| [Purpose]The purpose of this study was to observe the changes of cardiac tissue and cardiac microvessels in the HFpEF model of Dahl-ss rats,to explore the effect of shenyuanxize prescription on the expression of VEGF pathway in HFpEF model rats,and to find a new therapeutic target for the prevention and treatment of HFpEF by traditional Chinese medicine.[Method]1 Experimental grouping and sample collection.1.1 experimental grouping:Twenty SPF male Dahl-ss rats(180-200g)were randomly divided into control group,model group,SFXZF group and LCZ696 group.Control group were given 0.3% NaCl diet for 8 weeks.The ejection fraction retention heart failure model was established by feeding 8% NaCl high-salt diet for 8 weeks.After model established successfully,SFXZF group was gavaged shenfuxiongze prescription6.4g/ml once a day for 8 weeks.LCZ696 group was gavaged the Sacubitril/Valsartan for 60 mg/kg,once a day for 8 weeks.1.2 sample collection:The rats were tested for behavioral indicators.After the rats were anesthetized with pentobarbital sodium(30 mg/kg,intraperitoneal injection),M-type echocardiography,electrocardiogram,and blood from the abdominal aorta were reexamined.Take blood from the abdominal aorta,quickly take out the heart and remove large blood vessels,fat and other non-cardiac tissue.One part of the isolated heart tissue was fixed in a 4% paraformaldehyde solution and the other part was stored in a-80℃ refrigerator for later use.2 Detection indicators:2.1 Behavioral indicators:The general situation of rats in each group was observed,including the weigh of rats,anal temperature,running experiment and swimming exhaustion experiment.2.2 Detection of cardiac function:The change of LVEF was observed by M-type echocardiography.Ecg was used to measure the changes of heart rate and voltage time of each wave.Serum indicators of BNP and NT-pro BNP were detected by ELISA.2.3 Morphological detection:Morphological changes of cardiac myocytes and cardiac microvessels were observed by HE staining.Proliferation of collagen fibers in cardiactissue and perivascular cardiac vessels was observed by Masson staining.Immunohistochemistry detected the positive expression of VEGF,and immuno-fluorescence detected the expression of vWF and CD31.2.4 Western Blotting:The expression changes of cdc42,p38,MAPKAPK and HSP27 proteins in rat cardiac tissue of each group..[Result]1 Behavioral indicators:1.1 The weigh of rats:Compared with the control group,the weigh of rats in the model group was reduced(P<0.05)in the 8 weeks of high-salt diet with 8%NaCl,showing a trend of decline after a slow growth.After 8 weeks of drug intervention,compared with the model group,the weigh of rats in the SFXZF group and the LCZ696 group increased,showing an upward trend(P<0.05).The results showed shenfuxiongze prescription could improve the symptoms of emaciation in rats.1.2 Anal temperature in rats:Compared with the control group,the anal temperature of rats in the model group decreased(P<0.05).Compared with the model group,the anal temperature of rats in the SFXZF group and the LCZ696 group increased(P<0.05).Compared with the control group,there was no significant change in anal temperature in the SFXZF group and the LCZ696 group(P > 0.05).1.3 Rats running experiment:Compared with the control group,the running time of rats in the model group was shorter(P<0.05).Compared with the model group,the running time of rats in the SFXZF group and LCZ696 group was prolonged(P<0.05).Compared with the control group,there was no significant change in running time between the SFXZF group and the LCZ696 group(P > 0.05).1.4 Rats swimming experiment:Compared with the control group,the swimming exhaustion time of rats in the model group was shorter(P<0.05).Compared with model group,the swimming exhaustion time of rats in SFXZF group and LCZ696 group was prolonged(P<0.05).Compared with the control group,the swimming exhaustion time of rats in SFXZF group and LCZ696 group showed no significant change(P > 0.05).2 Detection of cardiac function:2.1 M-type Echocardiography of rats:Compared with the control group,the LVEF value of rats in the model group decreased(P<0.05).Compared with the model group,the LVEF value of rats in the SFXZF group and LCZ696 group increased(P<0.05).2.2 Rat electrocardiogram:Compared with the control group,the heart rate of rats in the model group increased,and the amplitude of p-wave increased,t-waveamplitude,PR interval amplitude(P<0.05).Compared with the model group,rats in the SFXZF group and LCZ696 group experienced decreased heart rate,p-wave decreased,t-wave increased and PR interval long(P<0.05).2.3 BNP and NT-proBNP:Compared with the control group,serum BNP and NT-proBNP were increased in the model group(P<0.05).Compared with the model group,serum BNP and NT-proBNP were significantly decreased in the SFXZF group and LCZ696 group(P<0.05).Compared with the control group,the serum BNP and nt-probnp of SFXZF group and LCZ696 group showed no significant changes(P>0.05).The results showed that shenfuxiongze prescription could improve the degree of heart failure in rats.3 Morphological detection:3.1 HE staining of rat heart tissue:In the control group,the cardiomyocytes were arranged neatly,the nuclei were basically the same size,and the cardiac fibers were clearly striated,without swelling or atrophy,with good continuity and relatively parallel distribution.The vascular wall structure is complete,thin and uniform,and the vascular intima is arranged regularly,showing a normal annular shape.Compared with the control group,the model group showed disordered arrangement of cardiomyocytes,disordered vascular wall structure,different thickness and nuclear size,and most of the myocardial fibers were broken and dissolved.The vascular lesions were severe and the vascular basement membrane was thickened.Compared with the model group,the myocardial cells in the LCZ696 group and the SFXZF group were in a more orderly arrangement,with basically the same size of myocardial cells,complete and regular vascular wall structure,uniform thickness,and relatively regular vascular annular shape.Compared with the model group,the vascular structural lesions in the model group were better.The results showed that shenfuxiongze prescription could promote the restoration of myocardial cells to normal,reduce the rupture and dissolution of myocardial fibers,and improve the injury of cardiac vascular endothelium3.2 Masson staining of rat heart tissues:In the control group,the cardiomyocytes were arranged neatly,and there was a small amount of blue collagen fibers in the interstitium and around the blood vessels.Compared with the control group,the visual field of the model group showed that a large number of collagen fibers were distributed in blue in the myocardial interstitium,and a large number of crisscrossed collagen fibers were observed around the blood vessels.The collagen fiber accumulation increased significantly and the degree of fibrosis significantly increased(P<0.05).Compared with the model group,collagen fibers in the LCZ696 group andthe SFXZF group were arranged in the interstitium of myocardial cells in a branching manner,the collagen fiber accumulation around the blood vessels was reduced,and the degree of myocardial fibrosis was significantly reduced(P<0.05).See figure 8 for details.shenfuxiongze prescription can reduce the deposition of collagen fibers in myocardium of rats with cardiac ejection fraction retention heart failure and reduce the deposition of collagen fibers around their blood vessels.3.3 Immunohistochemistry of VEGF in rat heart tissue:A small amount of positive expression was observed around the myocardial vessel wall in the control group.Compared with the control group,there was a significant increase in the yellow-brown particles and the expression of VEGF protein in the model group(P<0.05).The difference was statistically significant.Compared with the model group,VEGF protein in the SFXZF group and LCZ696 group showed different degrees of decrease(P<0.05).The results showed that shenfuxiongze prescription could reduce the expression of VEGF protein.3.4 Immunofluorescence of vWF and CD31 in rat heart tissue3.4.1 vWF positive expression in heart tissues of rats in each group: Compared with the control group,vWF protein expression was increased in the model group(P<0.05),and the difference was statistically significant.Compared with the model group,the vWF protein expression in the SFXZF group and the LCZ696 group decreased(P<0.05),and the difference was statistically significant.Compared with the control group,there was no significant change in vWF protein expression in the SFXZF group and the LCZ696 group(P>0.05).3.4.2 CD31 positive expression in rat heart tissue in each group: Compared with the control group,CD31 protein expression was significantly increased in the model group(P<0.05).Compared with the model group,CD31 protein expression was significantly decreased in the SFXZF group and the LCZ696 group(P<0.05),and the difference was statistically significant.Compared with the control group,there was no significant change in CD31 protein expression in the SFXZF group and LCZ696group(P>0.05).4 Western Blotting:Western Blotting was used to detect the expression of proteins related to the VEGF pathway in the heart of rats,and to investigate the specific effect of shenfuxiongze prescription on the VEGF conduction pathway in HFpEF rats.Compared with the control group,p38,HSP27,cdc42 and MPKAPK proteins in the model group were all significantly up-regulated(P<0.05),while those in the SFXZF group and LCZ696 group were down-regulated(P<0.05).The resultsshowed that shenfuxiongze prescription could up-regulate the VEGF pathway p38,HSP27,cdc42 and MPKAPK proteins in HFpEF model rat cardiomyocytes.[In conclusion]1 shenfuxiongze prescription can significantly improve HFpEF in rats,improve cardiac microvascular injury,myocardial injury,and reduce the expression of collagen fibers in cardiac tissue and perivascular tissue of fibrotic rats.2 The mechanism of shenfuxiongze prescription for HFpEF intervention may be to improve cardiac microvascular disorders and reduce HFpEF by inhibiting the expression of proteins related to the VEGF pathway in myocardial tissue,such as p38,HSP27,cdc42 and MPKAPK proteins. |
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