Wei Zhang, Ying Wang, Wei Yang, Er-Nu Zheng, Xian-Ming Su
Department of Geriatric Cardiology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi, China
Running title: Xuezhikang inhibits inflammatory reaction
Disclosure of conflict of interest
None.
Abstract: Objective: To investigate the effect of Chinese prepared medicine Xuezhikang on inflammatory reaction of human U937 cells. Methods: Human U937 cells were cultured with 50 mg/L oxLDL for 0 hour, 4 hours, 8 hours, 16 hours and 24 hours. The content of IL-6 and TNF-α of the cultured medium were respectively assessed using Enzyme linked immunosorbent assay. Human U937 cells were cultured with different concentration of Xuezhikang (0.125 g/L, 0.25 g/L, 0.5 g/L, 1 g/L) and 1 μmol/L Atorvastatin for 12 hours then cultured with oxLDL for 24 hours. The levels of IL-6 and TNF-α of the cultured supernatants were measured. Results: oxLDL significantly upgraded the IL-6 and TNF-α production in human U937 cell medium. Xuezhikang suppressed the IL-6 and TNF-α secretion induced by oxLDL slightly. The effect of 1 g/L Xuezhikang is most significant (IL-6 and TNF-α respectively decreased by 25%, 24.4%, P<0.01) the same as the effect of 1 μmol/L Atorvastatin (IL-6 and TNF-α respectively decreased by 25.3%, 27.5%, P>0.05). Conclusion: Chinese medicine Xuezhikang can inhibits inflammatory reaction of human U937 cells stimulated by oxLDL.
Keywords: U937 cells, Xuezhikang, tumor necrosis factor-α, interleukin-6 Introduction
Inflammatory is the most important component of the progress of atherosclerosis [1]. The main pathological presentation of AS is the formation of plaque in arterial vessels. Monocytes migrate into the arterial intima and differentiate into macrophages. Macrophages take up oxidized low-density lipoprotein (ox-LDL) then differentiate into foam cells [2, 3]. The accumulation of foam cells in the artery wall causes the formation of fatty streak(s) then to plaque [4]. These are the pathological changes in the early stage of atherosclerosis. The uptake of ox-LDL by macrophages results in the production of numerous pro-inflammatory cytokines that can amplify the inflammatory response and promote the evolution of atheroma [5]. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) are two important pro-atherosclerotic cytokines secreted by ox-LDL-activated macrophages [6, 7]. A high circulating concentration of IL-6 is associated with an increased risk of coronary heart disease [8]. IL-6 can regulate the neutrophil transform to monocyte during inflammation. [9] TNF-α significantly increased the transcytosis of LDL across human umbilical vein endothelial cells (HUVECs) and stimulated an increase of subendothelial retention of LDL in vascular walls [10]. TNF-α also stimulate the adhesion of U937 cells to HUVECs. TNF-α antagonists may have a beneficial effect on preventing the progression of subclinical atherosclerosis [11]. Statins possess the great level of evidence regarding lipid lowering. More and more evidence certificate that patients using statins may have effects beyond lipid lowering, such as anti inflammatory, and then prevent the incidence of death caused by CHD [12].
Xuezhikang is an extract of Chinese red yeast rice (RYR). Several studies comparing RYR with statins, show that RYR have the same effect of lipid lowering [13] and fewer side effects [14]. RYR may become a potential substitute for statin, and help patients to achieve their lipid goals while reducing side effects from statins. Zhao [15] et al. demonstrated the effectiveness of RYR in decreasing C-reactive protein and improving flow-mediated vasodilation in patients with CHD. Whether xueshikang have the same effect of anti inflammatory as statins is unknown. This study investigated the effect of xuezhikang on the level of IL-6 and TNF-αsecreted by U937 cell which cultured with oxLDL, then compared it with the effect of Atorvastatin. And explore whether Xuezhikang prevent atherosclerosis development through reducing the inflammatory reaction.
Materials and methods
Materials
Human monocyte cell line-U937 were purchased from Shanghai Cell Biology; RPMI1640 medium were purchased from Thermo Fisher Biochemical Products Co., Ltd.; Fetal calf serum were purchased from Shanghai Biological Technology Co., Ltd. Super research; oxidized low-density lipoprotein were purchased from Beijing Association of Health Biosciences; Xuezhikang provided by Beijing University WeiXin biotechnology Limited company, according to Japanese Masayoshi Yamashiki’s method [16]; Atorvastatin 20 mg purchased from American Pfizer Pharmaceutical Co. LTD.
Cell culture
Normal growth’s U937 cell was maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. Medicine preparation
Adding Xuezhikang crude drugs 10 g into 500 mL PBS, then putting the solution in 37°C shaking table 2 hours, until complete dissolution. Adjusting the PH value of the solution to 7.2~7.4 and making 20 g/L traditional Chinese medicine original fluid. Grinding Atorvastatin 20 mg to powder and joining it into the dimethylsulfoxide, using 7.33 ml PBS fluid dissolved fully, adjusting the pH value to 7.2~7.4 and making 2 mmol/L original fluid. The subpackages were preserved at 4°C.
Experiment group
Take the experiment according to the following grouping:
(1) oxLDL group(control): The U937 cell were cultured in the medium with 50 mg/L oxLDL for 0 h, 4 h, 8 h, 16 h, 24 h.
(2) Xuezhikang group: Confluent U937 were cultured in the medium with a concentration gradient of Xuezhikang (0.125 g/L, 0.25 g/L, 0.5 g/L, 1 g/L) for 12 hours. Then replacing the medium which contain 50 mg/L oxLDL and continuousculturing the cells for 24 hours.
(3) Atorvastatin group: Confluent U937 were cultured in the medium with a concentration of the Atorvastatin (1 μmol/L) for 12 hours, then replacing the medium which contain 50 mg/L oxLDL and continuous to culturing the cells for 24 hours.
Experimental methods
After the experiments, supernatants were collected and the amount of IL-6 and TNF-α was quantified using specific enzyme-linked immunosorbent assay (ELISA) according to instructions provided by the manufacturer (R&D Systems Inc Company).
Statistical analysis
Statistical analysis was performed with SPSS11.0 software. The data were presented as mean ± SEM () standard deviation. Statistical significance was defined as P<0.05.
Results
oxLDL promotes the U937 cell to release IL-6 and TNF-α
The IL-6 content was 272.3±1.5, 427.5±1.4, 550.3±1.2 and 700.6±1.8 pg/ml for 4 h, 8 h, 16 h and 24 h affected by oxLDL, compared to the control group (89.3±1.6 pg/ml) increasing to 2 times, 3.8 times, 5.2 times and 6.9 times. Each group had the significant difference (P<0.01). The TNF-α content was 296.4±1.5, 542.6±1.7, 721.3±1.7 and 899.3±1.9 pg/ml for 4 h, 8 h, 16 h and 24 h affected by oxLDL, compared to the control group (103.2±1.7 pg/ml) increasing 1.9 times, 4.3 times, 6.0 times and 7.7 times, each group had the significant difference (P<0.01) (Table 1).
Table 1. Time-course effects of oxLDL on IL-6 and TNFα (n=6, )
Group |
the content of IL-6 (pg/ml) |
the content of TNF-α (pg/ml) |
oxLDL 0 h |
89.16±1.6 |
103.2±1.7 |
oxLDL 4 h |
273.5±1.5▲,★ |
296.4±1.5▲,★ |
oxLDL 8 h |
427.5±1.4▲,★ |
542.6±1.7▲,★ |
oxLDL 16 h |
550.3±1.2▲,★ |
721.3±1.7▲,★ |
oxLDL 24 h |
700.6±1.8▲,★ |
899.3±1.9▲,★ |
▲P<0.01 vs. 0h; ★P<0.01 vs. other groups.
IL-6 and TNF-α secretion of U937 cell in different density Xuezhikang
The IL-6 content was 624.5±1.8, 542.6±1.2, 572.4±1.4 and 529.3±1.5 pg/ml giving Xuezhikang by 0.125 g/L, 0.25 g/L, 0.5 g/L and 1 g/L, compared with the control group (700.6±1.8 pg/ml) to reduce 10.8%, 22.6%, 18.3% and 25% (P<0.01). The TNF-α content was 835.7±1.4, 802.4±1.6, 745.3±1.3 and 679.5±1.7 pg/ml giving Xuezhikang by 0.125 g/L, 0.25 g/L, 0.5 g/L and 1 g/L, compared with the control group (899.3±1.9pg/ml) to reduce 7.1%, 10.8%, 17.1% and 24.4% (P<0.01); the IL-6 content was 523.4±1.4 pg/ml giving 1 μmol/L Atorvastatin, compared with the control group to reduce 25.3% (P<0.01). The TNF-α content was 652.4±1.1 pg/ml giving 1 μmol/L density Atorvastatin, compared with the control group to reduce 27.5% (P<0.01). The IL-6 and TNF-α content in 1 g/L Xuezhikang was similar to 1 μmol/L Atorvastatin (P>0.05) (Table 2).
Table 2. Effects of Xuezhikang and Atorvastatin on cell secreting IL-6 function (n=6, )
|
control |
Atorvastatin (1 μmol/L) |
Xuezhikang (g/L) |
|
0.125 |
0.25 |
0.5 |
1 |
|
IL-6 |
700.6±1.8 |
523.4±1.4▲,★ |
624.5±1.8▲,※ |
524.6±1.2▲,※ |
572.4±1.4▲,※ |
529.3±1.5▲,※ |
|
|
TNFα |
899.3±1.9 |
652.4±1.1▲,★ |
835.7±1.4▲,※ |
802.4±1.6▲,※ |
745.3±1.3▲,※ |
679.5±1.7▲,※ |
|
▲P<0.01 vs. control; ★P<0.01 vs. each group of Xuezhikang; ※P<0.01 vs. other groups of Xuezhikang.
Discussion
OxLDL is one of the most important factors in chronic inflammatory reaction process. IL-6 and TNF-α are two traditional cell inflammatory factors that are well established as vital factors in determining the risk of coronary heart disease and pathogenesis of atherosclerosis. They are mainly secreted by the macrophage. Ridker et al [17] confirmed that serum TNF-a level of the male stabilization patients with myocardial infarction were positively correlated with the risk of recurrence coronary events. TNF-α could stimulate the endothelial cell produce the E-selec element, and stimulated the smooth muscle cell to produce IL-6, then stimulated liver cell to produce the C-response protein. TNF-α might play a central role in the inflammatory regulating process. Ridker et al [18] also proved that the serum IL-6 heighten level in the healthy crowd wall increase the risk of occurring myocardial infarction obviously in future, therefore IL-6 was independence dangerous factor to forecast the cardiovascular event.
Currently accepted statins not only adjusted the lipid, also had anti-oxidant, anti-inflammatory and inhibited platelet aggregation. Traditional Chinese medicine Xuezhikang an extract of Chinese red yeast rice includes statins material, also contains a variety of effective ingredients, such as 8% of the unsaturated fatty acid (mainly for linoleic acid, oleic acid and hexadecanoic acid and stearic acid etc), enable it to adjust the lipid and anti-atherosclerosis. The clinical research demonstrated that Xuezhikang could reduce the danger of 45% in coronary disease event such as acute cardiac arrest and sudden death, coronary disease death danger dropping 31%, re infarction danger dropping 56%, the total mortality reducing 33% [19]. Red yeast rice has also been studied head to head with statins and was shown to be noninferior in reducing cholesterol levels and cardiovascular risk [20]. It seems to be a safe, viable option in patients unable to tolerate statins because of myalgias and/or other statin-related side effects.
This study demonstrated that ox-LDL treatment significantly up-regulate secretion of TNF-α and IL-6 in macrophage-like cells, this conform to the previously reported [21]. And this study demonstrated the inhibitory effect of xuezhikang and Atorvastatin on TNF-α and IL-6 expression in U937 cells in culture. This inhibitory effect of the xuezhikang on TNF-α and IL-6 production is revealed a dose-dependent. Given that TNF-α and IL-6 are proinflammatory cytokines that play an important role during macrophage foam cell formation and atherosclerotic development and progression [22, 23]. These findings suggest a possible inhibitory effect of xuezhikang on the pathogenesis of atherosclerosis as well as statins.
References
[1] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340: 115-126.
[2] Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol 2010; 7: 77-86.
[3] Bobryshev YV. Monocyte recruitment and foam cell formation in atherosclerosis. Micron 2006; 37: 208-222. [4] Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med 2002; 8: 1235-1242.
[5] Miller YI, Viriyakosol S, Worrall DS, Boullier A, Butler S, Witztum JL. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler hromb Vasc Biol 2005; 25: 1213.
[6] Jovinge S, Ares MP, Kallin B, Nilsson J. Human monocytes/macrophages release TNF-alpha in response to Ox-LDL. Arterioscler thromb Vasc Biol 1996; 16: 1573-1579.
[7] Wang YC, Hu YW, Sha YH, Gao JJ, Ma X, Li SF, Zhao JY, Qiu YR, Lu JB, Huang C, Zhao JJ, Zheng L, Wang Q. Ox-LDL Upregulates IL-6 Expression by Enhancing NF-κB in an IGF2-Dependent Manner in THP-1 Macrophages. Inflammation 2015; [Epub ahead of print]
[8] Analysis C. Interleukin-6 Receptor Mendelian Randomisation. The interleukin-6 receptor as a target for prevention of coro-nary heart disease: a Mendelian randomisation analysis. Lancet 2012; 379: 1214-1224.
[9]Kaplanski G1, Marin V, Montero-Julian F, Mantovani AMantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol 2003; 24: 25-29.
[10]Zhang YZ. TNF-alpha promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: Crosstalk between NF-kappa B and PPAR-gamma. J Mol Cell Cardiol 2014; 72: 85-94
[11] Tam LS (Tam, Lai-Shan). Can suppression of inflammation by anti-TNF prevent progression of subclinical atherosclerosis in inflammatory arthritis? Rheumtology 2014; 53: 1108-1119.
[12] Artom N, Montecucco F, Dallegri F, Pende A. Carotid atherosclerotic plaque stenosis: the stabilizing role of statins. Eur J Clin Invest 2014; 44: 1122-34.
[13] Yang CW, Mousa SA. The effect of red yeast rice (Monascus purpureus) in dyslipidemia and other disorders. Complement Ther Med 2012; 20: 466-74.
[14] Liu J, Zhang J, Shi Y, Grimsgaard S, Alraek T, Fonnebo V. Chinese red yeast rice (Monascus purpureus) for primary hyperlipidemia: a meta-analysis of randomized controlled trials. Chinese Medicine 2006; 1: 4.
[15] Zhao SP, Liu L, Cheng YC, Shishehbor MH, Liu MH, Peng DQ, Li YL. Xuezhikang, an extract of cholestin, protectsendothelial function through antiinflammatory and lipid-lowering mech-anisms in patients with coronary heart disease. Circulation 2004; 110: 915-920.
[16]Yamashiki M, Nishimura A, Suzuki H, Sakaguchi S, Kosaka Y. Effects of the Japanese Herbal Medicine “Sho-Saiko-To” (TJ-9) on In Vitro Interleukin-10 Production by Peripheral Blood Mononuclear Cells of Patients With Chronic Hepatitis C. Hepatology 1997; 25: 1390-1397.
[17]Ridker PM, Rifai N, Pfeffer M. Elevation of tumor necrosis alpha and increased risk of recurrent coronary enents after myocardial infarction. Circulation 2000; 101: 2149-2153.
[18] Ridker PM, Rifai N, Stampfer M. Plasma concertration of interleukia 6 and the risk of future myocardial infarction among apparently healthy men. Circulation 2000; 101: 1767-1772.
[19]Liu ZL. China coronary secondary prevention study (CCSPS). Chin J Cardiol 2005; 33: 109-115.
[20]Megan E, Musselman RS, Pettit KL, Derenski A. Review and Update of Red Yeast Rice. Complementary & Alternative Medicine 2012; 17: 33-39.
[21]Hulthe J, Fagerberg B. Circulating Oxidized LDL Is Associated With Subclinical Atherosclerosis Development and Inflammatory Cytokines (AIR Study). Arterioscler Thromb Vasc Biol 2002; 22: 1162-1167.
[22] Persson J, Nilsson J, Lindholm MW. Interleukin-ibeta and tumour necrosis factor-alpha impede neutral lipid turnover in macrophage-derived foam cells. BMC Immunol 2008; 9: 70.
[23] Kang MW, Song HJ, Kang SK, Kim Y, Jung SB, Jee S, Moon JY, Suh KS, Lee SD, Jeon BH, Kim CS. Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production. Korean J Physiol Pharmacol 2015; 19: 229-34.