| Purpose: This research aimed to study the relationship between Wnt signaling pathway and the effect of BuShen YiQi HuoXue traditional Chinese medicine Compound under the theory which the inspirit of kidney responds to ectogenic stimuli.Strengthening the inspirit of kidney can regulate the activity of Wnt signaling to prevent Wnt pathway over-excited,result in preventing cardiac remodeling and improve the therapeutic effect of chronic heart failure.Through establishing the model of chronic heart failure rat,we studied the effects of lisinopril and BuShen YiQi HuoXue traditional Chinese medicine Compound on the ligands and antagonists of Wnt signaling pathway in rats’ serums,and we also tested the effects of lisinopril and BuShen YiQi HuoXue traditional Chinese medicine Compound on the key proteins of Wnt signaling pathway in chronic heart failure rats’ myocardial tissues,in order to find out the relationship between Wnt signaling pathway and the therapeutic effect of chronic heart failure with BuShen YiQi HuoXue traditional Chinese medicine Compound.Materials and Methods:1.20 rats without any treatment were randomly selected from 100 rats as blank control group.While the rest 80 rats were subjected to ligation of anterior descending branch of left coronary artery established as chronic heart failure model.The 80 rats were intraperitoneal injection anesthesia with 10% Chloral hydrate 0.3~0.4mL/100 g.The supine position were fixed on the operating table and after tracheal intubation through the glottis,the rats were connected to small animal ventilator(ventilator parameters were breathing frequency 80~100 times per minute and ventrilator tidal volume 6~8 mL).The needle electrode were injected into the limbs of the rats to observe and record the electrocardiograms during the surgery.The third or forth rib was positioned in the iodine liquor disinfection operation area,and the skin and pectoralis major muscle were cut layer by layer through the third intercostal space of the left chest to expose the ribs.After thoracotomy,the anterior descending branch of left coronary artery was found between the left atrium and pulmonary conus,and was ligated with thread 2~3 mm below its origin point.0.1mL lidocaine was administered on the surface of the heart to prevent ventricular arrhythmia.Closed the chest and used thread to close the chest layer by layer.When the rats restored spontaneous stable breathe,stoped the small animal ventilator and pulled out the tracheal intubation needle.Myocardial infarction was determined by the elevation of ST segment of lead on electrocardiogram,which indicated that the operation was successful.As prophylactic anti-infective treatment,40,000 units penicillin sodium was injected into each rat per day lasting for 3 consecutive days.After one week of routine feeding,the surviving rats were starved with half food.At the same time,they swam exhaustively per day for 4 weeks.During the training period,the model rats showed signs as mental malaise,decreased activity,hair removal and interlaryngeal wheezing,while the rats’ left ventricular ejection fractions(LVEF)< 45% were detected by echocardiography as the chronic heart failure model established.Then these chronic heart failure model rats were included in the next experimental subjects.2.The blank group and the model group rats were with distille water to saline irrigation.The lisinopril western medicine group was given to lisinopril 1.5mg/kg/d,while the BuShen YiQi HuoXue traditional Chinese medicine group was given 9.2g raw Chinese drug kg/d.The two group drugs were dissolved in 3mL distilled water to gavage stomach once per day.Because the rats in the model group and the treatment group died in different numbers during intragastric administration period,12 rats in each group were selectede by random number table to test in the later experiment.After 28 days of continuous administration and 12 hours of fasting treatment,all rats were killed.Blood was collected from abdominal aorta for 5 mL per rat,coagulated naturally for 20 minutes at room temperature,and separated for 20 minutes at 3000 rpm.The supernatant was collected carefully.3.By enzyme-linked immunosorbent assay,the standard samples and the rats’ samples were diluted,sampled,incubated,mixed with liquid,washed,enzymatic and colored.The absorbance of each pore was measured at 450 nm wavelength.Finally,the actual concentration of the sample was calculated.The serum levels of NT-proBNP and cTnI in four groups of rats were detected,then observed the change trend of rats’ serum NT-proBNP and cTnI in each group.4.The classical Wnt signaling ligand Wnt3 a,non-classical Wnt signaling ligands Wnt5a/Wnt11 and Wnt signaling pathway antagonists DKK1,sFRP1 and WIF1 in rats’ serums in four groups were detected by enzyme-linked immunosorbent assay.5.Western blot analysis was used to detect the levels of Dvl3,GSK3β Ser9 phosphorylation and β-catenin protein in rats’ myocardial tissues in four groups.The effects of the disease of chronic heart failure and therapeutic results of chronic heart failure with lisinopril or BuShen YiQi HuoXue traditional Chinese medicine Compound on the key proteins levels or activities of Wnt signaling pathway were analyzed.Results:1.Detection of serum NT-proBNP and cTnI levels of rats in each group.1.1 Compared with the blank group,the serum NT-proBNP level in the model group was significantly higher(P<0.01),and the serum NT-proBNP level in the BuShen YiQi HuoXue group or the serum NT-proBNP level in the lisinopril group was separately significantly higher than that in the blank group(P<0.01),which proved that the serum NT-proBNP level was increasing in chronic heart failure.Compared with the model group,the serum NT-proBNP levels in the BuShen YiQi HuoXue group and the lisinopril group were both significantly lower(P<0.01),but there was no significant difference in the serum NT-proBNP levels between the BuShen YiQi HuoXue group and the lisinopril group(P>0.05).1.2 Compared with the blank group,the level of serum cTnI in the model group was significantly higher(P<0.01),while the level of serum cTnI in the BuShen YiQi HuoXue group or the level of serum cTnI in lisinopril group was separately significantly higher than that in the blank group(P<0.01),suggesting that the level of cTnI in chronic heart failure was increasing.Compared with the model group,the levels of serum cTnI both in the BuShen YiQi HuoXue group and in the lisinopril group were significantly lower with statistical significance(P<0.01).There was no significant difference in the levels of serum cTnI between the BuShen YiQi HuoXue group and the lisinopril group(P>0.05).2.The expressions levels of Wnt signaling ligands and antagonists in serums of rats in each group.2.1 The expression level of classical Wnt signaling ligand Wnt3 a in the serums of rats in each group.Compared with the blank group,the content of Wnt3 a in the serum of the model group decreased significantly with difference(P<0.01).Compared with the model group,the level of Wnt3 a in the serums of rats in the western medicine group increased after treatment with lisinopril,and the level of Wnt3 a in the serums of rats in the traditional Chinese medicine group also increased significantly after treatment with BuShen YiQi HuoXue traditional Chinese medicine(P<0.01).Compared with the western medicine group,Wnt3 a content in serums of rats in the traditional Chinese medicine group was significantly higher with statistical significance(P<0.01).2.2 The expression levels of non-classical Wnt signaling ligands Wnt5 a and Wnt11 in serums of rats in each group.Compared with the blank group,the level of Wnt5 a in serums of rats in the model group was significantly lower,which had statistical difference(P<0.01).Compared with the model group,the level of Wnt5 a in serums of rats in the western medicine group was increased after treatment with lisinopril,and the level of Wnt5 a in serums of rats in the traditional Chinese medicine group was also increased after treatment with BuShen YiQi HuoXue traditional Chinese medicine,which had statistical difference separately(P<0.01).Compared with western medicine group,Wnt5 a content in serums of rats in the Chinese medicine group was significantly higher than that in the western medicine group with statistical significance(P<0.01).Compared with the blank group,the level of Wnt11 in the serums of rats in the model group was significantly lower,which had statistical difference(P<0.01).Compared with the model group,the level of Wnt11 in the serums of rats in the western medicine group was increased after treatment with lisinopril,and the level of Wnt11 in the traditional Chinese medicine group was also significantly increased after treatment with BuShen YiQi HuoXue traditional Chinese medicine,which had statistical difference separately(P<0.01).Compared with the western medicine group,the level of Wnt11 in the Chinese medicine group was significantly higher than that in the western medicine group with statistical significance(P<0.01).2.3 The expression levels of Wnt signaling pathway antagonists DKK1,sFRP1 and WIF1 in the serums of rats of each group.Compared with the blank group,the level of serum Wnt signaling antagonist DKK1 protein in the model group was significantly higher,which had statistical difference(P<0.01).Compared with the model group,the levels of serum DKK1 in the western medicine group treated with lisinopril and in the traditional Chinese medicine group treated with BuShen YiQi HuoXue traditional Chinese medicine were both higher,which had statistical difference separately(P<0.01).Compared with the western medicine group,the serum DKK1 protein level in the Chinese medicine group decreased more significantly(P<0.01).Compared with the blank group,the level of sFRP1 protein in serums of rats in the model group was significantly higher(P<0.01),which had statistical significance.Compared with the model group,the levels of sFRP1 in serums of rats in the western medicine group treated with lisinopril and in the traditional Chinese medicine group treated with BuShen YiQi HuoXue traditional Chinese medicine were significantly lower,which had statistical difference separately(P<0.01).Compared with the western medicine group,the level of sFRP1 protein in serums of rats in the traditional Chinese medicine group was significantly lower,which had statistical difference(P<0.01).Compared with the blank group,the level of WIF1 protein in the serums of rats in the model group was significantly higher,which had statistical difference(P<0.01).Compared with the model group,the levels of WIF1 protein in the serums of rats in the western medicine group treated with lisinopril and in the traditional Chinese medicine group treated with BuShen YiQi HuoXue traditional Chinese medicine were significantly lower,which had statistical difference separately(P<0.01).While the level of WIF1 protein in the serums of rats in the traditional Chinese medicine group was lower than that in the western medicine group,which had significantly statistical difference(P<0.01).2.4 In this study,the levels of Wnt signaling pathway ligands in the serums of rats decreased and the levels of Wnt signaling pathway antagonists in the serums of rats increased significantly in chronic heart failure model,while the levels of Wnt signaling pathway ligands and antagonists recovered obviously after treatment with BuShen YiQi HuoXue traditional Chinese medicine and western medicine lisinopril,and the regulation of BuShen YiQi HuoXue traditional Chinese medicine on Wnt signaling ligands and antagonists was significantly higher than that of lisinopril treatment.The results indicated that the active state of Wnt signaling pathway could sensitively reflect the therapeutic effect of chronic heart failure.Compared with the western medicine lisinopril,the prescription of BuShen YiQi HuoXue traditional Chinese medicine may contain active molecules that directly regulate classical Wnt signaling pathway and non-classical Wnt signaling pathway.It was confirmed for the first time that the prescription of BuShen YiQi HuoXue traditional Chinese medicine could play a role in treating chronic heart failure by regulating the activity of Wnt signaling pathway.3.The expression levels and phosphorylation levels of key proteins in Wnt signaling pathway of rats in each group.3.1 The expression level of Dvl3 protein in myocardial tissues of rats in each group.Compared with the blank group,the level of Dvl3 protein in myocardial tissues of rats in the model group was significantly higher,which had statistical difference(P<0.01).Compared with the model group,the levels of Dvl3 protein in myocardial tissues of rats in the western medicine group and in the traditional Chinese medicine group were significantly lower,which had statistical difference separately(P<0.01).Compared with the western medicine group,the level of Dvl3 protein in myocardial tissue of rats in the traditional Chinese medicine group was significantly lower,which had statistical difference(P<0.01).3.2 The expression level of phosphorylated GSK3β protein in rat myocardial tissues in each group.Compared with the blank group,the level of p-GSK3β protein in rats’ myocardial tissues in the model group was significantly increased,which had statistical difference(P<0.01).Compared with the model group,the levels of p-GSK3β protein in rat myocardial tissues in the western medicine group and in the traditional Chinese medicine group were significantly decreased,which had statistical difference separately(P<0.01).Compared with the western medicine group,the level of p-GSK3β protein in rat myocardial tissues in the traditional Chinese medicine group was significantly decreased,which had statistical difference(P<0.01).3.3 The expression level of β-catenin protein in myocardial tissues of rats in each group.Compared with the blank group,the level of β-catenin protein in myocardial tissues of rats in the model group increased significantly,which had statistical difference(P<0.01).Compared with the model group,the level of β-catenin protein in myocardial tissues of rats in the western medicine group did not decrease(P>0.05),while the level of β-catenin protein in myocardial tissues of rats in the traditional Chinese medicine group decreased significantly(P<0.01).Compared with the western medicine group,the level of β-catenin protein in rat myocardial tissues decreased significantly,which had statistical difference(P<0.01).3.4 This study explored the molecular mechanism of BuShen YiQi HuoXue traditional Chinese medicine regulating Wnt signaling pathway activity in vivo.It found that BuShen YiQi HuoXue traditional Chinese medicine had the strongest influence on Dvl protein expression level,followed by GSK3β protein activity(i.e.phosphorylation state)and β-catenin protein expression level.The results suggested that BuShen YiQi HuoXue traditional Chinese medicine may play a directly and effectively regulatory role in the level of Dvl protein.And BuShen YiQi HuoXue traditional Chinese medicine as a multiple compound drug contained many components and played a regulatory role in different molecular levels of Wnt signaling pathway.It was also noted that although the use of lisinopril as the western medicine group treatment could negatively regulate the level of Dvl protein and GSK3β phosphorylation,it had no effect on the level of core protein β-catenin in classical Wnt signaling pathway,suggested that lisinopril may affect the level of Dvl protein level and GSK3β activity through indirect regulation.It further verified the specificity of Wnt signaling pathway regulated by BuShen YiQi HuoXue traditional Chinese medicine,and provided further important clues and basis for the next step to find out the active molecules in BuShen YiQi HuoXue traditional Chinese medicine,which could regulate Wnt signaling pathway.Conclusions:1.The rat model of chronic heart failure was established by anterior descending branch of left coronary artery ligation and starvation combined with exhaustive swimming training.The therapeutic effects of BuShen YiQi HuoXue traditional Chinese medicine and lisinopril on chronic heart failure were evaluated by detecting the levels of NT-proBNP and cTnI in rat serums.It was found that the therapeutic effect of BuShen YiQi HuoXue traditional Chinese medicine on chronic heart failure was comparable to that of lisinopril.2.The level of Wnt3 a,a classical Wnt signaling ligand in serums of rats with chronic heart failure,increased significantly after treatment with BuShen YiQi HuoXue traditional Chinese medicine.It suggested that classical Wnt signaling pathway participated in the pathological process of chronic heart failure,and BuShen YiQi HuoXue traditional Chinese medicine may play a role in treating chronic heart failure by regulating the activity of classical Wnt signaling pathway.The serum levels of non-classical Wnt signaling ligands Wnt5 a and Wnt11 in rats with chronic heart failure also increased significantly after treatment with BuShen YiQi HuoXue traditional Chinese medicine.These data suggested that non-classical Wnt signaling pathway could also participate in the pathological process of chronic heart failure,and BuShen YiQi HuoXue traditional Chinese medicine may play a role in treating chronic heart failure by regulating the activity of non-classical Wnt signaling pathway,too.The effects of BuShen YiQi HuoXue traditional Chinese medicine on Wnt signaling ligands Wnt3 a,Wnt5a and Wnt11 were significantly better than those of lisinopril,suggested that BuShen YiQi HuoXue traditional Chinese medicine may directly regulate classical Wnt signaling pathway and non-classical Wnt signaling pathway to play a role in the treatment of chronic heart failure,while lisinopril may only indirectly affected classical Wnt signaling pathway and non-classical Wnt signaling pathway.3.BuShen YiQi HuoXue traditional Chinese medicine could significantly reduce the levels of Wnt signaling pathway antagonists DKK1,sFRP1 and WIF1 proteins in serums of rats with chronic heart failure,indicated that BuShen YiQi HuoXue traditional Chinese medicine could play a role in treating chronic heart failure by regulating the expression of Wnt signaling pathway antagonists.4.BuShen YiQi HuoXue traditional Chinese medicine significantly decreased the level of Dvl3 protein,inhibited the phosphorylation of GSK3β at Ser9 site and significantly decreased the level of β-catenin protein in myocardial tissues of rats in the traditional Chinese medicine group.All these results indicated that BuShen YiQi HuoXue traditional Chinese medicine may directly regulate the activity of classical Wnt signaling pathway,and had its therapeutic effect on chronic heart failure.After treatment with lisinopril,only Dvl3 protein level and GSK3β Ser9 phosphorylation level were down-regulated in myocardial tissues of rats in western medicine group,while the level of core protein β-catenin protein in classical Wnt signaling pathway was not changed compared with that in model group,suggested that the effect of lisinopril on Wnt signaling pathway may be indirect.The changes of Dvl,GSK3β and β-catenin were compared horizontally.BuShen YiQi HuoXue traditional Chinese medicine could regulate the level of Dvl protein most obviously,suggesting that BuShen YiQi HuoXue traditional Chinese medicine might specifically play a role mainly by regulating the level of Dvl protein. |
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