The Effect Of Biochanin A On The Cultured Rat Hippocampal Neurons And The Associated Crosstalk With Growth Hormone JAK/STAT Signal Pathway

Alzheimer’s disease(AD)is presenile dementia,which belongs to "dementia" and "stay" in Traditional Chinese Medicine(TCM) and is the most common form of dementia among older adults. Epidemiological studies have shown that in postmenopausal women, estrogen levels dropped significantly, the risk of AD greatly increased than before, and the prevalence and cognitive impairment was significantly higher than male. If perimenopausal women(especially after excision of ovarian women) were given hormone replacement therapy(HRT), the risk of AD could be greatly reduce and the cognitive function could be improved in patients with AD. Clinical practice has proved that women with long-term use of estrogen can increase the risk of breast cancer and endometrial carcinoma,so whether it is suitable for use is very controversial.Phytoestrogen is a kind of selective estrogen receptor modulators(SERMs), which are compounds that exert different biological effects on ER in different tissues and cells. A large number of preclinical medicine,clinical and epidemiological studies have shown that the role of phytoestrogen is similar to estrogen in in animals and humans, which can alleviate menopausal symptoms, and reduce the rate of endometrial cancer and breast cancer. Based on this, phytoestrogen can overcome the disadvantages of HRT treatment.Growth hormone(GH),simaliar to estrogen, one of important endocrine hormones in the body, has be proved to improve memory impairment induced by AD. GH binded receptor dimers and activated the JAK2.Then a series of signaling pathways including JAK/STAT pathway were motivated. In recent years, the interaction of GH and estrogen has been confirmed by many experiments, but the effect on the nervous system has been extremely rarely studied by far.Given the above, it is worth exploring that if biochanin A(a kind of phytoestrogen) can influence rat hippocampal neurons through regulating JAK/STAT pathway of GH.Our works includes three parts: 1.Effects of Biochanin A on Learning and Memory in Ovariectomized RatsRats of the sham group only underwent the resection of a bit fat. The rest groups all underwent the resection of bilateral ovaries. Vaginal smears were taken daily during the eighth to thirteenth postoperative day to observe the rate of successful model.All rats were randomly divided into six groups: sham group,model group,17β-estrodiol, bio A high-does group, bio A middle-does group, bio A low-does group. Intragastrically administration began 16 weeks after surgery for treating 4 weeks. The changes of body weight were monitored. The change of the spatial learning and memory function, and the improving effect of Biochanin A in adult OVX rats were observed through Morris water maze. After the treatment, the rat brain tissues of each group were perfused for fixation. Morphous of the hippocampus were observed after HE stained by microscope. Internal structure of hippocampal neurons was observed under transmission electron microscope. After the treatment, rat brain tissue samples of left rats were collected. Colorimetry method was used to test SOD and GSH-PX activity and MDA content in the left cerebral homogenate. Western Blot was used to examine the expression of PPAR gamma and MMP-2 in right hippocampus of rats. After the treatment, the histopathological changes in uterus and breast tissues were observed to probe its safety for therapy.The weight of all group rats was on the rise in the course of experiment, especially the model group rats increased rapidly. But compared with model group rats, body weight growth trends of administered groups were restrained. In water maze test,compared with the model group, the escape latency was shortened in the rats of bio A treatment groups and 17β-estrodiol group(P<0.05). On probe day, the times across platform and time percent during platform quadrant in administered groups were significantly higher than those in model group(P<0.05). Under the optical microscope, the arrangement of hippocampal neurons was tight with complete neuronal structure in sham group. It can cause apoptosis or necrosis, pyknosis of neuron, hyperchromatic cytoplasm, arranged disorder in model group. The number of necrosis nerve cell in bio A treatment groups was significantly reduced. Electron microscopy observation of the rat hippocampal neurons in each group: The rats in the sham group had complete neuronal structure and mitochondria membranes became visualized; in model group cell membrane dissolved, chromatin condensation and aggregation at the periphery of the nucleons were observed; compared with the model group, the bio A treatment groups were reduced to varying degrees, and there was a significant difference in high-dose group. After the rats were ovariectomized, the content of MDA in brain homogenate increased significantly, the activity of GSH-PX, SOD decreased significantly. Bio A groups could decrease the content of MDA, and increase the activity of GSH-PX, SOD. Compared with sham operation group, the expression of PPAR gamma decreased and MMP-2 increased obviously in model group. Bio A induced the elevation of PPAR gamma and decline of MMP-2. The breast tissues of ovariectomized rats were observed by HE staining: the number of acini decreased, tissue atrophy in model group, low-dose group and middle-dose group,but the situatiion reserved in high-dose group. 2. Effect and Mechanism of Biochanin A on Rat Hippocampal Neurons Injuries Impaired by H2O2New born hippocampus within 24 hours for primary neuronal cell cultures. Cultured for 10 days, with a neuron-specific marker MAP-2 identified neurons purity. To investigate the protective effect of bio A in different dose level(10-7,10-6,10-5,10-4,10-3 mol/L)on primary cultured rat hippocampal neurons through CCK-8 colorimetry. To investigate the injury action of H2O2 in different dose level(100,200,400,800μmol/L)on primary cultured rat hippocampal neurons through CCK-8 colorimetry. The hippocampal neurons of the neonatal wistar rats were cultured in vitro and divided randomly into the normal control group, H2O2 group, small-dose bio A group, middle-dose bio A group and large-dose bio A group.The bio A group were treated different dose bio A for 24 hours,except normal control group,other groups were given 300μmol/L H2O2 for 12 hours to investigate the activity of hippocampal neurons through CCK-8 colorimetry. The hippocampal neurons of the neonatal wistar rats were cultured in vitro and divided randomly into the normal control group, H2O2 group, small-dose bio A group, middle-dose bio A group and large-dose bio A group.The bio A group were treated different dose bio A for 24 hours,except normal control group,other groups were given 300μmol/L H2O2 for 12 hours to investigate the effects of bio A on neuronal apoptosis and reactive oxygen species(ROS) in hippocampal neurons with flow cytometry(FCM). The hippocampal neurons of the neonatal wistar rats were cultured in vitro and divided randomly into the normal control group, H2O2 group, small-dose bio A group, middle-dose bio A group and large-dose bio A group.The bio A group were treated different dose bio A for 24 hours,except normal control group,other groups were given 300μmol/L H2O2 for 12 hours. Bcl-2、Bax and Caspase-3 protein expression were tested by western blot method.After 10 d, primary cultured rat hippocmpal neurons cells tend to be mature. Under the microscope, cells showed cone, spindle shaped, triangular. Identified by MAP-2 immunofluorescence, reached a purity of more than 98%. Hippocampal neurons with different concentrations of bio A cocultured 24 h, hippocampal neurons grew well, the bio A concentration in the vicinity of 10-5 mol/L, neuronal activity was the strongest. Different concentrations of H2O2 and hippocampal neurons co-cultured 12 h, hippocampal neurons showed different degree of necrosis, shedding, along with the increase of H2O2 concentration, cell viability decreased significantly, when administered 300μmol/L after H2O2, cell death rate was about 30%. To observe the effect of bio A on the cell vitality in rat primary hippocampal neurons culture. Compared with H2O2 group, Bio A treatment groups could significantly suppress the injury action induced by H2O2,especially the large-dose bio A group(P<0.05). To investigate the effects of bio A on ROS in hippocampal neurons with FCM. Results showed that intracellular fluorescent intensity was markedly elevated due to H2O2, bio A inhibited H2O2-induced ROS content and showed a good dose-effect relationship. To investigate the effects of bio A on neuronal apoptosis in hippocampal neurons with Annexin V + PI flow cytometry. Results showed the number of apoptotic cells and necrotic cells was significantly increased induced by H2O2, bio A could inhibit neuronal apoptosis and necrosis. To investigate the effects of bio A on Bcl-2、Bax and Caspase-3 protein expression by western blot method. Results showed bio A clearly enhanced the protein expresion of Bcl-2 and decreased the protein expression of Bax and Caspase-3. 3. Regulation Mechanisms of Biochanin A on the Growth Hormone JAK/STAT Signal PathwayTo investigate the protective effect of GH in different dose level(5,25,125,250,500 ng/ml)in primary culture of rat hippocampal neurons for 24 hours through CCK-8 colorimetry. To investigate the effects of bio A and(or) GH on the cell vitality in primary culture of rat hippocampal neurons and observe the possible synergism between two drugs. Phosphorylated-STAT5(P-STAT5) and STAT5 protein levels were tested in primary culture of rat hippocampal neurons after 24 h of serum starvation and treated for 60 min with different doses of GH(0.1ng/ml–20ng/ml), or different time of GH(15min–90min) to observe GH the optimal concentration and action time. SOCS2 protein levels were tested in primary culture of rat hippocampal neurons after 24 h of serum starvation and treated for 60 min with different doses of bio A(10-11-10-7mol/L), or different time of bio A(15min–90min) to observe bio A the optimal concentration and action time. P-STAT5 and STAT5 protein levels were tested in primary culture of rat hippocampal neurons after 24 h of serum starvation and treated for 60 min with GH(5ng/ml), or bio A(10-8 mol/L), or bio A + GH. In this latter case bio A was added 60 min before GH. SOCS1, SOCS2 and SOCS3 mRNA and protein levels were detected in 24 h serum starved rat hippocampal neurons after 60 min treatment with Bio A(10-8 mol/L). SOCS2 mRNA and protein levels were detected in 24 h serum starved rat hippocampal neurons after 60 min treatment with GH(5ng/ml), or bio A(10-8 mol/L), or bio A + GH. In this latter case bio A was added 60 min before GH.It was testified that GH was nontoxic to rat hippocampal neurons in the range of 5 ng/ml-250 ng/ml. However, if GH concentration was over 250 ng/ml, the activity of hippocampal neurons began to decrease. The combined treatment with the two hormones(bio A+GH) had more significant proliferation effect on rat hippocampal neurons than either of the drugs alone had. It suggested that there was synergism between two drugs. STAT5 and P-STAT5 protein levels were response to increasing concentrations of GH(0.1–20 ng/ml) in rat hippocampal neurons after 60 min of treatment, which determined optimal concentration and effect time of GH. SOCS2 protein levels were response to increasing concentrations of bio A(10-11-10-7mol/L) in rat hippocampal neurons after 60 min of treatment, which determined optimal concentration and effect time of bio A. P-STAT5 and STAT5 protein levels were tested in primary culture of rat hippocampal neurons after 60 min GH(5 ng/ml) or bio A(10-8 mol/L) treatment. For the combined treatment with the two hormones, bio A pretreatment(10-8 mol/L) 60 min before GH induced a significant increase of P-STAT5 and STAT5 compared with GH alone. Treatment for 60 min with bio A(10-8 mol/L) did not modify neither protein levels nor RNAs of SOCS1 and SOCS3 in rat hippocampal neurons, while bio A significantly reduced SOCS2 protein level without affecting its mRNA level. Neither 60 min treatment with GH(5 ng/ml) or bio A(10-8 mol/L) nor the combined treatment with the two hormones induced any modification of SOCS2 mRNA, whereas bio A pretreatment 60 min before GH was able to induce a significant reduction of SOCS2 protein levels.Biochanin A has a protective effect on rat hippocampal neurons, which may related to increasing the antioxidative ability and decreasing the apoptosis of neurons. Bio A has no effects on breast tissue in some extent dose. Once dose beyond A certain range,the numbers of gland alveolus increase obviously and the glands expanded. The combined treatment with the two hormones(bio A+GH) has more significant proliferation effect on rat hippocampal neurons. Bio A amplifies intracellular GH signaling by favoring SOCS2 degradation through the proteasome machinery.