Morinda Officinalis Polysaccharides Attenuate Hypothalamus-pituitary-testicular Axis Of Varicocele & The Direct Effects Of Vasopressin On Secretion Of GnRH

Objective:1)To investigate the protective effects of Morinda officinalis polysaccharides on the hypothalamic-pituitary-testicular axis in varicocele.2)To explore the definite effect and mechanism of vasopressin（AVP）on synthesis and secretion activities of gonadotropin-releasing hormone（GnRH）neuron.Contents:1)The crude polysaccharides of Morinda officinalis were isolated and their effects on the testicular morphology,angiogenesis and neuroendocrine functions of the hypothalamus were observed.2)The evidence of direct action of AVP on GnRH neuron were observed in rat hypothalamus.The effects of AVP on synthesis of GnRH were studied in GT1-7 cells.Methods:1)Water-extraction and alcohol-precipitation method were performed to extract crude polysaccharides from Morinda officinalis.The extrctions were analyzed by ultraviolet,infrared spectroscopy,gel permeation chromatography（HPGPC）and monosaccharide derivatization.2)A rat experimental left varicocele model were established,and the crude polysaccharides were administered for 5 weeks after surgery.GT1-7 cells were used to study the effects of drug serum or AVP on GnRH neuron in vitro.3)The morphological characteristics of testis and hypothalamus were studied by hematoxylin-eosin（HE）staining,Nissl staining and Golgi staining.Serum levels of GnRH,AVP,luteinizing hormone（LH）,follicle stimulating hormone（FSH）and testosterone（T）were measured by enzyme-linked immunosorbent assay.Immunohistochemical staining,immunoblotting and real-time quantitative PCR（q-PCR）were used to detect the expression and transcription of proteins.4)Triggered by changes of urine/water ratio and blood pressures of varicocele,the levels of AVP and GnRH in serum and hypothalamus were observed,and the correlation between the two covariates were analyzed.The co-localization of AVP and GnRH was observed by proximity ligation assay（PLA）and immunoelectron microscopy.The effect of AVP on GnRH neurons was explored by proteomics analysis.Q-PCR and double luciferase reporter assay were used to detect the changes of GnRH transcription activity and promoter activity after AVP dosed.Cytotoxicity assay,cell cycle assay,intracellular Ca²⁺assay and ATP assay were performed to observe the general effects of AVP on GnRH neuron.Results:1)The content of carbohydrate in the extracted crude polysaccharide of Morinda officinalis（MOP）was 91%,and the protein or the nucleic acid was not visible in UV spectroscopy.The characteristic absorption peak of MOP was observed by infrared spectroscopy.HPGPC showed a high peak and the average molecular weight was 1141 Daltons,weight average ratio Mw/Mn was 1.444.Chemical composition of glucose:galactose:xylose:fructose:maltose:sucrose is 7.63:1.23:0.95:0.72:0.87:0.64.2)Compared with ELV group,50 mg/kg-100 mg/kg MOP significantly attenuated the sexual behavior of ELV rats,increased the number of bilateral epididymal spermatozoa and reduced sperm abnormality（P<0.05）.The levels of GnRH,FSH,LH and T in serum significantly increased by 50 mg/kg-100 mg/kg MOP administration（P<0.05）.50 mg/kg-100 mg/kg MOP repaired the bilateral testicular spermatogenic epithelial structure,reduced the abnormal apoptosis of spermatogenic cells,reduced edema and inhibited vascular proliferation of interstium.The effects of 100mg/kg MOP on attenuating ipsilateral testes were better than 50mg/kg MOP,which may relate to the relative up-regulation of VEGF and MMPs（P<0.05）.50 mg/kg-100 mg/kg MOP significantly increased the dendritic spine density of the hypothalamic neurons of ELV rats（P<0.05）,as well as the number of functional dendritic spines（P<0.05）.50mg/kg-100 mg/kg MOP significantly up-regulated the expression of Kiss1-GPR54 in the hypothalamus and increased the transcriptional activity and expression level of hypothalamic GnRH（P<0.05）.Serum of ELV rats treated with 50 mg/kg-100 mg/kg MOP increased the transcriptions of GPR54,Oct-1 and SCIP,promoted MAPKs phosphorylation and stimulated GnRH synthesis and release in GT1-7 cells（P<0.05）.3)Increased urine/water ratio and decreased blood pressures were observed in varicocele progression（P<0.05）.Both GnRH and AVP in serum and hypothalamus significantly decreased（P<0.05）,and the decrease of AVP was significantly earlier and more obvious than the decrease of GnRH（P<0.05）.The GnRH-AVP PLA positive signals were detected in the arcuate nucleus,and GnRH and AVP immunoreactive substances were also observed in the same neuron by immunoelectron microscopy.After treated by 50 pg/ml-200 pg/ml AVP for 6 hrs,the levels of ATP and Ca²⁺in GT1-7cells significantly increased（P<0.05）,as well as the GnRH transcription level and the GnRH promoter activity（P<0.05）.The levels of GnRH-related transcription factors,Oct-1 and SCIP,and phosphor-MAPKs also significantly increased（P<0.05）.More than 1000pg/ml AVP may induce Ca²⁺overload,ATP synthesis disorders and inhibit cell proliferation of GT1-7 cells.After blocking the GPR54 receptor,these effects of 50pg/ml-200 pg/ml AVP on GnRH synthesis was significantly inhibited（P<0.05）,while the cytotoxic effects of high concentration（1000 pg/ml or more）AVP showed no significant changes.In addition,PLA and immunoprecipitation revealed the possible protein-protein interaction between AVP and GPR54.ITRAQ analysis of GT1-7 cells before and after treated with 200 pg/ml AVP for 6 hrs found that AVP treated group andshamgroupdifferentiallyexpressedprotein-relatedpathwaysare cysteine-methionine metabolic pathway,fatty acid metabolic pathway,pyruvate metabolic pathway,fatty acid synthesis pathway,typeⅠdiabetes related pathway and olfactory transduction pathway.Conclusion:1)50mg/kg-100mg/kg MOP could effectively repair the reproductive function of varicocele,improve sperm quality,repair testicular injury,repair synaptic plasticity of neuron in hypothalamus,up-regulate Kiss1-GPR54 system and promote GnRH synthesis and secretion.2)AVP may act directly on GT1-7 cells,enhance GnRH promoter activity and promote GnRH synthesis and release,which may be achieved by direct or indirect interaction with GPR54 receptor.In addition,AVP may increase the expression of GPR54 receptor in GnRH neuron and enhance the role of Kiss1-GPR54 system,followed by increased synthesis and secretion of GnRH.