Synergistic effects of dietary oxolinic acid combined with oxytetracycline on nonspecific immune responses and resistance against Vibrio parahaemolyticus infection of white shrimp (Penaeus vannamei).

This study investigated the synergistic effects of oxolinic acid (OA) combined with oxytetracycline (OTC) on white shrimp (Penaeus vannamei). Disk diffusion susceptibility testing was performed to analyze the sensitivity of Vibrio alginolyticus and Vibrio parahaemolyticus to different concentrations of OA and OTC. The results revealed that 50 mg OA/L combined with 50 mg OTC/L exhibited stronger antibacterial effects on V. alginolyticus and V. parahaemolyticus. The results of in vitro tests indicated that cotreatment with OA and OTC significantly reduced superoxide anion production and phenoloxidase activity, but not phagocytic activity. Subsequently, feeding trials were performed to investigate the immunomodulatory effects and bioaccumulation of dietary OA combined with OTC on shrimp. The healthy shrimp (15.13 ± 1.02 g) were divided into four groups: control, 100 mg OA/kg combined with 50 mg OTC/kg, 50 mg OA/kg combined with 100 mg OTC/kg, and 50 mg OA/kg combined with 50 mg OTC/kg. The shrimp were sampled to determine innate immunity parameters and residual OA and OTC levels in the muscle during a 28-day feeding regimen; the shrimp were fed the experimental diet from day 1 to day 5 and a commercial diet from day 6 to day 28. Residual OA levels were considerably higher in the group fed 100 mg OA/kg combined with 50 mg OTC/kg compared with the other groups and peaked on day 4. The residual OA levels of all the groups were below the detection limit after without providing OA. The residual OTC levels of the group fed 50 mg OA/kg combined with 100 mg OTC/kg were considerably higher from day 1 to day 4. The residual OTC levels in all the groups decreased rapidly and could not be detected on day 28. The administration of 50 mg OA/kg combined with 100 mg OTC/kg exerted the least effect on the white shrimp. Moreover, the survival rates of the treatment groups after the V. parahaemolyticus challenge were higher than those of the control group, especially the group fed 50 mg OA/kg combined with 100 mg OTC/kg. This result indicated that the synergistic effects of dietary OA and OTC are safe and effective. Combination therapy is a new method of antibiotic use in aquaculture.

Stem Cells-Loaded 3D-Printed Scaffolds for the Reconstruction of Alveolar Cleft.

The advances in the field of tissue engineering and regenerative medicine have opened new vistas for the repair of alveolar clefts. However, the currently available biomaterials used for the repair of alveolar clefts have poor mechanical properties and biocompatibility, which hinders the treatment outcomes. Here, we aimed to develop 3D printed biomimetic scaffolds that fuses ß-tricalcium phosphate (ß-TCP) and bone marrow mesenchymal stem cells (BMSCs) for improving the repair of alveolar clefts. The methacrylate gelatin (GelMA) was mixed with ß-TCP for the preparation of GelMA/ß-TCP hybrid scaffolds via 3D printing platform and chemically cross-linking with UV light. The physicochemical properties of the hydrogel scaffolds were characterized. Moreover, the survival state, proliferation ability, morphological characteristics, and osteogenic induction of BMSCs were examined. The prepared hybrid scaffolds showed good biocompatibility and mechanical properties. BMSCs attached well to the scaffolds and proliferated, survived, differentiated, and stimulated osteogenesis for the reconstruction of alveolar clefts. We expect that use of the prepared hybrid hydrogel scaffold can improve the outcomes of alveolar cleft repair in clinic and expand the application of hybrid hydrogel in tissue engineering repair.

Bioaccumulation of arsenic and immunotoxic effect in white shrimp (Penaeus vannamei) exposed to trivalent arsenic.

Trivalent arsenic (As (III)) contamination in the marine environment can produce adverse effects in crustaceans. The present study investigated the chronic toxicity of As (III) in white shrimp (Penaeus vannamei) by analyzing the tissue bioaccumulation and non-specific immune responses. Shrimps were exposed to 0 (control), 50, 500, and 2500 µg/L of As (III) for 21 days. The results showed that the hepatopancreas was the main tissue of arsenic accumulation in white shrimp. The cumulative concentration of total arsenic and inorganic arsenic but not arsenobetaine was positively correlated with the exposure concentration. In vitro As (III) treatment (0-2500 µg/L) with haemocytes isolated from healthy shrimp did not cause the cytotoxicity, but this arsenic treatments inhibited the phagocytic rate and O2- production. Moreover, the decrease of total haemocyte count and the inhibition of phagocytic rate, phagocytic index, O2- production and phenoloxidase activity were observed in white shrimp under the exposure of As (III) over a period of 21 days. This study revealed that chronic As (III) stress could disturb arsenic metabolism and immune responses in P. vannamei.

Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus.

Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2-) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10-10,000 µg/L to determine cell viability, phagocytic activity, and O2- production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2- production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2- production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2- production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2- production.

Co-Ni layered double hydroxides wrapped on leaf-shaped copper oxide hybrids for non-enzymatic detection of glucose.

The leaf-shaped copper oxide (CuO) was first fabricated by a direct precipitation method. Then, the flake-shaped Co-Ni layered double hydroxides (CoNi-LDHs) were grown on the leaf-shaped CuO by using an electrodeposition method to form a unique nanostructured electrocatalyst. Due to the unique structural and compositional advantages, CoNi-LDHs wrapped around the leaf-shaped CuO exhibited good electrocatalytic characteristics. The optimized CuO/CoNi-LDHs exhibited improved non-enzymatic electrochemical sensing performance for glucose, with a reliable linear range (0.1 µM-0.384 mM) and a low limit of detection (0.065 µM, S/N = 3). The utilization of the CuO/CoNi-LDHs sensor for glucose detection in human serum was also corroborated, implying promising potential for glucose monitoring. Results demonstrated that the developed sensor provides new horizons for facile and sensitive glucose detection.

Moringa oleifera Leaves' Extract Enhances Nonspecific Immune Responses, Resistance against Vibrio alginolyticus, and Growth in Whiteleg Shrimp (Penaeus vannamei).

Moringa is widely known as a plant with high medicinal properties. Therefore, moringa has a high potential for use as an immunostimulant in shrimp. This study investigated the effect of a moringa water extract on the immune response, resistance against V. alginolyticus, and growth performance of whiteleg shrimp. To perform the in vitro assay, hemocytes were incubated with different concentrations of the moringa extract. Furthermore, the moringa extract was incorporated at 0 (control), 1.25 g (ME1.25), 2.5 g (ME2.5), and 5.0 g (ME5.0) per kg of diet for the in vivo assay. During the rearing period, immune responses, namely the total hemocyte count (THC), phenoloxidase (PO) activity, phagocytosis activity, superoxide anion production, and immune-related gene expression were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Growth performance was measured 60 days after the feeding period. Furthermore, the shrimp were challenged with V. alginolyticus after being fed for different feeding durations. The results of the in vitro assay revealed that 100-250 ppm of the moringa extract enhanced the PO activity, phagocytic rate (PR), and superoxide anion production. The findings of the in vivo assay demonstrated that the THC, PO activity, PR, and immune-related gene expression, including alpha-2-macroglobulin, prophenoloxidase II, penaeidin2, penaeidin3, anti-lipopolysaccharide factor, crustin, lysozyme, superoxide dismutase, and clotting protein, were higher in the group of ME.25 and ME5.0 than in the control and ME1.25 at several time points. Growth performance was significantly increased (p < 0.05) in the ME2.5 group compared to the control group. Furthermore, the dietary ME2.5 resulted in a higher survival rate compared to that of the control group after challenging with V. alginolyticus, especially at ME2.5 administered for 4 and 7 days. This study indicated that the incorporation of the moringa extract at 2.5 g per kg of diet enhanced the immune response, the growth performance of the whiteleg shrimp, and the resistance against V. alginolyticus infection.

Elevated estrogen receptor expression in hypothalamic preoptic area decreased by electroacupuncture in ovariectomized rats.

In the present study, effects of electroacupuncture (EA) on estrogen receptor alpha (ERα) and beta (ERß) mRNA and protein expression in the hypothalamus of ovariectomized (OVX) rats were detected by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot analysis. Gonadotropin-releasing hormone (GnRH) release and GnRH mRNA level in hypothalamic preoptic area (POA) were evaluated by push-pull perfusion and qRT-PCR. Our results showed that elevated mRNA and protein expression of ERα and ERß in hypothalamus were restrained following EA treatment in OVX rats. EA treatment also inhibited GnRH release and GnRH mRNA levels in OVX rats. These results provide novel evidence that EA treatment down regulates the expression of hypothalamic estrogen receptors (ERs), thus restores the response of GnRH neurons to estrogen depression, and partially resets the negative feedback of estrogen to hypothalamus-pituitary-ovary axis (HPOA) in OVX rats, which may be a critical mechanism for EA on female reproductive disorders.

[Effects of electroacupuncture on the expression of corticotropin-releasing factor (CRF) family and CRF receptor 1 in traumatic injury rats].

OBJECTIVE: To observe the effects of electroacupunture (EA) on the abnormal hypothalamus-pituitary-adrenal (HPA) axis and the expression of hypothalamic corticotropin-releasing factor (CRF) peptides family and CRF receptor (R)1 mRNA in traumatized rats, so as to study its underlying mechanism in improving traumatic injury. METHODS: Forty male Sprague-Dawley rats were randomly divided into normal control group (n=10), normal+ EA group (n=10), surgical trauma (model) group (n=10), and model+ EA group (n=10). Traumatic injury model was established by performing an exploratory laparotomy in the rats under anesthesia. EA (2 Hz/15 Hz, 1-2 mA) was administered for 30 min via two stainless steel needles inserted in "Zusanli" (ST 36) and "Sanyinjiao" (SP 6). Serum adrenocorticotropic hormone (ACTH) , corticosterone (Cort), luteinizing hormone (LH) and testosterone (T) contents were detected by double-antibody radioimmnoassay. Hypothalamic CRF and ORF R1 mRNA expression was assayed by reversed transcription polymerase chain reaction (RT-PCR) technique. RESULTS: The serum ACTH concentration decreased significantly in model group (P < 0.05) compared with those in normal control and normal+ EA groups, and was significantly higher in model+ EA group than that in model group (P < 0.05). No significant difference was found between normal control and normal+ EA groups in serum ACTH levels (P > 0.05). The serum Cort level increased significantly in model group (P < 0.05) compared with those in normal control and normal + EA groups, and was obviously lower in model+EA group than that in model group (P < 0.05). While serum LH and T contents had no apparent changes in the four groups (P > 0.05). The expression of hypothalamic CRF mRNA decreased significantly in model group (P < 0.05) compared with that in normal control group, but increased significantly in model + EA group (P < 0.05) compared with that in model group. However, no significant difference was found between normal control and normal + EA groups in the CRF mRNA expression level. The expression of hypothalamic Ucn 1 mRNA increased significantly in model group than that in normal control group (P < 0.05), and decreased considerably in model+ EA group than that in model group (P < 0.05). There were no significant changes in the expression of hyp, thalamic Ucn 2 mRNA, Ucn 3 mRNA and CRF R1 mRNA in the four groups (P > 0.05). CONCLUSION: EA of "Zusanli" (ST 36) and "Sanyinjiao"(SP 6) can improve surgical trauma-induced HPA disorders, which is probably related to its effects in upregulating CRF mRNA expression and downregulating Ucn 1 mRNA expression in the hypothalamus.

Effect of nourishing "Yin"-removing "Fire" Chinese herbal mixture on hypothalamic kisspeptin expression in female precocious rats.

AIM: The present study aims to investigate the effect of nourishing "Yin"-removing "Fire" herbal mixture, a Chinese herb-based formulation, on hypothalamic kisspeptin expression in danazol-induced female precocious model rats. MATERIALS AND METHODS: The female Sprague-Dawley rats were divided into intact normal (N), central precocious puberty (CPP) model (M), vehicle without CPP (V), CPP model exposed to herbal mixture (HM) and CPP model exposed to saline (S) groups. At postnatal day 5, a single subcutaneous injection of 300 microg of danazol was administered to induce CPP model rats. From P15, rats in the HM group were continuously gavaged with the 1 ml/50 g body weight mixture, until two consecutive regular estrous cycles were established. The hypothalamic Kiss-1 expression was detected by RT-PCR and immunohistochemistry. RESULTS: The day of vaginal opening and establishment of two regular estrous cycles were delayed in the HM group compared with M and S groups (P<0.05, respectively). The level of hypothalamic Kiss-1 mRNA and the number of kisspeptin-immunoreactive (kisspeptin-ir) cells in the arcuate nucleus (ARC), preoptic area (POA) and periventricular nucleus (PeN), were decreased significantly in the HM group compared with the M and S groups (P<0.01, respectively) on the day of onset-puberty. CONCLUSION: These results indicate that the kisspeptin signaling pathway might be involved in the effect of herbal mixture treatment on CPP.

[Effects of electro-acupuncture on signal transduction pathway of hypothalamic neuroendocrine system in ovariectomized rats].

OBJECTIVE: To compare the varieties and contents of the main nerval information molecules in perfusate from hypothalamic medial preoptic area (MPOA) of the rats in different sexual cycles and the ovariectomized rats treated by electro-acupuncture, so as to observe the similarities and differences of hypothalamic neuroendocrine signal transduction pathway under the physiological and pathological status, and to explore the mechanisms of neuroendocrine signal transduction of electro-acupuncture therapeutic effect in perimenopausal syndrome. METHODS: The stereo localization technique and push-and-pull perfusion of the rat brain nucleus were adopted for collecting the hypothalamic MPOA perfusate of the female rats with normal sexual cycle, and also for collecting the MPOA perfusate of ovariectomized rats after electro-acupuncture treatment as acupuncture perfusate (AP). After being respectively microinjected into MPOA of the ovariectomized rats, the influence of the different perfusates on vagina cytology and serum estradiol (E2) level was observed. The contents of gonadotropin-releasing hormone (GnRH), dopamine (DA), gamma-aminobutyric acid (GABA), glutamate (Glu), aspartate (Asp) and beta-endorphin (beta-EP) in the perfusate of each group were detected by radioimmunoassay or high performance liquid chromatography, and then the varieties and contents of these substances in the perfusate of each group were compared and analyzed. RESULTS: The contents of neural active substances including DA, GABA, Glu, and beta-EP in the perfusate from the rats' MPOA during different stages of sexual cycle showed some regular changes. After the perfusate was microinjected respectively into the MPOA of the ovariectomized rats, the changes of animal vaginal exfoliated cells and serum E2 level showed the similar four-stage cycle characteristics as normal rats; the changes of vaginal exfoliated cells and serum E2 level of the ovariectomized rats without electro-acupuncture treatment showed the acupuncture-like effects following the microinjection of AP in the MPOA. CONCLUSION: The information molecules in the perfusate from MPOA of rats in normal sexual cycle or electro-acupuncture-treated rats include classical neurotransmitters, amino acid neurotransmitters, and neuropeptides. Acupuncture may modulate the release and synthesis of these information molecules to normalize the subnormal function of MPOA in perimenopausal period. Thus, AP may be a neuroendocrine signal carrier of acupuncture effects for acupuncture signal transduction from one individual to another. This may provide a new thinking and method for studying acupuncture signal transduction of the acupuncture therapeutic effects on perimenopausal syndrome.

Neurochemical Mechanism of Electroacupuncture: Anti-injury Effect on Cerebral Function after Focal Cerebral Ischemia in Rats.

We explored the neurochemical mechanism of electroacupuncture's (EA) protective effect on brain function in focal cerebral ischemia rats, using cerebral ischemia/reperfusion rats established by the middle cerebral artery occlusion (MCAO) method. Adult male Sprague-Dawley rats were randomly divided into four groups: Sham, Sham+EA, MCAO and MCAO+EA. The rats in Sham+EA and MCAO+EA were accepted EA treatment at 'GV26' and 'GV20' acupoints for 30 min. Electric stimulation was produced by a G-6805 generator and neurological deficit scores were recorded. Mitochondria respiratory function and the activities of respiratory enzymes were measured by a computer-aided Clark oxygen electrode system. Results showed that EA treatment might reduce the neurological deficit score, and significantly improve respiratory control ratio (RCR), the index of mitochondrial respiratory function, and increase the activities of succinic dehydrogenase, NADH dehydrogenase and cytochrome C oxidase in the MCAO rats. Results suggest that EA might markedly decrease the neurological deficit score, promote the activities of respiratory enzymes and reduce the generation of reactive oxygen species (ROS), resulting in improvement of respiratory chain function and anti-oxidative capability of brain tissues in the infarct penumbra zone. This be a mechanism of EA's anti-injury effect on brain function in MCAO rats.

[Observation on the central afferent pathway of "Guanyuan" (CV 4) under normal and pathological states and the influence of electroacupuncture].

OBJECTIVE: To investigate the afferent pathways of "Guanyuan" (CV 4) under normal and pathological states and to observe the effect of electroacupuncture (EA) on them. METHODS: Twenty-four female SD rats were randomly divided into normal control (NC), NC + EA, ovariectomy (OVX) and OVX + EA groups, with 6 cases in each. Four weeks after OVX, Pseudorabies Virus (PRV, 10 microl liquid containing 10(8) plaque-forming units) was injected into CV4. Thirty minutes after PRV injection, EA (2 Hz, 2-3 mA) was applied to CV4 for 30 minutes, once daily for 3 days. Six hours after the third EA treatment, all the rats were perfused transcardially with 4% paraformaldehyde under anesthesia, followed by sampling the brain and spinal cord and cutting into sections (35 microm). Immunohistochemical method (rabbit anti-rat beta-galactosidase) was used to display PRV immunoreactive (PRV-IR) positive neurons. RESULTS: 1) After injection of PRV, PRV-IR positive cells widely distributed in the spinal cord (cervical, thoracic and lumbar segments), brain stem (nucleus of solitary tract, cuneate nucleus, gigantocellular reticular nucleus, nucleus of spinal tract of trigeminal nerve, nucleus raphe magnus, locus caeruleus, etc), hypothalamus and cerebral cortex in each group. 2) After OVX, apparently fewer PRV-IR positive cells were found in some nuclei as medial septum nucleus (MSN), arcuate nucleus (ARC), diagonal band nucleus (DBN), paraventricular nucleus (PVN) which have close relation with endocrine activity (P<0.05); and rarely seen in ventromedial hypothalamus (VMH) and lateral preoptic area (LPO) (P<0.01). But in OVX + EA group, PRV-IR positive cells in above regions or nuclei increased significantly (P<0.05 or P<0.01). No obvious difference was found between NC and NC+ EA groups in the number of PRV-IR positive cells (P>0.05). CONCLUSION: Following OVX, PRV-IR positive cells in neuroendocrine-related nuclei of hypothalamus reduced significantly in number; EA of "Guanyuan" (CV 4) can promote the recovery of these neurons in above mentioned nuclei.

Role of hypothalamus nociceptin/orphanin FQ in pre-ovulatory luteinizing hormone surge of estrogen and progesterone-primed, ovariectomized rats.

AIM: To investigate the role of hypothalamus nociceptin/orphanin FQ (OFQ) and its endogenous receptor, the opioid receptor-like1 receptor (ORL1 receptor) in the estrus cycle of female rats. METHOD: Radioimmunoassay was used to detect the effect of the intracerebroventricular (icv) administration of OFQ and/or the ORL1 receptor antagonist [Nphe1]Nociceptin(1-13)NH2, that is, NC13 on luteinizing hormone (LH) levels of estrogen- and progesterone (EBP)-primed, ovariectomized (OVX) rats (EBP-primed OVX rats). RT-PCR, Western blotting, and immunohistochemistry techniques were adopted to observe the changes of OFQ and the ORL1 receptor in the pre-optic area (POA) and the medial basal hypothalamus (MBH) of the estrus cycle of female rat. RESULTS: Pre-ovulatory LH surges in EBP-primed, OVX rats were significantly reduced by icv administration of 20 and 200 nmol OFQ (P<0.05), and the effect of 20 nmol OFQ could be abolished by pretreatment with 20 nmol NC13. The OFQ mRNA level in the POA on pro-estrus was lowered markedly compared to diestrus and estrus (P<0.05), while the mRNA and protein levels of the ORL1 receptor showed no significant changes in the POA and MBH across the estrus cycle. Meanwhile, the number of OFQ-immunoreactive neurons in the medial POA, ventromedial hypothalamus, and the arcuate nucleus on pro-estrus was significantly decreased compared to diestrus and estrus (P<0.05). CONCLUSION: The inhibitory effect of OFQ on the LH surge of EBP-primed, OVX rats and its downregulation in POA and MBH on pro-estrus suggests that it might play a negative modulatory role in the estrus cycle.

Expression of brain prolactin releasing peptide (PrRP) changes in the estrous cycle of female rats.

Prolactin releasing peptide (PrRP) is a neuropeptide with 31 or 20 amino acid residues and regarded as a potent and specific stimulator of pituitary prolactin. PrRP immunoreactive (PrRP-ir) neurons and mRNA are found in medulla oblongata and hypothalamus and the fibers containing PrRP are widely distributed in rat brains. Therefore, it is postulated that PrRP might act as a neurohormone or a neurotransmitter as well as a neuromodulator in the brain. In the present study, we probed the expression of brain PrRP in the estrous cycle of female rats and the relationship between brain PrRP and GnRH. Female rats were divided into four groups: the diestrus, the proestrus, the estrus and the metaestrus, which were identified by the vaginal cytological examination. Immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescent double labeling histochemistry combining confocal laser scanning microscope (CLSM) were used. The results showed that PrRP immunoreactive neurons in nucleus of solitary tract (NTS) and ventrolateral reticular nucleus (VLRN) in the proestrus were less than those in the diestrus, the estrus and the metaestrus. Similarly, the relative optical density of PrRP-ir fibers of the bed nucleus of stria terminalis (BST) in the proestrus was decreased compared with those in other three groups. However, the brain PrRPmRNA level was higher in the proestrus and estrus than those in the metaestrus and diestrus. We also observed the co-localization of GPR10-immunoreactive (GPR10-ir) and GnRH-immunoreactive (GnRH-ir) neurons in hypothalamic medial preoptic area (MPO). The present results provide morphological evidences that PrRP in the female rat brains might participate in the regulation of the rat estrous cycle at least in a direct way.

Characteristic of hypothalamic kisspeptin expression in the pubertal development of precocious female rats.

To investigate the potential role of kisspeptin in the advance onset of puberty in precocious puberty, model rats induced by danazol were used to study the developmental expression of hypothalamic kisspeptin. Kisspeptin immunoreactive cells were observed in the arcuate nucleus (ARC), periventricular nucleus (PeN) and preoptic area (POA) in model rats on the day of onset-puberty. On the day of post-puberty, however, the number of kisspeptin immunoreactive cells in ARC and PeN decreased while the number of those cells in POA increased. Kisspeptin immunoreactive cells were not detected in hypothalamus in both normal and model rats at their pre-puberty stages. Furthermore, the expression of hypothalamic Kiss-1 mRNA reached top on the day of onset-puberty in both of the normal and model rats, and the expression of Kiss-1 mRNA increased significantly in the model rats compared with those in the normal ones. Our results indicated that kisspeptin might involve in the advance onset of puberty in danazol induced female precocious model rats.

Electroacupuncture stimulates the expression of prolactin-releasing peptide (PrRP) in the medulla oblongata of ovariectomized rats.

Electroacupuncture (EA) in reproductive medicine has become established in Western medicine as a therapy over the last decade. EA performs a variety of neuromodulatory functions in the central nervous system (CNS). Prolactin-releasing peptide (PrRP) is a neuropeptide identified as an endogenous ligand for the orphan G protein-coupled receptor hGR3. PrRP can affect the function of hypothalamus-pituitary-ovary axis (HPOA) and hypothalamus-pituitary-adrenal axis (HPAA). The present study was undertaken to characterize the effect of EA on the expression of PrRP in the medulla oblongata in ovariectomized (OVX) rats by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). In addition, estrogen (E2) levels were detected by radioimmunoassay (RIA). The results suggest that EA significantly increase the blood level of E2 and the expression of PrRP in the medulla oblongata of OVX rats. The number of PrRP immunoreactive (ir) neurons was higher in the group ovariectomized with EA than that in the OVX group. The numbers of PrRP-ir neurons in intact (INT) and intact with EA (INT+EA) were not significantly different between the two groups. The expression of PrRP mRNA was increased in the OVX+EA group than that in the OVX group. These results suggest that the mechanism that EA improved reproductive disorders induced by ovariectomy in rats is related to the modulation of the blood E2 level and the expression of PrRP in the medulla oblongata.

[Effects of Chinese herbal medicine on modulating the course of puberty development in children with precocious puberty].

OBJECTIVE: To explore the therapeutic effects of Ziyin Xiehuo Recipe (ZYXHR) for nourishing yin and lowering fire and Yishen Tianjing Recipe (YSTJR) for nourishing kidney and replenishing essence on regulating the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis, and to reveal the mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development of children with precocious puberty. METHODS: The pubertal rats were fed with ZYXHR or YSTJR for 30 days, and the parameters of rats were monitored as the followings: The content of gonadotropin-releasing hormone (GnRH), the frequency and amplitude of GnRH impulse releasing, the releasing amounts of aminoacid neurotransmitters, and neuropeptide Y (NPY) and beta-endorphin (beta-END) in the gonadotrophic area of the hypothalamus were detected with neurobiological methods (push-pull perfusion, homogenate, incubation of brain slices, and immunohistochemical staining). The levels of gene and protein expressions of GnRH, growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and follicular stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) in adenohypophysis as well as insulin-like growth factor I (IGF-I) in metaphysis were determined with real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The ZYXHR could reduce the activity of GnRH neurons in hypothalamus through inhibiting the release of central exciting aminoacid neurotransmitters, whereas promoting the release of central inhibiting aminoacid neurotransmitters, NPY and beta-END in gonadotrophic area of hypothalamus. The expression levels of GnRH, FSH and LH mRNAs were down-regulated while the expression level of SS mRNA in hypothalamus was up-regulated in the ZYXHR-treated group. The GH mRNA in hypophysis and the IGF-I mRNA in metaphysis were both down-regulated by ZYXHR. The YSTJR could promote the activity of GnRH neurons in hypothalamus through inhibiting the release of NPY in gonadotrophic area of hypothalamus, up-regulating the expression levels of GnRH, FSH, LH and GH mRNAs in hypophysis, and IGF-I mRNA in metaphysis, while down-regulating the expression level of SS mRNA in hypothalamus. CONCLUSION: The ZYXHR and YSTJR could both regulate the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis through modulating the neuroendocrine regulation and the gene expressions of GnRH and SS in hypothalamus, GH, FSH and LH in hypophysis, and IGF-I in metaphysis. These may be the chief mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development and ameliorating the skeletal development in children with precocious puberty.

Involvement of nociceptin/orphanin FQ in release of hypothalamic GnRH mediated by ORL1 receptor in ovariectomized rats.

AIM: To investigate effect of the nociceptin/orphanin FQ (OFQ) on hypothalamus gonadotropin-releasing hormone (GnRH) release in ovariectomized (OVX) rats. METHODS: GnRH radioimmunoassay (RIA) was used to study the effect of OFQ on GnRH release in hypothalamus slices in vitro. Push-pull perfusion and intracerebroventicular (icv) injection were used to examine the effect of OFQ on GnRH release in the hypothalamus medial preoptic area (POA) in vivo. Ovariectomies were performed on female Sprague-Dawley rats, and their plasma luteinizing hormone (LH) levels were measured after icv injection of OFQ with or without [Nphe1]NC(1-13)NH2, a competitive antagonist of opioid receptor-like1 receptor (ORL1 receptor). Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the expression of the ORL1 receptor in rat pituitary. RESULTS: GnRH release from hypothalamus slices was inhibited 90 min after the administration of 2 mmol/L and 20 mmol/L OFQ (P<0.05). Accordingly, GnRH release from hypothalamus POA was also significantly reduced by the injection of 0.2 mmol/L and 2 mmol/L OFQ. Plasma LH levels were also decreased significantly 2 h after icv injection of 20 nmol OFQ in OVX rats (P<0.05) and this effect could be abolished by pretreatment with 20 nmol [Nphe1]NC(1-13)NH2, that is, NC13. More interestingly, plasma LH levels in OVX rats increased markedly 2 h after icv injection of 100 nmol and 200 nmol NC13. RT-PCR analysis further revealed that the ORL1 receptor was not expressed in the pituitary of OVX rats. CONCLUSION: Central administration of nociceptin/orphanin FQ might inhibit the release of hypothalamic GnRH and decrease the plasma LH levels through ORL1 receptors in OVX rats.

Evaluation of the true precocious puberty rats induced by neonatal administration of Danazol: therapeutic effects of nourishing "Yin"-Removing "Fire" Chinese herb mixture.

BACKGROUND: Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture, a traditional herb-based formulation, has been successfully used for the management of idiopathic true precocious puberty (IPP) for more than thirty years. Precocious puberty rat model by neonatal administration of Danazol was used to investigate the effects of the herb mixture on the advanced sexual development of the rats, and the expression of hypothalamic gonadotropin-releasing hormone (GnRH), which is the important regulator for the hypothalamus-pituitary-gonadal axis, particularly at puberty. METHODS: Female Sprague-Dawley rats were divided into five groups: intact normal (N), IPP model (M), vehicle with no IPP (V), IPP model exposed to herb mixture (HM) and IPP model exposed to saline (S). Rats at 5 days of age were given a single subcutaneous injection of 300 microgram of Danazol dissolved in 25 microliter vehicle of propylene glycol-ethanol (1:1, v/v), to establish the precocious puberty model. From the day 15, rats in HM and S groups were continuously fed with either Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture 2 ml or saline 2 ml, until 3 consecutive regular estrous cycles were established. The day of vaginal opening and the day of setup regular estrous cycle of the rats were observed. Blood concentration of estrogen was determined by radioimmunoassay. Immunohistochemistry and RT-PCR analysis were used to explore the expression of GnRH. RESULTS: The day of vaginal opening and first estrous showed significant advancement in M compared with N and V (p < 0.05, respectively). The blood estrogen level increased significantly in M compared with those in other groups (about 28 days of age, at the time of vaginal opening in M rats) (p < 0.05, respectively). GnRH cells in rostral medial septum (MS), Broca diagonal band nucleus (DBB) and the medial preoptic area (MPOA), were calculated. The number in M was less than those in N and V (p < 0.05, respectively). The number was significantly higher in HM than that in M (p < 0.05). The GnRH mRNA expression increased significantly in M compared with that in N and V (p < 0.05). CONCLUSION: The true precocious puberty model by neonatal administration of Danazol in female rats showed augmented expression of hypothalamic GnRH; the Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture down-regulated the increased GnRH expression, and significantly delayed the sexual development of the precocious puberty rat.

Circulating estradiol and hypothalamic corticotrophin releasing hormone enhances along with time after ovariectomy in rats: effects of electroacupuncture.

Repeated electroacupuncture (EA) stimulation is known to enhance the circulating level of estrogen in the ovariectomized rats by stimulating the activity of the hypothalamus-pituitary-adrenal axis. To explore the mechanism of EA in promoting the homeostasis in ovariectomized rats, the hypothalamic CRH content and its mRNA expression as well as the circulating estradiol in the rats along with time after ovariectomy were detected. Female rats were divided into fourteen groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M; intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M; OVX1M with EA (OVX1M+EA) and intact 1 month with EA (INT1M+EA). The blood concentrations of estradiol (E2) and total contents of hypothalamic CRH were measured by radioimmunoassay. The expressions of CRHmRNA were semiquantified by RT-PCR. The results showed that the blood E(2) concentrations increased gradually along with time after ovariectomy in the rats. The level was higher in OVX1M+EA than those in OVX1M, 2M, 3M (p < 0.05), which was near to the level in OVX6M. The total content (ng/g tissue) of hypothalamic CRH and the CRHmRNA expressions in the OVX6M rats were higher than other groups (p < 0.05). The CRH content or mRNA expression in the OVX1M+EA increased significantly compared with that in OVX1M (p < 0.05), and it was close to the level in the OVX4M and OVX5M. The results suggested that the circulating E2 and hypothalamic CRH enhances along with time after ovariectomy in rats, and the EA stimulation might speedup the process.