Terfezia boudieri and Terfezia claveryi inhibit the LPS/IFN-γ-mediated inflammation in RAW 264.7 macrophages through an Nrf2-independent mechanism

Desert truffles have been used as traditional treatments for numerous inflammatory disorders. However, the molecular mechanisms underlying their anti-inflammatory effects in RAW 264.7 macrophages have yet to be fully elucidated. The present study investigated the anti-inflammatory activities of two main desert truffles, Terfezia boudieri and T. claveryi, and the underlying mechanisms associated with their anti-inflammatory activities in RAW 264.7 macrophages stimulated with lipopolysaccharide/interferon-gamma (LPS/IFN-γ). Our results demonstrated that treatment with T. boudieri and T. claveryi extracts effectively suppressed the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 macrophages. Specifically, T. boudieri extract was found to reduce the production of nitric oxide and inhibit the expression of various pro-inflammatory markers, including inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α, and interleukin-6 (IL-6) at both the mRNA and protein levels. Similarly, T. claveryi extract exhibited comparable inhibitory effects, except for the expression of IL-6 and COX-2 at the protein level, where no significant effect was observed. Moreover, both studied extracts significantly downregulated the microRNA expression levels of miR-21, miR-146a, and miR-155, suggesting that T. boudieri and T. claveryi suppress the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 cells through an epigenetic mechanism. Furthermore, our study reveals a new mechanism for the anti-inflammatory properties of desert truffle extracts. We show for the first time that Terfezia extracts do not rely on the nuclear factor erythroid 2-related factor 2 pathway, previously linked to anti-inflammatory responses. This expands our understanding of natural product anti-inflammatory mechanisms and could have important implications for developing new therapies. To account for differences in truffle effects, extracts prepared were subjected to secondary metabolites profiling using UPLC-MS. UPLC-MS led to the annotation of 87 secondary metabolites belonging to various classes, including amino acids, carbohydrates, alkaloids, amides, fatty acids, sterols, and phenolic compounds. Therefore, these results indicate that T. boudieri and T. claveryi exhibit anti-inflammatory activities through suppressing multiple inflammatory mediators and cytokines and may be potential anti-inflammatory agents.


Identification of metabolites. Compounds were assigned by comparison of their retention times and
MS spectral data (accurate mass, isotopic distribution, and fragmentation pattern) in both negative and positive ionization modes with those reported for Terfezia species alongside searching the phytochemical dictionary database.
Cell viability assay. RAW 264.7 cells were seeded at a density of 1 × 10 5 cells/mL in a 96-well plate and cultured in DMEM, high glucose supplemented with 10% heat-inactivated FBS and 1% Pen/Strep for 4 h at 37 °C in a humidified 5% CO2 incubator. After removing the medium, the cells were treated with increasing concentrations of T. boudieri and T. claveryi extracts (5,10,20,40,80, and 160 μg/mL) for 24 h to determine the non-cytotoxic concentrations to be used in the study. The cell viability was measured with the MTT colorimetric assay. After the incubation period, the medium was decanted, and the cells were incubated with serum-free DMEM containing 1 mg/mL of MTT for 2 h at 37 °C in a humidified 5% CO2 incubator. MTT was reduced by NAD(P) H-dependent cellular oxidoreductase enzymes of the metabolically active cells into formazan crystals, which www.nature.com/scientificreports/ were then dissolved in DMSO. The absorbance of each group was measured at 570 nm using SPECTROstar ® Nano microplate reader (BMG LABTECH, Ortenberg, Germany), and the results were presented as the percentage of the control. In this study, RAW 264.7 cells were stimulated with LPS (100 ng/mL) plus IFN-γ (10 U/mL). Therefore, the cytotoxicity of T. boudieri and T. claveryi extracts in the presence of LPS/IFN-γ was also evaluated. RAW 264.7 cells were stimulated with LPS/IFN-γ (100 ng/10 U/mL), and co-incubated with the non-cytotoxic concentrations of T. boudieri and T. claveryi extracts 5, 10, and 20 µg/mL for 24 h in a 96-well plate. Additionally, the cells were treated with vehicle control, which was DMEM medium containing 0.05% v/v DMSO matching the maximum final concentration of DMSO in our treatments. Sulforaphane (SFN) (1 µM) was used as a positive control in all further experiments, so its cytotoxicity on RAW 264.7 cells with or without LPS/IFN-γ was also examined. The cell viability was then measured with MTT, as previously mentioned.
Nitrite assay. RAW 264.7 cells were seeded at a density of 1 × 10 6 cells/mL in a 96-well plate and incubated for 4 h. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. The nitrite accumulated in the culture medium was measured as an indicator of NO production using the Griess Reagent Kit by mixing 150 µL of the culture supernatant from each well with 130 µL of deionized water and 20 µL of Griess reagent and incubating at room temperature for 30 min in the dark according to the manufacturer's instructions. The absorbance of the mixture was measured at 548 nm using a SPECTROstar ® Nano microplate reader. The nitrite concentration in each sample was calculated based on a standard curve prepared with NaNO2.
Quantitative real-time polymerase chain reaction (qPCR). RAW 264.7 cells were seeded at a density of 1 × 10 6 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/ IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL for 6 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. Total RNA was extracted from the cells using QiAzol lysis reagent and used for cDNA synthesis. The cDNAs were synthesized from the total RNA using Thermo Scientific™ RevertAid™ First Strand cDNA Synthesis Kit for mRNA and miScript ® II RT Kit for the microRNA (miRNA). The qPCR analyses were then performed using PowerUp™ SYBR™ Green Master Mix to quantify the mRNA expression of iNOS, COX-2, TNF-α, IL-6, HO-1, OSGIN1, and GAPDH (as a housekeeping gene). Furthermore, the miScript SYBR ® Green PCR Kit was used to quantify the miRNA expression of miR-21, miR-146a, and miR-155, and RNU6-2 (as a housekeeping gene). The qPCR analyses were conducted using ABI Prism 7500 system (Applied Biosystems). The relative gene expression levels were determined by the 2 −ΔΔCT method. The final results were expressed as the fold change of target gene expression in a target sample (treated sample) relative to a reference sample (untreated control), normalized to a reference gene: GAPDH (for mRNA) or RNU6-2 (for miRNA) as the following: fold change = 2 −ΔΔCT , while ΔΔCT = ΔCT (treated sample) − ΔCT (untreated control), and ΔCT = CT (target gene) − CT (reference gene). The mRNA primers used in this study were designed using NCBI Primer-BLAST and are listed in Table 1. The miScript primer assays (miRNA-specific forward primers) were purchased from Qiagen. The miScript universal primer (miRNA reverse primer) was provided in the miScript SYBR ® Green PCR Kit. It was applied to all reactions allowing the detection of miRNAs in combination with miScript primer assays. Primer assays used in this study are listed in Table 2.  ACC TAC CAG CTC ACT CTGG  TGC TGA AAC ATT TCC TGT GCTGT   COX-2  CTC ACG AAG GAA CTC AGC AC  GGA TTG GAA CAG CAA GGA TTTG   TNF-α  GAA CTC CAG GCG GTG CCT AT  TGA GAG GGA GGC CAT TTG GG   IL-6  GAT GCT ACC AAA CTG GAT ATA ATC AG CTC TGA AGG ACT CTG GCT TTG   HO-1  CAC AGA TGG CGT CAC TTC GTC  GTG AGG ACC CAC TGG AGG AG   OSGIN1  CGG TGA CAT CGC CCA CTA C  GCT CGG ACT TAG CCC ACT C   GAPDH  CTT TGT CAA GCT CAT TTC CTGG  TCT TGC TCA GTG TCC TTG C   Table 2. Primer assays used for the qPCR analysis. www.nature.com/scientificreports/ Enzyme-linked immunosorbent assay (ELISA) for TNF-α and IL-6. RAW 264.7 cells were seeded at a density of 1 × 10 6 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/ IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5 and 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. After incubation, the supernatants were collected, centrifuged at 1000× g for 20 min at 4 °C, and then stored at − 80 °C. The samples were subsequently analyzed for TNF-α and IL-6 proteins. The absorbance was measured at 450 nm using SPECTROstar ® Nano microplate reader. The concentration of the mouse TNF-α and IL-6 proteins in each sample was calculated by comparing the absorbance of the samples to the standard curve.
Western blotting. RAW 264.7 cells were seeded at a density of 1 × 10 6 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. Total protein was extracted from the cells using an ice-cold cell lysis buffer containing a protease inhibitor cocktail.

Results
Metabolites profiling of T. boudieri and T. claveryi. To assess for differences in the two desert truffles' metabolite composition, a non-targeted metabolite profiling of their ethanolic extracts was employed. The secondary metabolites were tentatively identified in the two desert truffles, T. boudieri and T. claveryi, via highresolution UPLC-Orbitrap HRMS analysis. A total of 87 different metabolites belonging to various metabolite classes were annotated, including 21 amino acids and sugars, 11 alkaloids, 2 amides, 23 fatty acids/esters, 5 sterols, 4 phenolic, and 21 miscellaneous peaks, as listed in Table 3. Provided below are details for the identification of each metabolites class.
Amino acids and sugars. Truffles  Alkaloids and nitrogenous compounds. MS spectral analysis also revealed the presence of nitrogenous compounds or alkaloids typically derived from amino acids. Based on their even high-resolution masses and improved response in positive ionization mode, 11 alkaloids (peaks 22-32) were annotated. Peak 24, for example, was annotated as puniceusine F 35  Fatty acids/esters. In the late elution region of the chromatogram, several peaks were observed. MS/MS spectra (Rt 10-18 m min) in the extracts of T. boudieri and T. claveryi revealed the presence of several fatty acids annotated by high-resolution masses and much higher response in negative ionization mode as major peaks. MS/MS spectra also revealed several mono, di, and trihydroxy fatty acid conjugates assigned from their high-resolution masses and predicted formulas (35, 37, 40, 44, 45, 48, 49, 50, and 53) (Table 3). Negative ion MS was also revealed for several hydroxylated fatty acids. The major hydroxy fatty acids identified in both Terfezia species were annotated as hydroxy octadecanedioic acid (37), hydroxy-octadecadienoic acid (48), and hydroxyoctadecatrienoic acid (49)

Effect of T. boudieri and T. claveryi extracts on the viability of RAW 264.7 cells. Before examin-
ing the effect of T. boudieri and T. claveryi extracts on LPS/IFN-γ-induced inflammation in RAW 264.7 cells, an MTT assay was performed to determine the optimal concentrations for use in the study. RAW 264.7 cells were incubated with increasing concentrations of T. boudieri and T. claveryi extracts (5-160 μg/mL) in the presence or absence of LPS/IFN-γ (100 ng/10 U/mL) for 24 h. SFN (1 µM) was used as a positive control. Both extracts did not significantly affect the cell viability at concentrations below 40 μg/mL compared to the untreated control. However, cell viability was decreased by approximately 63% with T. boudieri and 46% with T. claveryi when the concentration was increased to 160 μg/mL, revealing that T. boudieri has more cytotoxic effect on RAW 264.7 cells (Fig. 1A,B). When the cells were co-incubated with LPS/IFN-γ, similar results were observed with no cytotoxicity at concentrations of 5, 10, and 20 µg/mL for both extracts (Fig. 1C). SFN (1 µM) alone and combined with LPS/IFN-γ did not show any cytotoxicity on RAW 264.7 cells (Fig. 1D). Therefore, T. boudieri and T. claveryi extracts were used at concentrations of 5, 10, 20 μg/mL in all subsequent experiments to investigate their effects on inflammation induced by LPS/IFN-γ in RAW 264.7 cells.

Effect of T. boudieri and T. claveryi extracts on nitrite production in LPS/IFN-γ-stimulated RAW 264.7 cells.
To investigate the anti-inflammatory effects of T. boudieri and T. claveryi extracts on nitrite production, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 24 h. SFN (1 µM) was used as a positive control. The nitrite levels in the cell culture medium were measured using the Griess Reagent Kit. As shown in Fig. 2, nitrite levels exhibited a substantial increase of 200% compared to the untreated control after stimulating cells with LPS/IFN-γ. However, treatment with both Terfezia extracts inhibited nitrite production in LPS/ IFN-γ-stimulated cells in a dose-dependent manner. T. boudieri extract at concentrations of 5, 10, and 20 µg/mL significantly inhibited nitrite production by 11, 31, and 45%, respectively, and T. claveryi at concentrations of 10 and 20 µg/mL showed statistically significant inhibition of nitrite production by 38 and 41%, respectively. SFN (1 µM) also decreased nitrite levels by 29%.

Effect of T. boudieri and T. claveryi extracts on the mRNA expression of iNOS in LPS/ IFN-γ-stimulated RAW 264.7 cells.
To investigate whether the reduction of nitrite production following treatment with Terfezia extracts was due to suppression of iNOS gene expression, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. iNOS mRNA expression was then determined using qPCR. As shown in Fig. 3  www.nature.com/scientificreports/ However, when the LPS/IFN-γ-stimulated cells were co-treated with Terfezia extracts, T. boudieri and T. claveryi at only 20 µg/mL significantly reduced the mRNA expression of TNF-α by 23, and 30%, respectively (Fig. 5A). Moreover, the mRNA expression of IL-6 was significantly downregulated by treatment with T. boudieri (5, 10, and 20 µg/mL) by 23, 25, and 30%, respectively, and also downregulated by treatment with T. claveryi (5, 10, and 20 µg/mL) by 20, 27, and 40%, respectively, as shown in Fig. 5B. Both extracts suppressed the LPS/IFN-γinduced upregulation of TNF-α and IL-6 mRNA expression levels in a concentration-dependent manner. SFN (1 µM) also achieved a statistically significant inhibition of TNF-α and IL-6 by around 58%.

Effect of T. boudieri and T. claveryi extracts on the secretion of TNF-α and IL-6 proteins in LPS/ IFN-γ-stimulated RAW 264.7 cells. To establish a relationship between the inhibitory effects of Terfezia
on TNF-α and IL-6 mRNA expression and their corresponding secretion levels, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5 and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 24 h. SFN (1 µM) was used as a positive control. The concentration of TNF-α and IL-6 proteins secreted into the cell culture supernatant was measured using ELISA. As shown in Fig. 8, the production of TNF-α and IL-6 proteins was significantly increased in the cell culture medium of LPS/IFN-γ-induced cells compared to the untreated controls. However, when the cells were co-treated with Terfezia extracts, T. boudieri at 20 µg/mL significantly reduced TNF-α and IL-6 production by 24, and 14%, respectively. It was also found that T. claveryi at 20 µg/mL and SFN (1 µM) displayed a significant reduction in TNF-α production by 27 and 34%, respectively, but they did not exhibit any significant inhibition of IL-6 production (Fig. 8A,B).  Fig. 9, LPS/IFN-γ significantly increased the expression of iNOS and COX-2 proteins by 251 and 274%, respectively, compared to the untreated controls. However, when the LPS/IFN-γ-stimulated cells were co-treated with Terfezia extracts, T. boudieri (20 µg/mL) significantly inhibited the iNOS and COX-2 expression by 46 and 36%, respectively. Similarly, T. claveryi (20 µg/mL) suppressed the expression of iNOS by 40%, but it did not demonstrate any significant effect on the COX-2 expression. SFN (1 µM) also inhibited the iNOS and COX-2 expression by 28 and 33%, respectively. The expression of the GAPDH, which was used as a loading control was not changed (Fig. 9A,B).

Discussion
Inflammation is a highly regulated process that can be triggered by noxious stimuli, such as pathogens and toxins. Inflammation is, therefore, the first immunological line of defense through which the body can remove infection and repair tissue damage. The extent of the inflammatory response is critical because failure to eliminate the inflammatory trigger during acute inflammation leads to chronic inflammation, autoimmune reactions, and severe tissue damage. NSAIDs are the most used drugs in treating inflammation-associated diseases but have serious side effects 13 . Thus, multiple studies have been performed to explore alternative anti-inflammatory medicines of natural origin without the side effects of NSAIDs. Natural products are considered potential sources of novel anti-inflammatory agents, which can contribute to developing innovative therapeutics 39 . Desert truffles were reported to treat several inflammatory diseases 25 . Nevertheless, the mechanisms behind their anti-inflammatory www.nature.com/scientificreports/ activities in RAW 264.7 macrophages remain unclear. Hence, the anti-inflammatory properties of two major desert truffles, T. boudieri and T. claveryi have been examined in the present study. Our metabolomic analysis of T. boudieri and T. claveryi revealed that their extracts contained several biologically active substances, including alkaloids, phenolics, amino acids, and fatty acids, which may be responsible for their anti-inflammatory activities. It has been reported from previous studies that natural alkaloids exhibited anti-inflammatory activities through various targets and cell signaling pathways 40,41 . Our results indicated the appearance of 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol) and piperlongumine alkaloids in the chemical profile of T. claveryi. In an in vitro study to evaluate the anti-inflammatory activities of tempol, it was determined that tempol treatment inhibited NO production in LPS-induced macrophages as well as suppressed the expression of cytokines (TNF-α, IL-1β, and IL-6) and inflammatory enzymes (iNOS, and COX-2) in IL-1βstimulated chondrocytes 42 . Similarly, in activated macrophages, Sun et al. demonstrated that piperlongumine or its analog inhibited LPS-induced production of NO and PGE2 by downregulating the expression of iNOS and COX-2, respectively. They also reported that piperlongumine analog reduced LPS-dependent induction of NF-κB and MAPKs 43 . Phenolics are among the most essential classes of secondary metabolites, present in the fungal fruiting bodies with proven anti-inflammatory activities 44 . From our analysis, T. boudieri was characterized by the presence of phenolic compounds, particularly scopoletin, which exerted anti-inflammatory activities in croton oil-treated mouse ears by reducing the overproduction of TNF-α and PGE2 45 . The anti-inflammatory actions of truffles can be attributed to their amino acid contents, which are associated with prostaglandin metabolism 46 . Pyroglutamic acid was identified in T. claveryi, and its derivatives significantly inhibited the secretion of NO, TNF-α, and IL-6 in LPS-treated RAW 264.7 macrophages 47 . On the other hand, T. boudieri was rich in stachydrine, which exhibited anti-inflammatory properties by suppressing the levels of NO, PGE2, iNOS, COX-2, TNF-α, and IL-6 as well as blocking the IL-1β-mediated potentiation of NF-κB pathway in IL-1β-induced chondrocytes 48 . Furthermore, T. boudieri contained N-acyl homoserine lactone, and analogues of this compound have been shown to inhibit cytokine release of IL-6 and IL-8 in different in vitro-stimulated cell lines 49 . Fatty  www.nature.com/scientificreports/ SFN was used as a positive control in this study because it possesses a potent anti-inflammatory activity, mediated through suppressing the TLR4 oligomerization 53 . Additionally, Heiss et al. indicated that SFN reduced the production of the inflammatory mediators, including NO, PGE 2 , and TNF-α, accompanied by downregulation of iNOS and COX-2 proteins in LPS-induced RAW 264.7 cells 54 . SFN-mediated anti-inflammatory activity has been attributed in part to the activation of the Nrf2, as reported by Lin et al. 55 . In addition to the previous studies, Saleh et al. demonstrated that SFN modulated the TLR-associated miRNAs, such as miR-146a and miR-155, in LPS/IFN-γ-induced RAW 264.7 cells 56 . As a result, SFN can be considered as a suitable benchmark for assessing the anti-inflammatory properties of other medications.
Macrophages play pivotal roles during the inflammatory response, such as phagocytosis of microbes, antigen presentation, and secretion of inflammatory mediators. Moreover, macrophages are essential for maintaining homeostasis and tissue regeneration after injury 57 . Therefore, in vitro models of macrophages are important tools in evaluating the efficacy of the anti-inflammatory drugs through assessing the inflammatory response and cytotoxicity. RAW 264.7 macrophage cells are generally used to investigate the anti-inflammatory properties of drugs, and significantly activated by LPS and/or IFN-γ 58,59 . IFN-γ is included in combination with LPS in the macrophage polarization, and the induction of macrophages with either one of them, leads to the secretion of several inflammatory mediators 60 . In the current study, to examine the effects of T. boudieri and T. claveryi extracts on the inflammatory pathways, RAW264.7 cells were stimulated with LPS (100 ng/mL) plus IFN-γ (10 U/mL). The rationale for selecting LPS/IFN-γ instead of LPS alone was based on our optimization experiments. We found that RAW 264.7 cells treated with LPS alone exhibited weak immune responses, and the addition of IFN-γ was able to potentiate the immune response by activating signaling pathways that lead to the production of inflammatory cytokines and NO.
The increased production of NO, as measured by the Griess assay, supported this finding. Our results align with the previously published study by Saleh et al., which has shown that LPS/IFN-γ produces a more robust immune response than LPS alone 56 . Initially, we determined the non-cytotoxic concentrations of T. boudieri, and T. claveryi extracts with or without LPS/IFN-γ in RAW 264.7 cells using an MTT assay. Our data showed that The inflammatory response is mediated by a wide range of mediators forming complex regulatory networks that prevent further tissue damage and restore the normal physiology of the inflamed tissues 2 . Once LPS/IFN-γ activates macrophages, the production of inflammatory mediators, including cytokines (e.g., TNF-α and IL-6), eicosanoids (e.g., prostaglandins), and NO is increased 61,62 . NO is a key signaling molecule that is vital to the inflammatory response. NO is released as a cellular signaling molecule to increase the vasodilation in blood vessels by activating iNOS, which subsequently leads to an apparent increase in the blood flow and recruitment of leukocytes to the region of inflammation 7,63 . Therefore, to investigate the anti-inflammatory effects of T. boudieri and T. claveryi extracts, we analyzed the NO production and the iNOS expression at the mRNA and protein levels in LPS/IFN-γ-stimulated RAW 264.7 cells. Since NO is rapidly oxidized to nitrite, the nitrite level in the culture medium was measured as an indicator of the NO production 64 . Here, we found that T. boudieri and T. claveryi extracts inhibited NO production in a dose-dependent manner, accompanied by a simultaneous reduction in the iNOS mRNA expression. These data were consistent with our Western blotting analysis, in which both extracts Prostaglandins are eicosanoid-derived molecules that participate in modulating numerous physiological processes, particularly during immune responses. Prostaglandins are produced through arachidonic acid metabolism by cyclooxygenases, which exist in two isoforms: COX-1 and COX-2. COX-1 is produced constitutively in most cells, whereas COX-2 is induced in response to inflammatory stimuli 65 . As a consequence, a plethora of pharmacological agents has been developed to suppress COX-2 activity and thereby mitigate the inflammatory response. Thus, to examine the suppressive effect of T. boudieri and T. claveryi extracts, we measured the mRNA and protein expression of COX-2 in LPS/IFN-γ-stimulated RAW 264.7 cells. Our study findings demonstrated that both extracts effectively reduced the mRNA expression of COX-2. Notably, T. boudieri exhibited more potent inhibitory action on both mRNA and protein levels. Taken together, these data suggest that the antiinflammatory properties of Terfezia extracts are associated with the suppression of COX-2 expression in LPS/ IFN-γ-stimulated macrophages.
Cytokines are essential signaling proteins regulating the crosstalk between different cell types involved in the immune and inflammatory response 12 . Among cytokines, TNF-α is the primary mediator of inflammation with several effects, including stimulating other cytokine secretion, activating cell adhesion molecules, and promoting cell growth and proliferation 3,12 . IL-6 is another important cytokine released during inflammation, and its dysregulation causes a variety of inflammatory disorders 66 . Hence, the dysregulation in the production of inflammatory cytokines is commonly related to inflammatory diseases, making them potential therapeutic targets. In the current study, we assessed the expression of pro-inflammatory cytokines TNF-α and IL-6 at the mRNA and protein levels to identify the possible effects of T. boudieri and T. claveryi extracts on the inflammation mediated by LPS/IFN-γ in RAW 264.7 cells.
Interestingly, T. boudieri extract decreased the expression of TNF-α and IL-6 in a dose-dependent manner at the mRNA and protein levels. In contrast, T. claveryi was found to decrease the mRNA expression of TNF-α and IL-6, along with the inhibition of TNF-α protein production, while no significant effect was detected on IL-6 protein levels. Consistent with our results, Darwish et al. revealed that T. claveryi displayed anti-inflammatory activity by reducing TNF-α, IL-1β, and IFN-γ in LPS-stimulated WBCs 67 . These results reveal that Terfezia extracts possess anti-inflammatory properties via suppressing the pro-inflammatory cytokines involved in the inflammatory process.
Following the earlier findings, it can be observed that T. claveryi treatment decreased the mRNA expression of COX-2 and IL-6 but did not show the same effect on the protein levels. Several possibilities could explain this discrepancy, including differences in the time frame of our measurements, post-transcriptional modifications, protein stability, and other regulatory factors 68 . Firstly, the time frame of our measurements could be a contributing factor. We measured the mRNA expression levels at 6 h after stimulation with LPS/IFN-γ, while protein expression levels were measured at 24 h. Therefore, it is possible that T. claveryi treatment had a transient effect on the mRNA levels that were not reflected at the protein expression levels at 24 h. Secondly, post-transcriptional modifications can affect mRNA's stability and translation efficiency, which may not always be reflected at the protein expression levels. After transcription, mRNA undergoes several post-transcriptional modifications such as splicing, polyadenylation, and transport from the nucleus to the cytoplasm, affecting its stability and translation efficiency 69 . Thirdly, protein stability can also contribute to the discrepancy between mRNA and protein expression levels. Even if the mRNA levels are decreased, the protein levels may remain stable due to post-translational modifications or other factors 68 .
Nrf2 is a key transcription factor, playing a central role in the inflammation signaling pathways and oxidative stress responses. Therefore, the present study examined whether the anti-inflammatory effects of T. boudieri and T. claveryi extracts were related to activation of the Nrf2 signaling pathway in RAW 264.7 cells induced by LPS/ IFN-γ through assessing the gene expression of Nrf2 target genes, HO-1 and OSGIN1. Inflammatory cells produce numerous inflammatory mediators, which subsequently attract more inflammatory cells to the site of injury, leading to increased oxidative stress levels. Meanwhile, persistent oxidative stress is associated with chronic inflammation. Nrf2 signaling pathway is also essential in reducing inflammation-related disorders, including atherosclerosis, asthma, autoimmune diseases, and rheumatoid arthritis 15 . Several studies have reported that the activation of the Nrf2 signaling pathway suppressed cytokines, chemokine, iNOS, and COX-2 secretion, which modulate the NF-kB and other inflammatory cascades that regulate the transcription and activity of downstream target proteins during the inflammation process 19 . Our preliminary results demonstrated that exposure to various concentrations (5, 10, and 20 µg/mL) of T. boudieri, T. claveryi, as well as SFN at a concentration of 1 µM, did not result in a significant upregulation of the mRNA expression of HO-1 and OSGIN1. Since SFN is known to be a potent activator of Nrf2, we initially expected it to upregulate the expression of Nrf2 target genes. However, we did not observe any significant effects on the expression of these genes at a concentration of 1 µM. Therefore, we increased the concentration of SFN to 5 µM to determine if this would lead to changes in Nrf2 target genes expression. The concentration of T. boudieri and T. claveryi was also increased to a non-cytotoxic concentration of 40 µg/mL, as assessed by MTT assay. As opposed to our initial results, we observed that SFN at a concentration of 5 µM activated the gene expression of HO-1 and OSGIN1 in our study. This is consistent with previous research by Doss et al., who reported a significant increase in HO-1 mRNA levels in erythroid cells treated with 5 µM SFN, whereas lower concentrations (100 nM-1 µM) did not produce the same effect 70 . On the other hand, treatment with T. boudieri and T. claveryi at concentration of 40 µg/mL did not show any significant induction on the gene expression of HO-1 and OSGIN. These results imply that the anti-inflammatory effects of Terfezia extracts are not mediated through Nrf2 activation.
In order to gain a deeper understanding of the anti-inflammatory mechanisms of Terfezia extracts, this study explored the effects of T. boudieri and T. claveryi extracts on the expression of inflammatory miRNAs, including www.nature.com/scientificreports/ miR-21, miR-146a, and miR-155 in RAW 264.7 cells activated with LPS/IFN-γ. The selection of these miRNAs in the study was based on their relevance to the TLR4 signaling pathway 71 . miRNAs play significant roles in regulating immune cell functions in both innate and adaptive immune systems through targeting inflammation-related pathways, such as TLR4 72 . miR-21 is a negative regulator of the TLR4-induced immune response that suppresses the NF-κB activity and promotes the production of the anti-inflammatory cytokine IL-10 by targeting the programmed cell death protein 4 (PDCD4) 73 . Furthermore, miR-146a was identified to be an NF-κB-dependent gene that targets interleukin-1 receptor-associated kinases1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) in the TLR4 signaling pathway, suggesting it as a negative regulator of the innate immune response 74 . Moreover, miR-155 is known to play a significant role in inflammation by targeting multiple proteins involved in the TLR4 signaling pathway 75 . Ceppi et al. reported that miR-155 negatively regulated the inflammatory response to LPS in monocyte-derived dendritic cells. This negative regulation by miR-155 was related to its ability to target TAB2, suppressing its activation of TAK1, and thus the NF-κB and MAPK 76 . The results of the present study demonstrated upregulation of miR-21, miR-146a, and miR-155 in response to LPS/IFN-γ stimulation, consistent with previous studies [77][78][79] . Interestingly, treatment with T. boudieri and T. claveryi extracts was observed to inhibit the LPS/IFN-γ-induced upregulation of these miRNAs in a concentration-dependent manner, suggesting that both Terfezia extracts suppress the inflammatory response in activated macrophages through regulating the expression of miR-21, miR-146a, and miR-155. Nrf2 activation is known to regulate inflammation by activating miRNAs, such as miR-21, miR-146a, and miR-155 80 . However, our results demonstrate that the anti-inflammatory properties of T. boudieri and T. claveryi are independent of Nrf2 activation. As such, the effects observed at the miR-21, miR-146a, and miR-155 are equally independent of any effects at the Nrf2 level. Although the specific mechanisms involved are not yet fully understood, our results suggest that these compounds may utilize alternative pathways to regulate inflammation. One possible mechanism is the inhibition of NF-κB signaling, a central inflammation regulator. While some natural compounds have been shown to inhibit NF-κB signaling, we did not investigate this pathway in our study.
Nevertheless, our findings are supported by similar results regarding the effects of some biologically active substances in T. boudieri and T. claveryi metabolome suggesting that NF-κB signaling may indeed play a role in the observed effects 43,48,50 . In addition, previous research from our laboratory has also suggested a role for Glycogen synthase kinase 3 (GSK3) in mediating anti-inflammatory response that is also Nrf2 independent 81 . As such, further research is necessary to determine the involvement of these pathways in the anti-inflammatory effects of T. boudieri and T. claveryi. Overall, our study suggests that these compounds may have the potential as alternative anti-inflammatory agents that act through non-traditional pathways.

Conclusion and future perspectives
In conclusion, our results suggest that targeting specific inflammatory mediators associated with TLR4-mediated signaling could be an effective therapeutic approach for mitigating inflammation induced by LPS/IFN-γ in RAW 264.7 macrophages. The current study highlights the potential anti-inflammatory properties of T. boudieri and T. claveryi extracts in regulating the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 macrophages through modulation of the TLR4 activation. The results of our experiments showed that treatment with these extracts led to a concentration-dependent reduction in the production of NO that coincided with the downregulation of iNOS at both the mRNA and protein levels. Both extracts downregulated the COX-2 mRNA expression, with T. boudieri further reducing the expression of COX-2 protein. Furthermore, T. boudieri extract dose-dependently downregulated TNF-α and IL-6 mRNA and protein levels, while T. claveryi extract showed significant inhibition of TNF-α and IL-6 mRNA expression without affecting IL-6 and COX-2 protein levels. This study also offers novel insights into the epigenetic suppressive properties of T. boudieri, and T. claveryi extracts on miR-21, miR-146a, and miR-155 expression in LPS/IFN-γ-induced RAW 264.7 macrophages, proposing that Terfezia extracts could exert anti-inflammatory activities by suppressing the miRNAs involved in the TLR4 signaling pathway. Additionally, our findings showed that T. boudieri and T. claveryi exhibited anti-inflammatory effects through an Nrf2-independent manner. Furthermore, our study demonstrated differences in the chemical composition and anti-inflammatory effects of T. boudieri and T. claveryi extracts, despite both species being desert truffles. These differences can be attributed to the distinct chemical compositions of the two species. These differences highlight the importance of identifying and characterizing the specific bioactive compounds in each species, which could have different pharmacological properties and therapeutic potential. Finally, the present study suggests that T. boudieri and T. claveryi could serve as promising alternative agents for treating inflammation. However, the efficacy and safety of these extracts in vivo should be evaluated using appropriate animal models in future studies.