Effect Of Liuwei Dihuang Pills On Apoptosis Of PC12 Cells Induced By H ₂ 2

Purpose: This study, taking Phosphoinositide 3-Kinases/protein kinase B(PI3K/Akt) signal pathway as the starting point, adopted cell culture, biochemical detection kits, RT-PCR and Western Blot and other technical means to explore and observe the classical prescription Liuwei Dihuang pills’ protective function on PC12 cell apoptosis damaged by H2O2 and reveal the action mechanism of Liuwei Dihuang pills on nerve cell apoptosis inhibition from the perspective of molecular biology, providing experimental basis and theoretical basis for treating alzheimer’s disease with Liuwei Dihuang pills.Materials and methods: The rats were randomly divided by weight into blank control group and Liuwei Dihuang pills group(rats in this group were treated with Liuwei Dihuang pills). Rats in Liuwei Dihuang pills group were given 10 m L/kg aqueous solution of Liuwei Dihuang pills in 0.75 kg/L crude drug concentration every morning and afternoon by gavage. Rats in the blank control group were given double distilled water with the same volume for 7 days by gavage. Then, abdominal aorta blood was collected 2 h after the first dose, and the blood kept stationary state for 2 hours and then centrifuged at 2000 r/min for 10 minutes to separate serum. After inactivating and 0.22μm filter membrane filtration sterilizating in 56 ℃ for 30 min, the serum was stored at –20 ℃. PC12 cells in the logarithmic phase were taken and vaccinated in 96-well microplate for culture in 200 μl solution at the density of 1×105/ml. After the cells grew to adherent to the wall, culture medium was replaced by no fetal bovine serum. After the cells were cultured for another 24 hours, H2O2 with different concentrations of 100, 150, 200, 250, 300, 350 and 400 μmol/L were added into cell medium. After intervention for 12, 24, 36, and 48 hours respectively, the best intervention time and dose of H2O2 inducing PC12 cell damage were determined. Rats serum from blank control group and rats from Liuwei Dihuang group pills group were selected to prepare respectively cell medium with 5%, 10% and 20% of serumconcentration. In 96-well microplate, the cells were vaccinated with the concentration listed above. The cells could be synchronized after 24 hours starvation, and then cell medium was added with corresponding serum concentration. The cells carried out pre-protection before modelling, and the survival rate of cells was tested by MTT method. The appropriate serum concentration and pre-protection time were selected. With the same method, the concentration of positive control medicine Vit E was conducted. The PC12 cells were divided into four groups:(1) control group,(2) model group,(3)serum containing Liuwei Dihuang pills group,(4) Vit E group. When the cells of each group had been vaccinated for 24 hours and DMEM without fetal bovine serum had been used to culture for 24 hours, DMEM medium containing 10% normal rat serum were used to culture group(1) and group(2) for 24 h, DMEM medium with serum containing Liuwei Dihuang pills was used to culture group(3) for 24 h and DMEM with 10% normal rat serum concentration and Vit E was used to culture group(4) for 24 hours. Then, H2O2 with concentration of 200μmol/L was added in each group to act for 24 hours, and the cell morphology of each group was observed under a microscope. Meanwhile, the cells and supernatant were collected and LDH, SOD, MDA and other biochemical indicators were detected. The objective was to find out whether serum containing Liuwei Dihuang pills plays a protective role in PC12 with hydrogen peroxide damage. The cells in the above four groups were collected for flow cytometry instrument determination and apoptosis related gene(caspase-8, caspase-9, caspase-3, Bax, Bad, Bcl-2) m RNA and protein determination. It aimed to find out whether the serum containing Liuwei Dihuang pills can inhibit PC12 cell apoptosis. The PC12 cells were divided into five groups:(1) control group,(2)model group,(3) serum containing Liuwei Dihuang pills group,(4)Vit E group and(5)PI3K inhibitor group. Group(1), group(2), group(3)and group(4) were treated as above, and PI3 K inhibitor was added in group(5) 30 minutes before H2O2 induces PC12 cell apoptosis. The cells were collected, total proteins of cells in each group were extracted, and the protein expressions of PI3 K, p-Akt, Akt T were measured by western blot. The total RNA was extracted from the cells in each group and m RNA expressions of PI3 K, Akt were measured by RT-PCR. All the above researches were used for finding out whether Liuwei Dihuang pills could affect theinhibition of PC12 cell apoptosis induced by H2O2 through PI3K/Akt signaling transduction pathway.Results: 1 Serum containing Liuwei Dihuang pills’ protective effect on PC12 cell induced by H2O2 1.1Serum containing Liuwei Dihuang pills effect on the proliferation rate of PC12 cell damaged by H2O2 After the screening, we determined 200μmol/l H2O2 acting on PC12 cell for 24 h as a stimulation condition of PC12 cell damage. The intervention by serum containing Liuwei Dihuang pills with different concentrations was done to protect cells. Compared with the model group, the serum containing Liuwei Dihuang pills with the concentration of 5% was used to provide protection for 24 h and 36 h, and cell proliferation rate showed an increasing trend while there was no significant difference(P>0.05); the serum containing Liuwei Dihuang pills with the concentration of 10% was used to provide protection for 24 h, cell proliferation rate increased significantly(P< 0.01); after the serum containing Liuwei Dihuang pills with the concentration of 20% was used to provide protection for 12 h, 24 h and 36 h, cell proliferation rate showed an increasing trend, but the difference was not significant(P>0.05). Finally, we select serum containing Liuwei Dihuang pills with the concentration of 10% provide protection for 24 h as the best treatment condition. 1.2 Serum containing Liuwei Dihuang pills’ effect on general form of PC12 cells damaged by H2O2 Under an inverted microscope, the cells of the blank control group showed fusiform, big cell body, extending and enlarging trunk and branches of cell body, close intercellular contacts, pseudopodia stretching out, a large number of cells, strong proliferation and diopter, and few floating cells off the wall; the cells in model group showed a shorter cell synapse and pseudopods disappearance, and some cells shriveled, turned round, fell off and floated in the culture medium, and the intercellular broadened, cells number became less, diopter weakened, and growing status worsen. In Liuwei Dihuang pills group and Vit E group, cells restretched out pseudopodia, intercellular distance narrowed, more cells grew adherent to the wall and floating cells decreased, diopter were stronger, and growing status became better. 1.3 Serum containing Liuwei Dihuang pills’ effect on LDH in PC12 cell medium damaged by H2O2 In cell medium from normal control group, LDH content is few; compared with it, LDH content in cell medium after H2O2 damage increased significantly(P<0.01); compared with model group, LDH content in Liuwei Dihuang pills group decreased significantly(P<0.01). 1.4 Serum containing Liuwei Dihuang pills’ effect on MDA content in PC12 cell damaged by H2O2 In normal control group, MDA content in cell is few; compared with it, MDA content in cell after H2O2 damage increased significantly(P<0.01); compared with model group, MDA content in Liuwei Dihuang pills group decreased significantly(P<0.01). 1.5 Serum containing Liuwei Dihuang pills’ effect on SOD activity of PC12 cell damaged by H2O2 In normal control group, SOD activity in cell is comparatively high; compared with it, SOD activity in cell after H2O2 damage decreased significantly(P<0.01); compared with model group, SOD activity in Liuwei Dihuang pills group increased significantly(P<0.01). 2 Serum containing Liuwei Dihuang pills’ effect on PC12 cell apoptosis caused by H2O2 damage 2.1 Observation of serum containing Liuwei Dihuang pills’ effect on PC12 cell apoptosis caused by H2O2 damage by means of Annexin V-EGFP/PI double staining After Annexin V-PE/ PI of cells in all groups were dyed, we put them under the inverted fluorescence microscope for observation, and found that in normal control group, there were less and weaker fluorescence and few apoptosis cells; in model group, there were stronger fluorescence, many dyeing cells and many apoptosis cells. Compared with model group, in Liuwei Dihuang pills group and Vit E, there were fewer dyeing cells and apoptosis cells. Compared with normal control group, apoptosis rate in model group increased significantly(P<0.01). Compared with model group, apoptosis rate in Liuwei Dihuang pills group deceased significantly(P<0.01), and decreased to 29.18% plus or minus 3.71%, 28.53% plus or minus 4.52% respectively, 2.2 Effects of serum containing Liuwei Dihuang pills on Bcl-2、Bad、Bax m RNA and protein expression of PC12 cells injuried by H2O2 Compared with normal control group, in model group, m RNA and protein expression ofBcl-2 decreased significantly(P<0.01); compared with model group, m RNA and protein expression of Bcl-2 in Liuwei Dihuang pills group increased significantly(P<0.01); Compared with normal control group, Bax, Bad m RNA and protein expression in model group were significantly higher(P<0.05, P<0.01); compared with model group, Bax, Bad m RNA and protein expression in Liuwei Dihuang pills group decreased significantly(P<0.05); 2.3 Serum containing Liuwei Dihuang pills’ effect on Caspase-9, Caspase-3, Caspase-8 m RNA and protein expression of PC12 cell damaged by H2O2 Compared with normal control group, Caspase-8, Caspase-9, Caspase-3 m RNA and protein expression in model group increased significantly(P<0.01); compared with model group, Caspase-8, Caspase-9, Caspase-3 m RNA and protein expression in Liuwei Dihuang pills group decreased significantly(P<0.01,P<0.05,P<0.01); 3 Serum containing Liuwei Dihuang pills’ effect on PC12 cells’ PI3K/Akt signaling pathway damaged by H2O2 Compared with normal control group, PI3 K, Akt m RNA and protein expression of PI3 K, Akt, p-Akt in model group decreased significantly(P<0.01); compared with model group, PI3 K, Akt m RNA and protein expression of PI3 K, Akt, p-Akt in Liuwei Dihuang pills group increased significantly(P<0.01); Compared with Liuwei Dihuang pills group, PI3 K, Akt m RNA and protein expression of PI3 K, Akt and p-Akt in PI3 K inhibitors group decreased significantly(P<0.01).Conclusion: 1 We use H2O2 exogenous oxidants to successfully simulate and establish stable PC12 cell oxidative damage model. 2 Serum containing Liuwei Dihuang pills can promote cell proliferation of PC12 dama ged by H202, in which serum containing Liuwei Dihuang pills with theconcentration of 10% used for protecting 24 h is most effective for increasing the proliferation rate of PC12 cell damaged by H202. 3 Serum containing Liuwei Dihuang pills play a protective role in PC12 cell apoptosis damaged by H2O2. It can protect the completeness of the membrane structure, reduce the change of cell morphology, reduce LDH leakage and MDA content in cells, increase SOD activity of cells, strengthen cell anti-oxidation ability and inhibit cell oxidative damage. 4 Serum containing Liuwei Dihuang pills can reduce apoptosis rate of PC12 cells dam aged by H202, for it plays a role in inhibiting cell apoptosis. 5 Serum containing Liuwei Dihuang pills can significantly reduce Bax, Bad m RNA and protein expression, and increase Bcl-2 m RNA and protein expression to exert its protection function of inhibiting cell apoptosis. 6 Serum containing Liuwei Dihuang pills can significantly reduce Caspase-8, Caspase-3, Caspase-9 m RNA and protein expression to exert its protection function of inhibiting cell apoptosis. 7 Serum containing Liuwei Dihuang pills can significantly promote protein expression of PI3 K m RNA, Akt m RNA, PI3 K, Akt and p-Akt of PC12 cell. PI3 K inhibitors LY294002 can significantly prevent the promotion of protein expression of PI3 K m RNA, Akt m RNA, PI3 K, Akt and p-Ak of PC12 cell by the serum containing Liuwei Ddihuang pills, indicating that Liuwei Dihuang pills play its role in inhibiting PC12 cell apoptosis through PI3K/Akt pathway to prevent the occurrence and development of AD.