| Objective: To explore the mechanism of angiogenesis mediated by regulation of Notch pathway by Jiawei Xuefu Zhuyu formula in Yang deficiency and blood stasis syndrome induced myocardial ischemia-reperfusion rats.By studying the macroscopic symptoms,Yang deficiency score,Whole Blood viscosity value,Histopathological morphology of myocardial ischemia and reperfusion in a rat model of myocardial ischemia with Yang Deficiency Blood Stasis Treated with Jiawei Xuefu Zhuyu formula,the protein expression of vascular endothelial growth factor VEGF and b FGF,and the inflammation related factor TNF-α,IL-6 and LDH,Notch signaling pathway related proteins including Notch1 and DLL4.Methods: A total of 60 Sprague Dawley(SPF)-grade SD rats were divided into 12 sham operated and 48 sham operated rats by random number table method,and the model rats underwent left anterior descending coronary artery ligation by freezing method combined with doxorubicin intraperitoneal injection for 6 W followed by 15 min reperfusion for 1 h.Sham animals only had a thoracentesis without ligation.Postoperatively,the model rats underwent the second grouping and were randomly divided into a model group,Jiawei Xuefu Zhuyu formula group,tanshinone group,and Jiawei Xuefu Zhuyu formula formula + dapt group.After fabrication,the rats were subjected to Yang deficiency score and measurement of Anal temperature and the macroscopies of the rats were observed.Rats were sacrificed 7 d after drug treatment in each group,and serum levels of tumor necrosis factor alpha(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA)、 Interleukin-6(IL-6)and lactate dehydrogenase(LDH)content,hematoxylin eosin staining(HE)were used to observe the pathological tissue morphology of myocardium,and the protein expressions of Notch1,Dll4,VEGF and b FGF were detected by Western blot(WB),Immunohistochemistry(IHC)was performed to detect newly generated MVD(micro vessel Density)in the ischemic myocardium in the border zone of myocardial infarction labeled with CD31.Results: 1.Macroscopic symptoms: in the sham operated group,the spirit were good,the shape were moderate,the weight were between 260 g and 330 g,the lips and tongue were bright red in color,the tongue body had no abnormal morphology,the hair were soft and emollient,the auricles,the limbs and tail were bright red in color,no abnormalities were observed,the temperament were mild,the stool were shaped,the colour of the stool were normal,the diet were normal,and the action were normal.In the model group,they were apathetic and lethargic,and had body wasting,their weight were between 220 g and 240 g,their abdomen were distended,their weight were reduced,their lips and tongue color were dark purple,their tongue were reddish violet,their hair were yellow and vertical,their extremities were pale and grayish and cool,they gathered and gathered daily,their stools were loose and loose,their stools were normal,their diet were reduced,and their activities were reduced.The mental status of rats in the Jiawei Xuefu Zhuyu formula group were better than that of the model group,with a moderate body shape,a body weight between 260 g and 300 g,dark red color of the lips and tongue,better turned tongue body deviation,significantly better spirit,more emollient hair than that of the model group,better turn of auricular ulcers,slightly irritable character,loose and loose stools,yellow stools,food intake in general,crawling slightly tended to the affected side,and the action was relatively convenient.Jiawei Xue Fu Zhu Yu Fang + dapt group are apathetic,lethargic,and body wasting,with body weight between 220 g and 260 g,distended abdomen,weight loss,dark purple lips and tongue color,red violet tongue coating,yellow and vertical hair,pale and graying hair on the extremities,daily aggregation and pile ups,loose stools,normal urination,reduced diet,and reduced activity.2.Anal temperature: before modeling,there was no significant difference in Anal temperature among rats in each group;After ostomy,compared with the sham operation,the rats in the surgery group had a significant decrease in Anal temperature,and the difference was statistically significant(P < 0.05);After drug administration,compared with the sham group,the anal temperature of the model group decreased(P < 0.05);Compared with the model group,the TCM group and the tanshinone group had increased Anal temperature,and the differences were statistically significant(P < 0.05,P < 0.01).3.Yang deficiency score: compared with the sham group,the prepared model rats of heart yang deficiency developed symptoms of Yang deficiency such as apathy,decreased activity,body huddling,and pile up,and the Yang deficiency score was performed by observing the performance of the rats in each group during the administration period.Compared with the sham group,the Yang deficiency score was significantly higher in the model group.Compared with the model group,the Yang deficiency scores were significantly lower in the Jiawei Xuefu Zhuyu formula group and the tanshinone group.4.Cardiac color ultrasound: compared with the sham group,the EF value of the model group was significantly lower and the difference was statistically significant(P < 0.05),compared with the model group,the EF value of the TCM group increased,the tanshinone group increased,and the EF value of the Jiawei Xuefu Zhuyu formula + dapt group decreased.5.Whole blood viscosity: compared with the model group,the whole blood viscosity in the Jiawei Xuefu Zhuyu group was significantly reduced at the cut rate of 1,50,200 S-1(P < 0.01);The tanshinone group was attenuated significantly at a shear rate of 1,50,200 S-1(P < 0.01,P <0.05),and the Jiawei Xuefu Zhuyu formula + dapt group was attenuated at a shear rate of 1,50,200 S-1(P < 0.01,P < 0.05).6.TNF-α,LDH and IL-6 content: the sera of rats in each group were analyzed by ELISA to detect TNF-α,LDH and IL-6 content,and the results showed that: In the model group compared with the sham group,Serum levels of LDH and TNF-α,IL-6 were significantly increased(all P< 0.01);Compared with the model group,the TNF-α,LDH and IL-6 in the Jiawei Xuefu Zhuyu formula group,Jiawei Xue Fu Zhu Yu formula + dapt group,serum levels decreased significantly(P < 0.01).7.HE staining: The myocardial cells of rats in the Sham surgery group had clear striations,and the fibers were arranged orderly in bundles.The nucleus was located in the middle of the fiber bundles,and the cytoplasm was full.The nucleoli of the nucleus were clear and full,without pathological changes or inflammatory infiltration.However,in the model group of rats,significant myocardial fiber breakage was observed,with irregular and irregular arrangement.There was more cell necrosis,unclear nuclear nucleoli,and a large amount of inflammatory infiltration,exhibiting the characteristics of ischemic damage.After the intervention of modified Xuefu Zhuyu Decoction,the myocardial injury of rats was lighter,the degree of inflammatory cell infiltration and muscle fiber edema was lower,the structure of nucleus and cytoplasm was normal,a large number of myocardial cells survived,and the ischemic injury was significantly reduced compared with the model group.The myocardial injury of rats in tanshinone group was mild,with only a small amount of inflammatory cell infiltration,a small amount of fiber swelling,regular arrangement of myocardial cells,normal nuclear and cytoplasmic structure,and less cell necrosis.In the modified Xuefu Zhuyu Decoction+DAPT group,the myocardial injury was serious,the arrangement of myocardial cells was irregular,the normal myocardial fibers were severely damaged,a large number of inflammatory cells infiltrated,and there were ischemic changes.8.Immunohistochemistry: for angiogenic factor labeling with CD31 on myocardial ischemic tissue of rats in each group,MVC of ischemic myocardial tissue was significantly increased in the model group compared with the sham group(P < 0.01);Compared with the model group,the expression of MVD increased in the ischemic myocardial tissues of rats in the tanshinone group,the Jiawei Xuefu Zhuyu formula group,and the Jiawei Xuefu Zhuyu formula + dapt group.(P <0.05 or P < 0.01);Compared with the tanshinone group,there was no significant difference in MVD in the ischemic myocardial tissue in the Jiawei Xue Fu Zhu Yu formula(P > 0.05),indicating that the Jiawei Xue Fu Zhu Yu formula can increase the microvessel density and promote revascularization of myocardial tissue in the border zone of myocardial infarction in acute MIRI rats,thus establishing a good collateral circulation,but the promoting effect can be similarly reversed by dapt,a specific inhibitor of the Notch signaling pathway,Inhibits angiogenesis.9.Western bolt: compared with the sham group,the protein expression levels of Notch1,Dll4,VEGF,and b FGF were up-regulated in the model group;Compared with the model group,the protein expressions of Notch1,Dll4,VEGF,and b FGF were increased in the Jiawei Xuefu Zhuyu formula group and tanshinone group;The protein expressions of Notch1,Dll4,VEGF,and b FGF were decreased in the Jiawei Xuefu Zhuyu formula + dapt group compared with those in the Jiawei Xuefu Zhuyu formula group.Conclusion: 1.Jiawei Xuefu Zhuyu formula improves symptoms of Yang deficiency and blood stasis,improves myocardial pathomorphological structure,and reduces TNF-α,LDH and IL-6 in serum in rats with myocardial ischemia-reperfusion caused by Yang deficiency and blood stasis,which showed some protective effects on myocardial tissue in myocardial ischemia-reperfusion injury model rats.2.Jiawei Xuefu Zhuyu formula is able to upregulate the protein expression of Notch1 and Dll4 and upregulate the protein expression of VEGF and b FGF in the myocardial ischemic area.Promotes angiogenesis.3.Jiawei Xuefu Zhuyu formula may promote angiogenesis and play a myocardial protective role by promoting the expression of angiogenic factors VEGF and b FGF through the Notch signaling pathway. |
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