Study Of The Effect And Mechanism Of Heshowuyin On Alleviating The Hippocampi Neurons Injury Induced By Aβ

Alzheimer’s disease (AD) is a common degenerative disease of the central nervous system. It is estimated that there are more than35million people suffering from Alzheimer’s around the world,and the death rate has been increasing, Therefore the study of the disease becomes one of the hotspots at home and abroad. The AD pathology is characterized by neurofibrillary tangles inside nerve cells (hyperphosphorylated Tau protein is the main ingredient) and extracellular senile plaques, which are mainly distributed at the front cortex and hippocampus. The main component of senile plaques is Aβ (β-amyloid protein). It is generated from β-amyloid precursor protein (Amyloid prescursor protein APP)’s abnormal shear and is the main factor which causes the AD pathology. Apoptosis is the main mechanism of which Aβ lead to hippocampal neuronal damage. Previous studies have been reported that many compound preparations mainly contain Polygonum Multiflorum have the protective effect against learning and memory deficits induced by Aβ in rat. It is used extensive for anti-age.But there is no report about the protective effect of Heshouwuyin against damaged hippocampal neuronal induced by Aβ.Objective:To investigate the protective effect of Heshouwuyin on primary hippocampal neuronal cells induced by Aβand its possible mechanisms.Method:1. Cultured hippocampal neurons were from newborn SD rats in24h.2. Model group:after the hippocampi was cultured8days,It is treated with Aβ1-40for48hours, the concentration of the AP1-40is10μmol/L.Heshouwuyin prevention group:after the hippocampi was cultured6days, It is pre-incubated with Heshouwuyin for48hours, the concentration of the Heshouwuyin is10%, then exposed Aβ1-40for48hours, Heshouwuyin treatment group:after the hippocampi was cultured6days, It is pre-incubated with Aβ1-40for48hours, then exposed in Heshouwuyin for48hours, Heshouwuyin control group:after the hippocampi was cultured8days, It is treated with Heshouwuyin for48hours. normal control group:It is cultured for10days, don’t need to add drug.3. Adopt Trypan Blue staining method to observe the change of the hippocampal:Cells’s survival rate. and the living cells can’t be stained. The Heshouwuyin’s effect on the hippocampal cell induced by Aβ is evaluated by the percentage of living cells in total cell.4. Adopt Hoechst33258staining to observe the apoptosis rate of the hippocampal cells: Hippocampal cells cultured10days are then stained by Hoechst33258, A fluorescence microscope is used to observe the dyed nucleus. We selected10visual fields randomly, Then mark the apoptosis rate.5. Detect the expression of Trxl in hippocampal cells of each group:Through immunohistochemistry staining to observe the expression of Trxl, the Trx1mRNA expression level of each group is detected by RT-PCR, while the Trxl protein level is detected by the Western Blot.Results:1. The change of the Cellular morphology The neurons of the control group and Heshouwuyin group grew well with big cell body and Clear boundary,they have long neurite, reticular branch connection; Cells of model group cannot adherence firmly and have lost the previous morphology, the cell body swelling, membranes ruptured in the model group Compared with the control group, surrounded by much cell debris, most neurite were broken into disconnected lines. Compared with model group,the cell body of Heshouwuyin prevention group and the Heshouwuyin treatment group is complete and most cells have connective neurite.2.The change of the Cellular survival rate Use the Trypan Blue staining to detect the survival rate of the model group and the blank control group. Comparison results showed a significant decrease of the model group (P<0.05). The survival rate of Heshouwuyin prevention group and the Heshouwuyin treatment group increased greatly compared with model group(P<0.05). There is no significant difference among Heshouwuyin prevention group, Heshouwuyin treatment group and blank control group (P>0.05)3.. Hoechst33258staining Compared with the blank control group, the apoptosis rate of the model group increase significantly(P<0.05),Compared with the model group, the apoptosis rate of the Heshouwuyin treatment and the Heshouwuyin prevention group reduced distinctly (P<0.05).There is no significant difference among Heshouwuyin prevention group, Heshouwuyin treatment group and blank control group (P>0.05)4. Results of Cellulal immunol staining The Trxl positive substances are brown, located in the cytoplasm, The staining results showed that compared with the blank control group, the model group’s Trxl expression decreased significantly(P<0.05), compared with model group, Heshouwuyin treatment group and the Heshouwuyin prevention group’s Trxl expression increased significantly(P<0.05). Compared with blank control group, Heshouwuyin treatment and the Heshouwuyin prevention group’s Trxl expression increased significantly(P<0.05) and there is no significant difference between Heshouwuyin treatment group and the Heshouwuyin prevention group.5. The results of Trxl protein and mRNA expression the Heshouwuyin treatment group and prevention group were significantly higher than the model group(P<0.05); the model group was much lower than the control group(P<0.05); there were no obvious differences between Heshouwuyin treated group and the Heshouwuyin prevented group (P>0.05), which is the same occasion between the control group and Heshouwuyin group (P>0.05)Conclusion:1. Heshouwuyin can reduce the hippocampal cell apoptosis induced by Aβ, and has a protective effect on primary hippocampal neuronal cells induced by Aβ.2. Heshouwuyin can increase the survival rate of hippocampal neuronal cells induced by Aβ.3. Heshouwuyin protect hippocampal neuronal cells through increase the Trxl expression level.