| Gastrodiae Rhizoma is the dry tuber of Gastrodia elata Bl.from orchid family.The flavour is sweet,and the nature of it is slightly warm.The effects include suppressing hyperactive liver,calming endogenous wind,relieving spasm and removing obstruction in the meridians.It commonly treats dizziness,headache,numbness of the limbs,hemiplegia,aphasia,convulsive epilepsy of children et al.It mainly contains phenols,organic acids,polysaccharides,sterols and other components,and gastrodin is the main active ingredient of them.Derivatives of gastrodin and gastrodigenin also display important pharmacological activities.Based on the above,the study was guided by Traditional Chinese Medicine theory.We optimized the extraction conditions of Gastrodiae Rhizoma by combining the chemical contents of it and the bioactivities in vitro.The rat atherosclerosis(AS)model induced by high fat diet and the model of oxygenized low density lipoprotein(ox-LDL)induced injury of human umbilical vein endothelial cells(HUVECs)were established.It explains the anti-atherosclerosis effect of Gastrodiae Rhizoma from various perspectives in vivo and in vitro,such as regulating lipid metabolism,antioxidantion,antiinflammation and protection of endothelial cells.In order to investigate its mechanism,the vascular injury model of Tg(fli-1:EGFP)y1 zebrafish induced by vascular endothelial growth factor receptor tyrosine kinase inhibitor Ⅱ(VRI)was established.By studying the protective effect on vascular endothelial injury,the anti-atherosclerosis mechanism of Gastrodiae Rhizoma had been explored,which provided a reference for exploring the effective mechanism of Traditional Chinese medicine on disease in multiple angles and links.The main contents concluded the following parts:Part one:Lliterature reviewThrough relevant literature at home and abroad,the chemical components,pharmacological activities,and clinical application of Gastrodiae Rhizoma were reviewed.In addition,the current treatments of AS of traditional Chinese medicine are also discussed,including the pathogenesis,and active ingredients,single herb and compound preparation which have the therapeutic effects on AS.Part two:Optimal Extraction Study of Gastrodiae Rhizoma by Response Surface Design with Integrated Phytochemical and Bioactivity EvaluationSix gastrodin-type components from Gastrodiae Rhizoma were identified and quantified by high performance liquid chromatography(HPLC),including gastrodin,gastrodigenin,parishin A,parishin B,parishin C and pari shin E.1,1-diphenyl-2-picrylhydrazyl(DPPH)free radical scavenging method was selected to evaluate antioxidant activity,and the effects of protecting cells was evaluated by the repair rate of HUVECs.The above activities were combined as biological activity.The results showed that the IC50 of Gastrodiae Rhizoma scavenging DPPH free radicals ranged from 21.23 to 44.65 mg/mL,and the increasing rate of HUVECs was between 18.13%and 38.65%.It indicated that Gastrodiae Rhizoma had antioxidant activity and could protect the injured endothelial cells.With the integrated bioactivity index and quantitative index,the extraction conditions were optimized and a quadratic polynomial model was obtained for the extraction of the gastrodin-type components in Gastrodiae Rhizoma by the aid of Box-Behnken Design(BBD)and response surface method(RSM).The optimum extraction conditions were listed as follows:ethanol concentration of 41%,liquid-solid ratio of 28.58 mL/g,soaking time of 23.91 h and extraction time of 46.60 min.So,the established method by integrating the phytochemical yields and in vitro bioactivities of Gastrodiae Rhizoma was feasible.Rart three:Effect of Gastrodiae Rhizoma on atherosclerosis model ratsAS is the pathological basis of a variety of cardiovascular diseases,and the pathogenesis of it is complex.In order to explore the regulate lipid metabolism of Gastrodiae Rhizoma on AS,the AS rat model was established by feeding with high fat diet.Sixty SD rats were randomly divided and after by feeding with high-fat diet to induce AS,they were treated with fenofibrate(0.03 g/kg/d)and Gastrodiae Rhizoma extract(0.25,0.5,1 g/kg/d)for 4 weeks.Then the samples of blood and liver tissues were collected for measurement.Animal experiments showed that the liver index of AS model rats increased significantly(P<0.01),and the levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),non-high-density lipoprotein cholesterol(non-HDL-C)and Atherogenic index(AI)in serum also increased significantly(P<0.01).Hemorheological indices showed that the value of high-shear viscosity,medium-shear viscosity,low-shear viscosity,plasma viscosity and red cell assembling index were significantly increased(P<0.01).According to pathological analysis,the thoracic aorta had obvious pathological damage,lipid infiltration,lipid accumulation and plaque.However,all the changes in vivo above were significantly ameliorated by the Gastrodiae Rhizoma extract treatment in dose-dependent manner,while the serum lipid levels and blood viscosity decreased,and pathological damage of liver and artery were repaired.It can be seen that Gastrodiae Rhizoma extract could improve the pathological injury of AS by regulating lipid metabo.After 4 weeks of intragastric administration,levels of oxidative stress,anti-inflammatory molecules and relative factors of vascular endothelium function in serum and liver tissues of rats were measured.The results showed that,Gastrodiae Rhizoma extract could significantly increase the levels of superoxide dismutase(SOD)and glutathione(GSH)in serum and liver tissues of AS model rats(P<0.01),and could also remarkably reduce levels of malonaldehyde(MDA)and lactate dehydrogenase(LDH)(P<0.01).And levels of endothelin 1(ET-1),tumor necrosis factorα(TNF-α),and interleukin 6(IL-6)significantly decreased,while nitric oxide(NO)content significantly increased.It can be seen that the Gastrodiae Rhizoma extract has the effect of anti-atherosclerosis by means of inhibiting the expression of inflammatory and oxidative stress factors.In summary,Gastrodiae Rhizoma extract has effect of anti-atherosclerosis by its antioxidant activity,anti-inflammatory activity and ability of protecting the endothelium against injury.Rart four:Antioxidant and anti-inflammatory effects of Gastrodiae Rhizoma in vitro and in vivo on AS modelIn addition,the results of in vitro experiments showed that the activity of damaged HUVECs cells decreased by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay after ox-LDL induced.The levels of SOD,GSH and NO in the supernatant of cells were significantly decreased(P<0.01),and the levels of MDA,LDH,ET-1,TNF-α and IL-6 were significantly increased(P<0.05,P<0.01).After intervention by Gastrodiae Rhizoma extract,the relative factors of oxidative stress,inflammatory reaction and vascular endothelium function were regulated.Gastrodiae Rhizoma extract could protect HUVECs injured by ox-LDL.Therefore,Gastrodiae Rhizoma extract has effect of anti-atherosclerosis by its antioxidant activity,anti-inflammatory activity and ability of protecting the endothelium against injury.Rart five:Mechanism of Gastrodiae Rhizoma treated AS based on vascular injury model of zebrafishThe Anti-atherosclerosis role and mechanism of Gastrodiae Rhizoma by repairing endothelial cells was explored,by establishing the vascular injury model of Tg(fli-1:EGFP)y1 transgenic zebrafish induced by VRI.After treated by 50,100,200 μg/mL of Gastrodiae Rhizoma extract,the intersegmental blood vessels(ISVs)of zebrafish was observed in each group.Then the numbers of IS Vs were counted and the index of IS Vs was calculated.In addition,the expression of related factors of vascular endothelial growth factor(VEGF)signaling of zebrafish,including antibody kdrl,vascular endothelial growth factor receptor 1(flt-1),von willebrand factor(vWF),Vascular endothelial growth factor A(VEGF-A)was detected by real-time fluorescent quantitative polymerase chain reaction(PCR).The experimental results showed that Gastrodiae Rhizoma extract could promote the growth of injured zebrafish ISVs.Compared with the model group,the index of ISVs increased significantly and the angiogenesis effect was notable(P<0.05,P<0.01).Real-time quantitative PCR showed that Gastrodiae Rhizoma extract could up-regulate the expression of kdrl,flt-1,vWF and VEGF-A.In conclusion,Gastrodiae Rhizoma can promote damaged intersegmental blood vessels by regulating relative factors of VEGF signaling,and the mechanism is related to anti-atherosclerosis effect of it. |
