| To observe the protective effect of the addition and sustraction of Achyranthes bidentatae in Tong-Sai-Mai preparation onto the normal PC12cells and the induction of different injury model factors in PC12cells and its mechanism in the ischemic stroke prevention. After PC12cells were routinely cultured, the MTT method of cell viability was used to detect the addition and substration of Achyranthes bidentatae in Tong-Sai-Mai preparation for the concentration screening. After determining the concentration of the drug’s safety role, the logarithmic phase of PC12cells were divided into normal control group, model group, Tong-Sai-Mai decoction with high dose group(5mg/ml), Tong-Sai-Mai decoction with middle dose group(0.5mg/ml),Tong-Sai-Mai decoction with low dose group(0.05mg/ml), Tong-Sai-Mai decoction without Achyranthes in high dose group(5mg/ml), Tong-Sai-Mai decoction without Achyranthes bidentatae in middle dose group(0.5mg/ml) and Tong-Sai-Mai decoction without Achyranthes bidentatae in low dose group(0.05mg/ml). Then, the MTT method was used to observe the effect of the addition and substration of Achyranthes bidentatae in Tong-Sai-Mai preparation onto PC12cells different cell injury model. The results showed that various concentrations of the addition and substration of Achyranthes bidentatae in Tong-Sai-Mai preparation on the PC12cells have normal proliferative activity. Among of the7groups of drugs concentrations, the3concentrations(0.05mg/ml,0.5mg/ml,5mg/ml)of the addition and substraction of the Achyranthes bidentatae in Tong-Sai-Mai DMEM solution were selected as low, medium and high concentrations for the following study. After the glutamate, sodium dithinite and potassium chloride irritant causing the PC12cells injury, all these injury models were found to reduce the cell viability. After co-treatment with different concentrations of the addition and substraction Achyranthes bidentatae in Tong-Sai-Mai preparations, the results revealed that the significant protective effects in PC12cells. When Tong-Sai-Mai with Achyranthes bidentatae group compared with Tong-Sai-Mai without Achyranthes bidentatae group, there were no significant differences, implying that the Achyranthes bidentatae played mediated negative results.Various concentrations of the addition and substraction of Achyranthes bidentatae in Tong-Sai-Mai preparation on the PC12cells survival had normal proliferative activity. When the Tong-Sai-Mai with Achyranthes bidentatae group compared with the Tong-Sai-Mai without Achyrathes bidentatae group, there was no significant difference. The addition and substraction of Achyranthes bidentatae in Tong-Sai-Mai preparation could significantly protect PC12cells injury, its mechanism was related to the inhibition of intracellular calcium overload against the free radical damage and the glutamate excitotoxicity. When the Tong-Sai-Mai with Achyranthes bidentatae group compared with the Tong-Sai-Mai without Achyranthes bidentatae group, there was no significant difference, proving the Achyranthes bidentatae in mediating was negative results.After PC12cells were selected routinely cultured, firstly the MTT cell viability was conducted to detect the hydrogen peroxide concentrations damage for fumble screening. After determining the hydrogen peroxide concentrations causing the PC12cells injury, the logarithmic phase of PC12cells were randomly divided into8groups such as normal group, hydrogen peroxide group, Tong-Sai-Mai decoction with high dose group(5mg/ml), Tong-Sai-Mai decoction with middle dose group(0.5mg/ml),Tong-Sai-Mai decoction with low dose group(0.05mg/ml), Tong-Sai-Mai decoction without Achyranthes bidentatae in high dose group(5mg/ml), Tong-Sai-Mai decoction without Achyranthes bidentatae in middle dose group(0.5mg/ml) and Tong-Sai-Mai decoction without Achyranthes bidentatae in low dose group(0.05mg/ml). After the200μmol/L H2O2was added to stimulate the PC12cells and incubated for4h, then the final concentration of drugs (5mg/ml,0.5mg/ml,0.05mg/ml) were added and continuously incubated and cultured for24h. The MTT assay was facilitated to measure and calculate the cell viability the protection rate of the drugs on PC12cells induced by hydrogen peroxide. Finally, the corresponding biochemical indicators like SOD, MDA, NO, LDH and Ca2+were measured. The results revelaed that with the increasing hydrogen peroxide concentration, the PC12cells viability reduced. The cell survival rate,52.3%was selected when the hydrogen peroxide concentration,200μmol/L was chosen as the damage model. The addition and subtraction of Achyranthes bidentatae in Tong-Sai-Mai decoction (5mg/ml,0.5mg/ml,0.05mg/ml) possess protective effects on H2O2-cellular injury model group induced to PC12cells injury. The hydrogen peroxide model group revealed that could reduce the SOD activity and SOD inhibition rate(P≤0.05,P≤0.01), conversely could increase the MDA,NO content, LDH activity and Ca2+levels(P≤0.05,P≤0.01).Each dose of drugs groups could increase the SOD activity and SOD inhibition rate, conversely could reduce the LDH activity, NO content, MDA and Ca2+levels when compared with the hydrogen peroxide model group showing the significant differences(P<0.05,P<0.01). But however, when the Tong-Sai-Mai with Achyranthes bidentatae groups compared with the Tong-Sai-Mai without Achyranthes bidentatae groups, there were no significant differences. The addition and subtraction of the Achyranthes bidentatae in Tong-Sai-Mai decoction revealed the significant protective effects towards the PC12cells injury, its mechanisms are closely-related to the calcium overload, antagonizing oxidative stress and others. When the Tong-Sai-Mai with Achyranthes bidentatae groups compared with the Tong-Sai-Mai without Achyranthes bidentatae groups, there were no significant differences, implying that the Achyranthes bidentatae in mediating with negative results.Tong-Sai-Mai decoction is a Chinese materia medica poly-herbal formulation that applied in treating the brain ischemia. Because it could repress the oxidative stress in vivo study, now we focused on the in vitro study to investigate the mechanism by targeting the oxidative stress dependent signaling. The relation between the neurogenesis and the reactive oxygen species(ROS) production remain largely unexamined. The PC12cells are the excitable cell types widely used as in vitro model for neuronal cells. Most marker genes that are related to neurotoxicity, apoptosis and cell cycles are easily expressed in these cells. The aim of the present study designed is to explore the cyto-protection of the addition and subtraction of Achyranthes bidentatae in Tong-Sai-Mai decoction against hydrogen peroxide(H2O2)-induced apoptosis and the molecular mechanisms underlying in PC12cells. Our findings revealed that H2O2significantly reduced the cell viability and induced apoptosis of PC12cells. The addition and subtraction of Achyranthes in Tong-Sai-Mai decoction protected PC12cells against H2O2-induced cytotoxicity and apoptosis not only by inducing the bcl-2expression, but also reducing the intracellular [Ca2+] concentration, lactate dehydrogenase(LDH) release, the increase of mitochondria membrane potential(MMP), the reduction expression of Caspase-3, bax and the overexpression of inducible nitric oxide synthase (INOS). But however, when the Tong-Sai-Mai with Achyranthes bidentatae groups compared with the Tong-Sai-Mai without Achyranthes bidentatae groups, there were no significant differences. The results of the present study suggested that the cyto-protective effects of the addition and subtraction of Achyranthes in Tong-Sai-Mai decoction might be mediated, at least in part, by the bcl-2-mitochondria-ROS-INOS pathway. When the Tong-Sai-Mai with Achyranthes bidentatae groups compared with the Tong-Sai-Mai without Achyranthes bidentatae groups, there were no significant differences, implying that the Achyranthes bidentatae in mediating with negative results. Due to its non-toxic characteristics, Tong-Sai-Mai decoction could be further developed to treat the neurodegenerative diseases which are closely-associated with the oxidative stress. |
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