Regulation of the kiss2 promoter in yellowtail clownfish (Amphiprion clarkii) by cortisol via GRE-dependent GR pathway.

Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of kiss gene. In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position -660 and -433 with GR1, and -912 and -775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position -573 in the presence of GR1 and by each GRE site located at position -883, -860, -851, and -843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.

Sexually dimorphic distribution of kiss1 and kiss2 in the brain of yellowtail clownfish, Amphiprion clarkii.

Kisspeptin system was shown to be a key factor in mediating social stress and reproduction. Yellowtail clownfish, Amphiprion clarkii, is a hermaphrodite fish, whose sex determination and gonadal development are affected by the social status of individuals. The yellowtail clownfish is a fantastic animal model to explore sex determination, but the social status and precise distribution of kiss mRNAs in the brain of this species are unknown. Hererin, a novel in situ hybridization technique, RNAscope, was used to investigate the distribution of kiss1 and kiss2 expressions in the brain of yellowtail clownfish. The coronal planes of brain showed that the kiss1 signal was mainly present in dorsal habenular nucleus (NHd) and kiss2 mRNA was widely expressed in telencephalon, midbrain, and hypothalamus, especially in dorsal part of the nucleus of the lateral recess (NRLd). Additionally, kiss1 and kiss2 signals have sexually dimorphic distribution. The kiss1 mRNA was distributed in NHd, the telencephalon, and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of females but in NHd and NDLIl of males. kiss2 signals were stronger in females than that in males. The distribution of kiss1 and kiss2 neurons in NHd of habenula and NRLd of hypothalamus may suggest that kiss genes associate environmental signaling and reproductive function in yellowtail clownfish.

Apoptosis induced by diallyl disulfide in human breast cancer cell line MCF-7.

AIM: To investigate the effect of diallyl disulfide (DADS), a component of garlic, on apoptosis in human mammary cancer cell line (MCF-7) and its mechanisms. METHODS: Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays. Morphology of apoptotic cells was detected by acridine orange and ethidium bromide staining. Apoptotic cells stained with propidium iodide were examined using flow cytometry. Protein levels were detected by Western blot analysis. RESULTS: DADS inhibited the proliferation of MCF-7 cells and induced the apoptotic ratio to increase rapidly. Cleavage of the caspase-3 and caspase-3 substrate poly(ADP-ribose) polymerase was observed in MCF-7 cells after 24 h of treatment with DADS. When the MCF-7 cells were treated with 200 micromol x L DADS, the stress-activated protein kinase extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase, was inhibited after 6 h; c-Jun N-terminal kinase (JNK), that is stress-activated protein kinase (SAPK), and p38 mitogen-activated protein kinase were activated after 6 h. CONCLUSION: These results suggest that DADS both inhibits the proliferation of MCF-7 cells and induces apoptosis of MCF-7 cells. The mechanisms may include the inhibition of ERK and the activation of the SAPK/JNK and p38 pathways.

[Efficacy and safety of Fufang Biejia Ruangan tablet in patients with chronic hepatitis B complicated with hepatic fibrosis].

OBJECTIVE: To study the clinical therapeutic effects and safety of Fufang Biejia Ruangan tablet (FBRt) in patients with chronic hepatitis B complicated with hepatic fibrosis. METHODS: Totally 420 patients were randomly divided into two groups, FBRt group (300 cases) were treated with Fufang Biejia Ruangan tablets and control group (120 cases) were treated with He Luo Shu Gan capsule, the patients in both groups were treated for 6 months. RESULTS: The cure rate and total effective rate of FBRt group were significantly higher than those of control group (55.67 percent and 81.67 percent vs. 15.8 percent and 60.00 percent, P less than 0.01). CONCLUSION: Fufang Biejia Ruangan tablet could alleviate clinical symptoms and hepatic fibrosis. Fufang Biejia Ruangan tablet is effective and safe in treatment of patients with chronic hepatitis B complicated with liver fibrosis.