Effect And Mechanism Of Benifiting Qi,Warming Yang And Activating Blood On Autophagy Mediated By MPT In Heart Failure

ObjectiveTo observe the effect of Xinyang tablet,the representative prescription of Benifiting Qi,Warming Yang and Activating Blood,on heart failure(HF)rats by using the methods of western blot,MPT open agent,fluorescent probe and transmission electron microscope and immunohistochemical staining.Meanwhile,it should be carry out that the discussion of the effect and mechanism of Benifiting Qi,Warming Yang and Activating Blood on autophagy mediated by MPT in heart failure.Methods1.Copying heart failure model on rats:after anesthesia,aorta abdominails upward double renal artery of rats were ligatured along with a infusion needle and aorta abdominails were narrowed to about 25%-30%.Rats model can be accepted after heart color Doppler ultrasound confirmed the diagnosis of HF 10 days later.2.Experimental animals and grouping:SPF SD rats,male,250±30g.60 rats were randomized into following groups:Control group:the abdominal aorta of rats were separated without constriction.Model group(heart failure group):rats were induced HF by abdominal aorta constriction.Xinyang tablet group:HF model was prepared,and Xinyang tablets were given by gavage in 3 levels of dose(low,middle,high)ATR pretreatment group:HF model was prepared,and Xinyang tablets(middle dose)were given by gavage after a ATR gavage.Control group and model group were given a gavage of normal saline of the same volume with the drug.3.Heart color Doppler ultrasound:EF and FS of Rats were tested by color Doppler ultrasound after anesthesia and skin preparation of chest.4.Western blot:Plasmosin were extracted by myocardial tissue homogenate joined with RIPA.According to the instructions of Beyotime ADA-PAGE gel preparation kit,prepared 10%separation gel and 5%upper gel,filled gel,filled samples,conducted electrophoresis,transfer,antibody incubation and developing.5.Immunohistochemical staining:the heart tissue were operated in accordance with the general steps.6.Transmission electron microscope(TEM):Observed autophagic vacuoles and mitochondria by transmission electron microscope after the fresh ventricular muscle tissues were fixed,dehydrated,embed,sectioned and stained.7.Ultraviolet spectrophotometer to detect mitochondrial permeability transition(MPT)of myocardial cells:the MPT was analyzed by Ultraviolet spectrophotometer at wavelength of 540nm.Results1.Results of heart color Doppler ultrasound:the level of EF and FS of HF group is obviously reduced compared with control group.And all Xinyang tablets group are increased significantly compared with HF group.2.The expression of beclin-1 and cathepsin-D tested by WB:the expression of both protein of HF group is much more obvious than control group.The expression of beclin-1 of middle and high dose group of Xinyang tablets and Capethsin-D of all Xinyang tablets are decreased obviously than HF group.3.The expression of beclin-1 and cathepsin-D tested by immunohistochemical staining:the expression signal is weakest in control group and high dose group is second weaker.The signal of HF group is strongest(++)in all groups.4.The TEM results:The number of autophagic vacuoles of control group obviously increases and decreases when given the treatment of Xinyang tablets.Mitochondria of myocardial cells of HF rats are more swelling than control group.And the swelling is subsided after obtain Xinyang tablets.5.The results of MPT:△A540nm of HF group is much larger than control group meanwhile middle dose and high dose is much smaller than HF group.6.The effect of Xinyang tablets to Beclin-1 and Cathepsin-D after givenATR:the expression of Beclin-1 and Cathepsin-d is much more obvious compared ATR plus Xinyang tablets group with Xinyang tablet group(middle dose).ConclusionAutophagy plays an important role in HF.Cardiac function decreases in HF rats when autophagy markedly increases.The expression of autophay protein and the number of autophagic vacuoles both decrease obviously when Xinyang tablets were given to HF rats.At the same time,the cardiac function of HF rats improves.All the above point out that Xinyang tablets could improve cardiac function of HF rats by the way of decreasing autophagy.MPT of HF rats decreases and the swelling of mitochondrial is subsided when obtain Xinyang tablets.When given Xinyang tablets(middle dose)after using dispark agent ATR,the expression of autophagy proteins increase significantly as compared those in Xinyang tablets group(middle dose).Those remind us that MPP disparks could restrain the function of Xinyang tablets to decrease the expression of myocardial cells’ autophagy.That means it is though controlling MPT that Xinyang tablets effect myocardial cells’ autophay in HF rats.