Cell Biochem Biophys DOI 10.1007/s12013-014-0320-y
ORIGINAL PAPER
In Vitro Cardioprotective Effect of Wusen Erlian Granules Through the Inhibition of Coxsackievirus B3 Replication Yong Cao • Lin Hao • Cong-hui Han Pei-ying Zhang
•
Ó Springer Science+Business Media New York 2014
Abstract Wusen Erlian Granules are a traditional Chinese medicine and we sought to determine the antiviral activity of Wusen Erlian Granules against Coxsackievirus B3 infection. First, cytotoxicity of Wusen Erlian Granules was determined in cultured cardiomyocytes isolated from day-old Wister rat pups. Later, cardiomyocytes were infected with Coxsackievirus B3 and the protective effect of Wusen Erlian Granules against cell injury was compared with that of ribavirin. Cell injury indicators including myoglobin, MB isozyme of Creatine Kinase, and cardiac Troponin were assessed by enzyme-linked immunosorbent assay (ELISA) and antiviral effect was assessed by MTT assay. We found that the 50 %-Toxic (TC50) and 50 %Effective (EC50) concentrations of Wusen Erlian Granules were 394.05 and 30.26 lg/ml, respectively. Following infection of cardiomyocytes with Coxsackievirus B3, cell injury index of Wusen Erlian Granules, as determined by ELISA, was 125 lg/ml which yielded significant protection from virus-induced cell damage. The antiviral activity of Wusen Erlian Granules i.e., therapeutic index in MTT assay was higher (13.02) than that of ribavirin (6.93). It was, therefore, concluded that the Wusen Erlian Granules exerted better antiviral effect than ribavirin using
Y. Cao The First College of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210029, China Y. Cao Xuzhou Hospital of Traditional Chinese Medicine, Xuzhou 221009, Jiangsu, China L. Hao C. Han P. Zhang (&) Xuzhou Central Hospital, The Liberation of South Road 199, Xuzhou 221009, Jiangsu, China e-mail: [email protected]
Coxsackievirus B3 in vitro infection model in terms of rat cardiomyocytes protection from virus-induced cell injury. Keywords Wusen Erlian Granules Traditional Chinese medicine Antiviral effect Viral myocarditis Coxsackievirus B3 Myocardial cell injury
Introduction Viral myocarditis (VMC) is associated with myocardial viral tropism, and is characterized by non-specific interstitial myocardial inflammation. VMC is a clinical syndrome induced by viral infection, leading to the pathologic involvement of multiple organs, especially the heart [1]. Although various viruses may cause myocarditis, most cases of viral myocarditis in the clinical settings are related to Coxsackievirus and Echovirus infections. Coxsackievirus group B has been recognized as the commonest pathogen involved in human myocarditis. In VMC, major lesions are observed in the heart muscle (myocardium); however, pericardium and endocardium may also be involved and it is recognized as one of the main factors leading to dilated cardiomyopathy (DCM). According to existing medical evidence [2], the two main factors related to VMC pathogenesis include: (i) direct viral infection; and (ii) immune response. Until date, no highly effective regimens or medications against the disease are available in China or abroad. Relevant therapies will need to primarily focus on the following two aspects: (i) viral infection; and (ii) myocarditis. Due to the limited efficacy of currently available antiviral therapy for VMC, we resorted to the strategic use of integrative therapy, comprising of both traditional Chinese medicine (TCM) and allopathic medicine as also
123
Cell Biochem Biophys
described elsewhere [3, 4]. TCMs have proven to be important alternative therapy for VMC [5, 6]. We earlier alluded to the beneficial effects of Wusen Erlian Granules [7]. Herein, we show the antiviral and cardioprotective effects of Wusen Erlian Granules in VMC using in vitro assays.
Materials and Methods Virus, Test Drugs and Other Reagents Coxsackievirus B3 isolates stock cultured in our laboratory were used against the test drug called Wusen Erlian Granules (10 g/pack). Ribavirin (150 mg per pack) was purchased from Gabriel Sichuan Pharmaceutical Co. China (Batch# 120702). RPMI-1640 cell culture medium was from Gibco; fetal bovine serum (FBS) was from Yinxiang Weiye Biological Engineering Co. Shandong, China; and other chemicals were purchased from Sinopharm Group Co. Shanghai, China. Mouse cardiac myoglobin (MYO), troponin, and creatine kinase ELISA kit were purchased from Wuhan Huamei Bioengineering Co. Hubei, China.
Myocardial Cell Harvesting and Culture All procedures involving experimental animals conformed to the international guidelines for ethical conduct in the care and use of animals and the experiments were conducted after approval from the institutional Committee on Animal Research and Ethics (CARE). Day-old Wistar rat pups with disinfected body surface were transferred to a sterile Petri dish inside biosafety cabinet. Hearts were removed following aseptic procedures and transferred to Petri dish, followed by removal of atrial tissue, pericardial tissue, and other connective tissues. The samples obtained were thoroughly washed using sterile phosphate buffered saline (PBS; pH 7.4) and were sliced into small pieces less than 1 mm3 size using scissors before addition of 5-fold 0.25 % trypsin solution. The resultant mixture was placed on orbital shaker at 37 °C for 25 min, with intermittent shaking after every 5 min to enhance tissue trypsinization effect. Later, 5 ml of culture medium was added to terminate the trypsin effect. Tissue content was collected and mixed with 5 ml of fresh medium and let stand at room temperature for 5 min. The tissue suspension was transferred to a centrifuge tube and myocardial cells were obtained by centrifugation at 1,200 rpm for 5 min (4 °C). The cells were seeded (1 9 106 cells/ml) in Petri plates and incubated for 24 h at 37 °C in 5 % CO2 in a humidified incubator. Half of top medium containing floating dead cells was removed after 24 h and replaced with fresh
123
medium, and the cells were cultured until 72 h to obtain monolayer. Cytotoxicity Assay of Wusen Erlian Granules For test drug preparation, Wusen Erlian Granules were dissolved in distilled water and heat sterilized. The solution strength was adjusted using sterile distilled water to yield the final drug concentration of 1 mg/ml. The Wusen Erlian concentration used in cardiomyocytes culture was 31.25 lg/ml. Ribavirin, included as a positive control, was also used at the same concentration of 31.25 lg/ml. Test and positive control drug preparations were added in triplicate (500 ll/well) to microplate wells, followed by addition of RPMI-1640 medium containing 8 % FBS (250 ll/well). The plates were incubated for 5 days at 37 °C in 5 % CO2 in a humidified incubator. Cell lesions were examined microscopically at each day and at the end of 5th day, MTT solution (5 mg/ml) was added (20 ll per well) and plates were incubated for another 4 h. Culture supernatants were discarded and 150 ll of DMSO was added to each well, followed by gentle shaking for 10 min in order for the crystalline material to be dispersed homogeneously. The chromogenic solution was collected from each well and transferred to a 96-well plate, and the absorbance was recorded at 570 nm wavelength (A570 values) using a microplate reader to calculate the 50 %Toxic Concentration (TC50). Coxsackievirus B3 Replication Inhibition by Wusen Erlian Granules Test and control drugs were prepared and added to triplicate wells of a micro-titration culture plate as described before, followed by addition of RPMI-1640 medium containing 8 % FBS (250 ll per well). Later, Coxsackievirus B3 preparation (100 copies of TCID50/ml) was added (250 ll per well). In parallel, non-viral control cell cultures were also established and the plates were incubated at 37 °C in 5 % CO2 in a humidified incubator. The cultures were examined microscopically each day for cell lesions and the supernatants from corresponding wells were collected as *90 % cells showed morbid changes in viral control wells. The supernatants were collected and centrifuged at 5,000 rpm for 5 min and used in ELISA to determine the myocardial injury index by following the manufacturer’s instructions. Briefly, the supernatants collected from each well were transferred to a new microplate (100 ll per well), reagents added as instructed, and incubated at 37 °C for 1 h. After 3 thorough washes, antibodies against MYO, MB isozyme of Creatine Kinase (CK-MB), and cardiac Troponin (cTn) were added (100 ll per well), and the resultant mixtures were incubated at 37 °C for 1 h. After another 3 washes, the staining reagent was
Cell Biochem Biophys
added and let stand for 5 min. After termination of chromogenic reaction, A540 values were determined by spectrophotometry. To determine the antiviral activity of Wusen Erlian Granules, 20 ll of MTT solution (5 mg/m) was added to each well and plates were incubated at 37 °C for 4 h. After end of incubation, supernatant was discarded and plates were washed thrice with PBS (pH 7.4), followed by addition of DMSO (150 ll per well). After gentle plate rocking for 10 min, the chromogenic solution was collected from each well into a new microplate and A570 values were determined using a microplate reader to calculate the TC50, 50 %-Effective Concentration (EC50), and Therapeutic Index (TI).
Results TC50s of Wusen Erlian Granules and Ribavirin The TC50s of Wusen Erlian Granules and ribavirin were 394.05 and 357.29 lg/ml, respectively. Inhibitory effects of Wusen Erlian Granules and ribavirin on Coxsackievirus B3 infection of rat cardiomyocytes As shown in Table 1, the cardiomyocyte injury or cytopathic effects caused by Coxsackievirus B3 infection were significantly reduced as Wusen Erlian Granules were used at the dose rate of 125 lg/ml; the concentrations of myocardial cell injury indicators, that is MYO, CK-MB, and cTn, were 133.39 ± 11.21, 246.11 ± 21.01, and 167.66 ± 15.22 ng/ml, respectively (All P-values were \0.05 compared with viral controls).
Comparative Antiviral Activities of Wusen Erlian Granules and Ribavirin As determined by in vitro assays, Wusen Erlian Granules had a superior antiviral activity (TI) than ribavirin (13.02 vs. 6.93). The EC50 values of Wusen Erlian Granules and ribavirin were 30.26 and 51.57 lg/ml, respectively (Table 2).
Discussion Despite the recent advances in research on viral structural and functional biology, pathogenesis of viral diseases and host immunity, and antiviral modalities, there is still much room for further studies to find more effective and potent therapeutic interventions for VMC. In this context, emerging studies are now exploring the alternatives to the conventional antiviral agents, such as TCMs [8, 9]. The pathogenesis of VMC is complicated and involves the virus-induced myocardial cell damage, activation of antiviral host immunity, and development of immune impairment over time; together with induction of pathophysiological changes following massive myocardial cell damage. Regarding TCM-based therapeutic options, it
Table 2 Antiviral activities of Wusen Erlian Granules and ribavirin against Coxsackievirus B3 as determined by in vitro cytopathic assay Group
TC50 (lg/ml)
EC50 (lg/ml)
TI
Ribavirin Wusen Erlian Granules
357.29 394.05
51.57 30.26
6.93 13.02
TC50 50 % toxic concentration, EC50 50 % effective concentration, TI therapeutic index
Table 1 Inhibitory effects of Wusen Erlian Granules and ribavirin on Coxsackievirus B3 infection of rat cardiomyocytes (ng/ml, n = 3) Group
Ribavirin
Wusen Erlian Granules
MYO Viral controls Cellular controls 500 lg 250 lg 125 lg 62.5 lg 31.25 lg 15.65 lg
CK-MB
156.83 ± 14.25* 1.54 ± 0.21
#
cTn
270.76 ± 20.45* #
5.83 ± 1.02
#
35.25 ± 4.23 *
190.36 ± 15.57* 4.72 ± 0.95
#
91.2 ± 7.51 *
#
CK-MB
156.83 ± 14.25* #
1.54 ± 0.21
#
68.14 ± 8.57 *
121.67 ± 12.2 *
76.56 ± 9.17 *
45.21 ± 9.54 *
105.55 ± 9.98 *
#
#
#
#
106.94 ± 11.59 * #
99.57 ± 11.15 * #
270.76 ± 20.45*
#
190.36 ± 15.57* 4.72 ± 0.95#
5.83 ± 1.02
#
#
53.39 ± 9.21 *
#
cTn
#
#
28.91 ± 5.98 *
#
MYO
#
142.28 ± 12.57#*
#
134.27 ± 13.54#*
#
180.39 ± 17.54 * 217.36 ± 19.77 *
133.39 ± 11.21 *
246.11 ± 21.01 *
167.66 ± 15.22#*
85.9 ± 12.88 *
134.35 ± 12.54 *
113.47 ± 10.21 *
153.9 ± 14.8*
264.6 ± 20.63*
183.69 ± 14.21*
109.84 ± 10.21#*
168.66 ± 15.32#*
124.56 ± 11.66#*
159.76 ± 13.69*
268.7 ± 21.32*
194.37 ± 14.54*
158.24 ± 14.21*
271.66 ± 21.01*
192.25 ± 12.35*
#
128.24 ± 14.21 *
#
204.86 ± 14.01 *
#
142.37 ± 11.98 *
MYO myoglobin, CK-MB MB isozyme of Creatine Kinase, cTn cardiac Troponin # Significantly different from viral controls * Significantly different from cellular controls
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Cell Biochem Biophys
is feasible to formulate the highly effective drug combinations through an integrative approach as the efficacy of mono-TCM is usually less than that of combo-TCMs. However, while resorting to combo-TCMs, care will have to be taken concerning the drug incompatibilities to avoid any untoward therapeutic reactions. At present, the data on TCM-related pharmacodynamics and pharmacokinetics remain limited. Herein, we studied the safety and antiviral efficacy of Wusen Erlian Granules, a Combo-TCM developed in our laboratory, as compared with ribavirin against Coxsackievirus B3. The main components of this formulation include different herbs, such as Ginseng, Salvia, Panax, figwort, Sophora, Berberine, Forsythia, Lotus seeds, Poria, Chinese Arborvilae seeds, prepared Liquorice root, Polygonatum odoratum, Tangerine peel, and Amber powder. We found that this combo-TCM preparation was useful to treat the qi and yin deficiencies, dysphoria, anxiety, and general body listlessness. As per the TCM therapeutic philosophy, its main indications included qi and yin deficiencies syndrome and heart palpitations. The clinical symptoms include irregular/rapid pulse, heart palpitations, inflamed tongue with redness, and papillary changes. In this combinational remedy, Ginseng benefits qi, figwort, and P. odoratum nourish yin, as well as promote body strength and replenish tissue fluids. Berberine, Forsythia, and Sophora were used to nourish and support cardiovascular system and alleviate cardiac discomfort/dysfunction; Poria, Chinese Arborvilae seeds, and Lotus were included to support cardiohepatic function, cure hematologic ailments, and induce general body tranquility. Salvia, Panax, and Amber powder also supported the hematologic system function. They were all used as minister drugs. Tangerine peel is known to improve splenic function and dispel dampness; also it regulates qi and promotes the hepatic activity. These ingredients combined with other yin-nourishing agents were expected to induce an optimal therapeutic response. Moreover, prepared Liquorice root could help regulate the gastric functions and relieve other intestinal co-morbidities. This agent was used to support all other TCM components and, therefore, it acted as the courier drug. Taken all above together, the intended therapeutic effects included improvement of cardiac function, qi and yin nourishment, and alleviation of palpitations and dysphoria. Previously, Zhang et al. [7] formulated and compounded Wusen Erlian Granules as a Comb-TCM with multiple beneficial effects. In extrapolation of this work, we herein determined the safety and antiviral efficacy of Wusen Erlian Granules in the in vitro model of VMC. In this study, rat cardiomyocytes were isolated and cultured for the use of various assays. Following myocardial cell infection with Coxsackievirus B3, the TC50 of Wusen Erlian Granules was determined to be 394.05 lg/ml. The data based on
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myocardial cell injury indices in ELISA showed that Wusen Erlian Granules concentration of 125 lg/ml significantly prevented the myocardial cell injury caused by Coxsackievirus B3 infection. MTT assay was used to measure the antiviral activity of Wusen Erlian Granules and the TI determined using in vitro model of VMC was 13.02; which was higher than that of ribavirin. A recent study by Yao et al. [10] alluded to the qi-replenishing, Yinnourishing, and blood tonic properties of the Coxsackie B viral myocarditis capsules (KCoxBJN). This study reported the similar antiviral effects of KCoxBJN and ribavirin in cardiomyocytes infected with Coxsackie B virus whereas we found that Wusen Erlian Granules had a better antiviral effect than ribavirin. However, our study is limited for being the preliminary work involving in vitro rat model study only; therefore, further in-depth studies will be required to validate these initial findings. Taken together, our data show that Wusen Erlian Granules exerted antiviral effect and protected rat cardiomyocytes from damage by Coxsackievirus B3 infection in vitro. Further studies including multiple animal models, other viral pathogens, and various cardio-immune function parameters will be required to extend the VMC research beyond this preliminary work. Acknowledgments This study was supported by Kangyuan TCM Science & Technology Innovation Funds (Grant # HZ1018KY). Conflict of interest The authors have no conflicts of interest involved.
References 1. Ku¨hl, U., & Schultheiss, H. P. (2009). Viral myocarditis: Diagnosis, aetiology and management. Drugs, 69, 1287–1302. 2. Westermann, D., Savvatis, K., & Schultheiss, H. P. (2010). Immunomodulation and matrix metalloproteinases in viral myocarditis. Journal of Molecular and Cellular Cardiology, 2010(48), 468–473. 3. Liu, J. P., Yang, M., & Du, X. M. (2004). Herbal medicines for viral myocarditis. Cochrane Database of Systematic Reviews, 3, CD003711. 4. Yip, A. Y., Loo, W. T., & Chow, L. W. (2007). Fructus schisandrae containing compound in modulating human lymphatic system-a phase I minimization clinical trial. Biomedicine and Pharmacotherapy, 2007(61), 588–590. 5. Yao, R. M., Guo, S. S., Ma, H. X., Xu, J., Xie, F., Wang, W., et al. (2013). The in vitro anti-CVB3 effect of total saponins in anti-Coxsackie B virus myocarditis capsules and its impact on cell activity. Journal of Traditional Chinese Medicine, 2013(28), 2104–2108. 6. Bao, J. T., Xu, J., Ma, H. X., Yao, R. M., Wang, W., & Xie, F. (2013). Antiviral effect of new compound anti-coxsackievirus B myocarditis capsule on coxsackievirus B3: In vitro study. Journal of Traditional Chinese Medicine, 2013(54), 953–957. 7. Zhang, P. Y., Xu, X., Sun, Z. H., & Zhu, G. W. (1996). Application of Wusen Erlian Decoction in treating 70 cases of viral
Cell Biochem Biophys myocarditis. Zhejiang Journal of Traditional Chinese Medicine, 1996(10), 447. 8. Zheng, S. Z., Shen, C. H., & Piao, H. Z. (2007). Effect of pulseactivating powder combined with roasted glycyrrhiza decoction on cultured rat myocardial cells infected with Coxsackie virus B3. Lishizhen Medicine Materia Medica Resolution, 2007(6), 27–28. 9. Zhang, Z. C., Yang, Y. Z., & Li, S. J. (2003). Effect of astragaloside on myocardial fibrosis in viral myocarditic mice. Journal of New Drugs and Clinical Remedies, 2003(22), 515–519.
10. Yao, R. M., Guo, S. S., Ma, H. X., Xu, J., Xie, F., Wang, W., et al. (2013). Studies of the anti-coxsackie B-3 viral myocarditis capsules including total saponins, antiviral activity, and myocardial cell viability. Journal of Chinese Medicine, 2013(7), 2104–2108.
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ORIGINAL PAPER
In Vitro Cardioprotective Effect of Wusen Erlian Granules Through the Inhibition of Coxsackievirus B3 Replication Yong Cao • Lin Hao • Cong-hui Han Pei-ying Zhang
•
Ó Springer Science+Business Media New York 2014
Abstract Wusen Erlian Granules are a traditional Chinese medicine and we sought to determine the antiviral activity of Wusen Erlian Granules against Coxsackievirus B3 infection. First, cytotoxicity of Wusen Erlian Granules was determined in cultured cardiomyocytes isolated from day-old Wister rat pups. Later, cardiomyocytes were infected with Coxsackievirus B3 and the protective effect of Wusen Erlian Granules against cell injury was compared with that of ribavirin. Cell injury indicators including myoglobin, MB isozyme of Creatine Kinase, and cardiac Troponin were assessed by enzyme-linked immunosorbent assay (ELISA) and antiviral effect was assessed by MTT assay. We found that the 50 %-Toxic (TC50) and 50 %Effective (EC50) concentrations of Wusen Erlian Granules were 394.05 and 30.26 lg/ml, respectively. Following infection of cardiomyocytes with Coxsackievirus B3, cell injury index of Wusen Erlian Granules, as determined by ELISA, was 125 lg/ml which yielded significant protection from virus-induced cell damage. The antiviral activity of Wusen Erlian Granules i.e., therapeutic index in MTT assay was higher (13.02) than that of ribavirin (6.93). It was, therefore, concluded that the Wusen Erlian Granules exerted better antiviral effect than ribavirin using
Y. Cao The First College of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210029, China Y. Cao Xuzhou Hospital of Traditional Chinese Medicine, Xuzhou 221009, Jiangsu, China L. Hao C. Han P. Zhang (&) Xuzhou Central Hospital, The Liberation of South Road 199, Xuzhou 221009, Jiangsu, China e-mail: [email protected]
Coxsackievirus B3 in vitro infection model in terms of rat cardiomyocytes protection from virus-induced cell injury. Keywords Wusen Erlian Granules Traditional Chinese medicine Antiviral effect Viral myocarditis Coxsackievirus B3 Myocardial cell injury
Introduction Viral myocarditis (VMC) is associated with myocardial viral tropism, and is characterized by non-specific interstitial myocardial inflammation. VMC is a clinical syndrome induced by viral infection, leading to the pathologic involvement of multiple organs, especially the heart [1]. Although various viruses may cause myocarditis, most cases of viral myocarditis in the clinical settings are related to Coxsackievirus and Echovirus infections. Coxsackievirus group B has been recognized as the commonest pathogen involved in human myocarditis. In VMC, major lesions are observed in the heart muscle (myocardium); however, pericardium and endocardium may also be involved and it is recognized as one of the main factors leading to dilated cardiomyopathy (DCM). According to existing medical evidence [2], the two main factors related to VMC pathogenesis include: (i) direct viral infection; and (ii) immune response. Until date, no highly effective regimens or medications against the disease are available in China or abroad. Relevant therapies will need to primarily focus on the following two aspects: (i) viral infection; and (ii) myocarditis. Due to the limited efficacy of currently available antiviral therapy for VMC, we resorted to the strategic use of integrative therapy, comprising of both traditional Chinese medicine (TCM) and allopathic medicine as also
123
Cell Biochem Biophys
described elsewhere [3, 4]. TCMs have proven to be important alternative therapy for VMC [5, 6]. We earlier alluded to the beneficial effects of Wusen Erlian Granules [7]. Herein, we show the antiviral and cardioprotective effects of Wusen Erlian Granules in VMC using in vitro assays.
Materials and Methods Virus, Test Drugs and Other Reagents Coxsackievirus B3 isolates stock cultured in our laboratory were used against the test drug called Wusen Erlian Granules (10 g/pack). Ribavirin (150 mg per pack) was purchased from Gabriel Sichuan Pharmaceutical Co. China (Batch# 120702). RPMI-1640 cell culture medium was from Gibco; fetal bovine serum (FBS) was from Yinxiang Weiye Biological Engineering Co. Shandong, China; and other chemicals were purchased from Sinopharm Group Co. Shanghai, China. Mouse cardiac myoglobin (MYO), troponin, and creatine kinase ELISA kit were purchased from Wuhan Huamei Bioengineering Co. Hubei, China.
Myocardial Cell Harvesting and Culture All procedures involving experimental animals conformed to the international guidelines for ethical conduct in the care and use of animals and the experiments were conducted after approval from the institutional Committee on Animal Research and Ethics (CARE). Day-old Wistar rat pups with disinfected body surface were transferred to a sterile Petri dish inside biosafety cabinet. Hearts were removed following aseptic procedures and transferred to Petri dish, followed by removal of atrial tissue, pericardial tissue, and other connective tissues. The samples obtained were thoroughly washed using sterile phosphate buffered saline (PBS; pH 7.4) and were sliced into small pieces less than 1 mm3 size using scissors before addition of 5-fold 0.25 % trypsin solution. The resultant mixture was placed on orbital shaker at 37 °C for 25 min, with intermittent shaking after every 5 min to enhance tissue trypsinization effect. Later, 5 ml of culture medium was added to terminate the trypsin effect. Tissue content was collected and mixed with 5 ml of fresh medium and let stand at room temperature for 5 min. The tissue suspension was transferred to a centrifuge tube and myocardial cells were obtained by centrifugation at 1,200 rpm for 5 min (4 °C). The cells were seeded (1 9 106 cells/ml) in Petri plates and incubated for 24 h at 37 °C in 5 % CO2 in a humidified incubator. Half of top medium containing floating dead cells was removed after 24 h and replaced with fresh
123
medium, and the cells were cultured until 72 h to obtain monolayer. Cytotoxicity Assay of Wusen Erlian Granules For test drug preparation, Wusen Erlian Granules were dissolved in distilled water and heat sterilized. The solution strength was adjusted using sterile distilled water to yield the final drug concentration of 1 mg/ml. The Wusen Erlian concentration used in cardiomyocytes culture was 31.25 lg/ml. Ribavirin, included as a positive control, was also used at the same concentration of 31.25 lg/ml. Test and positive control drug preparations were added in triplicate (500 ll/well) to microplate wells, followed by addition of RPMI-1640 medium containing 8 % FBS (250 ll/well). The plates were incubated for 5 days at 37 °C in 5 % CO2 in a humidified incubator. Cell lesions were examined microscopically at each day and at the end of 5th day, MTT solution (5 mg/ml) was added (20 ll per well) and plates were incubated for another 4 h. Culture supernatants were discarded and 150 ll of DMSO was added to each well, followed by gentle shaking for 10 min in order for the crystalline material to be dispersed homogeneously. The chromogenic solution was collected from each well and transferred to a 96-well plate, and the absorbance was recorded at 570 nm wavelength (A570 values) using a microplate reader to calculate the 50 %Toxic Concentration (TC50). Coxsackievirus B3 Replication Inhibition by Wusen Erlian Granules Test and control drugs were prepared and added to triplicate wells of a micro-titration culture plate as described before, followed by addition of RPMI-1640 medium containing 8 % FBS (250 ll per well). Later, Coxsackievirus B3 preparation (100 copies of TCID50/ml) was added (250 ll per well). In parallel, non-viral control cell cultures were also established and the plates were incubated at 37 °C in 5 % CO2 in a humidified incubator. The cultures were examined microscopically each day for cell lesions and the supernatants from corresponding wells were collected as *90 % cells showed morbid changes in viral control wells. The supernatants were collected and centrifuged at 5,000 rpm for 5 min and used in ELISA to determine the myocardial injury index by following the manufacturer’s instructions. Briefly, the supernatants collected from each well were transferred to a new microplate (100 ll per well), reagents added as instructed, and incubated at 37 °C for 1 h. After 3 thorough washes, antibodies against MYO, MB isozyme of Creatine Kinase (CK-MB), and cardiac Troponin (cTn) were added (100 ll per well), and the resultant mixtures were incubated at 37 °C for 1 h. After another 3 washes, the staining reagent was
Cell Biochem Biophys
added and let stand for 5 min. After termination of chromogenic reaction, A540 values were determined by spectrophotometry. To determine the antiviral activity of Wusen Erlian Granules, 20 ll of MTT solution (5 mg/m) was added to each well and plates were incubated at 37 °C for 4 h. After end of incubation, supernatant was discarded and plates were washed thrice with PBS (pH 7.4), followed by addition of DMSO (150 ll per well). After gentle plate rocking for 10 min, the chromogenic solution was collected from each well into a new microplate and A570 values were determined using a microplate reader to calculate the TC50, 50 %-Effective Concentration (EC50), and Therapeutic Index (TI).
Results TC50s of Wusen Erlian Granules and Ribavirin The TC50s of Wusen Erlian Granules and ribavirin were 394.05 and 357.29 lg/ml, respectively. Inhibitory effects of Wusen Erlian Granules and ribavirin on Coxsackievirus B3 infection of rat cardiomyocytes As shown in Table 1, the cardiomyocyte injury or cytopathic effects caused by Coxsackievirus B3 infection were significantly reduced as Wusen Erlian Granules were used at the dose rate of 125 lg/ml; the concentrations of myocardial cell injury indicators, that is MYO, CK-MB, and cTn, were 133.39 ± 11.21, 246.11 ± 21.01, and 167.66 ± 15.22 ng/ml, respectively (All P-values were \0.05 compared with viral controls).
Comparative Antiviral Activities of Wusen Erlian Granules and Ribavirin As determined by in vitro assays, Wusen Erlian Granules had a superior antiviral activity (TI) than ribavirin (13.02 vs. 6.93). The EC50 values of Wusen Erlian Granules and ribavirin were 30.26 and 51.57 lg/ml, respectively (Table 2).
Discussion Despite the recent advances in research on viral structural and functional biology, pathogenesis of viral diseases and host immunity, and antiviral modalities, there is still much room for further studies to find more effective and potent therapeutic interventions for VMC. In this context, emerging studies are now exploring the alternatives to the conventional antiviral agents, such as TCMs [8, 9]. The pathogenesis of VMC is complicated and involves the virus-induced myocardial cell damage, activation of antiviral host immunity, and development of immune impairment over time; together with induction of pathophysiological changes following massive myocardial cell damage. Regarding TCM-based therapeutic options, it
Table 2 Antiviral activities of Wusen Erlian Granules and ribavirin against Coxsackievirus B3 as determined by in vitro cytopathic assay Group
TC50 (lg/ml)
EC50 (lg/ml)
TI
Ribavirin Wusen Erlian Granules
357.29 394.05
51.57 30.26
6.93 13.02
TC50 50 % toxic concentration, EC50 50 % effective concentration, TI therapeutic index
Table 1 Inhibitory effects of Wusen Erlian Granules and ribavirin on Coxsackievirus B3 infection of rat cardiomyocytes (ng/ml, n = 3) Group
Ribavirin
Wusen Erlian Granules
MYO Viral controls Cellular controls 500 lg 250 lg 125 lg 62.5 lg 31.25 lg 15.65 lg
CK-MB
156.83 ± 14.25* 1.54 ± 0.21
#
cTn
270.76 ± 20.45* #
5.83 ± 1.02
#
35.25 ± 4.23 *
190.36 ± 15.57* 4.72 ± 0.95
#
91.2 ± 7.51 *
#
CK-MB
156.83 ± 14.25* #
1.54 ± 0.21
#
68.14 ± 8.57 *
121.67 ± 12.2 *
76.56 ± 9.17 *
45.21 ± 9.54 *
105.55 ± 9.98 *
#
#
#
#
106.94 ± 11.59 * #
99.57 ± 11.15 * #
270.76 ± 20.45*
#
190.36 ± 15.57* 4.72 ± 0.95#
5.83 ± 1.02
#
#
53.39 ± 9.21 *
#
cTn
#
#
28.91 ± 5.98 *
#
MYO
#
142.28 ± 12.57#*
#
134.27 ± 13.54#*
#
180.39 ± 17.54 * 217.36 ± 19.77 *
133.39 ± 11.21 *
246.11 ± 21.01 *
167.66 ± 15.22#*
85.9 ± 12.88 *
134.35 ± 12.54 *
113.47 ± 10.21 *
153.9 ± 14.8*
264.6 ± 20.63*
183.69 ± 14.21*
109.84 ± 10.21#*
168.66 ± 15.32#*
124.56 ± 11.66#*
159.76 ± 13.69*
268.7 ± 21.32*
194.37 ± 14.54*
158.24 ± 14.21*
271.66 ± 21.01*
192.25 ± 12.35*
#
128.24 ± 14.21 *
#
204.86 ± 14.01 *
#
142.37 ± 11.98 *
MYO myoglobin, CK-MB MB isozyme of Creatine Kinase, cTn cardiac Troponin # Significantly different from viral controls * Significantly different from cellular controls
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Cell Biochem Biophys
is feasible to formulate the highly effective drug combinations through an integrative approach as the efficacy of mono-TCM is usually less than that of combo-TCMs. However, while resorting to combo-TCMs, care will have to be taken concerning the drug incompatibilities to avoid any untoward therapeutic reactions. At present, the data on TCM-related pharmacodynamics and pharmacokinetics remain limited. Herein, we studied the safety and antiviral efficacy of Wusen Erlian Granules, a Combo-TCM developed in our laboratory, as compared with ribavirin against Coxsackievirus B3. The main components of this formulation include different herbs, such as Ginseng, Salvia, Panax, figwort, Sophora, Berberine, Forsythia, Lotus seeds, Poria, Chinese Arborvilae seeds, prepared Liquorice root, Polygonatum odoratum, Tangerine peel, and Amber powder. We found that this combo-TCM preparation was useful to treat the qi and yin deficiencies, dysphoria, anxiety, and general body listlessness. As per the TCM therapeutic philosophy, its main indications included qi and yin deficiencies syndrome and heart palpitations. The clinical symptoms include irregular/rapid pulse, heart palpitations, inflamed tongue with redness, and papillary changes. In this combinational remedy, Ginseng benefits qi, figwort, and P. odoratum nourish yin, as well as promote body strength and replenish tissue fluids. Berberine, Forsythia, and Sophora were used to nourish and support cardiovascular system and alleviate cardiac discomfort/dysfunction; Poria, Chinese Arborvilae seeds, and Lotus were included to support cardiohepatic function, cure hematologic ailments, and induce general body tranquility. Salvia, Panax, and Amber powder also supported the hematologic system function. They were all used as minister drugs. Tangerine peel is known to improve splenic function and dispel dampness; also it regulates qi and promotes the hepatic activity. These ingredients combined with other yin-nourishing agents were expected to induce an optimal therapeutic response. Moreover, prepared Liquorice root could help regulate the gastric functions and relieve other intestinal co-morbidities. This agent was used to support all other TCM components and, therefore, it acted as the courier drug. Taken all above together, the intended therapeutic effects included improvement of cardiac function, qi and yin nourishment, and alleviation of palpitations and dysphoria. Previously, Zhang et al. [7] formulated and compounded Wusen Erlian Granules as a Comb-TCM with multiple beneficial effects. In extrapolation of this work, we herein determined the safety and antiviral efficacy of Wusen Erlian Granules in the in vitro model of VMC. In this study, rat cardiomyocytes were isolated and cultured for the use of various assays. Following myocardial cell infection with Coxsackievirus B3, the TC50 of Wusen Erlian Granules was determined to be 394.05 lg/ml. The data based on
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myocardial cell injury indices in ELISA showed that Wusen Erlian Granules concentration of 125 lg/ml significantly prevented the myocardial cell injury caused by Coxsackievirus B3 infection. MTT assay was used to measure the antiviral activity of Wusen Erlian Granules and the TI determined using in vitro model of VMC was 13.02; which was higher than that of ribavirin. A recent study by Yao et al. [10] alluded to the qi-replenishing, Yinnourishing, and blood tonic properties of the Coxsackie B viral myocarditis capsules (KCoxBJN). This study reported the similar antiviral effects of KCoxBJN and ribavirin in cardiomyocytes infected with Coxsackie B virus whereas we found that Wusen Erlian Granules had a better antiviral effect than ribavirin. However, our study is limited for being the preliminary work involving in vitro rat model study only; therefore, further in-depth studies will be required to validate these initial findings. Taken together, our data show that Wusen Erlian Granules exerted antiviral effect and protected rat cardiomyocytes from damage by Coxsackievirus B3 infection in vitro. Further studies including multiple animal models, other viral pathogens, and various cardio-immune function parameters will be required to extend the VMC research beyond this preliminary work. Acknowledgments This study was supported by Kangyuan TCM Science & Technology Innovation Funds (Grant # HZ1018KY). Conflict of interest The authors have no conflicts of interest involved.
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