Neuroprotective Effects Of Prenylflavnoid Estrogen Receptor Modulators And Differentiation Stimulatory Effects On Embryonic Stem Cells Into Neurons

Estrogen receptor modulator (ERM) is a type of ligands that could bind with estrogen receptors and induced agonistic or/and antagonistic activities. It could be divided into three subtypes—agonist, antagonist and estrogen receptor selective modulators (SERMs). Estrodial is the most potent agonist of estrogen receptor. It plays a critical role in the development of central nerve system and function maintenance of neuronal cells. The activities of SERMs are closely associated with the endosomatic level of estrogens and differ a lot in a tissue or cell-specific manner. The researches about exogenous SERMs on nerve system were seldom reported till now.Herb Epimedii is a traditional Chinese tonic Herbal medicine. It exerts the benefits on cardiovascular system, bone, immunological and endocrine systems etc. Icariin, a major active component from Epimedii, is a prenylflavonoid compound. Icaritin and desmethylicaritin, as the derivatives and endosomatic metabolites of icariin are also the components from Epimedii. In our studies, structural modification was carried on to obtain ICT and DICT, as the estrogen like effect alteration represented between daidzein and its precursor. MCF-7 proliferation assay (E-SCREEN method) was used to evaluate the estrogen like activities of the three compounds and the related mechanisms were explored. Additionally, the structure-activity relationships of prenylflavonoid estrogen receptor modulators were further explored in the second part of the dissertation.It is demonstrated that the endosomatic disrupt level of estrogens was closely associated with many refractory complicated diseases, e.g. degeneration. And estradiol possessed critical activities in neuroprotection and neurodevelopment. The further experiments explored the possible effects of prenylflavonoid estrogen receptor modulators on neuroprotection and neurodevelopment, which may provide evidences of possible precautions and cautions effects on neurodegenerative diseases. Chapter 1 Estrogen like activities of icaritin and desmethylicaritin and the related mechanismsTo evaluate the estrogen like activities of icariin, icaritin and desmethylicaritn and explore the related mechanisms, MCF-7/BOS cell proliferation assay, known as the E-SCREEN method, together with ER responsive gene detection method, was used to screen the estrogenic compounds. Cell viabilities were evaluated by MTT method, Cell cycle analysis were performed by Flow Cytometry, and the estrogen receptor responsive genes were detected by RT-PCR. ICI 182,780 [7a-[9(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl)-estra-1,3,5(10)-triene-3,17h-d iol)], the specific estrogen receptor antagonist, was used as a tool drug to identify the estrogen receptor dependent manner. The results demonstrated that icaritin and desmethylicaritin could significantly stimulate the proliferation of MCF-7/BOS significantly, which represented estrogen like effects, while icariin was inert. Cell cycle analysis revealed that the proliferation stimulatory effect was associated with a marked increase in the number of MCF-7/BOS cells in S phase, with effects similar to those of estradiol. These actions were dose dependent (range from 0.1 mol/L to 10 mol/L) and could be significantly inhibited by the ER antagonist. The ER responsive genes of progesterone receptor and PS2 mRNA levels were increased by the treatment of icaritin or desmethylicaritin within 24 h and the effects were also reversed by ICl 182,780. It could be concluded that icaritin and desmethylicaritin are novel herbal derivative estrogen receptor modulators and their estrogenic effects are mediated by the estrogen receptor.Chapter 2 Estrogen like activities evaluation and structure-activity relationship research of prenylflavonoid derivativesBased on the results of different potencies with different structures in ICA, ICT and DICT, a series of prenylflavonoids were synthesized and the structure-activities relationship was explored by above mentioned biological methods and receptor-ligand docking experiments. Results showed that the prenylflavonoids generally possess the estrogenic activities. Isobavachin was most potent and icariin was inert, which could be ordered in the following order: Estrodial > IBA > 4',5,7-Dihydroxy-8-prenylflavone > 4',7-Dihydroxy-8-prenylflavone > DICT > ICT > ICA (inert). Cell-cycle analysis revealed that the stimulatory effects of these compounds toward cell proliferation were elicited at the G1-S checkpoint. ER-responsive gene (pS2, PgR) and protein (PgR) expression was also detected. The mRNA and protein-expression levels for pS2 and PgR were up-regulated by the compounds in a dose-dependent manner. These effects could also be blocked by ICI 182,780, which could therefore conclude that the estrogen-like effects of these prenylflavonoids were mediated primarily through ERs. Furthermore, to explore the structure-activity relationship based on the estrogen receptor and detailed molecular mechanisms among the prenylflavonoids, protein-ligand docking simulations were carried out by using the DS-MODELING software package. It demonstrated that the activities were closely related with the binding affinity between the prenylflavonoid and ER. ICA was inert because of the steric hindrance produced by the bulky group which prevents the required docking to the ligand binding domain. The variance of activities of other prenylflavlnoids was induced by the alteration of subsituents and the prenyl substituent is an important symbol. This part of work demonstrated that prenylflavonoids were generally possess estrogen like activities and also explained some of the structure activity relationships.Chapter 3 Neuroprotective effects of prenylflavonoid estrogen receptor modulator icaritin against Aβpeptide induced neurotoxicity in primary cultured rat neuronal cellsIn recent years, the neuroprotective effects of estrogen against neurodegenerative diseases have been focused on. However, there are hot debates on the position and mechanisms of the therapy. The therapeutic effects of estrogen on Alzheimer's disease (AD) have not been confirmed yet. Considering the neuroprotective effects of estradiol, the potential therapeutic effects and related mechanisms of prenylflavonoid estrogen receptor modulators against AD were explored.The nerve loss caused byβ-amyloid is a hallmark and pathological mechanism of Alzheimer's disease and Aβ_25-35 is the toxic fragment ofβ-amyloid. In our study, the model of Aβ_25-35 induced neurotoxicity on primary cultured rat neuronal cells was used to assess the neuroprotective effects and mechanisms of the compounds. The primary cultured neuronal cells were identified by immunocytochemistry, and the morphological changes were evaluated by microscopic observation. MTT method and lactate dehydrogenase (LDH) assay were used to assess the viability of neuronal cells, AO/EB staining was used to detect the apoptosis, JC-1 staining was performed to assess the alteration of membrane potential (△ψm), and WESTERN BLOT was used to assay the protein expression of the cultures. The results showed that icaritin could protect the neuronal cells against the insult induced by AP25-35 in a dose-dependent manner, characterized by increased cell viability, decreased lactate dehydrogenase (LDH) release, and neuronal DNA condensation. The effects were associated with both the loss of membrane potential and the alteration of the expression of Bcl-2 family proteins. Furthermore, the neuroprotective effects of icaritin mentioned above were estrogen receptor dependent due to the partly blockage effects by estrogen receptor antagonist ICI 182,780. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibitor PD 98059 weakened the protective effects, which implied that mitogen-activated protein kinase/extracellular signal-regulated kinase pathway may also be involved in and partly contributed to the neuroprotective effects of icaritin.Chapter 4 Differentiation stimulatory effect of prenylflavoild ERM icaritin on mouse embryonic stem cells into neuronal cellsAD is one of the most complex and refractory disease hallmarked by the loss of cholinergic nerve and cell replacement therapy is the promising treatment. The embryonic stem cells, qualified with pluripotency, could differentiate into neuro-precursor cells or neuronal cells, which would be valuable in cell therapy on neurodegenerative diseases. The drug interventional potential in cell therapy was also focused on in recent years. The efforts were mainly focused on simplifying the procedure of the differentiation in vitro, reducing the differentiational duration and controlling the consistency. Moreover, it has also been demonstrated that certain small molecular chemicals could control the fate of ES cells by regulating the niche invironment of ES cells.The previous experiments demonstrated that icaritin could protect neuronal cells through ER pathway and ERK-MAPK pathway. It indicated that there may be some similar mechanisms existing in the differentiation of embryonic stem cells into neuronal cells. In our study, the model of embryonic stem cells differentiating into neuronal cells was used to assess the differentiation stimulatory effects of prenylflavonoid ERMs. The bioinformatics and mechanisms were also explored at the same time. Retinoic acid was used as positive drug and an existing 4-4+ protocol was used in the model. Cell morphological observation and immunocytochemistry study were used to assess the morphological changes during the development of ES cells. The neuronal precursor cells and neurons were immunomarked by nestin andβ-Tublin III, which were quantitated by flow cytometric assay in the treatment of drugs. RT-PCR and Western Blot were performed to detect differentiation dependent genes and proteins during the development of ES cells. The results demonstrated that ICT (0.01-1μmol/L) and RA (0.1μmol/L) could stimulate the differentiation of embryonic stem cells into neuronal cells. The differentiated neuronal precursor cells (d8+0) and neuronal cells (d8+10) increased significantly induced by RA or icaritin compared with control group. RT-PCR analysis of development related genes showed that the expression of mesodermal and ectodermal development marker genes (Fgf and Branchy) expression could be enhanced and elongated by the treatment of RA and icaritin. Western Blot analysis showed that the expression of neuronal protein markerβ-Tublin III and choline acetyltransferase (ChAT) increased sequentially in the later stage of differentiation, which also indicated that RA and icaritin possessed the effects of differentiation stimulatory effects. During the differentiation, icaritin and RA induced a decreased level of p-p38 and a slightly increased p-ERK. Considering the critical molecular switch role of p38 in the development of embryonic stem cells, the results showed that icaritin may exert the differentiation stimulatory effects via inhibiting the phosphorylation of p38.To prove the common rules of the activities of the prenylflanoid derivatives, icaritin and isobavachin were also evaluated in the experiments and it showed that isobavachin could stimulate the differentiation of embryonic stem cells into neurons. But icariin was inert. The results indicated that the estrogenic activities of prenylflavonoid ERMs may be involved in the differentiation stimulatory effects and it is a common effect of the compounds.Chapter 5 Biological assay guided isolation of prenylflavanoid ERMs from Chinese herbal medicine Psoralea corylifoliaIt was demonstrated in the previous experiments that isobavachin has potent estrogenic activity. Considering icaritin derivating from Epimedii and Psoralea corylifolia was the natural origin of isobavachin, biological activities guided isolation of prenylflavanoid estrogenic components was performed to research the fundamental basis of tonic herbal medicines.The crude product of Psoralea corylifolia was extracted by ethanol. The methods of liquid-liquid extraction, column chromatography, HPLC analysis, LC-MS and NMR were used to isolate and identify the constituents. E-SCREEN method based estrogenic activity evaluation was performed to guide the isolation of active components (Bio-guided). In our studies, 4 estrogenic compounds were isolated and three of them are prenylflavones (Neobavaisoflavone, 8-Prenyldaidzein, Isobavachalcone). The study is an attempt to explore the pharmacodynamic fundament of traditional Chinese tonic herbal medicines.Conclusion1. Prenylflavonoids such as icaritin, desmethyicaritin and isobavachin possess estrogenic activities. The effects were mediated by estrogen receptors and regulated by the estrogen receptor responsive genes and cell cycle phases. Prenylflavonoids could be considered as a new type of estrogen receptor modulators derivated from Chinese Herbal Medicines.2. There were some characteristics in the structure-activity relationships of the prenylflavonoids. Prenyl group is a common symbol of the structures of prenylflavonoid estrogen receptor modulators. The location and hydrophobicity of substituent groups could affect the estrogen like potency of the compounds by mediating the affinity between the ligands and receptor. The affinity between the ligands and receptor is a critical factor in determining the activities of the compounds. The steric hindrance produced by glycoside moieties which prevented docking to the estrogen receptor made it inert.3. Icaritin could protect the primary cultured neuronal cells against induced neurotoxicity and these effects could be partly blocked by estrogen receptor antagonist ICI 181,780 and ERK-MAPK inhibitor PD 98059. The mechanism was associated with the Bcl-2 upregulation and blockage of mitochondria apoptosis pathway.4. Icaritin and isobavachin could stimulate the differentiation of mouse embryonic stem cells into neuronal cells. Prenylflavonoids could be considered as a potential differentiation simulator. The mechanism was associated with estrogen receptor but not estrogen receptor dependent completely. The phosphorylation level of p38 is a critical factor in differentiation stimulatory effects of icaritin.5. The prenylflavanoid compounds was widely distributed in the tonic herbal medicine such as Herb Epimedii and Psoralea corylifolia, estrogen receptor modulators from which may be the phymacodynamic foundation of therapeutic effects of Chinese medicine.