Protective Effect of Theaflavin-3-digallate on Lipopolysaccharide-induced...

[Full title: Protective Effect of Theaflavin-3-digallate on Lipopolysaccharide-induced Inflammation Injury in Macrophage Cells]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2017.980.989 Research Article Protective Effect of Theaflavin-3-digallate on Lipopolysaccharide-induced Inflammation Injury in Macrophage Cells 1 Yanting Wu, 1 Fujun Jin, 1 Junwei Liu, 1 Danlin Zheng, 2 Yiliang Wang, 1 Feiyan Wu, 1 Yexuan Zhu and 1 Yifei Wang 1 Guangzhou Jinan Biomedicine Research and Development Center, Institute of Biomedicine, College of Life Science and Technology, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China 2 College of Pharmacy, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China Abstract Background and Objective: Inflammation is a defensive response against a multitude of harmful stimuli and stress conditions such as tissue injury and is one of the most common pathological processes of human diseases. Tumor Necrosis Factor- " (TNF- " ), interleukin-1 $ (IL-1 $ ) and interleukin-6 (IL-6) play important roles during the inflammatory state. The aim of this study was to investigate the suppressive effects of theaflavin-3-digallate on lipopolysaccharide (LPS)-induced inflammation in U 937 and RAW 264.7 cells Materials and Methods: The U 937 and RAW 264.7 cells were treated with 100 ng mL G 1 LPS in the presence of theaflavin-3-digallate (6.25-50 µM). The qRT-PCR assay was used to test the levels of TNF- " , IL-1 $ and IL-6 in LPS-treated Phorbol Myristate Acetate (PMA)-primed U 937 and murine RAW 264.7 cells and Western blot assays were used to identify potential mechanism of antiinflammatory potential of the theaflavin-3-digallate. Significance between groups data were analyzed by Graph Pad Prism 5.0, using Studentʼs t-test, One-way ANOVA. Results: The present study has clearly shown that theaflavin-3-digallate markedly decreased the mRNA expression of TNF- " , IL-1 $ and IL-6 in a dose-dependent manner. Moreover, western blot analyses demonstrated that theaflavin-3-digallate also blocked phosphorylation of I 6 B from cytosolic fraction and reduced LPS-induced nuclear accumulation of transcription factor nuclear factor-kappa B (NF- 6 B) p 65. Conclusion: These results demonstrated that theaflavin-3-digallate might abolish inflammation in RAW 264.7 macrophages and PMA-primed U 937 cells via NF- 6 B pathway Key words: Theaflavin-3-digallate, inflammation, lipopolysaccharide, tumor necrosis factor, interleukin-1 $ , interleukin 6 Received: April 21, 2017 Accepted: June 22, 2017 Published: October 15, 2017 Citation: Yanting Wu, Fujun Jin, Junwei Liu, Danlin Zheng, Yiliang Wang, Feiyan Wu, Yexuan Zhu and Yifei Wang, 2017. Protective effect of theaflavin-3-digallate on lipopolysaccharide-induced inflammation injury in macrophage cells. Int. J. Pharmacol., 13: 980-989 Corresponding Author: Yifei Wang, Guangzhou Jinan Biomedicine Research and Development Center, Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, 510632, People Republic of China Tel/fax: +86 20 85223426 Copyright: © 2017 Yifei Wang et al. This is an open access article distributed under the terms of the creative commons attribution License, which permitsunrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 13 (8): 980-989, 2017 INTRODUCTION Theaflavin-3-gallate (TF-2 a) is one kind of theaflavin in black tea. The TF-2 a has been applied for antioxidant, antitumor, anti-virus in cell cultures 1-3 . Nevertheless, the underlying mechanisms of the suppressive effects of TF-2 on LPS-induced inflammation remain unknown Numbers of human diseases even cancer are associated with inflammation. Inflammation is a defensive biological reaction involving various cells and cytokines. During the process of inflammation, many immune cells especially macrophages, hyper-secrete pro-inflammatory cytokines, which further accelerate inflammation reaction. Immune cells play crucial roles in eliminating exogenous pathogens and release various chemokine and pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF)- " , interleukin (IL)-1 $ and IL-6 4 . As one of the key downstream mediators in inflammation, the pro-inflammatory cytokine TNF- " controls inflammatory cells as well as influencing the processes of inflammation 5 . There have few IL-1 $ in cells of healthy persons, however, the level of IL-1 $ in monocytes from patients of inflammatory diseases is elevated significantly 6-11 Furthermore, IL-1 $ requires a series of other intracellular cytokines, such as TNF- " , IL-1 " , IL-6 and IL-18 before it trigger inflammation. Therefore, once the release of IL-1 $ increased rapidly, there is more serious inflammation 12 . Worse still, TNF- " and IL-1 $ also activate other transcription factors to further induce the secretion of IL-6. As reported by previous study, TNF- " , IL-1 $ and IL-6 play a key role in activating the host response through inducing an acute inflammatory response and alerting immune system to perform its function 4 TNF- " , IL-1 $ and IL-6 are also related to generic Mitogen-Activated Protein Kinase (MAPK) and NF- 6 B signaling pathways. The MAPK pathway is associated with cell life cycle, which can be activated by lipopolysaccharide (LPS) 13 . The signaling pathway includes extracellular signal-related kinases 1/2 (ERK 1/2), c-Jun N-terminal kinases (JNK), p 38 and ERK 5 14 These MAPK signaling pathways are vital for LPS-induced production of TNF- " , IL-1 $ and IL-6 in activated macrophages Suppression of any of these MAPK pathways is enough to block the LPS-induced increasing secretion of TNF- " 15 Furthermore, MAPKs are also related to the activation of nuclear factor-kappa B (NF- 6 B) 16 . The MAPKs and NF- 6 B can work synergistically to cause the increasing expression of pro-inflammatory genes and the secretion of cytokines 17 During inflammatory response, NF- 6 B is phosphorylated and translocate into the nucleus, then it induces the expression of genes relevant to inflammatory responses 18,19 . Therefore, MAPK and NF- 6 B signaling pathway have been recognized as key target for treatment of inflammation and inflammatory diseases This study aimed is to investigate the effects of TF-2 a on the cytokine levels in LPS-treated Phorbol Myristate Acetate (PMA)-primed U 937 and murine RAW 264.7 cells, to elucidate its potential mechanism of action MATERIALS AND METHODS Chemicals and reagents: The TF-2 a (>98% purity) was purchased from Pi and Pi Technology Inc. (Guangzhou, China), then solubilized in dimethyl sulfoxide (DMSO) and diluted with medium as needed. The final DMSO concentration was 0.1% (v/v). Dexamethasone (DEX) was obtained from Guangzhou Overseas Chinese Hospital. The 3-(4,5-Dimethyl-2-thiazolyl)- 2, 5-diphenyl-2 H-tetrazolium bromide (MTT) and LPS (Escherichia coli 055:B 5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal Bovine Serum (FBS), Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbeccoʼs modified Eagle medium (DMEM) and penicillin-streptomycin mixture were bought from Invitrogen (Carlsbad, CA, USA). All antibodies used were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). All other reagents were of pharmaceutical grade Cell culture: The U 937 human leukemia and RAW 264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured either in RPMI-1640 or in DMEM with 10% FBS and penicillin (100 U mL G 1 ) and streptomycin (100 µg mL G 1 ) in a suitable humidified incubator with 5% CO 2 atmosphere at 37 E C Cytotoxicity assays: The MTT assays were conducted to assess the cytotoxicity of TF-2 a towards U 937 human leukemia and RAW 264.7. Cells were cultured overnight in 96-well plates in 100 µL of growth media (RPMI-1640 or DMEM with 10% FBS) and exposed to a series concentrations of TF-2 a for 48 h. Then, 10 µL of 5 mg mL G 1 MTT solution was added to each well and the plate was incubated in the dark for 4 h. The MTT solution was then discarded and 100 µL of DMSO was added to each well. The plates were gently shaken for 15 min at room temperature, before the optical density at 570 and 630 nm was recorded with an enzyme immunoassay reader (Bio-Rad, Hercules, CA, USA). The normalized cell viability at each TF-2 a concentration was determined by comparison to TF-2 a-free cells 981

Int. J. Pharmacol., 13 (8): 980-989, 2017 LPS-induced inflammation assays: In vitro models of inflammation, such as LPS-treated, PMA-primed U 937 human leukemia and murine RAW 264.7 cells have been widely used to investigate anti-inflammatory effects of small-molecule compounds 20-23 . Therefore, these two models were used in this study to assess the anti-inflammatory activity of TF-2 a. To examine the inhibitory effects of TF-2 a on LPS-induced TNF- " , IL-1 $ and IL-6 expression in LPS-induced cells, two approaches were used in this study. The following experiments were performed to study LPS-induced inflammation in U 937 and RAW 264.7 cells: (1) U 937 cells were primed with PMA (10 ng mL G 1 ) for 48 h. The medium was discarded and cells were washed three times with PBS. A range of TF-2 a concentrations with LPS induction medium (100 ng mL G 1 ) in serum-free RPMI-1640 medium was added and cells were cultured for 24 h before total RNA isolation, (2) U 937 cells were primed with PMA (10 ng mL G 1 ) for 48 h. The medium was discarded and cells were washed three times with PBS. A range of TF-2 a concentrations in complete RPMI-1640 medium was added for 4 h. The medium was then discarded and cells were washed three times with PBS. The serum-free medium was then replaced with the LPS induction medium (100 ng mL G 1 ). The cells were cultured for 2 h before total RNA isolation. (3) RAW 264.7 cells were cultured in 12-well plates to 70~80% confluence. The medium was discarded and cells were washed three times with PBS. A range of TF-2 a concentrations with LPS medium (100 ng mL G 1 ) in serum-free DMEM was added and cells were cultured for 6 h before total RNA isolation. (4) RAW 264.7 cells were cultured in 12-well plates to 70~80% confluence. The medium was discarded and cells were washed three times with PBS. A range of TF-2 a concentrations in complete DMEM was added for 6 h. The medium was then discarded and cells were washed three times with PBS. The serum-free medium was then replaced with LPS induction medium (100 ng mL G 1 ). The cells were cultured for 6 h before total RNA isolation. Real-time PCR: Total RNA was extracted using the TRIzol® universal reagent (TIANGEN, Beijing, China) and RNA concentrations were measured using a Nanophotometer™ P-Class P 330 (Implen, Munich, Germany). The extracted RNA (1,000 ng) was reverse-transcribed using a PrimeScript™ RT master mix kit (Takara Bio, Kusatsu, Japan). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays were performed using SsoFast™ Eva Green® supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The sequences of the primers used were as follows: Mouse TNF- " (F: 5'-CAAAATTCGAGTGAC AAGCCTG-3' and R: 5'-GAG ATCCATGCCGTTGGC-3'), mouse IL-1 $ (F: 5'-ATGCCACCTTTTGACAGTGAT-3' and R: 5'-TGC GAGATTTGAAGCTGGAT-3'), mouse IL-6 (F: 5'-TCCATCCAGTTG CCTTCTTG-3' and R: 5'-GAA GTCTCCTCTCCGGACTT-3') and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (F: 5'-TGTGTCCGTCGTGGATCT GA-3' and R: 5'-CCTGCTTCA CCACCTTCTTGA-3'), human TNF- " (F: 5'-GGGCCTGTACCTCAT CTA CT-3' and R: 5'-TGACCTTGGTCTGGTAGGAG-3'), human IL-1 $ (F: 5'-TGATGGCTTATTACAGTGGCAATG-3' and R: 5'-GTA GTGGTGGTCGGAGATTCG-3'), human IL-6 (F: 5'-CCACTCACC TCTTCAGAACG-3' and R: 5'-CATGTCTCCTTTCTCAGGGC-3') and human GAPDH (F:5'-AGCCTCAAGATCATCAGCAAT G-3' and R: 5'-CACGATACCAAAGTTGTCATGGAT-3'). The relative expression was determined by the Ct method and normalized to the GAPDH expression Western blot assays: PMAprimed U 937 cells and RAW 264.7 cells were treated with LPS (100 ng mL G 1 ) alone or in combination with various concentrations of TF-2 a. An hour later, the cells were lysed in a 1% Sodium Dodecyl Sulfate (SDS) solution containing 2 mM proteinase inhibitor, phenylmethanesulfonyl fluoride (Invitrogen, Carlsbad, CA, USA). The lysate was heated for 10 min at 100 E C, then centrifuged at 12,000 rpm for 15 min at 4 E C and the supernatant was collected. The protein concentration was determined using a bicinchoninic acid assay kit (Beyotime, Jiangsu, China). The protein samples were resolved by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline-Tween 20 (TBS-T) [150 mM NaCl, 50 mM Tris-HCl, pH 7.6 and 0.1% (v/v) Tween-20] for 1 h at room temperature and then incubated with primary antibodies overnight at 4 E C. The membranes were then incubated with secondary antibodies diluted in 5% dried milk in TBS-T for 1 h. The immunoblots were visualized using the enhanced chemiluminescence reagent (Beyotime). The Image J software was used to quantify the band intensity. The signal intensity of each protein was normalized to that of GAPDH Statistical analysis: Results were presented as Mean±SEM and analyzed using Graph Pad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Significance between groups data were analyzed by Graph Pad Prism 5.0, using Studentʼs t-test, one-way ANOVA. A significant difference was indicated by p-values less than 0.05, 0.01, or 0.001 24 RESULTS Cytotoxicity of TF-2 a towards U 937 and RAW 264.7 cells: Results of TF-2 a cytotoxicity tests shown in Fig. 1 b-c indicated 982

Int. J. Pharmacol., 13 (8): 980-989, 2017 HO HO HO HO HO OH OH OH OH OH OH O O O O O (a) 200 150 100 50 0 0.00 3.12 6.25 12.50 25.00 50.00 100.00 200.00 Concentration (µM) C el l v iab ilit y ( % ) (b) 110 100 90 80 70 60 0.00 3.12 6.25 12.50 25.00 50.00 100.00 200.00 Concentration (µM) C ell v ia b ility ( % ) (c) Fig. 1(a-c): (a) Chemical structure of theaflavin-3-digallate (TF-2 a), Cytotoxic effect of TF-2 a on (b) PMA-primed U 937 and (c) RAW 264.7 Data are presented as the Mean±SEM of at least three independent experiments that the half-lethal concentrations of TF-2 a towards U 937 and RAW 264.7 cells were all over 200 µM, suggesting that TF-2 a (3.12~50 : M) was fairly non-toxic to cells TF-2 a inhibited mRNA expression of TNF- " , IL-1 $ and IL-6 in PMA-primed U 937 cells: As shown in Fig. 2 a-b, compared with those of control group, TNF- " , IL-1 $ and IL-6 levels were increased obviously ( # p<0.05, ## p<0.01 or ### p<0.001) after LPS stimulus. Interestingly, levels of TNF- " , IL-1 $ and IL-6 in PMA-primed U 937 cells were all significantly inhibited (*p<0.05, **p<0.01 or ***p<0.001) by TF-2 a in a dose-dependent manner when co-administered with LPS in PMA-primed U 937 cells (Fig. 2 a). However, compared with the LPS-treated control, the mRNA expressions of TNF- " , IL-1 $ and IL-6 almost were not inhibited by TF-2 a, when pretreated of four hours (Fig. 2 b). These results suggested that co-treated with TF-2 a and LPS, TF-2 a might exhibit inhibitory effect on LPS-induced increased release of inflammatory cytokines in PMA-primed U 937 cells TF-2 a inhibited mRNA expression of TNF- " , IL-1 $ and IL-6 in LPS stimulated RAW 264.7 macrophages: Following the observation that TNF- " , IL-1 $ and IL-6 production was inhibited by TF-2 a in PMA-primed U 937 cells, qRT-PCR analysis were carried out to determine whether the inhibition of TNF- " , IL-1 $ and IL-6 production by TF-2 a in the LPS-stimulated RAW 264.7 cells. As mentioned methodology, two approaches were also used in this study. In response to LPS treatment, mRNA expressions of TNF- " , IL-1 $ and IL-6 were significantly induced ( # p<0.05 or ### p<0.001); However, co-treatment with TF-2 a inhibited this up-regulation in a dose-dependent manner (***p<0.001) (Fig. 3 a). In addition, pretreatment with TF-2 a markedly suppressed the levels of TNF- " , IL-1 $ and IL-6(*p<0.05, **p<0.01 or ***p<0.001), respectively (Fig. 3 b). The results indicated that TF-2 a can down-regulate LPS-induced increased production of TNF- " , IL-1 $ and IL-6 in RAW 264.7 cells in different treatment ways TF-2 a didnʼt suppress the activation of MAPK signaling pathway in vitro: The MAPK pathways are also involved in inflammation response. Suppression of any of three MAPK pathways is enough to block the LPS-induced increasing secretion of TNF- " , IL-1 $ and IL-6 by LPS 15 . Thus, to clarify the anti-inflammatory mechanism of TF-2 a, Western blot analysis 983

Int. J. Pharmacol., 13 (8): 980-989, 2017 8 6 4 2 0 ** * * * ## C ont ro l LP S 6 25 µM 12 .5 0 µM 25 00 µ M 50 00 µ M Combined (24 h) R e lativ e ex p ress io n o f TN F- /GA P D H  (a) 50 40 30 20 10 0 ### R elati v e e x p re ss io n o f IL -1 /G A PD H  * * * *** Co nt ro l L PS 6 25 µM 12 .50 µM 25 .0 0 µM 50 00 µM Combined (24 h) 6 4 2 0 ** ## ** ** ** R el ati ve exp res si o n of I L -6 /G A P D H C on tro l LP S 6 25 µ M 12 .5 0 µ M 25 00 µM 50 00 µM Combined (24 h) 2.5 2.0 1.5 1.0 0.5 0.0 R e la ti v e e x p re ss io n o f T N F- /G A P D H  (b) Co nt ro l LP S 6 25 µ M 12 .50 µ M 25 .0 0 µ M 50 .0 0 µ M Pretreatment (4 h) 6 4 2 0 * # R elati v e e x p re ss io n o f I L- 1 /GA P D H  C on tro l L PS 6 25 µM 12 50 µM 25 00 µM 50 00 µM Pretreatment (4 h) 25 20 15 10 5 0 *** ### R elati v e e x p re ss io n o f I L -6 /G AP DH Co nt ro l L PS 6 25 µ M 12 50 µ M 25 00 µ M 50 .00 µ M Pretreatment (4 h) Fig. 2(a-b): Effects of TF-2 a on LPS-induced mRNA expressions of TNF- " , IL-1 $ and IL-6 in PMA-primed U 937 cells, (a) PMA-primed U 937 cells were treated with LPS only or in combination with different concentrations of TF-2 a for 24 h, (b) Before treatment with LPS for 2 h, PMA-primed U 937 cells were pretreated with different concentrations of TF-2 a for 4 h Gene expression was measured using qRT-PCR using GAPDH as reference. Data are presented as the Mean±SEM of three independent experiments # p<0.05, ## p<0.01, ### p<0.001 compared with the control group; *p<0.05, **p<0.01, ***p<0.001 compared with the LPS-treated group was conducted to assess the effects of TF-2 a on MAPK activation. However, as shown in Fig. 4 b, TF-2 a didnʼt inhibit any of the three MAPK pathways phosphorylation in RAW 264.7 cells. Furthermore, only p-JNK and p-Erk 1/2 were blocked by 50 : M TF-2 a treatment in PMAprimed U 937 cells (Fig. 4 a). These results suggested that TF-2 a didnʼt through 984

Int. J. Pharmacol., 13 (8): 980-989, 2017 Co nt ro l LP S 6 25 µ M 12 .5 0 µM 25 .0 0 µM 50 .0 0 µM Combined (6 h) 25 20 15 10 5 0 *** ### (a) *** *** ** R ela ti v e ex p res si o n o f T N F -/ G A P D H  2000 1500 1000 500 0 *** ### R el at iv e e x p re ss io n o f IL -/ G A P D H  (b) *** ** C on tro l LP S 6 25 µ M 12 .5 0 µM 25 .00 µ M 50 .00 µ M Combined (6 h) 1500 1000 500 0 *** ### (e) *** *** *** R e la ti v e ex p res sio n o f IL -1 /G A P D H  Co ntro l LP S 6 25 µ M 12 .5 0 µM 25 .0 0 µM 50 .0 0 µM Pretreatment (6 h) 15 10 5 0 ** ### R ela ti v e ex p re ss io n o f TN F - /G A P D H  (d) * * * Co nt ro l L PS 6 25 µM 12 .5 0 µM 25 .0 0 µM 50 .0 0 µM Pretreatment (6 h) 80 60 40 20 0 *** ### (c) *** *** * Co nt ro l LP S 6 25 µ M 12 50 µM 25 .0 0 µ M 50 .0 0 µ M Combined (6 h) R el at iv e ex p res si o n o f IL -6 /G A P D H 20 15 10 5 0 ### R e la ti v e e x p re ssi o n o f IL -6 /GAP DH (f) *** *** *** *** C on tro l LP S 6 25 µ M 12 .5 0 µM 25 .0 0 µM 50 .0 0 µM Pretreatment (6 h) Fig. 3(a-b): Effects of TF-2 a on LPS-induced TNF- " , IL-1 $ and IL-6 mRNA expressions in RAW 264.7 macrophages, (a) RAW 264.7 macrophages were treated with LPS only or in combination with different concentrations of TF-2 a for 6 h, (b) Before treatment with LPS for 6 h, RAW 264.7 macrophages were pretreated with different concentrations of TF-2 a for 6 h Gene expression was measured using qRT-PCR using GAPDH as reference. Data are presented as the Mean±SEM of three independent experiments ### p<0.001 compared with the control group; *p<0.05, **p<0.01, ***p<0.001 compared with the LPS-treated group blocking the activation of MAPK signaling pathway to suppress the expression of TNF- " , IL-1 $ and IL-6 in vitro TF-2 a inhibited LPS induced NF 6 B activity in vitro: Under normal circumstances, NF- 6 B binds with I 6 B and is localized to 985

Int. J. Pharmacol., 13 (8): 980-989, 2017 Fig. 4(a-b): Effects of TF-2 a on the activation of MAPK in LPS-induced PMA-primed U 937 cells and RAW 264.7 macrophages Protein expression levels of JNK, p 38 and Erk 1/2 were determined by Western blot assay with GAPDH as internal control Fig. 5(a-b): Effects of TF-2 a on the activation of NF- 6 B in LPS-induced PMA-primed U 937 cells and RAW 264.7 macrophages NF- 6 B p 65 and phosphorylated I 6 B- " protein expression were measured using Western blot assay with GAPDH as internal control the cytoplasm. However, in activated macrophages, I 6 B is phosphorylated by I 6 B kinase and degraded rapidly, which causes NF- 6 B translocate into the nucleus. Furthermore, NF- 6 B regulates the transcription of TNF- " , IL-1 $ and IL-6 23,25,26 Thus, to find out whether TF-2 a blocks NF- 6 B (p 65) from translocating to the nucleus, western blot assays were conducted to analyze the protein levels of phosphorylated p 65 and I 6 B. The result in Fig. 5 showed that the protein levels of the p-p 65 was markedly increased after LPS stimulation, be indicative of LPS activated NF- 6 B signaling pathway. The NF- 6 B translocated from the cytoplasm to the nucleus. However, TF-2 a reduced the accumulation of NF- 6 B p 65 in the nucleus in LPS-induced PMA-primed U 937 cells (Fig. 5 a) and RAW 264.7 cells (Fig. 5 b) in a dose-dependent manner. In addition, phosphorylated I 6 B- " was inhibited by TF-2 a. These results suggested that TF-2 a prevented NF- 6 B activation in vitro DISCUSSION The lipopolysaccharide (LPS) endotoxin is a component of cell membrane of the Gram-negative bacterial. The LPS can induce the release of pro-inflammatory cytokines by macrophages and circulating monocytes, resulting in a transient immune activation, which is characterized by elevated levels of TNF- " , IL-1 $ and IL-6 27,28 . The TF-2 a has been obtained to elevate the apoptosis of cancer cells 29 . The TF-2 a also suppress tumor growth and metastasis of cancer cells 30 In this study, the results demonstrated that TF-2 a exhibits a 986 p-p 38 p 38 p-JNK JNK p-Erk 1/2 Erk 1/2 GAPDH U 937 cell 6.25 12.5 25 50 6.25 12.5 25 50 p-p 38 p 38 p-JNK JNK p-Erk 1/2 Erk 1/2 GAPDH LPS (100 ng mL G 1 ) TF-2 a (µM) LPS (100 ng mL G 1 ) TF-2 a (µM) RAW 264.7 cell (a) (b) U 937 cell 6.25 12.5 25 50 LPS (100 ng mL G 1 ) TF-2 a (µM) (a) p-I  B  NF-  B GAPDH 6.25 12.5 25 50 LPS (100 ng mL G 1 ) TF-2 a (µM) RAW 264.7 cell (b) p-I  B  NF-  B GAPDH

Int. J. Pharmacol., 13 (8): 980-989, 2017 positive protective effect against LPS-induced inflammation in vitro . The methods used in this study was just like the previous researchs 28,31 . Before starting to carry out the formal experiments, preliminary experiments were performed, the incubation times and the concentrations of TF-2 a were based on the results of preliminary experiments. As is known to all, MTT is a most commonly used method to monitor the cytotoxic effects the drugs and also a lots of articles used this method to verify the cytotoxicity of the small molecule compounds 32,33 . In order to avoid false positives, MTT assays were first carried out to confirm that the positive protective effects of TF-2 a were not due to its cytotoxicity. Inflammation is a protective biological response to cell or tissue injury caused by external pathogenic microorganisms, during which large quantities of pro-inflammatory cytokines are produced 23,34 . Out of the many inflammatory factors, TNF- " , IL-1 $ and IL-6 are always regarded as the most essential cytokines involved in inflammatory reactions and are released in response to bacterial endotoxins. Inhibition of the production of these cytokines is beneficial for the treatment of inflammation. Hence, they are ideal targets for novel anti-inflammatory agents. In the study, the results showed that TF-2 a strongly inhibited the LPS-induced increasing mRNA expressions of TNF- " , IL-1 $ and IL-6 in PMA-primed U 937 cells and RAW 264.7 cells. Due to the different methods of treatment, not all of the results showed correlation with increase in the dose of TF-2 a. Numerous experiments have proved that co-administration of TF-2 a and LPS is beneficial to the action of TF-2 a. Thus, this particular mode of treatment was one of the methods adopted in the following study It has been reported that MAPK and NF- 6 B signaling pathways regulate LPS-induced inflammatory reaction 35 . To illustrate the potential mechanisms by which TF-2 a exerts positive effect on LPS-induced inflammation, the inhibitory effect of TF-2 a on the activation of MAPK and NF- 6 B signaling pathways in LPS-induced PMA-primed U 937 cells and RAW 264.7 cells was verified. After exposing to LPS, both NF- 6 B and MAPKs signaling pathways were activated but co-treatment of TF-2 a and LPS only suppressed nuclear translocation of NF- 6 B (p 65) and reduced the phosphorylation of I 6 B- " in LPS-treated cells. These data indicated that TF-2 a exerts its anti-inflammatory activity through reducing the phosphorylation of I 6 B- " and the nuclear translocation of NF- 6 B. The underlying mechanism of the inhibitory effect of TF-2 a on LPS-induced inflammation may be the same as that of other theaflavins. It was reported that TF-3, another kind of theaflavin, exerts its anti-inflammatory effects via the regulation of NF- 6 B activation 28 . Similarly, rhein inhibited the production of pro-inflammatory cytokines and attenuated leukocyte infiltration by suppressing the activation of NF- 6 B 33 Together, these observations demonstrated that TF-2 a inhibits the activation of the transcription factor NF- 6 B signalling pathway in macrophages, thereby inhibiting the expression of pro-inflammatory cytokines. In turn, the decrease in the release of inflammatory cytokines, for example, TNF- " , would suppress the activation of NF- 6 B CONCLUSION AND FUTURE RECOMMENDATION It can be concluded that TF-2 a have a positive protective effect against LPS-induced inflammation. It could decrease the production of TNF- " , IL-1 $ and IL-6 by inhibiting the activation of the NF- 6 B signaling pathway. Moreover, TF-2 a had no side effects on the body. Therefore, TF-2 a promises to be developed into potential health care products and services for sub-health populations. However, further investigations, both in vitro and in vivo , are needed to confirm its anti-inflammatory effects In vitro, further examinations to explore clearer mechanism are indispensable. Besides, it needs to be made certain that the effect on animal models of inflammation as well as investigating different periods of treatment, the optimal concentration of TF-2 a and the difference between its activity in human and animals SIGNIFICANCE STATEMENTS This study discovers the possible protective effect of theaflavin-3-digallate on lipopolysaccharide-induced inflammation injury in macrophage cells. This study will help the researcher to know more about the pharmacological effects especially the anti-inflammatory effect of theaflavin- 3-digallate. 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