Effects of Astragalus injection and Salvia Miltiorrhiza injection on serum inflammatory markers in patients with stable coronary heart disease: a randomized controlled trial study

Background: Coronary heart disease (CHD) is a clinical syndrome caused by coronary atherosclerosis (AS) or functional changes of coronary arteries. Our previous experimental study found that the main component of Astragalus, Astragaloside IV, and the main component of Salvia Miltiorrhiza, Tanshinone IIA, can effectively improve myocardial ischemic injury. Methods: This study was a prospective, randomized, blinded, parallel design trial. A total of 160 eligible patients were randomized to a treatment group (in three groups) or the placebo control group in a 1:1:1:1 ratio using a central randomization system. Patients will receive appropriate treatment for 7 days within 24 hours after enrollment and follow-up for 28 days. Main evaluation indicators: cell count, serum high-sensitivity C-reactive protein (hs-CRP) level, monocyte chemoattractant protein 1 (MCP-1), Interleukin-6(IL-6), IL-1β, IL-8, IL-18, IL-10, Tumor necrosis factor(TNF-α), Oxidized low density lipoprotein(OX-LDL), exosome levels, and angina grade and Traditional Chinese Medicine (TCM) symptom changes scale. Secondary evaluation indicators: B-type natriuretic peptide (BNP) level, troponin (cTn), muscle enzyme isoenzyme (CK-MB), heart-type fatty acid binding protein (H-FABP), liver function, renal function, blood coagulation and the like. Adverse events will be monitored throughout the trial. Discussion: This is a randomized controlled trial of Chinese herbal extracts for the treatment of coronary heart disease. The results of this trial will provide valuable clinical evidence for the recommendations for disease management and identify its underlying mechanisms. CHD: Coronary heart disease; AS: Atherosclerosis; hs-CRP: high-sensitivity C-Reactive Protein; MCP–1: Monocyte Chemoattractant Protein 1; IL–6: Interleukin–6; IL–1β: Interleukin–1β; IL–8: Interleukin–8; IL–18: Interleukin–18; IL–10: Interleukin–10; TNF-α: Tumor necrosis factor; OX-LDL: Oxidized low density lipoprotein; TCM: Traditional Chinese Medicine; BNP: B-type natriuretic peptide; cTn: troponin; CK-MB: muscle enzyme isoenzyme; H-FABP: Heart-type fatty acid binding protein; Percutaneous Coronary Intervention; CABG: Coronary Artery Bypass Grafting; Stable Coronary Atherosclerotic Heart Disease; ASA: Aspirin Capsules; Angiotensin converting enzyme inhibitor; ARBs: Angiotensin-II receptor blockers; receptor blocker; ACS: acute coronary syndrome; Case Report Form; CRA: clinical research associate; PI: investigator;GCP: Good Clinical Practice; SFDA: State Food and Drug Administration; ICF: Informed Consent Form.

and causing acute and temporary ischemia and hypoxia of the myocardium. Coronary atherosclerosis (AS) is a serious disease that endangers human health. In recent years, studies have found that AS is not only a simple lipid deposit, but also an inflammatory disease. Local or systemic inflammatory response plays an important role in the development of AS and its complications [1] . Thus, anti-inflammatory measures may be one of the ideal and scientific means of treating atherosclerosis. In fact, many studies have shown that statins, angiotensin-converting enzyme inhibitors, antiplatelet agents, calcium channel blockers and other drugs have certain anti-inflammatory properties [2] .
As an indicator of inflammatory response, inflammatory factors are endogenous peptides mainly produced by immune cells with strong biological effects, which can mediate a variety of immune responses, which are closely related to the occurrence and development of coronary heart disease [3] . Studies have shown that hs-CRP one of the markers of inflammation, not only marks an increased risk of vascular events, but also helps to judge prognosis, risk stratification, and may be a potential target for the treatment of AS and its complications [4] . In other literatures, hs-CRP, IL-6, IL-8, TNF-α, and MCP-1 are closely related to the pathogenesis of coronary heart disease [5,6] . It can be seen that inflammatory factors participate in the occurrence and development of CHD, and explore the relationship between various inflammatory factors and coronary heart disease, which can have a deeper understanding of the pathogenesis of CHD; Quantitative measurement of various inflammatory factors can also help to dynamically understand the patient's condition. Assess the effects of treatment and then guide clinical practice [7] .
Coronary heart disease belongs to the category of "chest pain" and "heartache" in TCM.
TCM plays an important role in its diagnosis and treatment. In particular, "Yiqihuoxue" method is the most widely used [8] , and among them, Astragalus and Salvia are the most commonly used compatibility drugs. Our previous experimental studies investigated the roles of TCM in CHD [9][10][11][12] . It is shown that Astragalus membranaceus, the main component of Astragalus membranaceus, and Tanshinone IIA, the main component of Salvia Miltiorrhiza, can effectively improve myocardial ischemic injury [13] , and its mechanism is related to the inhibition of cell inflammatory reaction and apoptosis in myocardial ischemic injury [14] .
Based on the above understanding, we hypothesized from the clinical perspective, whether Astragalus injection, Salvia Miltiorrhiza injection or the combination of the two have certain anti-inflammatory properties, and then play a role in the treatment of coronary heart disease. Intendeds to explore the effects of Astragalus injection, Salvia Miltiorrhiza injection and their synergistic treatment on stable coronary heart disease from the anti-inflammatory advantage, and provide the basis and basis for its clinical application.

Study design and settings
Prospective observational studies, randomized, double-blind, parallel design, single-center studies. The study will collect patients in the following four districts: 1. Guangdong Guangzhou Traditional Chinese Medicine University Shunde Hospital; 160 patients will be recruited to meet the eligibility criteria. Subsequently, the participants were divided into four groups in a ratio of 1:1:1:1 (three groups of treatment groups: treatment group A: will receive conventional drugs plus Astragalus injection treatment; treatment group B: will receive conventional drugs plus Salvia Miltiorrhiza injection Treatment; treatment group C: will receive conventional drugs plus Salvia Miltiorrhiza injection plus Astragalus injection treatment), the control group will receive conventional drugs plus 0.9% NaCl injection. The study design is illustrated in Figure.1.

Objectives
This study intends to explore the effects of Astragalus injection, Salvia Miltiorrhiza injection and their synergistic treatment on stable coronary heart disease from the antiinflammatory advantage, and provide the basis and basis for its clinical application.

Research standard
All patients enrolled in the study should meet the following diagnostic criteria and inclusion criteria and sign an informed consent form.

Diagnostic criteria
Coronary heart disease diagnosis: (1) coronary angiography or coronary CTA confirmed at least one major branch lumen diameter stenosis more than 50%, clinical with or without angina, heart failure, arrhythmia, sudden death recovery. Clinical diagnostic criteria for chronic stable angina [15] : Chronic stable angina is a patient whose degree, frequency, nature, and predisposing factors of angina pectoris have not seizures are related to labor or emotional excitement, can be relieved after stopping to rest, sublingual nitroglycerin can quickly relieve symptoms within 2-5 minutes.

Inclusion criteria
The inclusion criteria are as follows: 1.
Meet the Stable coronary atherosclerotic heart disease (SCAD) diagnostic criteria;

2.
The medical history is more than three months, and the frequency of angina pectoris is ≥3 times in the past one week, and the severity of angina pectoris is grade I II/I III; 3. Age 18-75 years old; 4. SCAD patients were treated with any combination of cardiac-related drugs for more than three months prior to enrollment, including atorvastatin, ASA, ACEIs, ARBs, and beta-blockers;

5.
Sign the informed consent form.

Exclusion criteria
The exclusion criteria are as follows:

Randomization
Random numbers are generated using SAS 9.2 software according to the principle of randomization. In order to implement random hiding, the random sequence is kept by a special person. The "remote" random hiding method is used to determine that the basic information of the qualified subject will be transmitted by telephone to the person who keeps the random number, and then the treatment allocation plan of each patient is obtained. Random grouping according to a pre-defined random plan.

Blinding
Due to the special color problem of traditional Chinese medicine injection, this study is difficult to achieve double blindness. However, in order to avoid the bias in the process,  The conventional drugs used in the stable coronary heart disease test are as follows: Refer to 2013 ESC Guidelines for the diagnosis and treatment of stable coronary atherosclerotic heart disease (SCAD) [16] . The standard is given aspirin, clopidogrel, low molecular weight heparin, statins, nitrates, beta-blockers, ACEI and other drugs.

Pharmaceutical and combination methods
The methods of allocation and combination used in this test are as follows:  [17] and TCM symptom change scale [18] , see Table 1 and Table   2.

Safety outcome
Includes vital signs, blood routine, urine routine, fecal routine, liver function, renal function and coagulation. Monitor these indicators closely during this process.

Observation record of adverse events
Any adverse events that occur during the course of the trial, including laboratory abnormalities, must be carefully interrogated and tracked. All adverse events must be judged for their nature, severity, and drug relevance and are strictly recorded in the case report form.

Research process record points
Record the subject content and data at each time point according to the patient's hospitalization period (Figure. 2).

1.
Screening period (0 days): 24 hours before recruitment. The researchers at each clinical research center confirm and sign the complete CRF form, which cannot be modified after the trial, and the data management will save the submitted data. The entire research process must be conducted in a confidential manner, prohibiting the disclosure of any information to other parties, and the main researcher will eventually have access to all data.

Construction of research institutions
In the process of clinical research, multi-department construction and composition are required, and each department must be firmly cooperating to ensure the completion of Ethical issues

Ethics statement
In clinical trials, the main researcher is required to follow the Helsinki Declaration and ethical principles. The investigator must fully respect the subject's choices, and must not force the subject to perform the trial. The subject has the right to participate voluntarily, voluntarily provide and sign a written informed consent form, and may withdraw at any time. In the process, each subject will be randomly assigned a random number to ensure random and blind implementation during the trial. In addition, researchers must protect the privacy, confidentiality, and confidentiality of the subject.  Table 3).

Informed consent form
The informed consent form is completely read and signed by the patient or his representative. The written must be complete. After signing, the main researcher will collect and save it. The researcher will save the informed consent form for review and test.

Discussion
Coronary heart disease is a clinical syndrome caused by coronary atherosclerosis or coronary functional changes, resulting in narrowing of the lumen of the blood vessels, insufficient blood supply to the coronary arteries, and thus cardiac load, causing acute, transient ischemia and hypoxia of the myocardium [19] . Atherosclerosis is one of the main causes of coronary heart disease and cerebrovascular disease, global morbidity and mortality. Whether through medical thrombolysis or interventional or surgical bypass surgery, coronary artery recanalization will directly face myocardial ischemic injury.
Therefore, myocardial ischemic injury has become a major clinical problem, which has attracted wide attention from scholars all over the world [20] . However, there is still no definitive and effective clinical method for the prevention and treatment of myocardial ischemic injury. Therefore, it is of great value to develop effective drugs for preventing and treating myocardial ischemic injury.
Recent studies have shown that chronic inflammation of the intima may play an important role in the pathogenesis of coronary atherosclerotic heart disease. There is a low degree of chronic inflammatory disease in atherosclerosis, the so-called vascular injury-response hypothesis suggests that endothelial dysfunction alters the anti-inflammatory properties of the endothelium. Inflammation plays a major role in the development of atherothrombotic thrombosis and triggers cardiovascular events. Since the 1990s, researchers have linked cardiovascular events to chronic inflammation in clinical research [21] . The role of inflammation in the development of atherosclerosis and sensitivity to predicting cardiovascular events has been identified. Current research has confirmed that the inflammatory process is involved in the atherosclerotic initiation, progression and secondary lesions [22] .
Traditional Chinese medicine has accumulated rich experience in the prevention and treatment of coronary heart disease. With the development of integrated Chinese and western medicine and the gradual deepening of research, TCM has played its own advantages in clinical practice and achieved good efficacy, and its unique TCM believes that the basic pathogenesis of coronary heart disease is qi deficiency and blood stasis, and the origin and symptoms of this disease are deficiency and excess. Compatible with this pathogenesis [20] .Salvia Miltiorrhiza injection is a traditional Chinese medicine preparation, and its main components are Salvia Miltiorrhiza and antibalm. According to the relevant studies of modern medicine, Salvia Miltiorrhiza can enhance the cardiac function, improve the myocardial contraction strength, increase the blood supply to the myocardium, expand the blood v Essels outside the heart, and have a very good antiinflammatory effect [23] . Salvia Miltiorrhiza injection has the effect of promoting blood circulation and removing blood stasis. Relevant studies have found that static dripping Salvia Miltiorrhiza injection can significant reduce inflammatory factors in patients With acute coronary syndrome [24] . It has also reported that in patients with acute myocardial infarction, intravenous Salvia Miltiorrhiza injection can reduce the level of hsCRP and improve the symptoms of chest tightness and pain [25] . Astragalus injection has a good effect Of increase myocardial contractility, and has a good protective effect on myocardial cells, and effectively improves the cardiovascular function of patients. The drug can also strengthen the phagocytic function of macrophages, improve the conversion rate of T cells, promote the synthesis of interferon, improve The activity of interleukin, and has the effect of immune regulation, antiviral replicatio I, allergy inhibition and bacteriostasis. It has been reported that ulinastatin combined with Astragalus injection has a good clinical effect on inflammatory reaction after PCI, which can reduce the expression level of inflammatory factors in patients and reduce the myocardial injury caused by ischemia and reperfusion, And has important clinical application value [26] .
Our previous experimental study found that [13,27] , the main component of Astragalus, Astragaloside IV, and the main component of Salvia Miltiorrhiza, Tanshinone IIA, can effectively improve myocardial ischemic injury, and its mechanism and cellular inflammation in myocardial ischemic injury the inhibition of death is related. However, from a clinical point of view, we are still not very clear about the combination of Astragalus injection, Salvia miltiorrhiza injection or the two, whether it also has certain anti-inflammatory properties, thereby improving coronary heart disease. This study will explore the effects of Astragalus Injection, Salvia Miltiorrhiza Injection and their synergistic treatment on stable coronary heart disease from the anti-inflammatory advantage, and provide the basis and basis for its clinical application.       Content and points of data capture

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