3.1. Evaluation of STLPRE phenolic content, antioxidant and anti-inflammatory properties
Homeostasis disorders of reactive oxygen and nitrogen species (ROS, RNS respectively) are generally considered to be associated to many maladies such as cancer, diabetes, atherosclerosis and neurodegenerative disorders (Fransen et al. 2012). Since plants’ bioactive compounds can scavenge free radicals, we thought it was worthy to assess antioxidant capacity of polyphenol-rich STLPRE. Amounts of total phenolics content (TPC), total flavonoids and tannins content in STLPRE are presented in Table 2. High TPC and total flavonoid content were found in STLPRE (57 mg GAE g− 1DE and 12.3 mg CEg− 1DE, respectively). However, tannins were not detectable by the vanillin assay. Phenolics are well known for their antioxidant activities due to their redox properties and can be reduce radicals primary by two mechanisms: transfer of single electron and hydrogen atom transfer (Ozgen et al. 2006). DPPH·, ABTS and FRAP assays are three tests with a good repeatability that are frequently used for phyto-extracts. As shown in the Table 2, STLPRE exhibited a wide range and a potential antioxidant capacity to quench radicals. This antioxidant property relied mainly on the presence of flavonoids such as catechin hydrate and isorhamnetin 3-O-rutinoside, phenolic acids (p-coumaric acid), two alkaloids (salsoline and fraxidin) and other aromatic compounds (resorcinol and catechol). The Presence of p-coumaric acid belonging to the hydroxycinnamic (CH-CH-COOH) group in STLPRE contributes to its antioxidant efficacy and sustain contention from previous studies carried out by Razzaghi-Asl et al. (2013). On the other hand, flavanols (catechin) and flavonols (isorhamnetin 3-O-rutinoside) which a free OH in the C-3 position have high capacity to scavenge DPPH radicals and protect the body against oxidative damages caused by ROS (Burda and Oleszek 2001). Indeed, the antioxidant activity of STLPRE could be also due to the alkaloids; and previous studies demonstrated S. oppositifolia, S. soda, S. tragus and S. kali are rich in salsoline alkaloids exerting antioxidant and several biological potentials (Boulaaba et al. 2019; Tundis et al. 2009).
Table 2
Total phenolic (TPC), Flavonoids and Tannins contents determined by colorimetric methods, bioactive compounds identified by HPLC and total antioxidant activity (TAA), ferric reducing antioxidant potential (FRAP), DPPH and ABTS radical scavenging activities of STLPRE.
TPC (mg GAE g− 1 DE) | 57.2 ± 0.12 |
Total flavonoids (mg CE g− 1 DE) | 12.3 ± 0.05 |
Total tannins (mg CE g− 1 DE) | No detected |
Bioactive compounds | Regression equation | Correlation Coefficient (R2) | Quantification (mg/g residue) |
Salsoline | y = 2,9264x + 4,4496 | 0.995 | 0.063899 |
Fraxidin | y = 4,009x + 9,6042 | 0.998 | 0.162715 |
Resorcinol | y = 8,5601x-4,0288 | 0.998 | 0.091101 |
Catechol | y = 5,1024x-1,0136 | 0.998 | 0.212244 |
Catechin | y = 3,6327x + 1,7743 | 0.998 | 0.913154 |
p-coumaric acid | y = 9,8458x-19,395 | 0.995 | 0,426149 |
Isorhamnetin 3-O rutinoside | y = 2,9952x-10,173 | 0.998 | 1.073685 |
TAA (mg GAE g− 1 E) | 9.275 ± 0.16 |
DPPH (IC50, mg/ml) | 0.65 ± 0.02 |
ABTS (IC50, mg/ml) | 3.25 ± 0.06 |
FRAP (EC50 mg/ml) | 6.565 ± 0.24 |
Abbreviations: mg GAE g− 1 DE, mg Gallic Acid Equivalent per gram Dry Extract; mg CE g− 1 DE, mg (+)-Catechin Equivalent/g of Dry Extract; IC50, half-maximal inhibitory concentration; EC50, Effective Concentration; DPPH, 2,2-diphényl-1-picrylhydrazyl; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). |
In the subsequent part, we set out to investigate the effect of STLPRE on nitric oxide (NO) production by LPS-stimulated RAW264.7 cells. First, cell viability assay was performed to ensure the non-cytotoxicity of the extract. As shown in Fig. 1a, STLPRE until 50 µg/ ml concentration does not affect macrophage cell viability. Hence, 10 and 30 µg/ml concentrations of STLPRE were preferentially used for subsequent experiments. LPS treatment drastically enhanced NO production by RAW264.7 cells (Fig. 1b). This high NO production can lead to an oxidative damage, as NO acts with free radicals to produce the highly reactive peroxynitrite, leading to cell damage. Interestingly, STLPRE significantly decreased LPS-induced NO production by RAW 264.7 in a dose dependent manner. These results highlighted a potent anti-inflammatory property of STLPRE. Indeed it has been reported that NO molecules can directly be scavenged by phenolic compounds (Vanacker et al. 1995). The inhibitory effect of STLPRE on NO production, is in line with a previous reports where phenolic compounds were shown to prevent oxidative stress and inflammation (Liu et al. 2002). There are several reports on the phenolic strategies leading to the reduction of NO and peroxinitrite cytotoxicity by proceeding as alternative substrates for nitration, as it has been demonstrated for phenolics from the hydroxycinnamic group such as p-coumaric and ferulic acids, or by reducing RNS as in the case of catechol structures (e.g. caffeic acid) (Choi et al. 2002; Pannala et al. 1998).
It is well known that the induction of NO is related to the stimulation of inducible nitric oxide synthases (iNOS) expression through the activation of the transcription factor NF-κB. On the one hand, in unstimulated RAW 264.7 cells, STLPRE increased iNOS mRNA level more than 20 fold of the control; and the NO production follows the same evolution as shown in Fig. 1(b and c). On the other hand, while the LPS enhanced drastically the iNOS levels by ~ 6400% of the control, STLPRE was able to suppress 70% of iNOS mRNA expression in LPS-stimulated RAW 264.7. Thus, it seems that STLPRE could influence macrophages in a double way: it was able to activate pro-inflammatory macrophages in resting conditions; conversely, it counteracted the NO production in RAW 264.7 cells stimulated by LPS. These results suggest that STLPRE could stimulate macrophages as well as re-establish normal immune function and homeostasis.
3.2. STLPRE affects immune responses by regulating pro and anti-inflammatory gene expression
Macrophages function, phenotype and polarization can be influenced by several microenvironment signals. Thus, cell shape also varies according to macrophage phenotype (McWhorter et al., 2013). Figure 1d illustrates that the unstimulated RAW 264.7 cells had a small round shape, whereas LPS-stimulated RAW 264.7 cells (M1-polarized) exhibited an irregular and more flatten form with the presence of pseudopodia and vesicles. This observation demonstrates that treatment of cells with STLPRE has significantly changed the cell morphology which became similar to cells treated with LPS.
To ascertain whether STLPRE had effectively influenced macrophage immune response, we compared the mRNA expression of pro-inflammatory M1-markers (IL-1β, IL-6, IL-12, TNF-α and Arg2) and anti-inflammatory M2 markers (Arg1, CD206, IL-10 and TGF-β) markers in stimulated and unstimulated RAW cells (Figs. 2 and 3).
In restring condition, RT-PCR analysis showed that STLPRE increased significantly pro-inflammatory markers levels as compared to the control. Analysis of M2 markers revealed that there was no significant variation in the TGF-β and IL10 mRNA expression associated with a reduction in Arg1 and CD206 as compared to negative control. Arg1 and CD206 are not expressed in M1 macrophages and therefore serves as a useful marker for M2 macrophages (Choi et al., 2010). This suggests that in the absence of LPS-stimulation, STLPRE treatment induces macrophage polarization into M1 phenotype.
LPS treatment considerably increased pro-inflammatory mRNA expression when compared to control cells. IL-1, IL6 and IL12 mRNA expression in LPS-stimulated cells were ~ 60%, 186% and 244%, respectively, higher than of those in STLPRE-treated cells (30 µg/ml). Therefore, STLPRE co-treatment of LPS-stimulated cells was able to regulate the inducible gene expression of pro-inflammatory cytokines in a dose-dependent manner. For anti-inflammatory cytokines, we did not detect any noticeable effect of STLPRE on LPS-stimulated cells mRNA expression.
We further examined the effect of STLPRE treatment on NF-κB (p65) and MAPKs activation. Western blot analysis (Fig. 4) has confirmed the double-action of STLPRE regarding immune response in RAW 264.7 cells. With LPS stimulation, we observed a decrease of mainly NF-κB and ERK phosphorylation. Nevertheless, in restring conditions, STLPRE treatment led to a significant increase in the phosphorylation of both NF-κB (p65) and MAPKs (ERK, JNK, and p38). Hence, activation of NF-κB and MAPKs signaling pathways could be responsible of the pro-inflammatory cytokines rise and consequently macrophage polarization (Han et al., 2013; Mosser and Edwards, 2008). These results are sustained by works indicating that M1 macrophage polarization are associated with JNK and NF-κB pathways (Fu et al., 2017). Moreover, it has been shown that defection in NF-κB activation in tumour-associated macrophages is in correlation with the weakened immune defense against tumor progression and that restitution of NF-κB activity in these macrophages is a possible strategy to restore M1 immunostimulation and cytotoxicity against tumors. This findings corroborate with previous studies indicating that restoration of M1 phenotype could provide therapeutic action in tumour-bearing mice (Saccani et al., 2006). Activation of macrophages and its polarization status has become recognized as a critical determinant in many diseases including cancer, several infections, allergic, autoimmune and metabolic diseases (Murray et al., 2014).
3.3 Potential in vitro and in vivo antitumoral activities of STLPRE
To evaluate antitumor potentiality of STLPRE, we used B16 cell line derived from C57BL/6 mouse spontaneous cutaneous melanoma. B16 cells proliferation were evaluated after 24 h treatment by increasing concentrations of STLPRE. Cell viability was then determined by crystal violet assay. As shown in Fig. 5, tumor cell viability was markedly reduced in a dose-dependent manner. At a dose of 40 µg/ ml, the proliferation of B16 cells was decreased by 50%. As a result, STLPRE seems to exert an inhibitory effect on the growth of murine B16 melanoma cell line. To evaluate the indirect effect of STLPRE on B16 growth, RAW 264.7 cells were treated with non-cytotoxic concentrations of STLPRE for 6 h. The medium was changed and cells were incubated for 24 h. Then, supernatants were collected and used to treat B16 cells. Interestingly, STLPRE induced B16 cell death in a dose dependent manner, suggesting that the phenolic extract could trigger cytotoxicity in melanoma cells through macrophage activation.
To assess the in vivo anti-tumor activity of STLPRE, B16 cells-transplanted C57BL/6 mice received STLPRE for 24 days, and the mortality was then scored. The survival rate in the untreated group was 60% at the end of experiment. However, in STLPRE-treated group, 100% of mice survived (Fig. 6a). We noticed that tumor growth was significantly curtailed in STLPRE-treated mice when compared to controls. This inhibition started to be significant at the 19th days after B16 injection and was maintained until the end of experiment where STLPRE diminished 80% tumour-size when compared to control group (Fig. 6b). This deceleration of tumor progression was confirmed by the fact that tumor weight after sacrifice was three fold more important in control group in comparison with treated one (Fig. 6c). All data highlights the potential of STLPRE to inhibit B16-tumor growth in C57BL/6 mice. Next, we extracted total mRNA from spleen mice and we investigated several cytokines expression by RT-PCR. The expression of IL-1, IL-12 and IFN-γ was significantly increased by STLPRE oral treatment (Fig. 6d). In fact, skin cutaneous melanoma is an extremely aggressive tumor and its progression is mostly expedited by the immunosuppression exerted by cancer cells (Romano et al., 2018). There is increasing support that stimulation of several cytokines production, such IFN- γ, is crucial in immunotherapy against cancer. Indeed, T cells produce IFN- γ and other cytokines in tumor microenvironment leading to tumor cells death (Ribas and Wolchok, 2018). We have found that STLPRE induced Wistar rat splenic-T cells when they were activated with anti-CD3/anti-CD28 (data not shown). Spleen is a secondary lymphoid organ that was qualified as a reservoir for tumor associated macrophages and neutrophils precursors that have a crucial function in controlling the tumor development (Mantovani et al., 2014). Several lines of evidence show that spleen-derived macrophages are readily polarized and have the plasticity to convert their phenotype (Mulder et al., 2014). Our results showed that STLPRE enhanced the production of cytokines, which are known to promote the M1 polarizations. Many studies proved that it is feasible to re-instruct macrophages and to encourage their tumoricidal capacity. A main enhancer towards M1 form is IFN- γ that plays a crucial role in immune defense against tumors (Mantovani et al., 2014). In addition, the expression of IL-1β is explicitly sensitive to STLPRE treatment. Actually, this cytokine is strongly involved in innate immune recognition, obviously in the Pattern Recognition Receptors (PRR) activation (Cantuária et al., 2018). Moreover, Haabeth et al. (2016) have demonstrated the key role of IL-1 in the activation of immunity mediated by T-cells against both cancer and pathogens. In fact, it was shown that the inhibition of IL-1α and IL-1β cell reception has dampened activation of macrophages and their tumoricidal activity. It was shown also that IL-12 plays a crucial role in the control of many disorders like cancers and infections. It is considered as a promising agent in immunotherapy and it synergizes with other cytokines for amplifying immunoregulatory actions (Akiyama et al., 2000; Hamza et al., 2010).