into hemolymph and ultimately accumulates in eggs. The ApmVg transcript can be detected in the fat bodies of female pupae four days after treatment with 20-hydroxyecdysone and shows an expression pattern distinct from that of vitellogenin (Vg), which is detectable throughout diapausing and in post-diapause development. ApmVg decreased dramatically during embryonic development. These results represent the first study of mVg outside M. sexta and B. mori and provide insight into the physiological role and evolution of mVgs.

The complete mitochondrial genome of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae).

The complete mitochondrial genome (mitogenome) of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae) was determined. The circular genome is 15,345 bp long, and presents a typical gene organization and order for sequenced mitogenomes of Bombycidea species. The nucleotide composition of the genome is highly A+T biased, accounting for 79.86%. The AT skew of the genome is slightly negative, indicating the occurrence of more Ts than As, as found in other Saturniidae species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI and COII, which are tentatively designated by CGA and GTG, respectively, as observed in other insects. Four of 13 PCGs, including COI, COII, ATP6, and ND3, harbor the incomplete termination codons, T or TA. With an exception for tRNASer(AGN), all other tRNAs can form a typical clover-leaf structure of mitochondrial tRNA. The 359 bp A+T-rich region of S. c. cynthia contains non-repetitive sequences, but harbors several features common to the Bombycidea insects, including the motif ATAGA followed by a poly-T stretch of 19 bp, a microsatellite-like (AT)7 element preceded by the ATTTA motif, and a poly-A element upstream tRNAMet. The phylogenetic analyses support the morphology-based current hypothesis that Bombycidae and Saturniidae are monophyletic. Our result confirms that Saturniini and Attacini form a reciprocal monophyletic group within Saturniidae.