Clinical effects of tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine in the treatment of AVMC and its effects on serum TNF-α, IL-18 and IL-35.

Clinical effects of tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine in the treatment of acute viral myocarditis (AVMC) were investigated. Eighty-six patients with AVMC treated in Dongying City People's Hospital from August 2016 to July 2017 were selected and randomly divided into control group (n=43) and observation group (n=43). Patients in control group were treated with tanshinone IIA sodium sulfonate, while those in observation group were treated with trimetazidine and levocarnitine. The curative effect and improvement in clinical symptoms were compared between the two groups of patients, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of heart-type fatty acid-binding protein (H-FABP), creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) in patients after treatment. Besides, the changes in levels of serum tumor necrosis factor-α (TNF-α), interleukin (IL)-18 and IL-35 were detected via ELISA. The total effective rate of treatment in observation group was significantly higher than that in control group (p<0.05). The improvement in clinical symptoms in observation group was significantly superior to that in control group (p<0.05). After treatment, levels of H-FABP, CK-MB and cTnI in observation group were obviously lower than those in control group (p<0.05). At 3, 5 and 7 days after treatment, the levels of TNF-α and IL-18 in both groups of patients were decreased compared with those before treatment, but the level of IL-35 was increased compared with that before treatment, and changes in observation group were more significant than those in control group (p<0.05). Tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine has definite curative effects in the treatment of patients with AVMC, which can alleviate myocardial injury with higher safety, and effectively mitigate the inflammatory response in patients, so it is of great clinical significance.

RCT Entities:   Population Interventions Outcomes

Introduction

Acute viral myocarditis (AVMC) is one of the common clinical diseases of the cardiovascular system. It is caused by the invasion of exogenous viruses, such as coxsackievirus, herpes virus, adenovirus, and rubella virus leading to myocardial necrosis, inflammatory cell infiltration and cardiac dysfunction (1). Early clinical manifestations of AVMC are mainly heartache, chest tightness, palpitation, fatigue and cold symptoms, leading to misdiagnosis and missed diagnosis easily. With the progression of disease, arrhythmia, heart failure and even sudden death will occur if not treated in time (2). Clinically, AVMC is usually treated with light diet, nutritional support and bed rest combined with anti-infection, anti-virus and anti-arrhythmia (3). Heart-type fatty acid-binding protein (H-FABP), creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) are the markers of myocardial injury, which can reflect the degree of myocardial damage in patients with AVMC (4). In this study, AVMC patients were treated with tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine, the curative effect was observed, and dynamic changes in tumor necrosis factor-α (TNF-α), interleukin (IL)-18 and IL-35 before and after treatment were analyzed, so as to provide a theoretical basis for the development of AVMC standardized therapeutic regimen.

Materials and methods

General data

A total of 86 patients with AVMC treated in Dongying City People's Hospital (Dongying, China) from August 2016 to July 2017 were selected. Inclusion criteria: i) Patients meeting the diagnostic criteria of AVMC (5); ⅱ) patients with abnormality in R wave-led ST-T segment shown in electrocardiogram for >4 days; and ⅲ) patients who signed the written informed consent. Exclusion criteria: i) Patients with severe renal insufficiency, or a history of heart failure; and ⅱ) patients who were allergic to experimental drugs or pregnant patients. Patients enrolled were divided into control group (n=43) and observation group (n=43) using a random number table. There were no statistically significant differences in general data between the two groups of patients (p>0.05), and data were comparable (Table I). The study was approved by the Ethics Committee of Dongying City People's Hospital. Written informed consents were signed by the patients or their guardians.

Table I.

Comparison of baseline data between the two groups of patients.

	Groups (n=43)
Item	Control	Observation	t/χ2	P-value
Sex (male/female)	22/21	20/23	0.046	0.829
Age (years)	40–80	40–85
Average age (years)	38.34±5.47	39.15±5.56	0.681	0.497
BMI (kg/m2)	22.23±3.15	22.56±3.18	0.483	0.630
NYHA grading
(n, %)
Grade II	13 (30.23)	11 (25.58)	0.058	0.810
Grade III	30 (69.77)	32 (74.42)

BMI, body mass index.

Methods

Treatment

All patients rested in bed and received nutritional support. In control group, tanshinone IIA sodium sulfonate (approval no. NMPN H31022558; Shanghai First Biochemical & Pharmaceutical Co., Ltd., Shanghai, China) was injected intramuscularly once a day (80 mg each time). In observation group, based on the treatment in control group, trimetazidine [approval no. NMPN H20055465; Servier (Tianjin) Pharmaceutical Co., Ltd., Tianjin, China] and levocarnitine (approval no. NMPN H19990372; Shenyang First Pharmaceutical Co., Ltd., of Northeast Pharmaceutical Group, Shenyang, China) were used for treatment, in which trimetazidine tablets were taken at a draught 3 times a day (1 pill each time), and levocarnitine oral liquid was taken during meals. Adults took levocarnitine orally 3 times a day during meals (1 g each time), while the initial dose was 50 mg/kg for children, and it was slowly increased to 100 mg/kg according to the tolerance of children, but the total dose per day was not >3 g. One course of treatment lasted for 14 days for both groups of patients.

Detection of relevant indexes

After one course of treatment, 3 ml venous blood was collected from patients and centrifuged at 1,000 × g at 4°C for 15 min to collect the supernatant. The levels of H-FABP, CK-MB and cTnI were detected via enzyme-linked immunosorbent assay (ELISA), and relevant kits were provided by RapidBio Systems, Inc. (Tucson, AZ, USA). According to the manufacturer's instructions, a microplate reader (Jiangsu Potebio Co., Ltd., Jiangsu, China) was used to read the optical density (OD) value at a wavelength of 450 nm, and the H-FABP, CK-MB and cTnI concentrations were calculated.

Before treatment and at 3, 5 and 7 days after treatment, 4 ml central venous blood was drawn from patients (fasting for >8 h) in the morning, and centrifuged at 1,000 × g at 4°C for 20 min (centrifugal radius of 15 cm). The supernatant was taken and stored in a refrigerator at −80°C. The levels of serum TNF-α, IL-18 and IL-35 were measured via ELISA, and relevant kits were provided by Sangon Biotech Co., Ltd. (Shanghai, China). According to the manufacturer's instructions, the microplate reader was used to read the OD value, and the TNF-α, IL-18 and IL-35 concentrations were calculated.

Evaluation indexes

Evaluation criteria of therapeutic effect

i) Remarkably effective: Clinical symptoms in patients basically disappeared after treatment, and heart rate and R wave-led ST-T segment returned to normal; ⅱ) effective: Clinical symptoms in patients were significantly alleviated after treatment, and improvement degrees in heart rate and R wave-led ST-T segment were >80%; and ⅲ) ineffective: clinical symptoms in patients were not improved or even aggravated after treatment (6). The improvement in symptoms, including palpitation, chest tightness, chest pain, and fatigue, in both groups of patients after treatment were observed, and the incidence rates of adverse reactions, including headache, insomnia, abnormal liver function and abnormal kidney function, in both groups after treatment were also observed.

The concentrations of H-FABP, CK-MB and cTnI in patients after treatment were detected via ELISA, and levels of TNF-α, IL-18 and IL-35 before treatment and at 3, 5 and 7 days after treatment were also detected via ELISA.

Statistical analysis

Statistical Product and Service Solutions (SPSS) 19.0 software (IBM Corp., Armonk, NY, USA) was used for data processing. Measurement data are presented as mean ± standard deviation (SD), and t-test was used. Enumeration data are presented as percentage (%), and Chi-square test was used. P<0.05 suggested that the difference was statistically significant.

Results

Comparison of curative effect between the two groups of patients

After one course of treatment, the total effective rate of treatment in observation group was significantly higher than that in control group (p<0.05) (Table II).

Table II.

Comparison of curative effect between the two groups of patients.

Groups	No.	Remarkably effective	Effective	Ineffective	Total effective rate
Observation	43	31 (72.09)	10 (23.26)	3 (4.65)	41 (95.35)
Control	43	19 (44.19)	14 (32.56)	10 (23.26)	33 (76.74)
χ2			4.754
P-value			0.029

Comparison of improvement in symptoms between the two groups of patients after treatment

After one course of treatment, the improvement in clinical symptoms in observation group was significantly superior to that in control group (p<0.05) (Table III).

Table III.

Comparison of improvement in symptoms between the two groups of patients after treatment.

Groups	No.	Palpitation	Chest tightness	Chest pain	Fatigue
Observation	43	1 (2.32)	0 (0.00)	0 (0.00)	1 (2.32)
Control	43	8 (18.60)	6 (13.95)	6 (13.95)	9 (20.93)
χ2		4.468	4.479	4.479	5.545
P-value		0.035	0.034	0.034	0.019

Levels of H-FABP, CK-MB and cTnI in both groups of patients after treatment

After one course of treatment, levels of H-FABP, CK-MB and cTnI in observation group were obviously lower than those in control group (p<0.05) (Table IV).

Table IV.

Comparison of levels of H-FABP, CK-MB and cTnI between the two groups of patients.

Groups	No.	H-FABP (µg/ml)	CK-MB (U/ml)	cTnI (ng/ml)
Observation	43	0.83±0.14	6.67±3.23	0.08±0.02
Control	43	1.12±0.38	17.27±3.64	0.23±0.07
t-test		4.696	14.283	13.511
P-value		<0.001	<0.001	<0.001

H-FABP, heart-type fatty acid-binding protein; CK-MB, creatine kinase-MB; cTnI, cardiac troponin I.

Comparison of incidence rates of adverse reactions between the two groups of patients

The incidence rates of adverse reactions had no significant differences between the two groups of patients (p>0.05) (Table V).

Table V.

Comparison of adverse reactions between the two groups of patients (n, %).

Groups	No.	Headache	Insomnia	Abnormal liver function	Abnormal kidney function
Observation	43	1 (2.32)	2 (4.65)	1 (2.32)	1 (2.32)
Control	43	3 (6.98)	4 (9.30)	0 (0.00)	2 (4.65)
χ2		0.262	0.179	0.001	0.001
P-value		0.609	0.672	0.999	0.999

Comparison of TNF-α levels between the two groups of patients

The TNF-α levels had no significant difference before treatment between the two groups of patients (p>0.05). At 3, 5 and 7 days after treatment, TNF-α levels in both groups were significantly decreased, and they were decreased more obviously in observation group than that in control group (p<0.05) (Table VI).

Table VI.

Comparison of TNF-α levels between the two groups of patients (ng/l).

Groups	Νο.	Before treatment	3 days after treatment	5 days after treatment	7 days after treatment	F	P-value
Observation	43	84.28±5.25	53.38±3.18	39.23±3.63	13.38±3.26	67.302	<0.001
Control	43	83.84±5.48	60.43±3.42	48.75±3.82	27.43±3.52	39.412	<0.001
t-test		0.380	9.899	11.846	19.203
P-value		0.705	<0.001	<0.001	<0.001

TNF-α, tumor necrosis factor-α.

Comparison of IL-18 levels between the two groups of patients

The IL-18 levels had no significant difference before treatment between the two groups of patients (p>0.05). At 3, 5 and 7 days after treatment, IL-18 levels in both groups were significantly decreased, and they were decreased more obviously in observation group than that in control group (p<0.05) (Table VII).

Table VII.

Comparison of IL-18 levels between the two groups of patients (pg/ml).

Groups	Νο.	Before treatment	3 days after treatment	5 days after treatment	7 days after treatment	F	P-value
Observation	43	165.75±7.25	92.64±4.28	66.36±3.27	23.52±3.16	69.622	<0.001
Control	43	166.82±7.57	118.56±4.63	77.47±3.36	39.56±3.74	54.652	<0.001
t-test		0.669	26.957	15.539	21.482
P-value		0.505	<0.001	<0.001	<0.001

IL, interleukin.

Comparison of IL-35 levels between the two groups of patients

The IL-35 levels had no significant difference before treatment between the two groups of patients (p>0.05). At 3, 5 and 7 days after treatment, IL-35 levels in both groups were significantly increased, and they were increased more obviously in observation group than that in control group (p<0.05) (Table VIII).

Table VIII.

Comparison of IL-35 levels between the two groups of patients (pg/ml).

Groups	Νο.	Before treatment	3 days after treatment	5 days after treatment	7 days after treatment	F	P-value
Observation	43	46.43±3.24	86.82±3.79	117.45±5.32	137.27±7.64	71.622	<0.001
Control	43	47.62±3.58	58.67±3.23	96.35±5.25	121.67±7.23	62.735	<0.001
t-test		1.616	37.069	18.512	9.725
P-value		0.110	<0.001	<0.001	<0.001

IL, interleukin.

Discussion

In AVMC, virus infection leads to myocardial damage in patients, thus inducing abnormal myocardial metabolism, cardiac dysfunction and other myocardial diseases (7). The mechanism of occurrence and development of AVMC is mainly as follows: Persistent virus infection in acute phase mediates the immune injury of the body, thus damaging the microvessel and myocardium of patients (8). If not treated in time, the disease will result in arrhythmia, heart failure, or sudden death. There is no specific medicine in the clinical treatment of AVMC, and the main treatment method is bed rest to reduce the patient's heart burden, combined with antiviral and anti-infective treatment. At present, there is no systematic and standardized therapeutic regimen (9).

The treatment principle of AVMC is to reduce myocardial apoptosis with immune regulation through the anti-virus and symptomatic treatment, thereby improving myocardial energy metabolism and restoring normal cardiac function of patients (10). Results of this study showed that after one course of treatment, the total effective rate of treatment in observation group was significantly higher than that in control group, and the improvement effect on clinical symptoms was significantly superior to that in control group (p<0.05). This is because tanshinone IIA sodium sulfonate can act on myocardial cells and block the Ca2+ channel of myocardial cells, thus effectively preventing the influx of Ca2+, enhancing the cell strength of patients and reducing the loss of myocardial cells, ultimately relieving the clinical symptoms of AVMC (11). Trimetazidine is a kind of piperazine compound that can optimize myocardial energy metabolism, and enhance cardiac contractility, improve myocardial oxygen supply and promote functional recovery of myocardial cells by inhibiting β oxidation of free fatty acids (12). Levocarnitine is a natural substance that can regulate lipid metabolic disorders and effectively improve cardiac dysfunction, which reduces myocardial damage by scavenging free radical accumulation, thereby promoting cardiac function recovery in AVMC patients (13).

H-FABP is a kind of low-molecular-weight cytosolic protein that exists in myocardial cells, which is often used as an index of early myocardial damage (14). CK-MB is one of the four isoforms of creatine kinase, and it is related to the muscle contraction and intracellular energy transfer, which is clinically used as an index of myocardial damage (15). Besides, cTnI is an inhibitor protein in troponin-tropomyosin regulatory complex, as well as a marker of myocardial damage (16). Results of this study showed that after treatment, levels of H-FABP, CK-MB and cTnI in observation group were significantly lower than those in control group (p<0.05). This is because tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine has more obvious effects than tanshinone IIA sodium sulfonate alone, which, at the same time, can regulate the body's immunity, provide energy for myocardium, improve myocardial contractility, reduce myocardial apoptosis, increase myocardial oxygen supply, improve myocardial ischemia and hypoxia, and protect myocardium, thereby reducing the leakage of H-FABP, CK-MB and cTnI from myocardial cells and release of them into the peripheral blood. The decreased expression of H-FABP, CK-MB and cTnI indicate that myocardial damage in AVMC patients is improved to a certain extent.

TNF-α is a key inflammatory mediator core that is produced the earliest in the body, which can initiate and trigger inflammatory response and cause cascade reactions, and are of importance for neutrophil aggregation-induced damage (17). IL-18 is a pro-inflammatory cytokine that plays an important role in infection, inflammation and immune diseases (18). IL-35 is a heterodimer that is specifically produced by Treg cells, which is a cytokine necessary for Treg cells to exert the maximal inhibitory effect (19). Results of this study revealed that levels of TNF-α and IL-18 in both groups were decreased at 3, 5 and 7 days after treatment compared with those before treatment, but the level of IL-35 was increased compared with that before treatment, and changes in observation group were more obvious than those in control group (p<0.05). This is because when AVMC occurs, myocardium will be in ischemia and hypoxia status, so interstitial cells and myocardial cells will secrete a large amount of TNF-α, and apoptosis of myocardial cells will be induced, resulting in myocardial injury, activating inflammatory response and mediating IL-18 secretion. At the same time, viral replication triggers programmed cell death, leading to cardiac dysfunction, destroying immune system and weakening anti-inflammatory effect, thus downregulating the expression of IL-35 (20). After treatment with tanshinone IIA sodium sulfonate combined with trimetazidine and levocarnitine, Treg cell proliferation is promoted, thus effectively regulating the body's immune response, upregulating IL-35 expression, controlling the development of AVMC, and inhibiting the inflammatory response. With the passage of treatment time, the expression of TNF-α and IL-18 are decreased, the number of inflammatory cells is decreased and the pathological injury of myocardium is improved.

In conclusion, the application of trimetazidine and levocarnitine based on the treatment of AVMC patients with tanshinone IIA sodium sulfonate can effectively improve the clinical symptoms of patients, protect the damaged myocardial cells and reduce the degree of inflammatory response. Due to the small sample size in this study, data bias was inevitable, so the sample size still needs to be expanded for further observation.