The Protection Of Gengnianchun Decoction From Amyloid Beta Protein-induced PC12 Cell Injury

The perimenopausal syndrome, such as vasomotion, mental and psychological symptoms as well as osteoporosis, degeneration of study and memory ability, even Alzheimer's disease occur with ovary aging in menopausal period.The therapy of AD is just at the symptomatic, and the estrogen and traditional Chinese medicine (TCM) get more and more attention. For the increasing risk of endometrial carcinoma, breast cancer and stroke companying estrogen replacement therapy, HRT is limited. A lot of clinical and experimental studies have shown that TCM is effective in treatment of perimenopausal syndrome resulting from estrogen decreasing without apparent side effects, which gives a new therapeutics for AD. According to TCM theory, tonifing Kidney is appropriate to improve the learning and memory in menopause.The principle of GNC decoction is nourishing the Kidney and the Liver and clearing Heart-fire. It is verified that this decoction is clinically effective, especially improving the memory. Our previous study has found that GNC appears to good effects in controlling the neuro-endocrino-immune network by up-regulating the expression of estrogen receptor, enhancing the OVX rats' manifestation in Morris water maze test, and GNC might increase the RNA and protein expression of ERβin hippocampus, and regulate the cholinergic neuron function so as to enhance the learning and memory ability.We are to investigate the roles of GNC decoction and its monomers in neuroprotection and try to find the mechanism in the present study.Part I Protective Effects of Gengnianchun Decoction on PC12CellsObjective To observe and compare the protective effects of Gengnianchun (GNC) Decoction and its main monomers on PC12 cells.Methods The serum of the rats was got after perfusion of GNC granula to OVX SD rats. PC 12 cells were treated with the serum or main monomers i.e. Paeoniflorin(Pae), Timosaponin A-III(Tim), Berberine Hydrochloride (Ber) and Icariine (Ica) that are the main substances in GNC decoction, respectively, and then analyzed by MTT assay for cell viability to find the proper culturing concentration and duration.Results Comparing with serum from normal rats and control group, the serum with GNC Decoction reinforced PC12 cells activity, with the strongest effect in 20% concentration of the serum at 24 h, 48 h, 72 h. Culturing with herb monomers show little difference except the Ber at the concentration of 1umol/L and mixed fluid of herb monomers at the concentration of degree 3 ( including Ber1umol/L,Tim1umol/L,Pae 1g/L,Ica 1ng/ml) which seems increased but can't make sure. Therefore, we choose the herbage containing serum and the herb monomer and mixed ones to culture the target cells.Conclusions GNC Decoction might exert a potential neuroprotective effect. The serum with GNC Decoction was found to be stronger than the main monomers.Part II PC12 Cells injury induced by AP25-35Objective To establish the AD model in vitro by Ap25-35-induced PC12 cells injury.Methods PC12 cells were incubated by different concentration of Aβ25-35 for 24 hours, 48 hours, respectively, and the cell livability was determined by MTT assay. Meanwhile, cellular morphological change was observed by phase contrast microscope.Results After PC12 cells were treated by different concentrations of 5,10,20,40μmol/ L of Aβ25-35 for 24 hours and 48 hours, the cell viability decreased, and the injury cells were more in number and nuclei contrasted, which shows obviously dose and time-dependent. Therefore, the AD model in vitro has been built successfully.Conclusions A|325-35 can inhibit the viability and promote its apoptosis of PC12 cells in a dose and time-dependent manner, and the PC12 cell injury may be as AD model.Part III Protective effects of Gengnianchun Decoction on PC12 cells injury induced by Aβ25-35 and its signal transductionObjective To investigate whether GNC can ameliorate the apoptosis of neuron and what are the effective ingredients. To elucidate which signaling transduction pathway mediates its anti-apoptosis action.Methods The Ap25-35-induced PC12 cell injury were cultured with GNC-containing serum and its monomers such as Paeoniflorin(Pae), Timosaponin A-III(Tim), Berberine Hydrochloride (Ber) and Icariine (Ica) that are the main substances in GNC decoction. The early apoptosis was determined by AnnexinV-FITC / PI flow cytometry. The protein expression of caspase-3, bax, bcl-2, p-ERK1/2 and ER subtypes were measured by western blotting.Results It has been found by MTT assay that the GNC-containing serum protects PC12 cells from the Aβ25-35-induced injury that is similar with NGF and that the monomers from GNC also protects the cells from injury (P<0.05), which suggests that GNC-containing serum, Ber, and herb monomer mixture all improve the cell viability of PC12 cells.It has been found by FCM that the GNC-containing serum and monomers of GNC inhibits the cell apoptosis induced by Aβtreatment, which suggests that the GNC-containing serum, Ber, herb monomer mixture all protects from the Aβ-induced apoptosis of PC12 cells.From the result of Western-blotting we can find there is much more expression of bcl-2 than bax in GNC-containing serum treatment compared to the Aβ-induced P12 cell injury. The expression of caspase-3 is decreased significantly in the treatment with the GNC-containing serum in comparison with the Aβ-induced P12 cell injury, which suggests that the GNC-containing serum protects P12 cell from the apoptosis induced by Aβthat is superior to the monomers of GNC.Also it can be found from the result of Western-blotting that there is no ERαband expressed in PC 12 cells. However, the GNC-containing serum increases significantly expression of ERβin the injured P12 cells induced by Aβ, suggesting the herb can elevate the expression of ERβin PC 12 cell.Western-blotting also shows that the expression of p-ERK1 / 2 is increased in the injury cells treated with GNC-containing serum, suggesting the herb can increase the expression of p-ERK1 / 2 in the Aβ-induced injured P12 cells.Conclusions The serum containing GNC, and its monomers can inhibit P12 cell apoptosis induced by Aβand protect cell from damage, which may be via ERβand MAPK pathway. Part IV Protective effects of Gengnianchun Decoction on Aβ-induced PC12 cells injury by ERβand its signal transductionObjective To probe into which signaling transduction pathway is involved in GNC's anti-apoptosis.Methods The early apoptosis was determined by AnnexinV-FITC / PI flow cytometry. The protein expression of caspase-3, bax, bcl-2, p-ERK1/2 and ER subtypes were measured by western blotting. The protein expression was measured again after pre-treating respectively by ER antagonist ICI182, 780 and blocker of MAPK.Results Compared with the GNC+Aβ, MAPK blocker, ER antagonist abolishes p-ERK increase and the PC12 cell early stage apoptosis protection induced by GNC and its monomer mixture (P<0.01), and inhibits bcl-2, ERβexpression, and enhances bax, caspase3 expression in the cells, which suggests that ERβand MARK pathway is involved in the protection of GNC from P12 cell apoptosis.Conclusions Up-regulation of ER and MAPK pathway and down-regulation of apoptosis signal pathway are involved in the protection of GNC from PC12 cell injury induced by Aβ. The up-regulation MAPK pathway by GNC may be via ERβ-mediated.