Preliminary To Study The Protective Effect Of The Total Flavonoids From Rosa Laevigata Michx Fruit On Cardiovascular And Cerebrovascular Diseases

Objective: The aim of the study was to evaluate the protective effect and thepossible mechanism of the total flavonoids （TFs） from Rosa Laevigata Michx fruit（RLMF） on cardiovascular and cerebrovascular diseases （CCVD）.Methods: This study was carried out as follows. In vitro test: First, the protectiveeffect of the TFs on the injury of HUVECs induced by H₂O₂was investigated. The besttreatment time and concentration of H₂O₂, and the optimal pretreatment time andconcentration of the TFs were optimized. Then, the cell cycle was detected by flowcytometry and the protective effect of TFs on nucleus was observed by DAPI staining.The ΔΨm was evalutated by Rhodamine123staining and the mitochondrialmicrostructure was observed by TEM. Furthermore, the levels of ROS, GSH, NO andthe activities of Caspase-3and-9were also detected. In addition, the expressions ofsome related proteins were evaluated by western blotting （Proaspase-3, Bcl-2, Bak, Bax,Bid and p53）. In vivo tests:（1） KM mice were intragastrically administered with the TFs（25,50and100mg/kg） for ten days, and aspirin was used as the positive control. Then,the time of breath, the content of MDA, the activities of SOD and GSH-PX were alldetermined. The histopathology of brain was made, and the expressions of someproteins related to mitochondrial pathway were evaluated （Proaspase-3, Bcl-2, Bak,Bax and Bid）.（2） KM mice were intragastrically administered with the TFs （25,50and100mg/kg） for ten days, and the lengths of the blacktail induced by carrageenin for24,48and72h were measured.（3） Wistar rats were oral administered with the TFs （20,40and60mg/kg） for ten days. Then, the wet and dry weights of thrombus in arteriovenousshunt thrombosis test were measured.Result: In vitro experiment, the inhibition rate of HUVECs incubated with100μM H₂O₂for2h was reached to63%. However, this damage was protected by pretreatment with the TFs for1h. The TFs decreased the cell rate of S phase to22.56%,28.61%and29.56%compared with model group （38.19%）. The TFs suppressed nuclearmorphological damage and inhibited the collapse of ΔΨm. Compared with model group,the relative fluorescence intensity and the morphology of mitochondria increased andchanged well in the tested groups. The excessive ROS generation was also attenuatedby the TFs. And the levels of GSH in the TFs groups were1.14,1.40（p <0.05） and1.60（p <0.01） times than H₂O₂group, while there was no difference on the NO levelbetween each group. In addition, compared with model group, the TFs depressed theactivities of Caspase-3and-9, increased the expressions of Procaspase-3and Bcl-2, anddecreased the expressions of Bak, Bax, Bid and p53. In vivo tests, compared with modelgroup, the TFs significantly increased the time of breath, the activities of SOD andGSH-PX, and reduced the content of MDA. Furthermore, the expressions of Bax, Bakand Bid were all significantly down-regulated, while the expre-ssions of Procaspase-3and Bcl-2were up-regulated. The TFs significantly reduced the length of black-tail, thewet and dry weight of thrombus in a dose-dependent manner.Conclusion: In the experiments, the protective effect and possible mechanism ofthe TFs on CCVD in vivo and in vitro were investigated. These results indicate that theTFs has excellent activities on CCVD, which means that the TFs should be developed tobe a useful drug for the prevention and treatment of CCVD.