Protective Effects Of Salidroside From Traditional Chinese Medicine On Oxidative Stress

ObjectiveOxidative stress and peroxide accumulation are the primary causes of various disasters (cerebral ischemia, Alzheimer, cerebral atherosclerosis and so on). H₂O₂ is one of the most active oxygens which closly related to oxidative stress reaction. Its primary outcome OH- has very strong oxidizability, based on this feature, it can invade cells, moreover, it can destroy the balance between active oxygen and anti-oxidation metabolization, and it can decline activity of antioxidase, meanwhile, it can damage the biomacromolecule in the organism as lipid, protein, and DNA, which can cause the change of enzymatic activity, metabolic disturbance, the damage of DNA and apoptpsis. The organism itself has the ability to fix the the damage of oxidation. For example, superoxide dismutase(SOD)and peroxidase (POD) are two primary antioxidases in the organism, and their functions are cleaning the free radical in the organism. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) is a multiple-function protein. It plays an important role in adjusting and controlling many biological processes, such as DNA base excision repair(BER), the regulation of redox of transcription factor and the regulation of reactive oxygen species. It is an important antioxidase enzyme which it can fix DNA oxidative damage. Accordingly, improving the activity of antioxidase enzyme can benefit the organism to prevent and cure the disasters mentioned above.Based on the research in recent years, a lot of Chinese medicines have special features like antihypoxic, anti-oxidation and improve hemorheology which can benefit human body, but its anti-oxidation mechanism is still under study. This paper builds the cell model of screening Antioxidants, and the result comes from screening out dozens of Chinese medicines. Based on the experiment, this paper also argues about the anti-oxidation irritability and its mechanism of the primary ingredient of Chinese medicine Salidroside. The paper states the relationship between the effection of APE/Ref-1, hydrogen peroxide and the cell damage of PC12 cell. It provides theory evidence to the prevention and therapy of age-realated cardiovascular diseases and nervous system diseases caused by the damage of oxidative stress.Methods1. Building the model of antioxidants screen: using the cell named PC12, incubated the cell in different densities of H₂O₂ (set 0, 50, 100, 200, 300, 400μM) for one hour, and using the density of 200μM H₂O₂ to react 30 minutes, 1 hour, 2 hour, 4 hours, 6hours, 12 hours, and then, screened out by MTT colorimetry assay. The research selects Vitamin E as positive control medicine, and then builds cell model which is used for the screening of antioxidants.2. The screen of anti-oxidation Chinese medicines ingredients: utilizing the anti-oxidation model built in last step, the researcher uses different densities of Chinese medicine and the cell of PC12 to incubate together for 30 minutes first, and then add H₂O₂ (the density is 200μM) to react for one hour. The researcher uses 50 different kinds of Chinese medicine ingredient to process the screen of anti-oxidation protection capability.3. The discussion of Sal's anti-oxidation protecting and its mechanism: selecting extractive form Chinese medicine Salidroside based on its feature of anti-oxidation, do a further discussion about its anti-oxidation protecting feature. What is the mechanism of Sal's anti-oxidation? The researcher hypothesizes that the anti-oxidation protecting feature of Sal relates to improving the toleration of the oxidative damage, alleviating the cell damage and accelerating cell multiplication. For discussing the protecting mechanism, the experiment selects three different densities of Sal (1,10,100μM) to intervene PC12 cell for 30 minutes, and then sets two different groups which includes normal matched groups (adds the same size cell culture fluid with the group of damage) and the group of oxidative damage (adds H₂O₂ density of 200μM and reacts for one hour). The researcher uses different methods as MTT, observation of cytomorphology and Typan blue to observe the affection of anti-oxidation. The researcher uses the detection of BrdU flow cytometry method to detect cell multiplication, and uses Western Blot method to observe the change of APE/Ref-1's expression, analyzes the possible mechnism of Sal's anti-oxidation damage mechanism.Results1. MIT method detects that the result of PC12 cell processing H₂O₂ oxidative damage. The experiment shows that the different densities of H₂O₂ (200, 300, 400μmol/L) could incur the damage of cell after one hour. The livability of the cell obviously declines, and the level of damage depends on the density of the solution. At the other hand, by using 200μM H₂O₂, the level of damage depends on the length of time period. Positive comparison medicine Vitamin E could alleviate the cell damage caused by oxidation. Moreover, it has dosage dependency. Based on the above conclusions, the researcher successfully builds the screen model of anti-oxidation damage.2. Based on the model built by last step, the medicine with different densities are co-incubated with PC12 for 30min, then treats with H₂O₂ of 200μM for 1h, and then screened out from 50 types of Chinese medicine component. The researcher finds 34 kinds of efficient medical composition in total, from which the researcher selectes the one having the most significant anti-oxidation effect– Sal as the target for the next step.3. The anti-oxidation effects of Sal and the study of its protection mechanism(1) Compared with normal group, MIT results shows that the live probability of PC12 cells decreased dramatically after treating with H₂O₂ of 200μM for 1h. Sal with concentration of 0.5μM and 1μM would slightly increase the live probability of PC12 cells after damage; Sal with density of 10μM and 100μM would greatly increase the live probability of PC12 cells; while the protection effects of Sal of 200μM and 400μM were not obvious.(2) The morphology results by inverted phase contrast microscope shows that the oxidation damage could result in the swell of cells, and some shows necrosis symptoms, like void and etc. inside the cells. Sal with density of 1,10,100μM could all alleviate the morphological changes in the cell after oxidation damage, like lessening cell swelling, maintaining the cell structure and etc. (3) The results by Trypan blue counting shows that the mortality of PC12 after damage increased apparently compared with the normal group. However, the Sal with concentration of 1,10,100μM would suppress the mortality of cells after oxidation damage, and this suppression effect is concentration dependent.(4) The BrdU results by flow cytometry method shows that the damaged PC12 cells appeared to be proliferating after Sal treatment compared to the ones in normal groups. The proliferation rate is proportional to the Sal density, further proving that Sal has the cell proliferation effect.(5) Western Blot method is applied to observe the APE/Ref-1 expression changes inside cells. The results shows that H₂O₂ damage could dramatically decrease the APE/Ref-1 expression.Conclusion1. The researcher has built the model of anti-oxidative damage screen and uses it for a large quantity of medical screen to select 34 types of anti-oxidation compositions out of 50 medical components.2. Sal has the protective effect to H202-induced oxidation damage on PC12 cells.3. The researcher first proves in the cell level that the anti-oxidative stress of Sal might be related to its cell proliferation function.4. The researcher has validated the relation between APE/Ref-1 expression and anti-oxidative stress protective function in the PC12 cell anti-oxidative stress model, but it still needs further evidence to prove that whether the anti-oxidation damage function implements by the affection to APE/Ref-1 expression or not.