| Objective:To explore the effects of female rats continusely exposed to light on reproductive endocrine rhythms and the functions of Chinese medicine with sequential interference by nourishing yin and invigorating yang.Methods:Select 32 rats of 3 weeks weaning female SD(continuous artificial illumination model rats). Randomly divided them into blank group, model group, and Chinese medicine group.3 groups of rats were observed, early sexual maturity, continuous cell detecting exfoliated vaginal. Recording the estrous cycle, continuous light for 30 days, in proestrus at 2:00,14:00 was the opening test records of behavioral data, at the same time, orbital blood detection of melatonin and sexual hormone. Light after 3 months, proestrus 2:00 a.m. to record again opening test, orbital blood, detection of melatonin,14:00 were guillotined from the carotid blood detection of sex hormone, melatonin. Operation the removal of ovaries rats, HE staining, expression profiling, according to gene expression spectrum detection results for RT-PCR and immunohistochemical identification of related factor gene expression. Results:the light early models of rats appeared to extend the early maturation, estrus, prolonged diestrus, gradually appeared irregular cycle, the circadian melatonin levels were decreased to 14 in the afternoon, the obvious differences, there is statistical significance; T, PRL, LH were markedly increased in the sex hormone, T, there were significant differences in PRL. Continuous light for 3 months to give the Yin and Yang of Chinese medicine after sequential serum melatonin, three groups were decreased, but no differences between the groups, sex hormone level is 50 days decreased, but there was no difference among groups. HE staining showed:film membrane thickening, interstitial cell hyperplasia, follicular development stagnation; cortex of all follicles are follicular dysplasia, granular cell reduction by extrusion was flat or cuboidal, reduce the number of mature follicles, no advantage. All corpora lutea showed diffuse reticular, microcapsule structure of sieve, severe dilatation, no ovulation, luteinization follicles in situ and form large screen mesh, microcystic structure of corpus luteum granulosa cells in the corpus luteum, significantly reduced. Chinese medicine treatment group of ovarian HE staining degree of hyperplasia of interstitial cells between visible membrane reduced, follicular development stagnation; but no dominant follicle, follicular dysplasia ratio compared to the model group decreased, the follicle granulosa cell layers than those in the model group increased slightly, still no trace of follicle ovulation, luteinization formed in situ, screen mesh, microcystic luteal structure than the model group slightly reduced, normal luteal ratio increases. Gene expression profile analysis showed: continuous artificial light spectrum has obvious effect on rat ovarian gene expression, the emergence of a large number of genetic differences, given to adjust reproductive rhythm of the Chinese medicine, deviation of 1067 differential genes have been adjusted, the expression of 611 genes increased, decreased the expression of 456 genes. Differential expression of biological process gene combined with pathway analysis results show that:the gene network of nourishing yin and tonifying yang sequential herbs on LHR, EGFR, MT receptor pathway regulates the expression effect is obvious. Immunohistochemistry results showed that, with the intervention of traditional Chinese medicine after as ovarian expression of Cry2 protein in rat obvious difference (P＜0.05); traditional Chinese medicine increased increase the expression of MT1 protein (P<0.05); the expression of LHR protein had no significant difference (P> 0.05), Chinese medicine can significantly increase the expression of EFGR (P<0.05). RT-PCR results showed that:the traditional Chinese medicine intervention significantly up-regulated after rat models of ovarian Cry2mRNA, expression of MT1mRNA, CYP17AlmRNA, EGFmRNA, EGFRmRNA gene (P＜0.05).Conclusions:1, continuous artificial illumination caused a clear effect on ovarian rhythm, and with the illumination time prolonged injury aggravated gradually, composed of gonadal function excitement early turned down, from vaginal smears to histology, serum melatonin, ovarian gene expression profiling, protein expression had some reaction.2, sustained by artificial light with the extension of time, TCM pathogenesis by "hyperactivity of Yang due to yin deficiency" into "two Yin and Yang deficiency".3, the traditional Chinese medicine of nourishing yin and tonifying yang sequential meets the pathogenesis characteristics, by nourishing kidney essence, tonifying kidney yang sequential administration, improve the proportion of dominant follicle and mature corpus luteum, improve the function of ovary.4, the Chinese medicine did not directly increase the serum melatonin levels, but raised its receptor and precursor levels, enhance its biological effects.5, the Chinese medicine to improve the continuous artificial illumination mechanism of rat ovarian rhythm is relatively complex, obtain evidence of short-term treatment of traditional Chinese medicine although not in the vaginal smears and serum sex hormone, but from the histology, expression profiling technique by means of gene expression, have strong evidence for gene expression in ovarian and related factors, to illustrate the traditional Chinese medicine intervention is multiple level, multiple target points, multi way. |
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