Screening Five Qi-Tonifying Herbs on M2 Phenotype Macrophages

Tumor-associated macrophages (TAMs) with M2 phenotype play an essential role in tumor microenvironment (TME) during the progression and development of numerous cancers and associated with poor prognosis. Thus, regulation of TAMs polarization emerged as a new strategy for tumor immune therapy. According to Traditional Chinese Medicine (TCM) theory, herbs with Qi-tonifying character are involved in improving the defense capacity of immune system. In this study, we screened extracts and ingredients from five Qi-tonifying herbs exhibiting an inhibitory effect on M2 polarization of murine macrophages RAW264.7 induced by IL-4 and IL-13. Among these candidates, total flavonoids from Glycyrrhiza Radix et Rhizoma (TFRG) and ethanol extract of Ginseng Radix et Rhizoma significantly inhibited the expression of Arginase-1 (Arg-1) (above 90% at 100μg/mL), one of the phenotype markers of M2 macrophages. The inhibition of total saponins of Ginseng Radix et Rhizoma, ethanol extract of Cordyceps, ethanol extract of Acanthopanacis senticosi Radix et Rhizoma Seu caulis, and ethanol extract of Astragali Radix reached above 50% at 100μg/mL. The inhibition of ingredients including glabridin, isoliquiritin apioside, lysionotin, cordycepin, astragaloside IV, and calycosin reached above 50% at 50μM. Then, we investigated the molecular mechanisms of TFRG. TFRG abolished the migration of murine breast cancer 4T1 stimulated by the conditioned medium from M2 macrophages (M2-CM). In addition to Arg-1, TFRG also antagonized the IL-4/13-mediated mRNA upregulation of the M2 markers including found in inflammatory zone 1 (FIZZ1), chitinase-3-like protein 3 (YM1), and mannose receptor (CD206) and upregulated the expression of inducible nitric oxide synthase (iNOS), one of the M1 markers. The further exploration showed that TFRG decreased the phosphorylation of STAT6 and increased the expression of miR-155. Our study provides a series of potential immune regulating natural products from five Qi-tonifying herbs on M2 phenotype. For instance, TFRG suppressed M2 polarization of macrophages partly by inactivating STAT6 pathway and enhanced the level of miR-155 to regulate the expressions of M1 and M2 markers.


Introduction
Macrophages are essential immune cells in protecting human body from external threats such as bacterial and virus threats, while their functions switch to associate tumor development under tumor microenvironment as tumor associated macrophages (TAMs). TAMs are involved in micrometastasis niches and immune escape [1]. The population of TAMs is negatively correlated with clinical prognosis in many kinds of malignant tumors. So TAMs are considered as a new therapeutic target in the field of tumor immune therapy [2].
Macrophage polarization is two extremes of situations after mononuclear cell activation, and various microenvironment signals induce its polarization. According to different functional characteristics and its induced consequence, macrophages are usually divided into M1 and M2 [3]. Classically activated macrophages (M1) are activated by T helper (Th)-1 cytokines such as interferon gamma (IFN-) and bacterial wall components lipopolysaccharide (LPS). These macrophages have antitumor effect due to the cell toxicity of nitric oxide induced by inducible nitric oxide synthase (iNOS). Alternatively activated macrophages (M2) are activated with T helper (Th)-2 cytokines such as IL-4 and/or IL-13 [4], which have opposite function to that of M1.
Arginase-1 (Arg-1) presents in both murine and human with M2 induction while being very low in M1 phenotype; 2 Evidence-Based Complementary and Alternative Medicine  [5]. Inhibition of STAT6 in TAMs attenuates tumor growth and metastasis [6]. Numerous recent studies reveal that some microRNAs (miRNAs) are involved in macrophage polarization by regulating transcription factors [7]. Among these miRNAs, miR-155 targeted IL-13R 1 via STAT6 signal pathway to suppress M2 phenotype of macrophages [8].
According to TCM, constant motion and transformation of Qi maintain body internal balance. And the occurrence of tumor is caused by the deficiency of Qi. One of the key principles to cancer intervention guided by TCM is strengthening Qi. Qi can improve the immune system against pathogenic factors. Qi-tonifying herbs or their formulas are usually used to prevent cancer recurrence and metastasis of postoperative cancer patients in China [9].
So, we selected five Qi-tonifying herbs with high frequency in clinical practice against cancers and screened their effects on M2 macrophages.

Preparation of Extracts from Five Chinese Herbs.
To prepare ethanol extracts, 100 g of each dried herbs was sliced and extracted with 1 L of 70% ethanol at 80 ∘ C for three times. Obtained ethanol extracts were evaporated under reduced pressure at temperature 60 ∘ C and stored at -80 ∘ C. Extracts were dissolved with DMSO before use. TFRG was obtained as described previously [10], and no endotoxin existed in it by endotoxin test.

Cell
Culture. Mouse macrophage cell line RAW264.7 and mouse breast cancer cell line 4T1 were obtained from ATCC. RAW264.7 cell and breast cancer 4T1 cell were cultured in RPMI-1640 medium which contained 10% FBS, placed in saturated humidity, 5% CO 2 incubator, and incubated at 37 ∘ C constant temperature. Take 10 to 15 generations of cells for these experiments.

Preparation of Conditioned
Medium. RAW264.7 cells were seeded in 30mm dish and cultured overnight. Then cells were treated with/no different concentrations (25, 50 g/mL) of TFRG and IL-4 (20 g/mL)/IL-13 (20 g/mL) induction for 24 h in serum free medium. The culture supernatants (conditioned medium, CM) were collected and centrifuged to remove cellular components and stored in 4 ∘ C before use.

Cell Viability Assay.
Cell viability was measured using MTT assay [10].  2.6. Trans-Well Migration Assay. Cell migration assay was performed using the Trans-well system (24-wells, 8-m pore size, Corning Costar, Lowell, MA, USA). Briefly, 4T1 cells were harvested and suspended in serum-free medium; then 5 × 10 4 cells were added to the upper wells. 600 L conditioned medium from each group was added to the lower chamber.
After treatment for 24 h, the cells attached to the lower surface were fixed with methanol and stained with 0.1% crystal violet. The number of cells that migrated was counted in five randomized fields using an Olympus light microscope.

qRT-PCR.
Total RNA was extracted from cells using TRIzol regents according to the manufacturer's protocol. The sequences of these primers are as shown in Table 2. The level of GAPDH mRNA was used as an internal standard and Applied Biosystems 7500HT Fast Real-Time PCR System was used to perform the reactions. PCR reaction conditions were the following: 95 ∘ C for 10 minutes, 50 ∘ C for 2 minutes, 95 ∘ C for 15s, 60 ∘ C for 60s, and 40 cycles.

TaqMan5
MicroRNA Real-Time RT-PCR Assays. Mmu-miR-155 and the housekeeping gene U6 were carried out according to the manufacturer's protocol [11]. These samples were measured in triplicate cases. U6 endogenous control was used for normalization, and expression levels were presented as 2 −ΔΔCT with standard deviation.

Western Blot Analysis.
Cell extracts were collected using RIPA buffer and separated on 4−12% SDS-PAGE gels followed by the transfer to PVDF membrane. The membranes were blocked with 5% skim milk and incubated with the appropriate primary antibodies. Antibodies used include Arg-1, iNOS, phosphor-STAT6, STAT6, and GAPDH.

Statistical
Analysis. Data were reported as the mean ± standard deviation for each individual experiment. Statistical analysis was performed using Student's t-test. P < 0.05 was considered as significant.

Effects of Herb Extracts and Ingredients on Arg-1 mRNA
Expression and Cell Growth. These extracts and ingredients were evaluated for their effects on Arg-1 mRNA expression in IL-4/IL-13-induced murine RAW264.7 macrophages and cell growth on unstimulated macrophages with the aid of qRT-PCR and MTT. A wide range of inhibitions in Arg-1 mRNA expression was observed with these candidates with no cell cytotoxicity (Table 3). TFRG and ethanol extract of RS showed the strong inhibition of Arg-1 mRNA expression (above 90% at 100 g/mL). The inhibition of total saponins of RS, ethanol extract of DCXC, ethanol extract of CWJ, and ethanol extract of HQ reached above 50% at 100 g/mL. The inhibition of ingredients including glabridin, isoliquiritin apioside, lysionotin, cordycepin, astragaloside IV, and calycosin reached above 50% at 50 M.

TFRG Abolished Migration of 4T1 Induced by M2CM.
Since TFRG showed the strongest inhibition of Arg-1 mRNA among these extracts without cytotoxicity, we wondered that whether TFRG can influence the function of M2 macrophage. Accumulated evidence suggests that M2 macrophages assist cancer cell to metastasis and worsen TME [12]. The conditioned medium from M2 macrophages after IL-4/IL-13 induction (M2CM) significantly improved the migration ability of breast cancer 4T1 cells in vitro. The conditioned medium from TFRG pretreatment M2 macrophages reduced the increased migration ability of 4T1 cells in a dosedependent manner (Figure 1).

TFRG Suppressed Arg-1 Expression at mRNA and Protein
Level. Arg-1, a marker for the M2 subset macrophages, hydrolyzes L-arginine into urea and ornithine and inhibits nitric oxide-mediated pathways by competing with iNOS for the same substrate L-arginine [13]. In resting macrophages, low expression of Arg-1 was detected, and the expression of Arg-1 was significantly increased after IL-4/IL-13 induction towards M2 phenotype. Thus, we investigated the possibility that TFRG blocked Arg-1 expression. To test this possibility, we determined the effect of TFRG on Arg-1 expression in induced RAW264.7 cells. Results from qRT-PCR and western blotting revealed that TFRG downregulated Arg-1 expression in a dose-dependent manner at levels of gene and protein ( Figure 2).

TFRG Downregulated M2 Related Markers and Upregulated M1 Marker iNOS.
Since TFRG is sufficient to reduce the expression of Arg-1 in M2 macrophages, we wondered whether the expression of other M2 markers can be also reduced by TFRG. We found that TFRG downregulated the M2 markers FIZZ1, YM1, and CD206 in IL-4/13 derived M2 macrophages (Figures 3(a)-3(c)). In addition, the M1 marker iNOS was impaired in macrophages towards M2, and TFRG restored the production of iNOS under the induction of M2 (Figure 3(d)).

TFRG Blocked IL-4/IL-13-Induced STAT6 Activation.
STAT6 signaling pathway is associated with the regulation of M2 polarization in macrophages induced by cytokines IL-4 and IL-13 [14]. To explore potential relationship between TFRG and STAT6 signaling pathway, we determined the effect of TFRG on the phosphorylation status of STAT6 in IL-4/IL-13-induced RAW264.7 cells. Western blotting showed that the level of phosphorylated STAT6 was much higher in induced M2 cells in comparison with unstimulated cells. Treatment of TFRG dependently decreased the level of phosphorylated STAT6 (Figure 4(a)). The result indicated that TFRG might exert its inhibition of M2 macrophages by the interference with STAT6 signaling pathway.

TFRG Induced miR-155 Expression in M2 Macrophages.
Recent evidence has demonstrated that miR-155 is involved in M2 polarization [15]. To determine the effect of TFRG on miR-155 under the induction of IL-4/IL-13 in RAW264.7 cells, we pretreated cells with TFRG prior to induction. qRT-PCR showed that IL-4/IL-13 led to the decrease of the expression of miR-155 comparing with unstimulated cells. TFRG dramatically increased the level of miR-155 in RAW264.7 cells challenged by IL-4/13. It suggested that TFRG may prevent M2 polarization via miR-155 (Figure 4(b)).

Discussion
Despite the effectiveness of conventional therapies that include surgery, radiotherapy, and chemotherapy that focus on tumor cells, the survival time of cancer patients is still unsatisfying after clinical treatment. TME and its components play a critical role in recurrence and metastasis [16].
Considering the essential role of macrophages in the progression of tumor, an increasing attention has been exerted on regulating their polarization to help better clinical diagnosis, prognosis, and therapeutic applications [17]. Natural products from TCM are always evaluated on proliferation of tumor cells, and further efforts should be made to evaluate their regulation on immune cells such as TAMs. TCM theory was developed during thousand years of clinical experience fighting against diseases, and the key principle of TCM cancer treatment is to strengthen Qi. Herbs with Qi-tonifying characteristic such as RS, DCXC, and HQ have been used with high frequency to ameliorate symptoms of insufficiency and reduction of Qi in cancer patients during tumor development or chemotherapeutic drugs administration [18]. However, Qi-tonifying herbs usually have no or less cell cytotoxicity to cancer cells. Thus, we hypothesize that Qitonifying herbs may have effect on M2 phenotype regulation to improve TME.
Arg-1 is well established as a major marker of M2 macrophages. Tumors of Arg-1-deficient mice were about half Evidence-Based Complementary and Alternative Medicine the size comparing with those of wild-type mice, suggesting that Arg-1 in TAMs has an essential role in tumor growth by producing polyamines to promote cell proliferation [19].
In this present study, taking advantage of the M2 phenotype cell model induced by IL-4/IL-13 on macrophage RAW264.7, we evaluated extracts and ingredients from five Qi-tonifying herbs for their inhibitory effects on IL-4/IL-13induced Arg-1 expression. We revealed that 6 extracts and 6 compounds markedly inhibited polarization of macrophages toward M2 phenotype via inhibiting Arg-1 mRNA.
Among them, TFRG showed the strongest inhibitory effect on Arg-1 mRNA. The mechanisms of TFRG in regulating M2 polarization events need to be explored. Previous studies showed that TFRG could inhibit the growth of tumor on nude mice bearing breast cancer [10]. Nevertheless, it remains unknown whether TFRG could inhibit breast cancer by targeting TAMs. So, we evaluated the regulatory effect of TFRG on TAMs in vitro. Macrophages in tumor tissues were mostly polarized to M2 phenotype. The M2-polarized macrophages derived by IL-4, IL-13, IL-10, and glucocorticoids express high levels of Arg-1, CD206 (MRC1), TGF , MMPs, FIZZ1, and YM1, which could promote the metastasis and angiogenesis of tumors [20]. Figure 1 showed that RAW264.7-M2CM attracted 4T1 to elevate its migration, a decreased chemotactic effect of TFRG treated M2-CM to 4T1 breast cells. Figure 2 demonstrated that TFRG could regulate the polarization of macrophages by decreasing the expression of M2-phenotype markers Arg-1 at levels of gene and protein.
Studies have shown that the expression of Arg-1 and iNOS is competitive regulation by Th1 and Th2 cytokines. Arg-1 is induced in response to Th2 cytokines such as IL-4 or IL-13, while iNOS is associated with Th1 cytokines including IFNwith/not LPS. Arg-1 competes with iNOS for they both can utilize L-arginine as substrate to produce L-ornithine and urea or L-citrulline and NO, respectively [21]. Increased Arg-1 activity reduced availability of L-arginine as the substrate for iNOS in M2 macrophages to limit the antitumor production NO of iNOS. TFRG not only decreased the Arg-1 expression, but also increased the expression of iNOS at the same time (Figures 2 and 3).
The STAT6 signaling pathway is well recognized for its role in the expression of Arg-1 on M2 macrophages induced by IL-4 [22]. By examining the effect of TFRG on the phosphorylation status of STAT6, we found that the level of phosphorylated STAT6 was reduced notably by TFRG (Figure 4(a)). Current studies have revealed a critical role of miR-155 in M2 polarization [23]. Figure 4(b) showed that the level of miR-155 was upregulated by TFRG. We reason that TFRG at least partially acts through increasing the amount of miR-155 in alternatively activated macrophages.
In conclusion, these active extracts and compounds from Qi-tonifying herbs based on M2 macrophage cell model screening may be useful in anticancer therapy by targeting the immunosuppressive activity of TAMs. TFRG is a potential candidate agent for anticancer therapy due to its antitumor effect and immunomodulation, which could inhibit the promoted migration of 4T1 by M2-CM and suppress the polarization of M2, partly via regulating STAT6 signaling pathway and miR-155 expression.

Data Availability
The data used to support the findings of this study are included within the article.