Protective Effects of Salvianolate on Myocardial Injury or Myocardial Infarction after Elective Percutaneous Coronary Intervention in NSTE-ACS Patients: A Randomized Placebo-Controlled Trial

To evaluate the protective effects of salvianolate on percutaneous coronary intervention (PCI) related myocardial injury or myocardial infarction after elective PCI in non-ST-segment elevation acute coronary syndrome (NSTE-ACS) patients. A total of 149 patients with NSTE-ACS who underwent elective PCI were enrolled. The patients were randomly allocated in a 1:1 ratio to the salvianolate group (74 cases) or the control group (75 cases). After exclusion criteria of coronary angiography, 60 patients with PCI therapy remained in the salvianolate group and 68 in the control group. The incidence and the severity of PCI related myocardial injury or myocardial infarction, in addition to major adverse cardiac events (MACEs) during 1 year follow-up after PCI were studied between the two groups. Multivariate logistic regression analysis was used to determine the independent factors for PCI related myocardial injury or myocardial infarction after elective PCI. Compared with the control group, salvianolate treatment reduced the incidence of PCI related severe myocardial injury or myocardial infarction (11.7% vs. 26.5%, P=0.035). The rate of MACEs or all-cause death within 1 month or 1 year after the procedure was not significantly different between the two groups. Conclusions: Periprocedural treatment with salvianolate reduces the incidence of PCI related severe myocardial injury or myocardial infarction, although it does not influence clinical prognosis. [Chinese clinical trial registry: ChiCTR1800016992]

In recent years, percutaneous coronary intervention (PCI) has increased rapidly and has played an important role in reducing coronary stenosis and alleviating ischemic symptoms. However, PCI related myocardial injury or myocardial infarction (MI) occurs approximately 20%-40% in stable coronary artery disease (CAD) and 40%-50% in MI patients who received elective PCI. (1) Zeitouni, et al (2) has reported that PCI related MI could negatively impact on clinical prognosis. Patients with periprocedural MI and myocardial injury had a higher rate of cardiovascular events at 30 days and 1 year after PCI. (2) Yet, there have been no proven effective therapies to reduce periprocedural MI or injury safely and economically. It is challenging but also has significant benefit to find any therapy to alleviate PCI related MI or myocardial injury, fi nally to improve clinical prognosis. Salvianolate is a Chinese medicine extracted from Salvia miltiorrhia. Previous studies have confirmed that salvianolate has multiple effects including anti-inflammation, (3) anti-platelet (4) and coronary microcirculation improvement, (5) which could be a good candidate to improve PCI related myocardial injury or MI. Till now, there has been no clinical data about whether salvianolate can reduce incidence or severity of PCI-related myocardial injury or MI in non-ST-segment elevation acute coronary syndrome (NSTE-ACS) patients undergoing elective PCI. We designed this randomized placebo-controlled ©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 Supported by the National Natural Science Foundation of China (No. 81673701 and No. 81573710) trial to investigate its clinical effects on PCI related myocardial injury or MI, hoping to provide evidence for its clinical use in such circumstances.

METHODS Population
All NSTE-ACS patients aged between 18 and 90 years from October 2016 to June 2017 admitted to Huashan Hospital (Shanghai, China) were screened for this study. All patients enrolled have received elective PCI and had normal cardiac troponin T (cTnT) or elevated cTnT with stable or declining tendency prior to PCI. Written informed consent for PCI and this clinical study was obtained by all participants. Exclusion criteria were the followings: (1) increased pre-PCI cTnT values (rising above 20% of the previous value) or undetermined tendency; (2) severe liver dysfunction (ALT/AST >3 times of normal value, but only AST elevation is not excluded); (3) severe renal dysfunction (creatinine clearance rate <30 mL/min); (4) no informed consent; (5) patients with coronary artery bypass grafting history.

Study Design
Eligible patients were enrolled by clinical doctors and randomized as the salvianolate group or the control group via simple random classifi cation. Patients in the salvianolate group received intravenous salvianolate solution (Shanghai Green Valley Pharmaceutical Co. Ltd., China, batch No.1604132) 200 mg diluted in 100 mL normal saline. Patients were given a total of three doses during the perioperative period. The first dose was given before PCI as pretreatment (2 h before PCI at a rate of 60 mL/h), the other two doses were given post PCI to explore its potential cardioprotective effects during the PCI related myocardial injury (24 and 48 h post PCI). The dosage was based on pharmacological information and consistent to the dose which is used in other studies. Patients in the control group received intravenous saline with the same volume and rate. All enrolled patients were blinded to salvianolate treatment and received standard medical care in accordance with the guidelines for the management of patients with NSTE-ACS, such as anti-platelet, anticoagulation therapy, lipid-lowering therapy, angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), or other antihypertensive therapy if clinically indicated. PCI was performed according to the current guidelines for the management of acute coronary syndromes. (6) The specific intervention strategies were dependent on cardiologists' discretion. All patients were followed up in outpatient service or telephone interview at 1 month and 1 year after PCI. This study was also approved as a registered study by the Chinese clinical trial registry (No. ChiCTR1800016992).

Sample Size Estimation
The estimated number of participants in each group was set at 52 by referring to the literature (β=0.2, α=0.05, P1=0.27, P2=0.07), (7) with the percentage of PCI related myocardial injury as the primary outcome measure. In addition, considering a 10% drop-out rate, the planned enrollment for each group was ultimately decided to be 58 subjects, with a total of 116 subjects.

Study Defi nition
PCI related myocardial injury or MI was diagnosed according to the third universal definition of MI. (8) PCI related MI meets the criteria including: (1) cTnT values >5 times of upper reference limit (URL) after PCI if patients with normal baseline cTnT values; (2) post-PCI cTnT values rising >20% of pre-PCI value if patients with elevated pre-PCI cTnT values but cTnT levels are stable or falling. In addition, at least one of the following criteria must be present: (1) evidence of prolonged ischemia (>20 min) as demonstrated by prolonged chest pain; (2) ischemic ST changes or new pathological Q waves; (3) angiographic evidence of a fl ow limiting complication including loss of patency of a side branch, persistent slow-flow, no-reflow, or embolization; (4) imaging evidence with lack of viable myocardium or new regional wall motion abnormality. For patients who have isolated cTnT elevation after PCI, they can only be diagnosed as PCI related myocardial injury.
All PCI-related myocardial injury was further categorized into mild myocardial injury or severe myocardial injury. Mild myocardial injury was defi ned as increase of post-PCI cTnT values <5 times URL with normal baseline values or a rise of post-PCI cTnT values <20% of the baseline value with elevated pre-PCI cTnT. Severe myocardial injury was an elevation of cTnT values >5 times with normal baseline values or a rise of post-PCI cTnT values >20% of the baseline value with elevated pre-PCI cTnT in the absence of ischemic, angiographic or imaging findings. Major coronary artery with stenosis more than 50% is defi ned as impaired vessel.

Measurements
Blood samples were collected in all enrolled patients before and at 8, 24 and 48 h after PCI to measure cTnT to diagnose PCI related myocardial injury or MI. Other baseline biochemical markers were also obtained.
Baseline characteristics including age, body mass index (BMI), past medical history and combined medication prescribed were collected. Angiographic features and procedural characteristics were recorded. Data of patients were collected under anonymous conditions at Huashan Hospital.

Study Endpoints
The primary endpoint of this study is the incidence of MI or severe myocardial injury after PCI. Secondary endpoints are all-cause death and incidence of major adverse cardiac events (MACEs), such as cardiovascular death, hospitalization for heart failure, MI, stent thrombosis, or target vessel revascularization within 1 year after PCI.

Statistical Analysis
Data from all participants were analyzed based on per-protocol (PP) analysis. Baseline clinical and demographic characteristics in addition to angiographic and intervention features were expressed as mean ± standard deviation (x -±s) or median according to their distribution. Normally distributed values were compared by Student t-test. Otherwise, Mann-Whitney U test was used. The differences of proportions were analyzed using the Chi-square test. Multivariate logistic regression was used to find independent protective factors for PCI related severe myocardial injury or MI. Different models will be used to testify salvianolate's effectiveness. The difference of prognosis between control group and salvianolate group was analyzed using Logrank test. Two-sided tests were used and values of P<0.05 were considered to be signifi cant. All analysis was conducted using SAS 9.4 software (SAS institute Inc., Cary, USA).

Patient Enrollment
There were 242 patients screened from October 2016 to June 2017. Among them, 43 patients were excluded because of emergent PCI, 21 patients were excluded for pre-procedural abnormal cTnT with undetermined trend, 23 patients were excluded due to severe hepatic or renal dysfunction, and 6 patients were excluded due to lack of informed consent ( Figure 1). There were totally 149 patients qualifi ed initially and salvianolate or saline was given during procedure. After coronary angiography (CAG), 21 patients were excluded since no PCI received. Finally 128 patients were enrolled with PCI therapy, 60 patients remained in the salvianolate group and 68 in the control group.

Baseline Characteristics
Demographic and clinical features of patients in control group and salvianolate group are shown in Table 1. Patients' age, BMI, concomitant disease, pre-PCI biomarkers such as cTnT, hs-CRP, NT-proBNP were not statistically different between the two groups (P>0.05). And combined medication of two groups did not show statistic difference either (P>0.05).

Coronary Angiography and PCI Characteristics
There was no significant finding in the number of impaired coronary artery vessels (P=0.835) or target vessels pre-PCI TIMI flow grade (P=0.214) between control group and salvianolate group. Similar results were found regarding number of coronary artery (P=0.682) or segments (P=0.849) received PCI, number of stents implanted (P=0.936), total stent length (P=0.735), or target vessels post-PCI TIMI fl ow grade (P=0.498, Table 2).

Primary End Point
There were 53 patients of no or mild myocardial injury (88.3%) and 7 patients of severe myocardial injury or MI (11.7%) in the salvianolate group. And 50 patients of no or mild myocardial injury (73.5%) and 18 patients of severe myocardial injury or MI (26.5%) in the control group. The univariate analysis showed that salvianolate treatment resulted in less severe myocardial injury or MI after PCI compared with the control group (11.7% vs. 26.5%, P=0.035). Multivariate analysis with different models all confirmed salvianolate treatment resulted in less Notes: PCI: percutaneous coronary intervention; cTnT: cardiac troponin T; hs-CRP: high-sensitivity C-reactive protein; NT-proBNP: N-terminal pro-B-type natriuretic peptide; ACEI: angiotensin-converting enzyme inhibitors; ARB: angiotensin receptor blockers

DISCUSSION
As a randomized placebo-controlled clinical study, we reported that salvianolate treatment during periprocedural period could reduce the severity of PCI related myocardial injury or MI in NSTE-ACS patients. NSTE-ACS represents the most frequent indication for CAG and PCI worldwide. According to clinical manifestations and GRACE score, many NSTE-ACS patients can receive elective PCI with stable or declining cTnT pre-PCI. (6) Elective PCI with stable or declining cTnT pre-PCI allows us to investigate incidence and severity of PCI related myocardial injury or MI as any new increase of cTnT after PCI reflects the damage from the procedure. Between control group and salvianolate group, there was no significant difference in baseline characteristics, therefore no noticeable selection bias in this study. For the primary end point, salvianolate treatment reduced severe myocardial injury or MI. After adjustment for many confounding factors, salvianolate treatment still showed signifi cant benefi ts after PCI, which confi rmed salvianolate treatment is a strong and independent Notes: Data are expressed as odds ratios (95% confi dence intervals). All variables are identifi ed as predictors for PCI related myocardial injury or myocardial infarction. Variable for groups: 1-salvianolate, 0-control; variables for other factors: 1-yes, 0-no.

Secondary End Points
There was no significant difference about incidence of all-cause death and MACEs at 1 month (P =0.930 and P =0.722 respectively) and 1 year (P=0.600 and P=0.596 respectively) after PCI between control group and salvianolate group (Table 4 and 5). Kaplan-Meier survival curves of 1 year without MACEs between two groups showed no signifi cant difference (90% vs. 86.8%, P=0.596, Figure 2).

Drug Safety
Salvianolate intravenous injection was safe in clinical application and no adverse reaction was observed during periprocedural period in our study. factor to improve PCI related myocardial injury or MI. PCI related myocardial injury or MI is crucial to influence clinical prognosis. (2) The pathogenesis of PCI related myocardial injury or MI includes vulnerable plaque disruption, distal embolization, (9,10) ischemia/reperfusion injury, oxidative stress, platelet activation, (11) inflammatory cytokines activation (12) or endothelial cell injury induced by balloon inflation or stent implantation during the procedure. When vascular endothelial cells are significantly injured, vasoconstricting factors and cell chemokines are released followed by collagen tissue exposed, (13) which leads to platelet activation, aggregation and adhesion. Finally thrombosis is formed which causes myocardial damage. These mechanisms may interact with each other, which can aggravate the fi nal outcome. (14) Salvianolate is extracted from Salvia miltiorrhiza which contains magnesium lithospermate B, rosmarinic acid and lithospermic acid. (15)(16)(17) Salvianolate treatment showed less severe myocardial injury or MI after PCI via its diverse pharmacological effects. Previous study showed that salvianolate inhibited human platelet activation via phosphodiesterase (PDE) or antagonizing P2Y12 receptor. (4) Salvianolate can also inhibit reactive oxygen species (ROS) production (18) and improve microvascular fl ow by inhibiting oxidative stress and apoptosis. (5) In addition, salvianolate can reduce serum interleukin-6, CRP levels or myocardial ischemia reperfusion injury induced by PCI in atherosclerosis rats. (3,19,20) Salvianolate treatment was safe and well tolerated among multiple clinical studies, including this study. Possible side effects, including headache, dizziness, or gastrointestinal reaction, were not different between salvianolate group and control group according to previous studies. (21) However, our study did not find significant difference in prognosis after salvianolate treatment during 1 year follow-up. The degree of myocardial protection by salvianolate might be not clinically potent enough to impact on clinical prognosis. Larger sample size and multi-center study is warranted to confi rm the benefits of salvianolate. We tested salvianolate with 200 mg daily for total 3 days in our study. In a previous study, 7 days of salvianolate infusion with 400 mg daily can protect contrast-induced nephropathy after PCI. (16) Another study with 400 mg daily for 7 days can attenuate microvascular dysfunction after cardiac ischemia and reperfusion which showed smaller infarct size and improved left ventricular systolic function. (5) With salvianolate treatment with 200 mg daily for 14 days, it can improve unstable angina pectoris with no significant difference of MACEs. (22) Longer course of treatment or higher dose of treatment can be a future direction.
In conclusion, our study reports that salvianolate treatment can alleviate PCI related severe myocardial injury or MI. It shines new approach to improve PCI related myocardial injury or MI, although salvianolate treatment does not improve clinical prognosis.

Author Contributions
Shen W is the guarantor of this article and contributed to the conception of scope and protocol. Shen W, Luo XP, Fan WH and Shi HM contributed to the design of the study. Ou Y, Sun SJ, Shen W and Shi HM performed the study and collected experiment data. Luo JF performed the statistical analysis. Shen YZ and Chen YF obtained funding for the study and Liu HY was responsible for providing salvianolate solutions used in the study.
Ou Y and Sun SJ drafted the initial and final manuscript. All authors have agreed and approved the fi nal manuscript.