Phyllolobium chinense Fisch Flavonoids (PCFF) Suppresses the M1 Polarization of LPS-Stimulated RAW264.7 Macrophages by Inhibiting NF-κB/iNOS Signaling Pathway

Background M1 macrophage plays an important role in inflammatory reaction. In this study, potential anti-inflammatory effect of Phyllolobium chinense Fisch flavonoids (PCFF) was assessed via Zebrafish acute inflammation model in vivo and LPS-induced pro-inflammatory M1 macrophage model in vitro. Methods The quality control of P. chinense Fisch flavonoids (PCFF) was analyzed by HPLC. Anti-inflammatory effect of PCFF on the acute injured zebrafish was evaluated by the migration of fluorescence labeled macrophages and neutrophils, and the gene expression of inflammatory factors. In addition, the anti-inflammatory mechanism of PCFF was investigated by the related gene expression and related signaling pathway regulation of pro-inflammatory mediators in LPS-induced pro-inflammatory M1 RAW264.7 macrophage. Results P. chinense Fisch flavonoids (PCFF) markedly suppressed macrophage and neutrophil migration and iNOS gene expression in acute injured zebrafish with tail-cutting. PCFF significantly inhibited NO overproduction and iNOS gene overexpression in LPS-sitimulated pro-inflammatory M1 RAW264.7 macrophages. What’s more, PCFF could evidently decrease p65 protein production, but had no effect on the production of P38, JNK and ERK1/2 proteins. Conclusion P. chinense Fisch flavonoids (PCFF) have a remarkable inhibitory effect on the inflammatory response in acute injured zebrafish and LPS-stimulated M1 RAW264.7 macrophage. The pharmacological mechanism may be related to the regulation of NO overproduction and the inhibition of NF-κB/iNOS signaling pathway.


INTRODUCTION
Phyllolobium chinense Fisch is the dry mature seeds of Astragalus complanatus R. Br. The main chemical constituents of P. chinense Fisch include flavonoids, triterpenoid saponins, sterols and so on. Among them, flavonoids are the main active ingredients of P. chinense Fisch. It has been showed that P. chinense Fisch flavonoids (PCFF) has many pharmacological effects, such as lowering lipid, bringing high blood pressure down, reducing blood sugar, inhibiting inflammation, improving immunity, etc. (Xu et al., 2018). However, the antiinflammatory mechanism of PCFF is still unclear.
Acute inflammation is the immediate and early response of the body to the stimulation of inflammatory factors, such as pathogens, damaged cells or irritants. The main characteristic of acute inflammation is exudative change centered on vascular response (Hwang et al., 2016). Excessive inflammation leads to the release of an enormous number of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6), which cause cell necrosis and tissue damage, thus aggravate inflammation (Medzhitov, 2008;Chu et al., 2020). Nitric oxide (NO) is a small molecule compound that regulates neurotransmission and inflammatory response (Lee et al., 2017). In mammals, NO is produced by three different nitric oxide synthases (NOS), namely inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS). iNOS gene is mainly expressed in activated macrophages (Janakiram and Rao, 2012). It is almost not expressed in normal cells, but only in inflammatory, hypoxic stress and other pathological conditions, which can stimulate the growth of inflammation and other diseases (Förstermann and Sessa, 2012;Xue et al., 2018). When iNOS gene expression is activated abnormally and NO is released in the wrong place or excessively produced in the body, it can increase inflammatory response by oxidative damage to healthy cells and tissues (Lange et al., 2009). Consequently, inhibiting the overexpression of iNOS gene and reducing the over-production of NO are important targets of anti-inflammatory drugs.
The purpose of this study was to investigate the inhibitory effect of PCFF on inflammation and its related mechanisms. The inhibition of PCFF on inflammation was studied by observing the effects of PCFF on macrophage migration in acute injured zebrafish embryos and LPS-induced over-expression of inflammatory mediators in RAW264.7 macrophages. Meanwhile, the mechanism of PCFF inhibiting iNOS gene overexpression was studied by analyzing the changes of major genes and proteins in MAPK and NF-kB signaling pathways. Antibodies including GAPDH (#2118), iNOS (#2982), P38 (#8690), ERK1/2 (#4695), JNK (#9258), I-kappa B (#4812), p65 (#3033), HRP-linked antibody (#7074) were purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphatase and protease inhibitors (Sangon Biotech, China). BCA protein assay kit was purchased from Biomiga, USA. SDS-PAGE and ECL were purchased from Beyotime. PVDF membranes was purchased from Millipore, USA. Trizol was purchased from Roche. PrimeScriptTM RT reagent kit with gDNA Eraser and SYBR Premix Ex TaqTM II were purchased from Takara

P. chinense Fisch Flavonoids (PCFF) Extract and Structural Analysis
Firstly, P. chinense Fisch (1 kg) were extracted for 2 h with 20 L boiling water. Secondly, the decoction was combined and concentrated to about 200 ml. Thirdly, the concentrated decoction was loaded on DM130 macroporous resin, which was then eluted by mobile phase composed of ethanol and water from 60 to 80%. Fourthly, the ethanol-eluted fraction was collected and concentrated to 200 ml, then ethanol was added to 1,000 ml. Finally, the precipitate in ethanol was removed and the upper clear liquid was concentrated to waterfreeno water. The powder obtained was P. chinense Fisch flavonoids (PCFF). Half of the powder is stored at −20°C for reserve. The other is dissolved in 20 ml DMSO and repeated separation on a C18 column. Finally, 180 mg Compound 1 and 210 mg Compound 2 were obtained. The structures of compounds 1 and 2 were identified by 1 H-NMR, 13 C-NMR and ESI-MS used Brucker AM-500, 600 NMR Spectrometer and Finnigan LCQ Deca XP respectively.
Preparation of PCFF solution: 20 mg PCFF was weighed and put in 5 ml capacity bottle. After dissolving in methanol, PCFF was filtered by 0.45 mm microporous membrane as test solution. The determination was carried out according to the above chromatographic conditions.
Preparation of reference solution: Complanatuside A and complanatuside B were selected as reference substances. The solution containing complanatuside A and complanatuside B were 0.42 mg and 0.68 mg per ml respectively, which were to be used.

Zebrafish Culture
The female (Cora1a: EGFP) and male (Lyz: DsRED) zebrafish were mating at a ratio of 2:2. The next morning, fish eggs were collected and cleaned with Embryo Medium, and incubated in 28.5°C incubators. Three days post-fertilization (3 dpf) zebrafish embryos were observed under fluorescence microscope, and both green fluorescence (macrophages' mark) and orange fluorescence (neutrophils' mark) Tg (Cora1a:EGFP/Lyz : DsRED) zebrafish were selected for subsequent experiments.

Acute Inflammatory Response in Zebrafish
The 3 dpf Tg (Cora1a: EGFP/Lyz: DsRED) zebrafish were placed in 6-well plates containing Embryo Medium, 10 zebrafish in each well. Zebrafish were divided into normal group, model group and PCFF (15, 30 mg/ml) treatment group, three wells per group. After anesthesia with 10× tricaine, the zebrafish tails of model and PCFF group were cut off. Then the zebrafish were treated without (normal, model) or with PCFF(15, 30 mg/ml) for 6 h in 28.5°C incubator. Then, the aggregation of macrophages and neutrophils in zebrafish were observed by fluorescence microscopy.

Real-Time RT-PCR Analysis of Inflammation Related-Genes in Zebrafish
The 3 dpf Tg (Cora1a: EGFP/Lyz: DsRED) zebrafish were placed in 6-well plates containing Embryo Medium, 10 zebrafish in each well. Zebrafish were divided into normal group, model group and PCFF (15, 30 mg/ml) treatment group, three wells per group.
After anesthesia with 10× tricaine, the zebrafish tails of control and PCFF group were cut off. Then the zebrafish were treated without (normal, model) or with PCFF (15, 30 mg/ml) for 6 h in 28.5°C incubator. After shaking zebrafish in PBST solution for 5 min and repeat for three times, the Trizol method was used to isolate total RNA from the zebrafish in each well. Then, 1 mg total RNA was reverse transcribed into cDNA, which was amplified by real-time PCR. The relative expression levels of target genes were quantified by housekeeping gene beta-actin and analyzed by 2 −DDCT method.
The sequences of primers (zebrafish) used for reverse transcription are listed below: Cell Culture Murine RAW264.7 macrophages were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (China). RAW264.7 macrophages were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were grown at 37°C in a humidified incubator with 5% CO 2 .

Greiss Method
The level of nitrite (NO 2− ) in the cell culture supernatant, stably generated during NO reactions, was assessed to determine the amount of NO production. Firstly, Raw264.7 macrophages (1 × 10 5 /well) were grown in 96-well plates. Cells were untreated (control), or treated with LPS (1 mg/ml) and PCFF at different concentrations (0, 25, 50, 100, 200, 400 mg/ml), three wells per PCFF concentration, for 24 h. Secondly, Griess reagent was prepared by mixing 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (dissolved in ddH 2 O) and 1% sulfanilamide (dissolved in 5% H 3 PO 4 ) in equal volume. Lastly, 100 ml of culture supernatant and 100 ml of Griess reagent were blended with a micropipette and set aside for 10 min at room temperature, then the absorbance at 540 nm was measured. A NaNO 2 standard curve was used to calculate nitrite concentration.

ELISA
The inhibitory effects of PCFF on pro-inflammatory cytokines produced by LPS-activated cells was detected by ELISA kit. Firstly, RAW264.7 macrophages (1 × 10 5 /well) were grown in 96-well plates. Cells were untreated (normal), or treated with only PCFF, only LPS (1 mg/ml), or both LPS and PCFF, 3 wells per group, for 24 h. Secondly, culture supernatants were collected and commercial ELISA kits were used to measure TNFa concentrations.

Real-Time RT-PCR Analysis of iNOS Gene in RAW264.7 Macrophages
Firstly, RAW264.7 macrophages (1 × 10 6 /well) were grown in 12-well plates. Cells were untreated (normal), or treated with only PCFF, only LPS (1 mg/ml), or both LPS and PCFF, three wells per group, for 24 h. Secondly, the Trizol method was used to isolate total RNA from the cells in each well. Thirdly, 1 mg total RNA was reverse transcribed into cDNA, which was amplified by real-time PCR. Finally, the 2 −DDCT method was used to analyze gene expression and GAPDH mRNA served as an internal control to quantify the levels of target mRNAs relatively. The cycling conditions were as follows: hold: 95°C for 10 s; cycling: 95°C for 5 s, 60°C for 30 s, 40 cycles; melt: 65-95°C.
The sequences of primers (mice) used for reverse transcription are listed below:

Western Blotting
Firstly, RAW264.7 macrophages (1 × 10 6 /well) were grown in 12-well plates. Cells were untreated (normal), or treated with only PCFF, only LPS (1 mg/ml), or both LPS and PCFF, three wells per group, for 24 h. Secondly, cells were harvested on ice and washed once with ice-cold PBS. Lysis buffer with phosphatase and protease inhibitors (Sangon Biotech, China) was added to lyse the cells. After incubating on ice for 30 min, cell extracts were centrifuged at 14,463×g in a refrigerated centrifuge (5418R, Eppendorf, Germany) at 4°C for 10 min to collect cell total proteins, the amount of which was quantified using a BCA protein assay kit. Thirdly, SDS-PAGE (10%) was used to separate proteins, which were electro-transferred to PVDF membranes. Membranes were blocked with 5% (wt/vol) dried skimmed milk for 1 h, and incubated with various specific primary antibodies, namely, anti-GAPDH, anti-iNOS, anti-p65, anti-P38, anti-ERK1/2, anti-JNK, to probe corresponding target proteins. Bound antibodies were detected using peroxidaseconjugated secondary antibodies, and the amount of bound antibody was assessed by enhanced chemiluminescence (ECL). Finally, Relative levels of target proteins were obtained based on the optical density of electrophoresis bands with GAPDH serving as an internal control.

Statistical Analysis
Data are presented as means (SD). Multiple comparisons were performed using the one-Way ANOVA test followed by Student-Newman-Keuls (SNK) and least significant difference (LSD) tests. P values <0.05 were considered to represent significant differences between means.

Extraction, Separation and Structural Analysis of P. chinense Fisch flavonoids (PCFF)
The molecular structures of compounds 1 and 2 were identified by 1 H-NMR, 13 C-NMR and ESI-MS (Supplementary S1 and S2). By comparing the chemical shifts with previous publications, we confirmed that the peak 1 is complanatosides B and the peak 2 is complanatosides A (Figure 1, Table 1). The content of Complanatoside A was 9.5% of total flavonoids of P. chinense Fisch, the content of Complanatoside B was 13.1%. It is suggested that the main constituents of P. chinense Fisch flavonoids (PCFF) are complanatosides A and complanatosides B, which can be used as quality control index for extracting PCFF.

Toxic Effect of PCFF on Zebrafish
The three days post fertilization (3 dpf) Tg (Cora1a: EGFP/Lyz: DsRED) zebrafish were treated with PCFF (15, 30, 50, 100 mg/ml) for 24 h to observe the survival of zebrafish. The results showed that the morphology and heart rate of zebrafish were normal when the concentration of PCFF was below 30 mg/ml. Beginning with 50 mg/ml concentration, part of zebrafish showed deformity, body bending and cardiac arrest in a dose-dependent manner ( Figure 2). Therefore, the concentration of PCFF at 15 and 30 mg/ml was selected for further study.

Effect of PCFF on Inflammatory Cells Migration in Zebrafish
The 3 dpf Tg (Cora1a: EGFP/Lyz: DsRED) zebrafish from Normal control group, Model group and PCFF (15, 30 mg/ml) treatment groups were photographed under fluorescence microscope. As showed in Figure 3, the distribution of neutrophils and macrophages in normal zebrafish is uniform; after tail cutting, a large number of neutrophils and macrophages gathered to the tail area to produce inflammatory reaction; and after treated with different concentrations of PCFF, the number of neutrophils and macrophages in the tail was decreased significantly in a concentration-dependent manner.

Inhibitory Effect of PCFF on Inflammatory Factors in Zebrafish
The iNOS, TNF-a and IL-1b levels in 3 dpf Tg (Cora1a: EGFP/ Lyz: DsRED) zebrafish were analyzed by RT-PCR. As showed in Figure 4, the expression of inflammatory genes, such as iNOS, TNF-a and IL-1b increased significantly after tail-cutting. PCFF can reduce the expression of iNOS mRNA, but has no inhibitory effect on TNF-a and IL-1b. Therefore, we try to further verify the anti-inflammatory mechanism of PCFF through in vitro experiments.  FIGURE 4 | Inhibitory effect of PCFF on expression of iNOS、TNF-a and IL-1b mRNA. Each value indicates the mean ± SD from three independent experiments. Compared with normal group, ## P < 0.01; compared with control group, *P < 0.05, **P < 0.01. NC group untreated; Model group cut tail; PCFF group added different dose PCFF after cut tail. Effects of PCFF on the Cytotoxicity of RAW264.7 Macrophages and the Production of NO In zebrafish experiments, we found that PCFF can reduce the activation of macrophages and the inflammatory response of the body. Therefore, we tried to elucidate the anti-inflammatory mechanism of PCFF in vitro. First, we used CCK-8 to detect the survival rate of RAW264.7 macrophages at different concentrations of PCFF (0-800 mg/ml). The results showed that PCFF had no cytotoxic effect on RAW264.7 macrophages when PCFF was less than 400 µg/ml ( Figure 5A). We further examined the effect of PCFF on NO production in LPS-induced M1 macrophages. The results showed that PCFF inhibited NO production in a dose-dependence manner, and the highest inhibition rate of NO production was 65.2% at 400 mg/ml ( Figure 5B). Therefore, the concentration of PCFF at 400 mg/ ml was selected for subsequent experiments.
Inhibition Effects of PCFF on iNOS Gene Expression in LPS-Stimulated M1 Pro-Inflammation RAW264.7 Macrophage Inducible nitric oxide synthase (iNOS) is the main catalytic enzyme for NO production during macrophage inflammation.
The results show that, PCFF can significantly reduce iNOS gene transcription ( Figure 6). Inhibitory Effect of PCFF on the Production of Inflammatory Factor TNF-a in LPS-Stimulated M1 Pro-Inflammation RAW264.7 Macrophage TNF-a is also an important inflammatory mediator and marker of M1 polarization of LPS-stimulated macrophages. The results showed that PCFF did not significantly inhibit TNF-a production in RAW264.7 macrophages induced by LPS (Figure 7). From here we see that PCFF may play an anti-inflammatory role by selectively inhibiting NO and iNOS gene production. Therefore, we attempt to elucidate the molecular mechanism of PCFF on inhibiting iNOS production in our follow-up studies.

PCFF Regulates iNOS Protein Expression Through NF-kB Signaling Pathway
MAPK and NF-kB signaling pathway are the main signaling pathways regulating iNOS gene expression. The results showed that LPS significantly increased the iNOS gene expression by stimulating p65 protein production in RAW264.7 macrophages, while PCFF significantly inhibited the excessive production of p65 ( Figure 8A). PCFF had no significant effect on the production of P38, JNK and ERK1/2 ( Figures 8B, C). These results suggested that PCFF could reduce iNOS gene overexpression by inhibiting the activation of NF-kB signaling pathway.

DISCUSSION
Inflammation is a basic defense response of the body against harmful stimuli, such as pathogens, damaged cells and wounds. However, this response generates pathological consequences that lead to inflammatory tissue damage, metaplasia, and a shift in homeostatic set points (Medzhitov, 2008). The zebrafish (Danio rerio), as a model organism, has been widely used in scientific research, including immunology (Rosowski, 2020;Sommer et al., 2020). Zebrafish larvae are suitable for investigating the innate immune response in vertebrates because their adaptive immune system matures in morphology and function 4-6 weeks after fertilization, while only innate immune response exists in larvae. In addition, transparency in zebrafish early life stages allows for useful real-time visualization (Novoa and Figueras, 2012). In this study, zebrafish embryos were traced with Tg (Cora1a: EGFP/Lyz: DsRED) double-labeled transgene line to detect macrophages and neutrophils. Macrophages were green fluorescence-labeled and neutrophils were red-green fluorescence-labeled. 3 dpf (days post fertilization) zebrafish were cut tail to induce inflammatory response. The migration of fluorescent labeled macrophages, neutrophils was observed and the inflammation related gene expression was detacted. Our results showed that P. chinense Fisch flavonoids (PCFF) could significantly decrease macrophages and neutrophils migration and alleviate iNOS gene expression, suggesting that PCFF could significantly inhibit the inflammatory response in vivo and the iNOS gene may be the key target of PCFF on the inhibition of macrophage activation.
Macrophages are innate immune cells widely distributed in the body and play an important role in inflammatory response (Yu et al., 2017;Lee et al., 2020). Macrophages can be divided into M1 proinflammatory type and M2 anti-inflammatory type according to the phenotype and function (Wynn et al., 2013;Orecchioni et al., 2019). Macrophages will polarize to M1 proinflammatory type after stimulated by lipopolysaccharide (LPS). Gene expression of inducible nitric oxide synthase (iNOS) was enhanced and iNOS increased significantly, which catalyzed L-arginine decomposition to produce excessive nitric oxide (NO). Nitrite peroxide produced by excessive NO and O 2− leads to cell death, extensive tissue damage and pathological changes through strong peroxidation. Therefore, the regulation of iNOS gene expression and activity is the most direct and key target in the treatment of inflammatory diseases involving NO. Our data demonstrated that P. chinense Fisch flavonoids (PCFF) could significantly reduce the excessive production of NO but not TNF-alpha in LPS-activated RAW264.7 macrophages, FIGURE 7 | Effects of PCFF on TNF-alpha secretion in LPS-induced RAW264.7 cells. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ## P < 0.01.
FIGURE 6 | Effect of PCFF on LPS-induced inflammatory factor iNOS gene in RAW264.7 cells. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ## P <0.01; compared with LPS group, **P <0.01.
suggesting that PCFF suppressed inflammation by inhibiting the production of NO. Further study dicovered that PCFF inhibited NO overproduction by significantly inhibiting iNOS gene expression.
The promoters of iNOS gene include identifying sites such as NF-kB, AP-1, STAT-1a and so on. Stimulated by LPS, many pathways such as NF-kB signaling pathway, MAPK signaling pathway and protein kinase C signaling pathway can be activated. After cascade amplification, iNOS gene expression is increased, resulting in excessive NO production (Ji and Vina, 2007;Li et al., 2009;Dai et al., 2011). MAPK signaling pathway is composed of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase and P38 MAPK (Lee et al., 2019). NF-kB is an important downstream target of the MAPK signaling pathway. The transfer of NF-kB dimer (mainly p65:p50 dimer) from cytoplasm to nucleus and will promote iNOS gene expression Zhou et al., 2020). P. chinense Fisch flavonoids (PCFF) could significantly inhibit the production of total p65 protein in LPS-stimulated M1 RAW264.7 macrophage, but had no effects on total P38, JNK and ERK1/2 protein. Because PCFF has a significant inhibition on the production of total p65 protein, so we did not further detect the phosphorylated p65. These results suggested that PCFF could inhibit iNOS gene overexpression by inhibiting the NF-kB signaling pathway, thus reducing NO overproduction and inhibiting the M1 pro-inflammatory polarization of LPS-stimulated RAW264.7 macrophage.
In conclusion, P. chinense Fisch flavonoids (PCFF) can reduce macrophage activation in zebrafish with acute injury, and alleviate inflammation by inhibiting iNOS gene expression. The inhibitory effect of PCFF on M1 pro-inflammatory polarization of macrophage in vitro can be achieved by regulating NF-kB signaling pathway to inhibit the activity of A B C FIGURE 8 | Effects of PCFF on NF-kB and MAPK signaling pathway proteins in LPS-stimulated RAW264.7 macrophages. (A) Effect of PCFF on p65 and iNOS proteins; (B) Effect of PCFF on JNK, P38 and iNOS proteins; (C) Effect of PCFF on ERK1/2 and iNOS proteins. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ## P < 0.01; compared with LPS group, **P < 0.01. iNOS and then down-regulate the expression of proinflammatory factor NO.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.