p53 Promotes Ferroptosis in Macrophages Treated with Fe 3 O 4 Nanoparticles

Background : Fe 3 O 4 nanoparticles (NPs, also known as iron oxide NPs; IONPs) have high biocompatibility and low biotoxicity. They are widely used in the field of biotechnology for targeted delivery, image formation, and photothermal therapy. NP biodistribution is determined by macrophage capture in vivo, and recently, the induction of macrophage polarization into the M1 phenotype by IONPs has become a hot topic in research. Previous research has shown that IONPs can induce ferroptosis of ovarian cancer cells and ischemic cardiomyocytes. In this study, we exposed macrophages to synthesized Fe 3 O 4 NPs (100 nm in diameter) and determined the effects of NPs in inducing cell death by RNA sequencing. Results: We observed that after 48 h exposure to NPs, there was a change in the macrophage phenotype and a reduction in cell viability. Then, we demonstrated that NPs could induce macrophage cell damage by increasing intracellular reactive oxygen species and by repressing the mitochondrial membrane potential. Furthermore, we investigated the underlying mechanisms of ferroptosis of macrophages using RNA sequencing and change in ultrastructural morphology, and found that ferroptosis was caused by the upregulation of p53 expression and inhibition of SLC7A11 expression, as their protein levels after 48 h exposure to Fe 3 O 4 NPs were consistent with erastin-induced ferroptosis. Conclusions: These results provide an insight into the molecular mechanisms underlying ferroptosis induced by Fe 3 O 4 NPs in macrophages and provide a basis for the biotoxicity study of Fe 3 O 4 NPs in vivo .

the capture of macrophage and subsequent degradation by macrophages [12]. However, except phenotypic changes, the effects of Fe3O4 NPs on macrophages remain poorly understood.
Ferroptosis is an oxidative type of regulated cell death caused by a metabolic disorder of intracellular lipid oxides and morphologically distinct from other forms of cell death. It was first formally described as an iron-dependent form of erastin-induced non-apoptotic cell death [13]. Ferroptosis is characterized by abnormal levels of iron ions and excessive reactive oxygen species (ROS) generation. When the antioxidant capacity of cells is weakened, ROS accumulates, leading to an imbalance in intracellular redox and the induction of ferroptosis. In addition to morphological changes, such as the rupture of the outer mitochondrial membrane, small mitochondria with a condensed membrane, and loss or decrease in mitochondrial cristae are also mediated by ferroptosis [14]. Ferroptosis induction is associated with multiple diseases and has recently gained substantial attention as a target for cancer treatment [14]. It is reported that iron oxide NPs induced ferroptosis in few cancer cells [17,18] and ischemic cardiomyocytes [19].
With respect to our current understanding of the mechanism underlying ferroptosis, glutathione peroxidase 4 (GPX4) reduces cytotoxicity by converting glutathione (GSH) to oxidized glutathione (GSSH), and the inhibition of GPX4 causes accumulation of lipid peroxides induces ferroptosis. The glutathione peroxidase pathway can directly inhibit GPX4, which is further regulated by system Xc-, an amino acid anti-transporter composed of heterodimeric disulfide bound to xCT (SLC7A11). GSH biosynthesis, cysteine availability, and the proper functioning of GPX4 are crucial for regulating ferroptosis [20,21]. Jiang et al. found that p53 promotes ferroptosis in fibroblasts, the human breast cancer cell line MCF7, and the human osteosarcoma cell line U2OS via trans-repression of solute carrier family 7 member 11 (SLC7A11) expression. This provided the first evidence for the induction 4 of p53 in response to ferroptosis [22,23]. At present, the regulatory effects of p53 in ferroptosis have only been detected in cancer cells [24,25].
Herein, we evaluated the effects of Fe3O4 NP treatment on macrophage phenotype. We observed that treatment with Fe3O4 NPs caused macrophages to polarize into the M1 phenotype, accompanied with a reduction in cell viability. Using RNA-seq, we found that NPs induced ferroptosis in macrophages via upregulation of p53 expression and downregulation of SLC7A11 expression. We used erastin-treated Ana-1 cells as positive control, and the ferroptosis of macrophages was verified by observing the changes in the ultrastructure of the mitochondria and by assessing the expression levels of TFR, p53, and xCT. Moreover, we demonstrated p53 regulation of ferroptosis in macrophages; this is the first evidence of p53 mediated ferroptosis in cells other than cancer cells.

Characterization of Fe3O4 NPs and effects on cell viability
Ultramicrographs (TEM, Fig. 1a; SEM, Fig. 1b) show that the NPs were spherical and uniform in shape. The hydrodynamic diameters of NPs were measured by dynamic light scattering (DLS; Nano ZS90, Malvern, England), and the average hydrodynamic diameters of NPs (Fig. 1c) were 104 ± 3.25 nm, in good agreement with the TEM results. The zeta potentials (Fig. 1d) were -28.03 ± 1.3 mV.
These results indicated that the NPs had excellent properties and stability in ultrapure water Ana-1 cells were exposed to various concentrations of NPs for 24 and 48 h, and cell viability was determined. As seen in Fig. 2a, NP treatment inhibited the proliferation of Ana-1 cells in a dose-and time-dependent manner. When the concentration was less than 400 μg/mL, there was no significant decrease in cell viability after treatment for 24 h. However, there was an approximately 8-10% reduction in the viability of cells. In figure 2b, when the exposure time was extended to 48 h, the cell viability decreased significantly (by approximately 25%) (p < 0.05). Therefore, in subsequent analyses, 400 μg/mL NPs was used. Cell viability was significantly inhibited at higher concentrations (500 μg/mL) (p < 0.01). Control cells cultured in nanoparticle-free medium were processed in parallel with the treatment groups. Results of the CCK-8 assay are expressed as percentage relative to the control. Data are expressed as means ± SEM, *p < 0.05; **p < 0.01 vs Control.

Effect of Fe3O4 NPs on macrophage phenotypes
TAMs can be functionally categorized into M2 macrophages [27]. M1 macrophages are characterized by a high expression of inducible nitric oxide synthase (iNOS), high levels of arginase-1 (Arg-1) and anti-inflammatory (IL-10) are the markers of M2 macrophages. Ana-1 cell line is an inactivated bone marrow monocyte-derived macrophage line and is classified as macrophages; however, its protein expression pattern is similar to that of the M1 phenotype .
Cells were incubated with gradient concentrations of NPs for 24 h and 48 h, while untreated cells were used as control. As shown in Fig. 3a, Arg-1 expression was extremely low in cells treated with NPs at concentrations of ≥200 μg/mL (p < 0.001). The expression levels of iNOS in cells treated with NPs at ≥300 μg/mL were 1.5-to 3-fold higher than those in the control (p < 0.05; p < 0.01). As shown in Fig. 3b, an RT-qPCR analysis revealed that the mRNA levels of iNOS at ≥400 μg/mL were significantly higher than those in the untreated cells (p < 0.05; p < 0.01). ARG-1 mRNA levels were significantly lower in all experimental groups than in the control group (p < 0.001), consistent with the results obtained by western blotting. The levels of IL-10 in all groups decreased markedly (p < 0.001), and low IL-10 expression is a characteristic feature of M1 macrophages. These results suggest that NPs at ≥300 μg/mL could polarize Ana-1 macrophages to the M1 phenotype, consistent with previous results [8,30]. Notably, the protein levels of macrophages treated with NPs for 24 h (Fig S1a) did not change significantly (p>0.05), suggesting that the macrophage phenotype did not change.
After the construction of the TAM models, they were incubated with or without NPs for another 24 h or 48 h. After incubating for 24 h (Fig S1b), the protein levels did not change significantly. The proteins in the 48 h group were examined by western blotting (Fig. 3c). Compared to levels in the control group, which were untreated cells, TAMs showed a significant increase (2-to 2.5-fold) in the expression of Arg-1 (p < 0.05). The levels of iNOS were lower in TAMs than in the control group, but the difference was not statistically significant, indicating that the TAM model contained a mixture of M1/M2 phenotypes [31]. iNOS expression levels in NP-treated TAMs were 4-to 5-fold higher than those in TAMs (p < 0.01), and Arg-1 expression levels were 5-to 7-fold lower (p < 0.01). We also examined the mRNA levels of iNOS, Arg-1, and IL-10 ( Fig. 3d). Significant reductions in the mRNA levels of Arg-1/iNOS (1.5-to 2-fold) and IL-10/iNOS levels (55-to 57-fold) were observed in the NP-treated TAMs compared with the control group (p < 0.01). These genes and protein expression analyses confirmed the polarizing effect of NPs on TAMs.

Uptake of intracellular iron 9
Ana-1 cells were incubated with NPs for 4, 8, 12, and 24 h at 37°C, and intracellular iron contents were measured by ICP-ACE (Fig. 4a). In all samples, the uptake of nanoparticles increased significantly in 8 h (140.29 pg/cell). The rate of uptake gradually slowed and reached a plateau at 12 h (134.74 pg/cell). This might reflect the depletion of free NPs in the culture medium. At 24 h, the decrease in the intracellular content may be due to exocytosis. The cellular NP content in the macrophages was visualized by Prussian blue staining (Fig. 4b), and the expected Prussian blue reaction was seen. We observed intracellular red fluorescence from lysosomes; the extensive colocalization of red fluorescence from the probes and NPs can be observed in Fig. 4c and d. These results indicated that NPs accumulated in lysosomes after internalization.

Mitochondrial damage triggered by Fe3O4 NPs
After NPs are degraded into iron ions in lysosomes, iron ions induce oxidative stress by generating ROS [31]. Ferroptosis is a type of ROS-dependent regulated cell death [31], mitochondrial damage is the most common feature of this process which caused by intracellular ROS [18]. As determined by flow cytometry (FCM) (Fig. 5a, b), the intracellular ROS content at 24 h was nearly 20-22% higher than that in the control group, with no significant difference. At 48 h, the contents were about 12% higher than those at 24 h, and 32-40% higher (p < 0.05). This means as the exposure time increased, intracellular ROS also increased. As shown in Fig. 5c, we compared MMP changes by the green/red fluorescence ratio. The 24 h group showed slight mitochondrial damage and relatively few inactivated cells, the ratio in the 48 h group was significantly higher than that in the control group (p < 0.005). As seen in

Ferroptosis of macrophages induced by Fe3O4 NPs
To further explore the influence of NPs on macrophages, the expression levels of all genes were evaluated by RNA-seq (Fig. 6a). We found that NPs induce ferroptosis by upregulating p53 and downregulating SLC7A11, a key component of the cystine/glutamate transporter. Transferrin receptor (TFR) mediates the entry of iron-containing ferritin into cells, and Feng et al. [34] have shown that TFR accumulation is a hallmark of ferroptosis. According to the RNA-seq results, NPs increased the level of TFR, consistent with the expression characteristics of ferroptosis.
A previous study has reported that the activation of p53 can induce ferroptosis by inhibiting system Xcactivity. Erastin is a typical ferroptosis-inducer, an oncogenic RAS-selective lethal small molecule [35]. Recent findings suggest that erastin can also activate p53 and enhance ferroptosis [36,37]. Therefore, we used erastin (25 μM)-treated Ana-1 cells for 24 h as a positive control for ferroptosis [38]. We determined the protein levels of TFR, p53, and xCT (SLC7A11) by western blotting to verify the sequencing results and further evaluate the ferroptosis pathway. As shown in

Ultrastructure of mitochondria in macrophage
Ultrastructural changes in the mitochondria, such as a volume reduction, outer mitochondrial membrane disruption, and disappearance of the mitochondrial cristae [40,41] are considered as hallmarks of ferroptosis [42]. As shown in Fig. 7b, shrunken mitochondria and fractured mitochondrial cristae are seen in the erastin-treated cells.
Ferroptosis can be characterized at a morphological level by the presence of shrunken mitochondria [22]. in addition to the normal mitochondria, ruptured mitochondrial cristae are also observed in the 24 h group (Fig. 7c). At 48 h (Fig. 7d), we observed 14 mitochondrial membrane disruption, disappearance of the mitochondrial cristae, and size shrinkage.

Discussion
As Fe3O4 NPs show high biodegradability, low biotoxicity, are easy to synthesize, and are magnetic by nature [43]; owing to these features, they are widely used in biotechnology and medicine.
NPs were originally developed for improving the contrast of MRI [44,45], that has been partially used in clinical applications [46]. Fe3O4 NPs are widely used in the magnetic hyperthermia treatment of tumors because of their high thermal effect in an oscillating magnetic field [47]. Because of their magnetic properties, NPs are easy to modify, and are therefore commonly used as drugs and gene to targeted delivery [48]. Notably, Fe3O4 NPs are also the first NPs that were found to have enzymatic activity [49]. These NPs possess antitumor effects that are mediated via the Fenton reaction [50], indicating their diverse applications in cancer treatment. Focusing on their ability to alter macrophage phenotypes, Zanganeh et al. [8] revealed that ferumoxytol inhibits cancer growth by inducing a pro-inflammatory immune response through M1 polarization. This study provides more evidence to the antitumor activity of Fe3O4 NPs. NPs are usually administered via the intravenous route for clinical applications [51]. Biodistribution studies have demonstrated that the uptake of NPs by reticuloendothelial system (RES)macrophages may be the ultimate fate for most NPs, followed by being dissolved and metabolized in the autolysosomes [51,52]. Although NPs are so widely used in the field of biotechnology, their cytotoxicity has not been fully explored.
TAMs can be functionally categorized into mixed type M1 and M2 macrophages, of which M2 accounts for a large proportion [53]. M1 and M2 macrophages are characterized by a high expression of nitric oxide (NO) producing iNOS and ornithine producing Arg-1, respectively, which correlate to either cytotoxic or repairing functions. TAMs are also known to express high levels of the anti-inflammatory cytokine IL-10. In this study, we found that the macrophage phenotypes changed following 48 h of exposure to NPs (Fig. 3), demonstrating that NPs not only polarized the macrophages into the M1 type, and also repolarized TAMs into M1 type, corroborating a previous report [8]. This implies that Fe3O4 NPs can be used for cancer treatment by means of immunotherapy.
Notably, as the exposure time increased from 24 h to 48 h, the viability of the macrophages decreased with phenotype change (Fig. 2). In order to evaluate the level of NP uptake, we measured the time gradient change of the intracellualr NPs (Fig. 4a). Consequently, we found that the cell uptake of NPs was maximum at 8 h, following which there was no significant increase in the NP content, suggesting that the reduction was partly mediated by exocytosis. Therefore, it was considered that the decrease in cell viability at 48 h was not related to the intracellular NP levels. We also found that intracellular NPs and lysosomes were colocalized and consider that the phagocytic capacity of macrophages and the concentration gradient between the outer and inner membranes are the underlying reasons for the colocalization. Supposedly, NPs entered the Ana-1 cells, where some of them were taken up by lysosomes while some were degraded into iron ions [54].
Excessive iron ions in cells can cause abnormal iron metabolism and oxidative stress, accompanying ROS production [32]. We found that the production of intracellular ROS increased proportionally to the exposure time of NPs, suggesting that the presence of NPs correlates with ROS production in the cells. ROS has always been known as an important mediator of inducing cell death in several biological and pathological conditions [55]. Mitochondrial damage often occurrs owing to ROS production and subsequently causes cell death, where decrease in MMP level is considered a hallmark in apoptosis or ferroptosis. In this study, we found no prominent change in MMP in the 24 h group, suggesting that accumulated ROS increased mitochondrial damage and further induced apoptosis or ferroptosis.
Ferroptosis is a newly described programmed cell death pathway that is iron-dependent and different from apoptosis, necrosis, and autophagy. Its most important characteristic is the production of ROS, which is mainly caused by dysregulated iron metabolism. To explore the effects of NPs on the macrophages, we sequenced the mRNA of the cells exposured to NPs for 24 h and 48 h. According to the RNA-seq results illustrated in Fig. 6a, NPs activated the ferroptosis pathway by inducing p53 expression after exposure for 48 h. The tumor suppressor gene, p53, plays an important role in the induction of ferroptosis by inhibiting the transcription of SLC7A11, which encodes the substrate-specific subunit of system Xc - [56,57], which is only found in cancer cells [58]. Jiang et al. [59] showed that p53 inhibits cystine uptake by transcriptionally suppressing SLC7A11 thereby leading to increased sensitivity of ROS-induced ferroptosis in p53-activated cells.
Erastin, also known as ferroptosis-inducer, can enhance the sensitivity of cancer cells to chemotherapy and radiation, suggesting that it can be used as a new anticancer drug [35]. Since erastin can mediate perroptosis through p53 and other molecules, we used it as a positive control for ferroptosis in this study. We found that TFR level, which is a marker of ferroptosis, was higher in the erastin-and NP-treated groups than that in control group, indicating that NP-induced ferroptosis led to decreased cell activity. The expression levels of p53 and xCT were similar to that of the erastin group, corroborating p53-mediated ferroptosis of macrophages. Currently, targeting TAMs by either eliminating them or inducing their repolarization into the M1 (tumor suppressive type) phenotype is currently a hot topic in antitumor immunotherapy research.

Conclusion
In this study, we found that NPs could induce phenotypic changes and ferroptosis in macrophages.
By analyzing the intracellular ROS levels and changes in MMP and mitochondrial morphology, we indicated that the ferroptosis-like cell death of macrophage increased as the exposure time increased.
Furthermore, we showed that NPs induced macrophage ferroptosis mediated by p53 using RNA-seq and western blotting. These results provide a basis for further investigations into the precise molecular mechanism and related signaling pathways involved in ferroptosis induced by Fe3O4 NPs in macrophages and allows for the determination of the cytotoxicity of Fe3O4 NPs in vivo. Importantly, this work can provide a basis for the application of Fe3O4 NPs in the medical field for the inhibition of tumor cell growth via the induction of ferroptosis in macrophages.

NP synthesis and characterization
Fe3O4 NPs (100 nm diameter) were fabricated by a hydrothermal method using FeCl3 and NaAc·3H2O. Briefly, FeCl3 was added to the solvent containing diethylene glycol and dissolved by ultrasonication. Then, NaAc·3H2O was added to this mixture and completely dissolved. The solution was autoclaved at 200°C for 12 h. Then the reactants were dried at 65°C after washed. The morphology and diameter of the NPs were determined using a transmission electron microscope (TEM Tecnai G2F30 S-TWIN; Hillsboro, NC, USA) and a scanning electron microscope (SEM S-4800; Hitachi, Tokyo, Japan). The particle size and zeta potentials of the NPs were measured using dynamic light scattering (Nano ZS90; Malvern, Inc., Malvern, UK).

Cell culturing
The mouse macrophage cell lines Ana-1 and lung cancer cell lines LLC (Lewis lung cancer cells) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cells were grown in RPMI 1640 (Gibco, New York, USA) culture medium supplemented with 10% FBS (Gibco) in a humidified atmosphere of 5% CO2 at 37°C.

Cell viability analysis
Cultured Ana-1 cells were centrifuged at 400 ×g for 5min, and counted. One hundred microliters of cell suspension (1 × 10 5 cells/mL) was prepared, added to a 96-well plate. NPs at concentrations of 200, 300, 400 and 500 µg/mL were added to the cultures. After 24 and 48 h of co-incubation, 10 μL of CCK-8 solution and 100 μL of culture solution were added to each well and incubated at 37°C for 1 h.
The absorbance of the solution was measured using a microplate reader (Epoch, Biotek, Winooski, VT, USA) at 450 nm.

Analysis of the effect of Fe3O4 NPs on macrophage phenotypes
To determine the effect of NPs on macrophages phenotypes. Ana-1 cells (1 × 10 6 cells/well) were seeded in 6-well plates and incubated for 12 h. Cells were collected for further analyses after exposed to 400 μg/mL NPs for 24 h and 48 h.

Construction of TAM model
The methods described by Wu et al. [60,61] were used to construct the TAM model. When LLC cells reached 70% confluence, the conditioned medium (CM) was stored at -80°C after harvested and centrifuged at 1000 × g for 5 min. Next, after 12 h incubation of Ana-1 cells (1 × 10 6 cells/well) in 6-well plates, the culture medium was replaced with CM (2 mL/well), followed by incubation for 48 h, after which Ana-1 cells became TAMs. Finally, TAMs were incubated with 400 μg/mL NPs in 2 mL of fresh culture medium. After exposure for another 24 h and 48 h, the cells were harvested for further analyses.

Uptake of Fe3O4 NPs
NP uptake was analyzed by measuring intracellular iron by inductively coupled plasma-atomic emission spectrometry (ICP-ACE Optima 7300 DV; PerkinElmer, Waltham, MA, USA). Briefly, Ana-1 cells and NP solution were co-cultured for 2, 4, 8, 12, and 24 h. After washing with PBS three times to remove free NPs, the cells were collected and counted in each sample, followed by cell pelleting by centrifugate at 400g ×g for 5min. Dry the pellets and dissolved them in 100 µL of 65% nitric acid for 2 h at 70°C. Then, 900 µL of distilled water was added, and the emission spectra of samples were analyzed.

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Ana-1 cells were seeded in 12-well plates with slides and incubated with a fresh cell culture medium containing NPs (400 μg/mL) for up to 8 h. following incubation, cellular NPs were visualized and localized using Prussian blue staining (Solarbio, Beijing, China) and Lyso-Tracker Red fluorescent probes (Beyotime, Shanghai, China) according to the manufacturers' instructions.
For Prussian blue staining, the cells were washed with PBS two times to remove the free NPs.
The slides with NP-loaded Ana-1 cells were immersed in ddH20 for 5 min before incubation in Prussian blue dying solution for 20 min. The slides were washed and counterstained with neutral red for 2 min before washing and mounting in an aqueous fixative. Images of slides were obtained using a light microscope (CX23; Olympus, Tokyo, Japan). The Lyso-Tracker Red staining for NPs intracellular localization, slides were incubated with 1 mL of Hank's balanced salt solution (HBSS, Beyotime) containing 50 nM Lyso-Tracker Red, which efficiently diffuses into living cells to visually detect lysosomes. Images were obtained using a fluorescence microscope (ELOS FL Auto; Thermo Fisher, Waltham, MA, USA).

Detection of ROS
Intracellular ROS was detected using the fluorescent probe DCFH-DA in the ROS Detection kit (Beyotime). The cells were collected by centrifugated at 400g × for 5min, resuspended in diluted DCFH-DA, and incubated at 37°C for 20 min. Cells were resuspended in PBS after centrifugated, the fluorescence intensity of the cells was analyzed using a flow cytometer (FCM LSRFormanc; BD, Franklin Lakes, NJ, USA). Rosup is a compound mixture with a concentration of 50mg/ mL which treated the cells for 20 min as a positive control reagent to facilitate the detection of ROS.

Analysis of mitochondrial damage
MMP was measured using an MMP Assay Kit (Solarbio) of tetraethylbenzimidazolyl carbocyanineiodide (JC-1). The cell suspension (1 × 10 6 cells/mL, 0.5 mL) supplemented. The cells were incubated with 0.5 mL of JC-1 at 37°C for 20 min. Based on the color change, MMP was detected using a fluorescence microscope, and the ratio of red/green fluorescence intensity was evaluated using a microplate reader (SYNERGY2, Bio-Tek, Winooski, VT, USA) to determine the degree of mitochondrial damage. Untreated cells were exposed to 10 μM carbonyl cyano-p-chlorophenylhydrazone (CCCP) for 20 min as a positive control; CCCP causes the complete loss of MMP.

RNA-sequencing
After cells were harvested, intracellular RNA was extracted using the TRIzol reagent (Takara, Tokyo, Japan), and RNA samples were analyzed by high-throughput transcriptome sequencing technology in Science and technology company (E-gene, Shenzhen, China).

Ultramicroscopic analysis of cells
The microstructure of the NP-treated Ana-1 cells were observed by a TEM. Briefly, Ana-1 cells were treated with NPs (400 μg/mL) for 24 h and 48 h or Erastin (25 μM; MCE, Princeton, NJ, USA) for 24 h. Cells were fixed in 4% glutaraldehyde for an additional 12-24 h followed by 4°C. Next, the cells were post-fixed with 1% OsO4 and embedded in Epon. Ultrathin sections were visualized using a Hitachi-7500 TEM (Hitachi, Tokyo, Japan). Erastin, an inducer of ferroptosis, was used as a positive control for induce of ferroptosis.

Western blotting
After cell lysis and protein extraction by total protein extraction kit (KeyGEN, Jiangsu, China).
The PVDF membrane with proteins was incubated with primary rabbit antibody specific for iNOS, Arg-1 (CST, Massachusetts, USA), TFR, p53, xCT (Abcam, Massachusetts, USA)and respective secondary antibody (1:4000). This was followed by visualization using an ECL system. Protein expression levels were standardized using tubulin or β-actin.

Reverse transcription-quantitative PCR (RT-qPCR)
The purified RNA was reverse transcribed using the Transcriptor cDNA Synthesis Kit (TaKara, Tokyo, Japan). The resultant cDNA was subjected to qPCR using the Light Cycler 96 (Roche, Basel, Switzerland). GAPDH used as a reference gene to analyze the iNOS, Arg-1, IL-10 gene quantitatively.
Relative gene expression was quantified using the 2 −∆∆Ct method.

Statistical analysis
Data are expressed as means ± standard error of mean (SEM) and all statistical analyses were performed using GraphPad Prism 8 (La Jolla, CA, USA). All reactions were performed in triplicate, and each experiment was repeated three times. Results were analyzed by a two-way anova.

Declarations
Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable
Availability of data and materials: We ensure the authenticity and repeatability of the data obtained by this research. All data generated or analyzed during this study are included in this published article.