Research On The Protective Effect Of Kang Chan Ning Decoction On The Damage Of PC-12Cell Induced By6-hydroxydopamine

Objective: By means of analysising the PC-12Cells’ rate of survival,apoptosis rate and Caspase-3’expression among different groups, I canresearch the protective effect of Kang Chan Ning decoction on PC-12cellinduced by6-Hydroxydopamine, then research the mechanism of action ofhow Kang Chan Ning decoction cure Parkinson Disease on apoptosis.Method: Fourty healthy male rats of Specific pathogen Free (SPF) wererandomly divided into two groups: the model group(10rats which just be given normal saline2ml twice/day), the drug serum group,(30rats which be given Kang Chan Ning decoction2ml twice/day). One week later, the blood,get from the forty rats’ aorta abdominalis, was stew, centrifuged, andfiltered to get the serum.2. Purchase a PC-12cell lines, and subculture thecell, cryopreserve some bottles of cells. When the cells grew to logarithmicphase, they could be digested and subcultured within36wells (150μl/well) in96-well plates. Every six wells were put under one group named normal cellsgroup, the high dose of decoction group (high dose of traditional Chinesemedicine (TCM) group), the middle dose of decoction group, the low doseof decoction group (low dose TCM group), the rat serum group and thedamage group. The rate of survival could be detected with the method of MTT.3When the cells grew to logarithmic phase, they could be digested andsubcultured within6wells (200μl/well) in6-well plates. Then every welladded2ml complete medium. Each well were put under one group.The rate ofearly apoptosis could be detected by flow cytometry. After testing the cells sixtimes, we can analysis the mean value and the standard deviation to describethe rate early apoptosis.4When the cells grew to logarithmic phase, as themethod above,the cells could be subcultured within6wells (200μl/well) in6-well plates. Then every well added2ml complete medium. Each well were put under one group. The expression of Caspase-3mRNA could be detected byreal-time fluorescence quantification RT-PCR. After testing the cells six times,we can analysis the mean value and the standard deviation of2－△△CTto describe the the expression of Caspase-3mRNA.Results:1By means of MTT, we can see the optical density(OD) of normal cellsgroup, the high dose of decoction group (high dose of traditional Chinesemedicine (TCM) group), the middle dose of decoction group are higher thanthe low dose of decoction group (low dose TCM group), the rat serum groupand the damage group. The optical density of the rat serum group and thedamage group were nearly the same. As the concentration of drug serumgrowing, the optical density grew either.2The rates of early apoptosis were detected by flow cytometry. The rateof the rat serum group and rate of the damage group was nearly the same value.As the concentration of drug serum growing, the rate of early apoptosisreducing with it. The normal cells group also exist early apoptosis cells andeven dead cells or small debris.3Based on the CT value showed in the end, we can get the value of2－△△CT.The mean value of the low dose of decoction group (low dose TCMgroup), the rat serum group and the damage group is higher than it of thenormal cells group, the high dose of decoction group (high dose oftraditional Chinese medicine (TCM) group), the middle dose of decoctiongroup. Compared with the the rat serum group, the expression ofCaspase-3mRNA of the high dose of decoction group decreased distinctly (P＜0.01)Conclusion：1Kang Chan Ning decoction can protect PC-12Cell from the damageinduced by6-Hydroxydopamine.2Kang Chan Ning decoction has the function of anti-apoptosis. Adjustand contro apoptosis-related genes Caspase-3maybe one of its mechanism of action.