Research method for Wnt signal channel regulation mechanism of myocardial infarction rats by astragalus membranaceus-ginseng qi-tonifying dropping pills and application of astragalus membranaceus-ginseng qi-tonifying dropping pills

Abstract

The invention provides a method for researching a Wnt signal channel regulation mechanism of a myocardial infarction rat by using radix astragali and radix salviae miltiorrhizae qi-tonifying dropping pill and application, and belongs to the technical field of traditional Chinese medicines. The research method comprises the following steps: step 1, randomly dividing prepared myocardial infarction rat models into a radix astragali-ginseng qi-tonifying dropping pill high-dose group, a radix astragali-ginseng qi-tonifying dropping pill medium-dose group, a radix astragali-ginseng qi-tonifying dropping pill low-dose group, a western medicine group and a model group, and respectively performing four batches of experiments in the first, fourth, seventh and fourteenth days; 2, index detection is carried out on the second day after the corresponding intervention time ends; and step 3, statistical analysis. Attention is given to a Wnt signal channel, regulation and control of the channel have important significance on secondary prevention of myocardial infarction, but at present, drug intervention researches aiming at the mechanism are not more, and the research takes Chinese patent medicines as test drugs, positive control is set, the treatment value of traditional Chinese medicines on myocardial infarction is explored, the advantages of the traditional Chinese medicines are explored, and a basis is provided for optimizing a clinical treatment scheme.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a research method and application of the Wnt signaling pathway regulatory mechanism of Qishen Yiqi Dropping Pills on rats with myocardial infarction. Background Art

[0002] Acute myocardial infarction (AMI) is a serious threat to human life and health. Due to the great progress of coronary revascularization, the mortality rate of primary acute myocardial infarction has been reduced. However, the long-term prognosis of surviving patients is not optimistic. They may suffer from reinfarction, heart failure or sudden death. Studies have found that the mortality rate of reinfarction is higher than that of primary infarction. Therefore, it is necessary to strengthen secondary prevention after myocardial infarction. Western medicine mainly uses conventional drug treatment: antithrombotic agents, statins, β-blockers and angiotensin-converting enzyme inhibitors. Although this regimen is effective, the current clinical use rate does not meet the expectations of the guidelines. The reasons may be: the use of antithrombotic agents has the risk of bleeding, statins may damage the liver, and β-blockers have contraindications. Because multiple drugs need to be taken at the same time, it also affects the patient's compliance to a certain extent.

[0003] The occurrence and development of myocardial infarction may be related to the signal transduction of multiple signal pathways. The Wnt signal pathway is the most popular one currently. The Wnt signal pathway is the most basic signal transduction pathway in humans. It regulates embryonic development and the occurrence and development of various diseases. It is closely related to myocardial infarction and participates in pathological processes such as atherosclerosis, damage repair and adverse remodeling after myocardial infarction, and heart failure.

[0004] The Wnt signaling pathway is a highly evolutionarily conserved complex protein network. It is divided into classical and non-classical transduction pathways according to the different signal transduction mechanisms of the proteins it expresses. The former mainly transduces signals through the activation of β-catenin; the latter relies on G protein to activate phospholipase C, increase the intracellular Ca2+ concentration, and release Ca2+ transduction signals, also known as the Wnt / Ca2+ pathway. It has been found that the Wnt signaling pathway plays a key role in the development of the heart, and plays an important role in the onset and healing of coronary heart disease, myocardial infarction, and cell therapy for myocardial infarction.

[0005] Coronary atherosclerosis is the most basic cause of myocardial ischemia and infarction. LRP5 and LRP6 are important receptor proteins of the classical Wnt signaling pathway. Studies have found that the plasma low-density lipoprotein and triglyceride levels of mice with LRP6 mutations are increased; LRP5 expression can be found in the arterial walls of high-cholesterol mice, and mild atherosclerosis is shown, indicating that the Wnt signaling pathway is involved in the regulation of lipid metabolism and affects the occurrence and development of atherosclerosis. Christiman et al. detected the expression of Wnt5a in atherosclerotic plaques in humans and mice at the same time, which is direct evidence of the association between the Wnt signaling pathway and atherosclerosis. Many evidences show that the classical Wnt signaling pathway is closely related to the early activation of the vascular endothelium, while the non-classical Wnt signaling pathway plays an important role in the endothelial inflammatory response, which will promote the occurrence of atherosclerosis. Montes et al.'s study confirmed that inhibiting the Wn / β-catenin signaling pathway can reverse the calcification of vascular smooth muscle cells; Ezan's study found that the β-catenin protein of the classical Wnt signaling pathway is highly expressed in vascular endothelial cells caused by vascular injury, and at the same time regulates vascular smooth muscle cell proliferation and apoptosis, which will promote the stabilization of atherosclerotic plaques.

[0006] After myocardial infarction, the heart itself plays a self-repair function. After myocardial infarction, the expression levels of many components of the Wnt signaling pathway will change to varying degrees. These changes are related to the time after the infarction, which means that the Wnt signaling pathway may be related to the healing after MI. In the experiment of Barandon et al., it was detected that the overexpression of sFRP1 caused the expression of interleukin-6 (IL-6) (pro-inflammatory factor) to decrease, while increasing the expression of IL-6 (anti-inflammatory factor), which will improve the hemodynamics of the infarct area, balance the inflammatory factors during injury repair, and reduce the scope of infarction. He et al. found that sFRP2 can reduce collagen deposition, prevent the thinning of the ventricular wall, reduce the scope of infarction, and improve cardiac function. Laeremans et al. found that UM206 can block Wnt3a / Wnt5a, so that it cannot bind to the receptor Fzd1 / 2, ultimately reducing the scope of infarction and effectively preventing the development of heart failure. The process of myocardial infarction is not only accompanied by irreversible necrosis of myocardial cells, but also by pathological apoptosis. The latest research suggests that secreted frizzled-related protein 1 (sFRP1) competes with Fz receptors for binding to Wnt3a protein, preventing the formation of Wnt3a-Fz-LRP5 / 6 complex, inhibiting the activity of Wnt signaling pathway, and thus reducing cardiomyocyte apoptosis and heart failure. Angiogenesis plays a huge role in the repair of myocardial injury and is a prerequisite for restoring blood perfusion in the infarct area after MI. In vitro experiments suggest that the Wnt-frizzled pathway is closely related to the formation of vascular endothelial cells. The proliferation of vascular endothelial cells in culture medium containing overexpressed Wnt1 protein is accelerated, while the gene transcription of Tcf / Lef (classical pathway receptor protein) is increased.

[0007] Left ventricular remodeling after myocardial infarction is a complex dynamic process involving the infarcted and non-infarcted areas, including changes in cardiac morphology, structure, size, and function. This change is initially intended to compensate for necrotic myocardial cells and maintain cardiac function, but will eventually lead to decompensation and affect the patient's long-term prognosis. Ahmad et al. found that the absence of myocardial glycogen synthase kinase 3α (GSK-3α) can alleviate myocardial remodeling after MI, thereby reducing systolic dysfunction and delaying heart failure. Hoehn et al. used transgenic technology to overexpress myocardial protein phosphatase 2A in a chronic MI mouse model and found that this would lead to poor myocardial remodeling, accompanied by the interruption of the classic Akt / GSK3 / β-catenin pathway, further indicating that the Wnt signaling pathway affects myocardial remodeling after infarction. Myocardial fibrosis is involved in the progression of ventricular remodeling, including reparative fibrosis in the infarcted area and reactive fibrosis in the non-infarcted area, accompanied by expansion and structural remodeling of the two areas.

[0008] Recent studies have shown that the reduction of myocardial fibrosis after MI is related to the downregulation of β-catenin, GSK-3β and Wnt1. The Wnt / β-catenin signaling pathway is an important mechanism for mediating tissue and organ fibrosis. Studies have found that CA125 can inhibit the phosphorylation and degradation of β-catenin mediated by GSK-3β, thereby increasing the level of β-catenin and affecting the process of myocardial fibrosis after myocardial infarction.

[0009] Intervention and regulation of the Wnt signaling pathway is of great significance for the secondary prevention and long-term prognosis of myocardial infarction, but current research is mostly focused on the correlation between the Wnt signaling pathway and myocardial infarction, and there are relatively few studies on drug intervention of the Wnt signaling pathway after myocardial infarction. The latest research shows that the Western medicine sacubitril valsartan has a certain effect in inhibiting the Wnt signaling pathway after myocardial infarction.

[0010] Myocardial infarction is called "true heart pain" in traditional Chinese medicine. Traditional Chinese medicine believes that the basic pathogenesis after myocardial infarction is mostly "Qi deficiency and blood stasis". A survey on the distribution of TCM syndromes in 7512 patients also confirmed this. Therefore, "invigorating Qi and activating blood circulation" is the basic treatment method for secondary prevention of myocardial infarction. Some researchers have comprehensively summarized and evaluated the evidence of Chinese medicine in preventing and treating myocardial infarction from basic research, case studies, clinical studies, meta-analysis and other aspects. The results suggest that Chinese medicine has certain advantages in the prevention and treatment of myocardial infarction, and the most obvious one is Chinese medicine that invigorates Qi and activates blood circulation.

[0011] Among the Chinese patent medicines currently used in clinical practice for secondary prevention of myocardial infarction, the one with good efficacy and a good research basis is Qishen Yiqi Dropping Pills. Its compatibility principle is in line with the basic pathogenesis after myocardial infarction. Clinical studies have shown that it has certain advantages in the prevention and treatment of myocardial infarction. Animal experiments have also clarified some of its mechanisms of action, but there have been no reports involving the Wnt signaling pathway. From the perspective of regulating the Wnt signaling pathway, further exploring the effects and specific mechanisms of Qishen Yiqi Dropping Pills on myocardial infarction can, on the one hand, provide more references for its clinical application, and on the other hand, help to improve the evidence chain for the use of Qishen Yiqi Dropping Pills for secondary prevention of myocardial infarction, which will be beneficial for the future systematic sorting out and comprehensive summary of relevant evidence materials, and lay a certain foundation for its going global. Summary of the invention

[0012] In view of the above-mentioned problems and in order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a research method and application of the regulatory mechanism of Qishen Yiqi Dropping Pills on the Wnt signaling pathway in rats with myocardial infarction, and to explore the regulatory rules of Qishen Yiqi Dropping Pills on the Wnt signaling pathway by observing the dynamic changes of relevant indicators in 4 batches; by analyzing the intrinsic connections between the research results, the specific mechanism and related factors of Qishen Yiqi Dropping Pills in regulating the Wnt signaling pathway are explored, providing a reference for further subsequent research.

[0013] The technical solution adopted by the present invention is: In the first aspect, the research method of the present invention for protecting the Wnt signaling pathway regulatory mechanism of Qishen Yiqi Drop Pills on rats with myocardial infarction comprises the following steps: Step 1: The prepared myocardial infarction rat models were randomly divided into a high-dose Qishen Yiqi Dropping Pills group, a medium-dose Qishen Yiqi Dropping Pills group, a low-dose Qishen Yiqi Dropping Pills group, a western medicine group, and a model group, and four batches of experiments were performed on the 1st, 4th, 7th, and 14th days of intervention respectively; Step 2: Conduct index testing on the second day after the corresponding intervention period ends; Step 3: Statistical analysis was performed using STAT professional statistical software. Statistical data were expressed as x±s. Statistical methods used included normality test, variance analysis, q test, rank sum test, and day test.

[0014] In one implementation, the high-dose group of Astragalus and Ginseng was given 600 mg / kg / d of Astragalus and Ginseng Yiqi Dropping Pills by gavage; the medium-dose group of Astragalus and Ginseng was given 300 mg / kg / d of Astragalus and Ginseng Yiqi Dropping Pills by gavage; the low-dose group of Astragalus and Ginseng was given 150 mg / kg / d of Astragalus and Ginseng Yiqi Dropping Pills by gavage.

[0015] In some implementations, in step 1, the intervention time on the first day is 24 hours after the completion of modeling.

[0016] In one part of the implementation, the intervention operation was as follows: the high-dose group of Qishen Yiqi Dropping Pills, the medium-dose group of Qishen Yiqi Dropping Pills, and the low-dose group of Qishen Yiqi Dropping Pills were gavaged with a suspension prepared according to the dosage, 2 mL / time, once a day; the western medicine group was gavaged with sacubitril / valsartan, and the sham operation group and the model group were gavaged with normal saline.

[0017] In some implementations, the indicator detection in step 2 includes: (1) Ultrasound evaluation of cardiac function and ventricular structure; (2) Blood was collected from the inner canthus of the eye, and the concentration of serum SFRP5 was detected by enzyme-linked immunosorbent assay; (3) The rats were killed, the hearts were removed, the infarcted myocardial tissue was dissected, and the infarct area was calculated; (4) Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the expression of Wnt-1 and β-catenin proteins and mRNA in infarcted myocardial tissue; (5) RT-PCR was used to detect the expression of Dishellved (dvl)-1 mRNA; Western-Blot was used to detect the expression of GSK-3β and sFRP-1 in the infarcted myocardium; (6) Analyze the relationship between cardiac function, ventricular structure, infarct size and various indicators in the Wnt signaling pathway.

[0018] In the second aspect, the present invention also protects the use of Qishen Yiqi Dropping Pills in the preparation of a drug for the prevention of the second stage of myocardial infarction. Beneficial Effects

[0019] 1. The present invention focuses on the Wnt signaling pathway. Regulating this pathway is of great significance for the secondary prevention of myocardial infarction. However, there are not many drug intervention studies targeting this mechanism. This study uses Chinese patent medicine as the test drug and sets a positive control to explore the therapeutic value of Chinese medicine for myocardial infarction, tap the advantages of Chinese medicine, and provide a basis for optimizing clinical treatment plans.

[0020] 2. By observing the dynamic changes of relevant indicators in 4 batches, explore the regulatory rules of Qishen Yiqi Drops on the Wnt signaling pathway; by analyzing the intrinsic connections between the research results, explore the specific mechanism and related factors of Qishen Yiqi Drops in regulating the Wnt signaling pathway, and provide a reference for further research.

[0021] 3. Combine the disease stage where TCM has advantages: the recovery period of myocardial infarction, with the Chinese patent medicine with a good research basis: Qishen Yiqi Drops, to further verify the advantages of Qishen Yiqi Drops in the secondary prevention of myocardial infarction, expand the mechanism of action of Qishen Yiqi Drops, improve the evidence chain of evidence-based medicine, and concentrate efforts to promote the internationalization of TCM. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Attached Figure 1 It is the technical circuit diagram of the present invention. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0025] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0026] The specific experimental contents of this embodiment are as follows: 1. Experimental methods 1.1 Grouping and Dosing Two hundred rats with successful modeling were randomly divided into high-, medium-, and low-dose groups of Qishen Yiqi Dropping Pills, a western medicine group, and a model group. Four batches of experiments were conducted on the 1st, 4th, 7th, and 14th days of intervention, with 10 rats in each group. Intervention began 24 hours after surgery. The high-, medium-, and low-dose groups of Qishen were converted to the corresponding dosage according to relevant standards and made into suspension for intragastric administration, 2 mL / time, once a day. The western medicine group was given sacubitril valsartan for intragastric administration. The sham operation group (the treatment method was the same as the model group, only the hanging line was not tied, and the model group and the sham operation group were provided by the laboratory) and the model group were given normal saline for intragastric administration. Four batches of experiments were completed on the 1st, 4th, 7th, and 14th days of intervention, respectively.

[0027] 1.2 Index detection On the second day after the corresponding intervention period (1st, 4th, 7th, 14th day), the indicators were tested: (1) Ultrasound evaluation of cardiac function and ventricular structure; (2) Blood was collected from the inner canthus of the eye, and the concentration of serum SFRP5 was detected by enzyme-linked immunosorbent assay; (3) The rats were killed, the hearts were removed, the infarcted myocardial tissue was dissected, and the infarct area was calculated; (4) Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the expression of Wnt-1 and β-catenin proteins and mRNA in infarcted myocardial tissue; (5) RT-PCR was used to detect the expression of Dishellved (dvl)-1 mRNA; Western-Blot was used to detect the expression of GSK-3β and sFRP-1 in the infarcted myocardium; (6) Analyze the relationship between cardiac function, ventricular structure, infarct size and various indicators in the Wnt signaling pathway.

[0028] 1.3 Statistical analysis STAT professional statistical software was used for analysis. Statistical data were expressed as x±s. Normality test, variance analysis, q test, rank sum test and day test were used for statistical methods. If the data conformed to normal distribution and variance was equal, variance analysis was selected, and q test was used for pairwise comparison. If the data did not conform to normal distribution, day test was used, and rank sum test was used for pairwise comparison. If P<0.01, it means that the difference is statistically significant. Pearson linear correlation was used for correlation analysis.

[0029] 2. Experimental results 2.1 Exploring the regulatory mechanism and rules of Qishen Yiqi Dropping Pills on the Wnt signaling pathway in rats with myocardial infarction 2.2 Analysis of the relationship between cardiac function, ventricular structure, infarct size and various indicators in the Wnt signaling pathway 3. Conclusion To explore the benefits of Qishen Yiqi Drops in the recovery period of myocardial infarction using a new and important mechanism, improve the evidence chain for its use in secondary prevention of myocardial infarction, and contribute to the modernization of traditional Chinese medicine.

[0030] The embodiments described above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belong to the protection scope of the present invention.

Claims

1. A method for studying the regulatory mechanism of Qishen Yiqi Drop Pills on the Wnt signaling pathway in rats with myocardial infarction, characterized in that: The following steps are involved: Step 1: The prepared myocardial infarction rat models were randomly divided into a high-dose Qishen Yiqi Dropping Pills group, a medium-dose Qishen Yiqi Dropping Pills group, a low-dose Qishen Yiqi Dropping Pills group, a western medicine group, and a model group, and four batches of experiments were performed on the 1st, 4th, 7th, and 14th days of intervention respectively; Step 2: Conduct index testing on the second day after the corresponding intervention period ends; Step 3: Statistical analysis was performed using STAT professional statistical software. Statistical data were expressed as x±s. Statistical methods used included normality test, variance analysis, q test, rank sum test, and day test.

2. The research method of the Qishen Yiqi Drop Pills on the Wnt signaling pathway regulation mechanism of rats with myocardial infarction according to claim 1, characterized in that: The high-dose group of Qishen was given Qishen Yiqi Dropping Pills 600 mg / kg / d by gavage; the medium-dose group of Qishen was given Qishen Yiqi Dropping Pills 300 mg / kg / d by gavage; the low-dose group of Qishen was given Qishen Yiqi Dropping Pills 150 mg / kg / d by gavage.

3. The research method of the Qishen Yiqi Drop Pills on the Wnt signaling pathway regulation mechanism of rats with myocardial infarction according to claim 1, characterized in that: In step 1, the intervention time on the first day was 24 hours after the completion of modeling.

4. The research method of the Qishen Yiqi Drop Pills on the Wnt signaling pathway regulation mechanism of rats with myocardial infarction according to claim 3, characterized in that: The intervention operation was as follows: the high-dose Qishen Yiqi Dropping Pills group, the medium-dose Qishen Yiqi Dropping Pills group, and the low-dose Qishen Yiqi Dropping Pills group were gavaged with a suspension prepared according to the dosage, 2 mL / time, once a day; the western medicine group was gavaged with sacubitril-valsartan, and the sham operation group and the model group were gavaged with normal saline.

5. The research method of the Qishen Yiqi Drop Pills on the Wnt signaling pathway regulation mechanism of rats with myocardial infarction according to claim 1, characterized in that: The indicator detection in step 2 includes: (1) Ultrasound evaluation of cardiac function and ventricular structure; (2) Blood was collected from the inner canthus of the eye, and the serum SFRP5 concentration was detected by enzyme-linked immunosorbent assay; (3) The rats were killed, the hearts were removed, the infarcted myocardial tissue was dissected, and the infarct area was calculated; (4) Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the expression of Wnt-1 and β-catenin proteins and mRNA in infarcted myocardial tissue; (5) RT-PCR was used to detect the expression of Dishellved (dvl)-1 mRNA; Western-Blot was used to detect the expression of GSK-3β and sFRP-1 in the infarcted myocardium; (6) Analyze the relationship between cardiac function, ventricular structure, infarct size and various indicators in the Wnt signaling pathway.

6. Application of Qishen Yiqi Dropping Pills in the preparation of secondary prevention drugs for myocardial infarction.

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