| Objective To investigate the gene expression of hypothalamic Ghrelin/GHSRl-a involved in the relationship between trophic status and HPGA during puberty onset, female precocious rat models were induced by subcutaneous injection of Danazol; and the rat models were administrated with Nourishing "Yin"-Removing "Fire" Chinese herbal mixture to oberseve its influence on gene expression of hypothalamic Ghrelin and GHSR1-a..Method40female SD rats aged3days were divided randomly into normal group (N group), precocious puberty model rat group (M group), model rat group fed with Chinese herbal mixture (TCM group) and model rat group fed with physiological saline (MS group),10rats per group. All the rats except those in N group were subcutaneously injected with300μg Danazol to induce precocious puberty on postnatal day5(P5). From P15, animals in the TCM and MS were administrated with Chinese Herbal Mixture and physiological saline respectively everyday, and all rats were weighed every5days.The vulva opening indicated rat’s puberty onset, and the time to kill anmals was based on the vulva opening of rats in M group, so all the rat vulvas in four groups were observed everyday from P20. The same number of rats in each group of N, MS and TCM were killed at the same time provided the vulvas of the rats opened group and they were killed immediately in M group. Gene transcription of hypothalamic Ghrelin and GHSR1-α was detected by Real-time PCR, and the levels of sexual hormones, hormones related to trophic status in blood and hypothalamic GnRH, Ghrelin and GHSR1-a were measured by RIA. Comparisons of data were conducted with one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) test or Kruskal-Wallis test followed by Mann-Whitney U-test by SPSS13.0. Result (1) Comparisons of the data of indices about puberty onset: the vulvas of the rats in M and MS group had opened until P23(Between P21and P23), which were earlier than those in TCM and N. Levels of blood E2(19.97±2.62pg/ml), LH(16.96±1.31mIU/ml), hypothalamic GnRH (18.15±3.67pg/ml)and uterus organ index (49.71±5.50) in TCM group, which were close to those in N group, were statisticlly lower than those in M (24.67±4.05pg/ml,69.34±9.60,18.83±1.11mIU/ml,23.81±3.99pg/ml)and MS (24.11+2.52pg/ml,66.53±6.87,18.81±1.08mIU/ml,22.70+2.52pg/ml)(P<0.05), and the rats in TCM group possessed a smaller ovary organ index in contrast to the ones in MS and N group.(2) Analysis of data concerning trophic status: the weight of rats in M on P15was heavier than those in N group, and the rats in TCM group had lighter weight on P15, P20and P23compared with the ones in M group. The levels of blood Leptin, Ghrelin and IGF-1were6.74+0.83ng/ml,67.08±16.17ng/mg, and262.60±28.77ng/ml respectively, which were comparable with those in M group (7.10±1.05ng/ml,70.36±13.82ng/ml,250.04±31.97ng/ml) and MS group (7.13±1.17ng/ml,82.70±16.56ng/ml,256.62±31.84ng/ml)(P>0.05), and there was no statistical correlation between the indices concerning puberty onset and the indices related to trophic status in every group.(3) Comparisons of the data on the hypothalamic Ghrelin and GHSRl-α: there was a decline in the levels of hypothalamic Ghrelin and GHSR1-αmRNA in M (0.82±0.03,0.91±0.05) group and MS (0.87±0.03,0.88±0.03) group in comparison to those in the N group (1.00±0.02,1.00±0.05)(P<0.01), however, the levels of hypothalamic Ghrelin and GHSRl-αmRNA in TCM group(0.96±0.05,1.00±0.02) were statistically higher than those in M group and MS group(P<0.0l). No statistical discrepancy existed in the levels of hypothalamic Ghrelin and GHSR1-a (numbers of active GHSR1-a and its affinity with Ghrelin)among four groups.Conclusion The rats exposed to Danazol on postnatal day5exhibited an increase in gene transcription of hypothalamic Ghrelin and GHSRl-a at the time of puberty onset. Chinese Herbs could up-regulate the gene transcription of hypothalamic Ghrelin and GHSR1-α, which might be an approach to delay the puberty onset. |
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