Multiple Beneficial Effects of Aloesone from Aloe vera on LPS-Induced RAW264.7 Cells, Including the Inhibition of Oxidative Stress, Inflammation, M1 Polarization, and Apoptosis

Aloesone is a major metabolic compound in Aloe vera, which has been widely used as a food source and therapeutic agent in several countries. Our recent study demonstrated that aloesone has anti-epileptic effects on glutamate-induced neuronal injury by suppressing the production of reactive oxygen species (ROS). Unless ROS are naturally neutralized by the endogenous antioxidant system, they lead to the activation of inflammation, polarization, and apoptosis. This study aimed to identify the multiple beneficial effects of aloesone and explore its molecular mechanism in macrophages. Hence, the murine macrophage cell line RAW264.7 was pretreated with aloesone and then exposed to lipopolysaccharides (LPS). The results demonstrated that aloesone, within a dosage range of 0.1–100 µM, dramatically decreased the LPS-induced elevation of ROS production, reduced nitric oxide (NO) release, inhibited the M1 polarization of RAW264.7 cells, and prevented cells from entering the LPS-induced early and late phases of apoptosis in a dose-dependent manner. Simultaneously, aloesone significantly decreased the mRNA expression of inflammation-related genes (iNOS, IL-1ꞵ, TNF-α) and increased the expression of antioxidant enzymes (Gpx-1 and SOD-1). The core genes HSP90AA1, Stat3, Mapk1, mTOR, Fyn, Ptk2b, and Lck were closely related to these beneficial effects of aloesone. Furthermore, immunofluorescence staining and flow cytometry data confirmed that aloesone significantly repressed the activation of mTOR, p-mTOR, and HIF-1α induced by LPS and inhibited the protein expression of TLR4, which is the target of LPS. In conclusion, aloesone demonstrated multiple protective effects against LPS-induced oxidative stress, inflammation, M1 polarization, and apoptosis in macrophages, suggesting its potential as a prodrug.


Introduction
Inflammation is the host's immune response to chemicals, physical injury, and infection. However, excessive inflammation induces an unpredicted increase in inflammatory mediators, such as cytokines (tumor necrosis factor-α (TNF-α), interleukins, inducible nitric oxide synthase (iNOS)), chemokines, and reactive oxygen species (ROS). Inflammatory mediators have implications in heart disease (ischemic heart failure and cardiac ischemia/reperfusion injury) [1,2], brain disorders (depression and anxiety) [3,4], and lung disease (chronic obstructive pulmonary disease and acute lung injury) [5,6]. Recent studies have confirmed that ROS could also cause oxidative stress, leading to the activation of

Aloesone Inhibited LPS-Induced Oxidative Stress in RAW264.7 Cells
Aloesone concentrations of 0.1, 1, 10, 100, and 1000 µM were used to verify its effect on the survival of RAW264.7 cells using the Cell Counting . Results demonstrated that these concentrations of aloesone did not affect the survival of RAW264.7 cells ( Figure 1A,B). According to our previous study, concentrations varying from 0.1 to 100 µM of aloesone were shown to ameliorate glutamate-induced neuron injury, and these concentrations were applied in subsequent experiments [24].
In the present study, pretreatment with aloesone for 2 h dramatically reduced the LPS-stimulated elevation of ROS production in a dose-dependent manner ( Figure 1C,D). In contrast, aloesone significantly increased the mRNA expression of Gpx-1 ( Figure 1E) and SOD-1 ( Figure 1F), which could clear the overloaded ROS, compared with the LPS group. These results confirmed the antioxidant effect of aloesone.
In contrast, aloesone significantly increased the mRNA expression of Gpx-1 ( Figure 1E) and SOD-1 ( Figure 1F), which could clear the overloaded ROS, compared with the LPS group. These results confirmed the antioxidant effect of aloesone. Results were expressed as mean ± standard deviation, ** p < 0.01, *** p < 0.001 compared with the control group; # p < 0.05, ### p < 0.001 compared with the LPS group. ns: no significance.

Aloesone Inhibited the M1-Polarization of RAW 264.7 Cells Induced by LPS
As shown in the micrographs ( Figure 3A), the administration of LPS for 12 h stimulated the polarization of RAW264.7 cells, with apparent antenna, a characteristic of the M1 phenotype, while aloesone inhibited this polarization. Furthermore, we confirmed the effect of aloesone on the polarization of RAW264.7 cells by detecting the specific surface phenotype marker of M1 (cluster of differentiation, CD86) [31]. The results demonstrated that LPS induced the membrane overexpression of CD86, while aloesone significantly inhibited the membrane expression of CD86, indicating that aloesone inhibited the polarization of RAW264.7 to M1 ( Figure 3B). Results were expressed as mean ± standard deviation, * 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 group.

Aloesone Inhibited the M1-Polarization of RAW 264.7 Cells Induced by LPS
As shown in the micrographs ( Figure 3A), the administration of LPS for 12 h stimulated the polarization of RAW264.7 cells, with apparent antenna, a characteristic of the M1 phenotype, while aloesone inhibited this polarization. Furthermore, we confirmed the effect of aloesone on the polarization of RAW264.7 cells by detecting the specific surface phenotype marker of M1 (cluster of differentiation, CD86) [31]. The results demonstrated that LPS induced the membrane overexpression of CD86, while aloesone significantly inhibited the membrane expression of CD86, indicating that aloesone inhibited the polarization of RAW264.7 to M1 ( Figure 3B).

Mammalian Target of Rapamycin (mTOR)/Hypoxia Inducible Factor-1α (HIF-1α) and TLR4 Are Involved in the Protective Effects of Aloesone Post LPS Stimulation
To explain the molecular mechanism of aloesone in oxidative stress, inflammation, M1 polarization, and apoptosis, overlapping genes were collected from Genecards and SwissTargetsPrediction. The results demonstrated that 86 genes were closely associated with the antioxidant stress, anti-inflammation, anti-polarization, and anti-apoptotic effects of aloesone ( Figure 5A,B), of which seven targets-heat shock protein HSP 90-alpha (HSP90AA1), signal transducer and activator of transcription 3 (Stat3), mitogen-activated protein kinase 1 (Mapk1), mTOR, fyn proto-oncogene (Fyn), protein tyrosine kinase 2 beta (Ptk2b), and lck proto-oncogene (Lck)-were the hub genes ( Figure 5C). Moreover, pathway enrichment analysis demonstrated that these seven hub genes were enriched in Th17 cell differentiation, PD-L1 expression, and PD-1 checkpoint pathways in cancer, acute myeloid leukemia, pancreatic cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, natural killer cell-mediated cytotoxicity, prostate cancer, T cell receptor signaling pathway, hypoxia inducible factor (HIF)-1 signaling, and phospholipase D signaling pathway ( Figure 5D). Results from immunofluorescent staining (IF) illustrated that aloesone significantly repressed the LPS-induced activation of mTOR ( Figure 6A,B), p-mTOR ( Figure 6A,C), and HIF-1α ( Figure 6A,D). Furthermore, aloesone decreased the membrane expression of TLR4, the specific receptor of LPS ( Figure 6E). These results suggest that the mTOR/HIF-1α pathway and TLR4 may be involved in the protective effects of aloesone. Results from immunofluorescent staining (IF) illustrated that aloesone significantly repressed the LPS-induced activation of mTOR ( Figure 6A,B), p-mTOR ( Figure 6A,C), and HIF-1α ( Figure 6A,D). Furthermore, aloesone decreased the membrane expression of TLR4, the specific receptor of LPS ( Figure 6E). These results suggest that the mTOR/HIF-1α pathway and TLR4 may be involved in the protective effects of aloesone. Molecules 2023, 28, x FOR PEER REVIEW 8 of 16

Discussion
Traditional medicine (TM) is commonly used worldwide. According to the prediction of the World Health Organization (WHO), 80% of the global population utilizes TM as a complementary or alternative medicine [32]. To date, many herbal extracts and specific natural compounds have shown anti-inflammation and antioxidant effects, including those in A. vera [33,34]. In the present study, we demonstrated that aloesone, a major metabolic compound of A. vera, has multiple protective effects against oxidative stress, inflammation, M1 polarization, and apoptosis.
Macrophages play a vital role in the pathogenesis of many chronic diseases, including fibrosis, asthma, and inflammatory bowel disease [35]. Macrophages are an important source of many key inflammatory cytokines that drive autoimmune inflammation, such as IL-12, IL-18, IL-23, and TNF-α. ROS are normally produced within the body in limited quantities and are important compounds involved in the regulatory processes of cell homeostasis and functions, including signal transduction, gene expression, and receptor activation [36]. An imbalance in ROS results in oxidative stress, which induces inflammation Results were expressed as mean ± standard deviation, *** p < 0.001 compared with the control group; ## p < 0.01, ### p < 0.001 compared with the LPS group.

Discussion
Traditional medicine (TM) is commonly used worldwide. According to the prediction of the World Health Organization (WHO), 80% of the global population utilizes TM as a complementary or alternative medicine [32]. To date, many herbal extracts and specific natural compounds have shown anti-inflammation and antioxidant effects, including those in A. vera [33,34]. In the present study, we demonstrated that aloesone, a major metabolic compound of A. vera, has multiple protective effects against oxidative stress, inflammation, M1 polarization, and apoptosis.
Macrophages play a vital role in the pathogenesis of many chronic diseases, including fibrosis, asthma, and inflammatory bowel disease [35]. Macrophages are an important source of many key inflammatory cytokines that drive autoimmune inflammation, such as IL-12, IL-18, IL-23, and TNF-α. ROS are normally produced within the body in limited quantities and are important compounds involved in the regulatory processes of cell homeostasis and functions, including signal transduction, gene expression, and receptor activation [36].
An imbalance in ROS results in oxidative stress, which induces inflammation by damaging DNA, proteins, and lipids [37,38]. Herein, synthesized aloesone repressed LPS-stimulated ROS production and induced the mRNA expression of vital antioxidant enzymes (Gpx1 and SOD1), suggesting the antioxidant stress effect of aloesone in RAW264.7 cells, which is consistent with a previous study in which aloesone scavenges radial DPPH and has high oxygen radical absorbance capacity at concentrations of 351 ± 35 µM and 66 ± 1 µM Trolox equivalents, respectively, in vitro [23].
Excessive ROS produced in the process of oxidant metabolism, as well as some natural or artificial chemicals, have been reported to stimulate macrophage M1 polarization, the proinflammatory phenotype [39], and subsequently initiate the inflammatory process. M1 macrophages tend to promote the synthesis and secretion of proinflammatory cytokines, such as iNOS, IL-1β, and TNF-α. These cytokines have also been documented to play critical roles in the inflammatory process, especially by causing macrophages apoptosis [40], leading to several chronic diseases [41]. In the present study, aloesone inhibited the M1 polarization of macrophages and alleviated the LPS-stimulated excessive release of NO and the overexpression of iNOS, IL-1β, and TNF-α in these cells, illustrating its anti-inflammatory effect. Furthermore, aloesone suppressed both the early and late phase of apoptosis. Overall, the multiple beneficial effects of aloesone on macrophages were confirmed.
Elucidating the mechanism of aloesone is vital for its further application. The mammalian target rapamycin (mTOR) is a serine/threonine kinase involved in gene regulation in inflammation [42]. The phosphorylation of mTOR can regulate the phosphorylation of various transcription factors, including p70S6K and 4E-BP1, which can further promote the expression of HIF-1α [43]. The mTOR/HIF-1α pathway participates in cellular responses, such as survival and polarization [44][45][46]. Aloesone inhibits the mTOR/HIF-1α pathway, which could be one of the potential mechanisms involved in its therapeutic effects regarding inflammation, oxidative stress, polarization, and apoptosis in RAW264.7 cells. Moreover, LPS binds to TLR4, leading to oxidative stress, inflammation, and M1 polarization. In the present study, we demonstrated that aloesone significantly decreased the membrane expression of TLR4, which can also be regulated by HIF-1α [47]. In contrast, the facilitation of HIF-1α by LPS is regulated by TLR4 [48]. Moreover, LPS-induced oxidative stress could induce HIF-1α expression and is central to determining the phenotype of macrophages [49]. Therefore, HIF-1α may be the core mediator of aloesone in protecting macrophages from oxidative stress, inflammation, polarization, and apoptosis.

Synthesis of Aloesone
Aloesone was synthesized in accordance with the methods of a previous study [50], and was obtained as a white solid powder. In brief, β-diketone was derived from an acetophenone derivative, by coupling it with 1, 3-dioxolane-proted acetoacetic acid, followed by treating it with hydrochloric acid and isopropanol to afford the aloesone. Nuclear magnetic resonance (NMR) spectra were obtained on a 400 MHz ECZ400S spectrometer (JEOL, Tokyo, Japan, 400 MHz for 1 H and 100 MHz for 13      according to the previously described method [51]. The survival rate was calculated by OD aloesone /OD control × 100%.

Groups
RAW264.7 cells were divided into six groups, as follows: (1) Control group: Cells were treated for 2 h with DMEM containing 0.1% DMSO as a vehicle, followed by DMEM with 0.1 M phosphate buffered saline (PBS, Gibco, New York, NY, USA) for 12 h. (2) LPS group: Cells were treated with DMEM containing 0.1% DMSO for 2 h, followed by DMEM with 1 µg/mL of LPS for an additional 12 h. (3) Aloesone groups: Cells were pretreated with various concentrations (0.1, 1, 10, and 100 µM) of aloesone in DMEM for 2 h, followed by DMEM with 1 µg/mL of LPS for an additional 12 h (Figure 8). Jose, CA, USA), according to the previously described method [51]. The survival rate was calculated by ODaloesone / ODcontrol × 100%.

Groups
RAW264.7 cells were divided into six groups, as follows: (1) Control group: Cells were treated for 2 h with DMEM containing 0.1% DMSO as a vehicle, followed by DMEM with 0.1 M phosphate buffered saline (PBS, Gibco, New York, NY, USA) for 12 h. (2) LPS group: Cells were treated with DMEM containing 0.1% DMSO for 2 h, followed by DMEM with 1 µg/mL of LPS for an additional 12 h. (3) Aloesone groups: Cells were pretreated with various concentrations (0.1, 1, 10, and 100 µM) of aloesone in DMEM for 2 h, followed by DMEM with 1 µg/mL of LPS for an additional 12 h (Figure 8).

Measurement of ROS generation
ROS accumulation was detected using a radical probe, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, Sigma, Saint Louis, MO, USA). RAW264.7 cells pretreated with aloesone and LPS were incubated with DCFH-DA (diluted to 1:1000 with serum-free medium) at 37 °C for 30 min in the dark. Then, the excess DCFH-DA that had not entered the cells was cleared using PBS. Thereafter, graphs were obtained by a magnification microscope (Zeiss X-Cite, Oberkochen, BW, Germany) and the mean fluorescence intensity was measured using flow cytometry (Agilent NovoCyte, Santa clara, CA, USA) at the fluorescein isothiocyanate (FITC) channel.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from RAW264.7 cells using TRIzol Reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Then, it was reversed to cDNA in a total volume of 20 µL (HiScript Reverse Transcrptase, Vazyme, Nanjing, China). PCR was performed using a real-time PCR system (Bio-Rad, Hercules, CA, USA), with the following amplification conditions: 95 °C initial denaturation for 5 min, followed by 39 cycles of 95 °C for 15 s and 60 °C for 30 s. Relative expression levels of the antioxidant enzymes Gpx-1 and SOD-1 were calculated based on the 2 −∆∆Ct method, according to the manufacturer's specifications, using the actin gene as a reference housekeeping gene. The sequences of primers used for qRT-PCR are shown below (Table 1), according to a previous study [52].

Measurement of ROS Generation
ROS accumulation was detected using a radical probe, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, Sigma, Saint Louis, MO, USA). RAW264.7 cells pretreated with aloesone and LPS were incubated with DCFH-DA (diluted to 1:1000 with serum-free medium) at 37 • C for 30 min in the dark. Then, the excess DCFH-DA that had not entered the cells was cleared using PBS. Thereafter, graphs were obtained by a magnification microscope (Zeiss X-Cite, Oberkochen, BW, Germany) and the mean fluorescence intensity was measured using flow cytometry (Agilent NovoCyte, Santa Clara, CA, USA) at the fluorescein isothiocyanate (FITC) channel.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from RAW264.7 cells using TRIzol Reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Then, it was reversed to cDNA in a total volume of 20 µL (HiScript Reverse Transcrptase, Vazyme, Nanjing, China). PCR was performed using a real-time PCR system (Bio-Rad, Hercules, CA, USA), with the following amplification conditions: 95 • C initial denaturation for 5 min, followed by 39 cycles of 95 • C for 15 s and 60 • C for 30 s. Relative expression levels of the antioxidant enzymes Gpx-1 and SOD-1 were calculated based on the 2 −∆∆Ct method, according to the manufacturer's specifications, using the actin gene as a reference housekeeping gene. The sequences of primers used for qRT-PCR are shown below (Table 1), according to a previous study [52]. Supernatant was used to detect the content of NO using a commercial kit based on the Griess reaction (Beyotime, Shanghai, China), as described previously [53]. The reaction was measured at 450 nm using a microplate reader (Spectra MAX 190).

Detection of mRNA Expression of Inflammation Associated Genes
Relative expression levels of inflammatory cytokines, including iNOS, IL-1β, and TNF-α, were detected by qRT-PCR and calculated based on the 2 −∆∆Ct method, according to the manufacturer's specifications, using the actin gene as a reference housekeeping gene.

Evaluation of Macrophage Polarization
The cells were washed with 0.1 M PBS, harvested using trypsin (Biosharp, Hefei, China), and centrifuged at 1500 rpm for 5 min at 4 • C. Aliquots of 100,000 cells were suspended in PBS and incubated with an FITC-conjugated monoclonal antibody against M1 marker CD86 (1 µg, Abcam, Cambridge, UK). The cells were resuspended with PBS and analyzed using a flow cytometer.

Detection of Apoptosis
The anti-appotic effect of aloesone was evaluated using the annexin V-FITC/propidium iodide (PI) apoptosis assay. Briefly, cells were harvested using trypsin and centrifuged at 1500 rpm for 5 min at 4 • C. Aliquots of 100,000 cells were suspended in 500 µL binding buffer and 5 µL staining reagent (Boster, Wuhan, Hubei, China). After incubation in the dark at 37 • C for 5 min, the fluorescent intensity of FITC and PI were analyzed by flow cytometry [54].

Membrane Distribution of TLR4
Cells were washed with PBS. After the cells were collected, the phycoerythrin-conjugated monoclonal antibody of TLR4 (1 µg per 100,000 cells, Santa Cruz) were applied to stain the cells. The cells were resuspended with PBS and analyzed using a flow cytometer.

Statistical Analysis
All data are expressed as mean ± standard deviation (SD). The normal distribution of data was tested using the Shapiro-Wilk test, after which data that distributed normally were analyzed by one-way analysis of variance (ANOVA) with Benjamini's test for multiple groups. Otherwise, non-normally distributed data were analyzed using the Kruskal-Wallis test. The results were considered to be significant with p < 0.05. Statistical analysis and figures were generated using GraphPad Prism (version 9.0.0).

Conclusions
In summary, we first demonstrated that aloesone significantly inhibited ROS production, NO release, and surface expression of CD86, and suppressed the early and late phase of apoptosis in RAW264.7 cells; this confirmed the multiple protective effects of aloesone on oxidative stress, inflammation, M1 polarization, and apoptosis of the macrophages. Furthermore, the mTOR/HIF-1α pathway and TLR4 were closely related to these effects. This study confirmed the potential use of aloesone as a therapeutic agent.
Author Contributions: Y.W. analyzed the data and wrote the manuscript; Z.X. and C.L. performed the cell experiments and analyzed the data; D.L., X.L., N.C. and X.W. performed the synthesis experiments. J.X., Y.L. and Q.L. designed the experiments and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare that they have no competing interest.
Sample Availability: Samples of the compound are available from the authors.