Odgovor hipotalamo-hipofiznog sistema pacova na izoflavone soje : morfofunkcionalna studija

Abstract

Izoflavoni soje, genistein i daidzein, se sve više koriste u preventivi i alternativnoj terapiji različitih patoloških stanja. Uticaj izoflavona soje na neuroendokrini sistem bio je, a i danas je, predmet istraživanja različitih studija. Veliki broj literaturnih podataka govori o uticaju izoflavona soje na reproduktivni sistem, s obzirom na njihovu estogensku i anti-estrogensku aktivnost. Ipak, podaci o njihovom uticaju na somatotropni i adrenokortikotropni sistem oskudni su, a neophodni, imajuću u vidu značaj ovih sistema za normalan razvoj i funkcionisanje organizma. Takoñe, mnogobrojne in vitro studije obezbeñuju samo limitirane informacije o potencijalnim efektima in vivo. Cilj ove studije je bio da se ispita uticaj hroničnih tretmana estradioldiproprionatom, genisteinom i daidzeinom na histološke, stereološke i biohemijske parametre somatotropnog i adrenokortikotropnog sistema orhidektomisanih adultnih pacova. Na početku eksperimenta adultni pacovi Wistar soja, stari 3 meseca, su podeljeni u dve eksperimentalne grupe: lažno orhidektomisanu (Sham) i orhidektomisanu (Orx) grupu. Dve nedelje nakon sham operacije ili orhidektomije pacovi su podeljeni u sedam eksperimentalnih grupa. Prvu (Sham1) grupu su sačinjavale sham operisane životinje subkutano (s.c.) tretirane odgovarajućom zapreminom sterilnog maslinovog ulja, medijumom estradiol-diproprionata (medijum I). Drugu (Orx1) grupu su sačinjavale orhidektomisane životinje s.c. tretirane odgovarajućom zapreminom medijuma I. U trećoj (Orx1+E) grupi orhidektomisane životinje su s.c. tretirane estradioldipropionatom u medijumu I (0,6 mg/kg t.m.). Četvrta (sham operisana - Sham2) i peta (orhidektomisana - Orx2) grupa s.c. su tretirane odgovarajućom zapreminom mešavine alkohola i sterilnog maslinovog ulja, medijumom genisteina i daidzeina (u odnosu 1:2; medijum II). Šestu (Orx2+G) grupu su sačinjavale orhidektomisane životinje s.c. tretirane genisteinom u medijumu II (30 mg/kg t.m.), dok je sedma (Orx2+D) grupa orhidektomisanih pacova s.c. tretirana daidzeinom u istom medijumu (30 mg/kg t.m.). Sve grupe životinja su dobijale tretman tokom tri nedelje (hroničan tretman), a žrtvovane su 24 h nakon poslednje primljene doze. Pre početka i nakon završenog tretmana izmerene su telesne mase životinja svih eksperimentalnih grupa, dok su mase hipofiza izmerene nakon žrtvovanja. Za lokalizaciju arkuatnog (Arc) i periventrikularnog (Pe) jedra, paravenrikularnog jedra (PVN) i eminencije medijane (ME) korišćeno je histološko bojenje krezil-ljubičastom bojom. Za obeležavanje oslobañajućih hormona hipotalamusa (oslobañajućeg hormona hormona rasta–GHRH, somatostatina-SS, kortikotropnog oslobañajućeg hormona-CRH) i ćelija adenohipofize (somatotropnih–GH i adrenokortikotropnih-ACTH) korišćeno je imunohistohemijsko bojenje. Histološki i imunohistohemijski obojeni preseci hipotalamusa, kao i imunohistohemijski obojeni preseci hipofiza stereološki su analizirani, uz pomoć newCAST stereološkog softverskog paketa. Dobijene su vrednosti: volumena Arc i Pe jedra i PVN, volumenske gustine SS neurona, volumena hipofize, volumenske i numeričke gustine GH i ACTH ćelija, apsolutnog broja i volumena GH i ACTH ćelija. Koncentracija GH i ACTH u cirkulaciji odreñena je biohemijskim metodama. Dobijeni podaci su statistički obrañeni. Orhidektomija je dovela do značajnog smanjenja (p<0.05) telesne mase adultnih pacova u odnosu na Sham grupu pacova. Hroničan tretman estradiol-diproprionatom je doveo do značajnog smanjenja (p<0.05) telesne mase u odnosu na Orx1 grupu pacova. Hroničan tretman genisteinom ili daidzeinom nije doveo do promene telesnih masa u odnosu na Orx2 grupu pacova. Orhidektomija adultnih pacova je dovela do smanjenja (p<0.05) apsolutne mase hipofize, dok je volumen hipofize ostao nepromenjen u odnosu na Sham grupu pacova. Tretman estradiol-diproprionatom je doveo do povećanja (p<0.05) apsolutne mase i volumena hipofize u odnosu na Orx1 grupu pacova. Tretman genisteinom je povećao (p<0.05) apsolutnu masu i volumen hipofize u odnosu na Orx2 grupu pacova. Tretman daidzeinom nije doveo do promena mase i volumena hipofize u odnosu na Orx2 grupu pacova. Orhidektomija je dovela do značajnog povećanja (p<0.05) volumena Arc jedra; i do značajnog smanjenja (p<0.05): imunoobojenosti GHRH neurosekreta u ME, volumenske gustine SS neurona u Pe jedru, imunoobojenosti SS neurosekreta u ME kao i volumenske i numeričke gustine GH ćelija; a nije uticala na volumen Pe jedra, apsolutni broj, volumen i intenzitet obojenosti GH ćelija i nivo GH u cirkulaciji, u odnosu na odgovarajuće parametre Sham grupe pacova. Nakon tretmana estradioldiproprionatom došlo je do povećanja (p<0.05) volumena Arc i Pe jedra, volumenske gustine SS neurona Pe jedra, intenziteta obojenosti SS neurosekreta u ME, inteziteta obojenosti GH ćelija i nivoa GH u cirkulaciji; i do smanjenja (p<0.05) volumenske i numeričke gustine GH ćelija; dok tretman nije uticao na intenzitet obojenosti GHRH neurosekreta u ME, apsolutni broj i volumen GH ćelija; sve u odnosu na odgovarajuće parametre Orx grupe. Hroničan tretman genisteinom je doveo do povećanja (p<0.05): volumena Arc jedra, intenziteta obojenosti GHRH neurosekreta u ME, volumenske gustine SS neurona Pe jedra, količine SS neurosekreta u ME, volumenske i numeričke gustine GH ćelija, apsolutnog broja i intenziteta obojenosti GH ćelija i nivoa GH u cirkulaciji; a nije uticao na volumen Pe jedra i volumen GH ćelija; sve u odnosu na odgovarajuće parametre Orx grupe. Hroničan tretman daidzeinom je doveo do povećanja (p<0.05): intenziteta obojenosti GHRH neurosekreta u ME, volumenske gustine GH ćelija, volumena i intenziteta obojenosti GH ćelija; do smanjenja (p<0.05) numeričke gustine i apsolutnog broja GH ćelija; a nije uticao na volumen Arc i Pe jedra, volumensku gustinu SS neurona Pe jedra, intenzitet obojenosti SS neurosekreta u ME i nivo GH u cirkulaciji; sve u odnosu na odgovarajuće parametre Orx2 grupe. Orhidektomija je dovela do značajnog povećanja (p<0.05): volumena PVN, volumenske gustine CRH neurona i duplo obeleženih CRH/cFos neurona u PVN, intenziteta obojenosti CRH neurosekreta u ME i nivoa ACTH u cirkulaciji; do značajnog smanjenja (p<0.05) volumenske i numeričke gustine ACTH ćelija i intenziteta obojenosti ACTH; dok apsolutni broj i volumen ACTH ćelija nisu promenjeni; sve u odnosu na odgovarajuće parametre Sham grupe pacova. Hroničan tretman estradiol-diproprionatom doveo do značajnog povećanja (p<0.05) volumenske gustine CRH/cFos neurona, intenziteta obojenosti CRH neurosekreta u ME, apsolutnog broja ACTH ćelija i nivoa ACTH u cirkulaciji; do značajnog smanjenja (p<0.05) volumenske i numeričke gustine i intenziteta obojenosti ACTH ćelija; dok nije uticao na volumen PVN, volumensku gustinu CRH neurona i volumen ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx1 grupe. Hroničan tretman genisteinom je doveo do povećanja (p<0.05): volumenske gustine CRH/cFos neurona PVN, volumenske gustine i apsolutnog broja ACTH ćelija i nivoa ACTH u cirkulaciji; do smanjenja (p<0.05) numeričke gustine i intenziteta obojenosti ACTH ćelija; dok nije uticao na volumen PVN, volumensku gustinu CRH neurona PVN, intenzitet obojenosti CRH neurosekreta u ME i volumen ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx2 grupe. Hroničan tretman daidzeinom je doveo do povećanja (p<0.05) volumenske gustine CRH/cFos neurona i nivoa ACTH u cirkulaciji; smanjenja (p<0.05) volumenske gustine CRH neurona; dok nije uticao na volumen PVN, intenzitet obojenosti CRH neurosekreta u ME, volumensku i numeričku gustinu, apsolutni broj, volumen i intenzitet obojenosti ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx2 grupe. Rezultati dobijeni u ovoj studiji ukazuju na stimulatorni efekat genisteina na histološke, stereološke i biohemijske parametre somatotropnog i adrenokortikotropnog sistema, dok daidzein pokazuje nešto manje izražen stimulatorni efekat na pomenute sisteme. Ova studija predstavlja solidnu osnovu za dalja istraživanja mehanizama delovanja genisteina, odnosno daidzeina na somatotropni i adrenokortikotropni sistem, kao i za njihovu potencijalnu terapijsku korisnost.

The soy isoflavones genistein and daidzein are being used increasingly as preventive or alternative therapeutics in various pathological conditions. The effects of soy isoflavones on the neuroendocrine system were, and still are, the subject of numerous studies. Literature data predominantly focus on their effects on the gonadotrophic axis, considering the soy isoflavones established estrogenic and antiestrogenic mode of action. However, data about their potential effects on the somatotrophic and adrenocorticotrophic systems are rather scarce, but nevertheless very meaningful, considering the importance of these systems for the normal development and function of an organism. Furthermore, in vitro studies provide only limited data, that cannot be fully extrapolated to in vivo conditions. The aim of this study was to investigate the effects of chronically applied estradiol dipropionate, genistein and daidzein on the histological, stereological and biochemical parameters of somatotrophic and adrenocorticotrophic systems in orchidectomized adult rats. Initially, adult (three months old) Wistar rats were divided into two groups: sham operated (Sham) and orchidectomized (Orx). Two weeks after the surgery, the rats were subdivided into seven experimental groups. The first group (Sham1) included sham operated rats subcutaneously (s.c.) treated with an adequate volume of sterile olive oil, the medium for estradiol dipropionate (medium I). In the second group (Orx1) were orchidectomized rats s.c. treated with an adequate volume of medium I. The third group (Orx1+E) was comprised of orchidectomized rats s.c. treated with estradiol dipropionate in medium I (0.6 mg/kg b.w.). The fourth (sham operated – Sham2) and fifth (orchidectomized – Orx2) group of rats were s.c. treated with adequate volumes of absolute ethanol and sterile olive oil mixture, the medium for genistein or daidzein (mixing ratio was 1:2; medium II). In the sixth group (Orx2+G) were orchidectomized rats s.c. treated with genistein in medium II (30 mg/kg b.w.), while the seventh group (Orx2+D) included orchidectomized rats s.c. treated with daidzein in the same medium (30 mg/kg b.w.). All the groups of animals received the treatment during three weeks (chronic treatment), and were sacrificed 24 h after the last injected dose. All the animals were weighed before and after the treatment, while the pituitary weights were measured after sacrificing. For the localization of arcuate (Arc), periventricular (Pe) and paraventricular (PVN) nuclei, as well as for median eminence (ME) cresyl violet histochemical staining was used. Hypothalamic releasing hormones (growth hormone releasing hormone – GHRH, somatostatin – SS, corticotrophin releasing hormone – CRH) and pituitary cells (somatotrophic – GH and adrenocorticotrophic – ACTH) were immunohistochemically (peroxidase-antiperoxidase) labeled. Histochemically stained and immunohistochemically labeled hypothalamic sections, as well as immunohistochemically labeled pituitary sections were stereologically analysed, using newCast stereological software package. The following values were obtained: the Arc, Pe and PVN nuclei volumes, volume density of SS neurones, volumes of the pituitaries, the volume and numerical density of GH and ACTH cells, as well as the absolute numbers and volumes of GH and ACTH cells. The circulating GH and ACTH were determined biochemically. The obtained data were statistically processed. Orchidectomy significantly (p<0.05) decreased the body weight of adult rats in comparison to Sham1 operated group. Chronic treatment with estradiol dipropionate caused a significant (p<0.05) decrease in body weight compared to the Orx1 group of rats. There were no changes in the same parameter after chronic treatments with genistein or daidzein, in comparison to Orx2 group. Orchidectomy of adult rats led to a decrease (p<0.05) in absolute pituitary weight, while the pituitary volume remained unchanged compared to Sham1 group. Treatment with estradiol dipropionate increased (p<0.05) absolute pituitary weight and volume, in comparison to Orx1 group of rats. Genistein treatment increased (p<0.05) absolute pituitary weight and volume compared to Orx2 group, while the same parameters remained unchanged after daidzein treatment. Orchidectomy provoked a statistically significant enlargement (p<0.05) of the Arc nucleus volume; a decrease (p<0.05) of: the GHRH and SS amounts in ME, the volume density of SS neurons in Pe nucleus, the volume and numerical density of GH cells, while orchidectomy did not affect: the volume of Pe nucleus, the absolute number, volume and intensity of staining of GH cells and GH level in blood, all corresponding to the appropriate parameters of Sham group. Administration of estradiol dipropionate to orchidectomized rats provoked an enhancement (p<0.05) of: Arc and Pe nucleus volume, the volume density of SS neurons in Pe nucleus, the SS amount in ME, the intensity of staining of GH cells and GH level in blood; and a decrease (p<0.05) of the volume and numerical density of GH cells; while the treatment did not affect: the GHRH amount in ME, the absolute number and volume of GH cells, all corresponding to the appropriate parameters of Orx group. Genistein treatment caused an increase (p<0.05) in: Arc nucleus volume, the GHRH and SS amounts in ME, the volume density of SS neurons in Pe nucleus, the volume and numerical density of GH cells, the absolute number and intensity of staining of GH cells, GH level in blood; while the treatment did not affect: the Pe nucleus volume and volume of GH cells, all corresponding to the appropriate parameters of Orx group. Daidzein treatment caused an increase (p<0.05) in: the GHRH amount in ME, the volume density of GH cells, volume and intensity of staining of GH cells; and a decrease (p<0.05) of: numerical density and absolute number of GH cells, while the treatment did not affect: Arc and Pe nucleus volume, the volume density of SS neurons in Pe nucleus, the SS amount in ME and GH level in blood, all corresponding to the appropriate parameters of Orx group. Orchidectomy provoked a statistically significant enlargement (p<0.05) of: the PVN volume, the volume density of CRH and CRH/cFos neurons in PVN, the CRH amount in ME and ACTH level in blood; a decrease (p<0.05) of: the volume and numerical density and intensity of staining of ACTH cells; while the treatment did not affect the absolute number and volume of ACTH cells, all corresponding to the appropriate parameters of Sham group. Administration of estradiol dipropionate to orchidectomized rats provoked an enhancement (p<0.05) of: the volume density of CRH/cFos neurons in PVN, the CRH amounts in ME, the number of ACTH cells and ACTH level in blood; a decrease (p<0.05) of : the volume and numerical density and intensity of staining of ACTH cells; while the treatment did not affect: PVN volume, the volume density of CRH neurons in PVN and volume of ACTH cells, all corresponding to the appropriate parameters of Orx group. The treatment with genistein caused an increase (p<0.05) in: the volume density of CRH/cFos neurons in PVN, the volume density and absolute number of ACTH cells and ACTH level in blood; a decrease (p<0.05) of: the numerical density and intensity of staining of ACTH cells; while the treatment did not affect: PVN volume, the volume density of CRH neurons in PVN, the CRH amounts in ME and volume of ACTH cells, all corresponding to the appropriate parameters of Orx group. Daidzein treatment caused an increase (p<0.05) in: the volume density of CRH/cFos neurons in PVN and ACTH level in blood; a decrease (p<0.05) of the volume density of CRH neurons in PVN; while the treatment did not affect: PVN volume, the CRH amounts in ME, the stereological parameters of ACTH cells, all corresponding to the appropriate parameters of Orx group. These results indicate stimulatory effects of genistein on the majority of examined histological, stereological and biochemical parameters of the somatotropic and adrenocorticotrophic systems, while daidzein has less pronounced stimulatory effects on the examined parameters. This study represents a solid basis for some further investigations aimed at establishing the precise mechanisms of genistein/daidzein actions on the somatotropic and hypothalamic–pituitary–adrenal systems and their potential therapeutic usefulness.