2 The Second Affiliated Hospital of Bengbu Medical College, Bengbu 233000, China;
3 The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Hefei 230032, China;
4 Institute for Liver Diseases, Anhui Medical University, Hefei 230032, China;
5 Anhui Provincial Hospital, Hefei 230001, China;
6 School of Pharmacy, The Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
Diversity and plasticity are two hallmarks of macrophages. Different stimulus induce macrophages to express distinct cytokines, surface markers, and activate arginine metabolic pathways that eventually exert different functions seen in inflammatory and non-inflammatory, tumor and non-tumor, immunogenic and tolerogenic settings [1-2]. Two well-esta blished polarized phenotypes are often referred to as tissue classically activated macrophages (M1) and alternatively activated macrophages (M2). M1-macrophages are induced by interferon-gamma (IFN-γ) alone or in combination with lipopolysaccharide (LPS) and produce high level of iNOS and the inflammatory cytokines such as IL-12, IL-1β, IL-6, and TNF-α. In contrast, M2-macrophages are induced by exposure to a variety of signals including the cytokines IL-4 and/or IL-13 and immune complexes and express high levels of Arg-1, IL-10, TGF-β and so on [3-4].
Macrophage activation phenotypes have been reported to maintain treatment for several diseases probably with their powerful functions. Mona AA et al.  reported that M2 cells might work to be tumor promotive in classical Hodgkin lymphoma (CHL) and M1 cells might be tumor suppressive in mixed cellularity type. According to the reports, insulin resistance and atherosclerosis induced by obesity were associated with macrophage infiltration into adipose tissue and a phenotypic switched from M2 to M1 [6-8]. On the other hand, macrophages, as the main adjustment cells, both M1- and M2-macrophage polarization occurred during occurrence and development of liver fibrosis and played the opposite effects . To the best of our knowledge, macrophage polarization played significant roles in many diseases and the polarization macrophage phenotype could turn into the novel therapeutic targets. Thus, searching the drug which could regulate the switch of M1 and M2 macrophage phenotypes has gained increasingly attention in recent years. The JAK/STAT signaling pathway affects cellular activities, such as proliferation, migration, growth, differentiation, and death . Studies have shown that macrophages with the stimulation of LPS dramatically activate the Janus kinase-signal transducers2 and activators of transcription3 (JAK2-STAT3) pathway to produce inflammatory cytokines and different macrophage phenotypes [11-12]. Recent studies have revealed that IFN-γ activates the JAK and the STAT-1 phosphorylation which leads to the M1-like macrophage polarization .
HDN (4'-methoxy-3', 5, 7-trihydroxy flavanone), a naturally occurring flavanone found in citrus fruit peels, has been known to exhibit several key biological and pharmacological properties. Previous studies have demonstrated that HDN has plays a significant role in anti-infla mmatory, anti-oxidant and anti-tumor [14-15]. However, its role in macrophage polarization remains poorly understood. In addition, its water solubility and bioavailability limited the medicinal, so we modified the structure of HDN. In previous studies, our team synthesized multiple derivatives of HDN and found that HDND-12, as shown in Fig. 1A, has better anti-inflammatory activity. The synthesis and analysis of HDND-12 had been reported in the manuscript by Wang et al. . The objective of this study was to demonstrate whether the HDND-12 plays an important role in the transformation of M1- and M2-like macrophage and to explore the corresponding mechanism, and our research focuses on the effect role of HDND-12 in M1 polarization.
HDND-12 was provided by the pharmacy of Anhui Me dical University. LPS (Escherichia coli 055: B5) and anddimethyl sulfoxide (DMSO) were purchased from Sigma Chemical (St.Louis, MO, USA). Recombinant Murine IFN-γ (20 μg) was purchased from PeproTech (Rocky Hill, NJ). AG490 inhibitor was purchased from Beyotime Inc (Shanghai, China). Rabbit anti-iNOS monoclonal antibodies were purchased from Cell Signaling (Beverly, MA, USA). Mouse monoclonal antibodies β-actin was purchased from Santa Cruz (California, USA) and Rabbit anti-Arg-1, JAK2, p-JAK2, STAT3, and p-STAT3 polyclonal antibody were purchased from Santa Cruz (California, USA). Arg-1, iNOS, TNF-α, IL-10, and GAPDH primers were produced by Sangon Biological and Technological Company (Shanghai, China). Enzyme Linked Immunosorbent Assay (ELISA) kit was acquired from Hushang Biotechnology Co., Ltd. (Shanghai, China). APC anti-mouse CD206 (MMR) and APC Rat IgG2a, κIsotype Ctrl were purchased from Biolegend (USA).Cell culture and treatment
RAW264.7, a cell line of mouse peritoneal macrophages, was obtained from Cell Bank of Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco' smodified Eagle's medium (DMEM; HyClone, USA) supplemented with 10% (V/V) heat-inactivated fetal bovine serum (Bovine, China), 100 U·mL–1 of penicillin (Gibco), and 100 μg·mL–1 of strep tomycin (Gibco). Cells were grown in a humidified 5% CO2 and 37 ℃ atmosphere. Cells were cultured by adding different concentrations for 24 h, or adding 1000 ng·mL–1 LPS and 10 ng·mL–1 IFN-γ for 12 h and then adding different concentrations for 24 h in the suitable conditions.Total RNA isolation and real-time quantitative PCR analysis
Total RNA was extracted from RAW264.7 cells by using TRIzol (Invitrogen) according to the manufacture's instruction. The first-strand cDNA was synthesized from total RNA using Thermoscript RT-PCR synthesis kit (Fermentas). The reaction mixture was prepared according to the manufacture's instruction using SYBRGreen qPCR Master Mix (QIAGEN). Real-time quantitative PCR was carried out under standard protocol using the following primers: GAPDH (forward: 5'-GGACCTCATGGCCTACATGG-3'; reverse: 5'-TAGGGC CTCTCTTGCTCAGT-3'), iNOS (forward: 5'-CTGCAGCAC TTGGATCAGGAACCTG-3; reverse: 5'-GGAGTAGCCTGT GTGCACCTGGAA-3'), Arg-1 (forward: 5'-TGACATCAAC ACTCCCCTGACAAC-3'; reverse: 5'-GCCTTTTCTTCCTT CCCAGCAG-3'), TNF-α (forward: 5'-TGTCCCTTTCACTC ACTGGC-3'; reverse: 5'-CATCTTTTGGGGGAGTGCCT-3'), IL-10 (forward: 5'-ACCTCCAGGACGACTTTGAT-3'; reverse: 5'- GCTGCCTGCTCTTACTGACT-3'). PCR was performed 95 ℃ for 10 min followed by 40 cycles at 95 ℃ for 15 sec and at 60 ℃ about 1 min by using Thermo Step One. Reactions were conducted three times, and threshold cycle values were normalized to GAPDH gene expression. The specificity of the products was determined by melting curve analysis. The ratio of the relative expression of target genes to GAPDH was calculated by the formula 2−ΔΔCt.Western blot analysis
Cells were harvested and homogenized using RIPA lysis buffer (Beyotime, China) and quantified with a Bicin Choninic Acid (BCA) protein assay kit (Boster, China). Total proteins were separated by denaturing 10% or 12% SDS-PAGE and subsequently electrophoretically transferred to a PVDF membrane (Millipore Corp, Billerica, MA, USA). The membranes were then incubated in TBST containing 5% skim milk at 37 ℃ for 3 h, and with specific primary antibodies at 4 ℃ overnight. The next day, the membranes were washed 3 times with TBS/Tween 20 (0.075%), followed by incubation with HRP-conjugated secondary antibodies (1 : 8000–1 : 10000) at 37 ℃ for 1 h. Proteins were visualized with ECL-chemi luminescent kit (ECL-plus, Thermo Scientific, USA). The bands were visualized by chemiluminescence reagents (Model No. ChemiQ 4600, Bioshine)Elisa assay
The levels of IL-12, IL-10, TGF-β and TNF-α in the supernatants of macrophages were determined using an enzyme-linked immunosorbent assay (ELISA) kit (R & D USA) according to the manufacturer's instruction.Flow cytometry analysis
The expression of CD206 on RAW264.7 cells treatment with HDND-12 was studied by flow cytometry. Purified cells following specific treatments were washed with normal saline twice and labeled with fluorescence-labelled antibody (APC- conjugated CD206 anti mouse antibody and isotype control) 1 μL for 15 min as manfacturer's recommendation. Then the mean fluorescence intensities were determined using quadrant statistics.AG490 treatment
RAW264.7 cells were seeded overnight in six-well plates and pretreated with AG490 (10 μmol·L–1) for 14 h, then treat it with or without M1 inducers (LPS and IFN-γ) for 12 h and HDND-12 until the processing time finished. The medium containing DMEM only was regarded as a control.Statistical analysis
Statistical analysis was completed with the Statistical package for Social Sciences (SPSS Inc., Chicago, IL, USA, version 13.0). The data were expressed as mean ± standard error (SE). The Bartlett's test was used to determine whether the data were heterogeneous or homogeneous. Statistical significance was determined by either the Student's t test for comparison between means or one-way analysis of variance with a post hoc Dunnett's test. Differences were considered significant at P < 0.05.Results HDND-12 affects macrophage polarization in RAW264.7 cells
MTT assay was used to screen the influences of HDND- 12 on the viability of RAW264.7 cells. We treated the RAW264.7 cells with different concentrations of HDND-12 (0, 25, 50, 75, 100, 200, 400 μmol·L–1) in the 24 h. As shown in Fig. 1B, we found HDND-12 (0–150 μmol·L–1) had no effects on RAW264.7 cells, but concentrations in the 200–400 μmol·L–1 range significantly reduced cell viability. This result revealed that HDND-12 in the concentrations of 0–150 μmol·L–1 had no toxicity to RAW264.7 cells. To elucidate the functional role of HDND-12 during macrophage polarization, RAW264.7 cells were stimulated by HDND-12 with different concentrations (0, 25, 50, 75, 100, 150 μmol·L–1) for 24 h, then we detected the markers of M1 and M2 (IL-12 and IL-10) in normal group and HDND-12 group (Fig. 2A), the expression of IL-10 significantly up-regu lated compared to the control group, and were much higher at 100 μmol·L–1. Then we chose 100 μmol·L–1 for 12, 24, 48 h (Fig. 2B), which peaked at 24 h after HDND-12 treatment. Ultimately we chose the appropriate concentrations (50, 100, 150 μmol·L–1) and time point (24 h) for the next studies. As illustrated in Fig. 3A, it is observed that the expression of IL-10 and TGF-β in HDND-12 concentration groups was increased strikingly compared with the control. In conjunction with RT-QPCR (Fig. 3B) and Western Blot (Fig. 3C) analysis, the expression of Arg-1, IL-10 mRNA and Arg-1 protein was also up-re gualted significantly compared with the control. In addition, FACS analyses (Fig. 4) showed that HDND-12 with different concentrations increased the mean fluorescence intensity (MFI) and percentage of CD206 cells in RAW264.7 and the changes of HDND-12 (100 μmol·L–1) were apparent especially. In light of these evidences, we suggested that HDND-12 might be a regulator in the transformation of M1- and M2-like macrophage.
To further elucidate the regulation role of HDND-12 in polarization macrophages, we chose the classic model in RAW264.7 cells, M1-like macrophage was induced by LPS (1000 ng·mL–1) and IFN-γ (10 ng·mL–1) for 12 h , and then stimulated with different concentrations of HDND-12 (0, 50, 100, 150 μmol·L–1) for 24 h. Interestingly, we found that the marker of M1-type was down-regulated in HDND-12 groups, suggesting that HDND-12 could inhibit the switch of M1-like macrophage. Western blot analysis showed that (Fig. 5) iNOS, the hallmark of M1 macrophage, was increased remarkably by LPS and IFN-γ treatment when compared with the normal. On the other hand, the expression of iNOS in the HDND-12 concentrations groups was decreased compared with the LPS and IFN-γ-induced cells. In light of these evidences, we suggested that HDND-12 could inhibit the shift of M1-like macrophages in RAW264.7 cells.
We have proved that HDND-12 could up-regulate the expres sions of M2 markers and inhibit M1 polarization of macrophage, however, the possible regulatory mechanism of HDND-12 in macrophage polarization is still unknown to date. It is well known that the JAK2/STAT3 pathway play an important role in macrophage inflammatory response and occupies a central role among the regulatory factors of macrophage pola rization [18-19]. Thus, we paid more attention to p-JAK2 and p-STAT3.
In order to examine whether JAK2 and STAT3 were keys in the process of the shift of RAW264.7 macrophage cells by HDND-12, JAK2, p-JAK2, STAT3 and p-STAT3 protein levels were analyzed by western blot. As shown in Figs. 6A, 6B and 6C, the expression of p-JAK2 was significantly down-regulated after stimulation with HDND-12 (50, 100, 150 μmol·L–1). Moreover, not only macrophage induced by LPS and IFN-γ significantly increased p-JAK2 protein expression, but also inhibited HDND-12-treated M1-type macrophage p-JAK2 protein level up-regulation. Furthermore, the expression of p-STAT3 was in accordance with the above results of p-JAK2. As indicated by Western Blot assay (Fig. 7), when we stimulated cells with AG490, the protein levels of iNOS and TNF-α were significantly reduced in LPS, IFN-γ, HDND-12 and AG490 stimulation group compare control group. Therefore, the above data indicated that the regulate role of HDND-12 in shift of M1- and M2-type macrophages may be, at least in part, mediated by JAK2/STAT3 signaling pathway.
Macrophages, which manifest great plasticity, are important components of the innate immune defense system and constitute an essential element of inflamed environment . Macrophages show significant heterogeneity in function and phenotype, which can shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, can regulate the initiation, development, and cessation of some diseases, such as inflammatory diseases, cancers, metabolic diseases and so on [21-22]. In recent years, macrophage polarization has gained increasing attention and looking for an operative medicine, which could target macrophage polarization and skew their phenotypes to adapt to the micro-environment, might hold great promise for the development of diseases.
HDN, a naturally occurring flavanone compound, was previously proved that HDN has several functions such as anti-oxidant, anti-tumor, anti-inflammatory, immune regulation and so on [23-24]. In the present study, we explored the regulation role of HDND-12 in the polarization of macrophage. HDND- 12 is one of the derivatives of hespe retin by structural modifications in order to improving its water solubility and bioavailability. In the current study, we tested the hypothesis that HDND-12 may be regulator in a prominent M2 macrophage phenotype and inhibitor in M1 ma crophage switch. In our study, we showed that HDND-12 led to a time- and dose-dependent increase in the expression of M2-type markers, such as Arg-1, IL-10 and so on. Additionally, we demonstrated that HDND-12 suppressed the expression levels of the markers of LPS and IFN-γ-induced M1-type macrophage including iNOS, IL-12. In addition, in order to explore the underlying mechanisms in RAW264.7 cells. We investigated HDND-12 was related to macrophage polarization and then observed the up-regulation of M2 markers at both the mRNA and protein levels after treatment with HDND-12 compared with normal RAW264.7 cells. These results proved that HDND-12 may be used as a useful drug for switching to M2-type. Moreover, LPS from Gram-negative bacteria and IFN-γ are the most potent innate immune activating and M1- type polarization stimuli known . LPS and IFN-γ exert their toxic effects by potently activating macrophage cells and activating TLR4 signaling, which could up-regulate the expression of M1 markers. On the contrary, we found HDND-12 down-regulated M1 related markers, such as iNOS, IL-12 and TNF-α. Finally, we study the possible mechanism about HDND-12 in regulating macrophage polarization. Various signaling pathways, including JAK/STAT, SOCS2/SOCS3, TLR4, and IRF5/IRF4, were reported to regulate macrophage activation and polarization [25-27]. Specifically, JAK/STAT signaling plays a central role in macrophage polarization and a growing body of evidences suggests that IFN-γ was one of the most potent endogenous macrophage-activating factors . Thus, we explored whether HDND- 12 regulate the JAK/STAT signaling pathway Intriguingly, HDN could attenuate the phosphorylation level of JNK in lipopoly saccharide-induced H9C2 cardiomyocytes . So we wonder if HDND-12 may function in JAK/STAT signaling pathway through the expression of phosphorylation level of JAK2 and STAT3, respectively. We found the expressions of p-JAK2 and p-STAT3 were up-regulated in M1-like macrophage and down-regulated in HDND-12-induced groups, which suggested the inhibitor role of HDND-12 to M1-like macrophage in RAW264.7 cells may be correlated with the JAK2/STAT3 pathway. Which puzzleed us was that some publications had already reported that JAK2/STAT3 signaling were activated in M2-like macrophage. But in our study the JAK2/STAT3 signaling were activated in M1-like macrophage, so we need more research to explain this problem. Additional, we pre-treated the cells with AG490 (10 μmol·L–1) for 14 h, then treat it with or without M1 inducers (LPS and IFN-g) for 12 h and HDND-12 until the processing time finished. It was noteworthy that the protein levels of iNOS and TNF-α were significantly reduced in LPS, IFN-γ, HDND-12 and AG490 stimulation group compare with LPS, IFN-γ and HDND-12 stimulation group. Taken together, HDND-12 could regulate the switch the ratio of M1-type and M2-type macrophages and the underlining mechanisms might be contributed to its beneficial effects on JAK2/STAT3 signaling.
In this study, we explored the potential role of HDND-12 in macrophage polarization and how did it work in RAW264.7 cells. However, macrophage polarization as related diseases' therapeutic target is in infancy, and there are little effective drugs against it. Traditional Chinese medicine is an attractive therapeutic direction, and this study demonstrated HDND-12 is a good beginning but also need further research. In future work, the important areas should focus on exploring the full spectrum of macrophage polarization, searching new drugs with regard to the transformation of polarization, and ultimately helping treat related diseases.
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