Anti-Inﬂammatory and Neuroprotective Effect of the Anti-Obesity Dietary Supplement Dekosilhue ® in an In Vitro Model of Neuroinﬂammation

: Obesity is now recognized as a global health problem and has reached epidemic proportions, affecting both developed and developing countries. This condition is associated with an increased incidence in central nervous system (CNS) disorders. The intake of foods or supplements with anti-inﬂammatory activity could potentially lead to a lower impact of obesity on the functionality of CNS. Therefore, in this work we tested the effect of a dietary supplement, DEKOSILHUE ® (DSK), containing polyphenols enriched herbal extracts and successfully used for improving the control of body weight, in an in vitro inﬂammatory model. LPS-stimulated immortalized microglia BV2 cells have been used as a model of neuroinﬂammation. The stimulation with LPS increased the release and activation of several neuroinﬂammatory mediators, such as IKB α , HDAC-1, and IL-1ß. DSK reduced the levels and the activation of all these pro-inﬂammatory markers. In addition, DSK increased the expression of the anti-inﬂammatory cytokine IL-10, promoting an anti-inﬂammatory response. Finally, we showed that the anti-inﬂammatory effect of DSK can prevent the neurotoxicity related to microglia hyperactivation. In conclusion, DSK could potentially help in controlling neuroinﬂammation CNS, reducing microglia pro-inﬂammatory activation.


Introduction
Obesity is now recognized as a global health problem and has reached epidemic proportions, affecting both developed and developing countries. The average global body mass index (BMI) has increased from 21.7 in 1975 to 24.2 in 2014. If this trend continues, it will exceed the upper limit of normal (BMI = 24.9) to overweightness (BMI = 25.0-29.9), then to obesity (>30.0), and the prevalence of global obesity is expected to be around 20% by 2025 [1]. Although genetic obesity can be found in some patients, it remains relatively rare [2] and predominantly results from environmental factors, such as diet type or physical activity level. This condition is associated with an increased incidence in central nervous system (CNS) disorders, such as dementia, stroke, depression, and Alzheimer's disease [3]. In particular, the type of diet correlates with major disorders in obese individuals. For example, following a "Western diet" alters glucose transporter function at the hippocampal level with severe cognitive deficits and increased anxiety levels [4], while a diet high in sugar predisposes to the development of depression. Although these diseases have different etiologies and pathophysiological manifestations, they all share a neuroinflammatory component [5] and obese patients represent a population that is more prone to develop central disorders [6]. Increased adipose tissue affects the normal function of many organs, Nutraceuticals 2022, 2 23 but more importantly, it promotes the release of substances with proinflammatory activity. Obesity causes increased levels of circulating fatty acids, endotoxins (e.g., LPS), and inflammatory mediators, altering the normal function of many organs. This inflammation can also affect the CNS leading to the appearance of neuroinflammation, resulting from hyperactivity of microglial cells (CNS resident immune cells), mainly affecting the hippocampus, amygdala, and cerebral cortex, leading to severe cognitive deficits and major alterations in mood [7]. The intake of foods or supplements with anti-inflammatory activity could potentially lead to a lower impact of obesity on the functionality of peripheral organs and higher nervous centers, with undoubted benefits in counteracting cognitive decline and states of anxiety and depression [8]. Among the substances of natural origin, those rich in polyphenols, such as green tea and coffee, have proved to be among the most promising in counteracting neuroinflammation and memory disorders [9]. Therefore, a product used for weight control and able to hamper neuroinflammatory processes, could help prevent CNS disorders related to obesity. On these bases, in this work, we tested the effect on an in vitro model of neuroinflammation of a dietary supplement, namely DEKOSILHUE ® , based on aqueous extracts of herbal extracts used to promote the metabolism of carbohydrates and lipids for improving the control of body weight and sense of hunger.

Sulforhodamine B (SRB) Assay
Cell viability was assessed by SRB assay. BV2 (2 × 10 5 cells in 200 µL) were seeded in 96-well plates, each well corresponding to a different treatment: control (CTRL), LPS, DSK 1, 10, 100, and 1000 µg/mL with or not LPS. The CTRL group was BV2 cells no treated with LPS. Serum-free medium was added to the wells followed by trichloroacetic acid, used as fixative (250 mg/mL TCA, Sigma-Aldrich). The plate was then incubated at 4 • C for 1 h. This was followed by 3-5 washes with double sterile water. The day after, a solution of SRB dissolved in 1% acetic acid was added to each well and incubated for 30 min at room temperature. Then, 3-5 washes with acetic acid were performed. Then, TRIS HCl solution (pH = 10) was added and incubated for 5 min. Finally, the absorbance was recorded at a wavelength of 570 nm. The treatments were performed in six replicates in three independent experiments, and cell viability was calculated by normalizing the values to the control's mean [11].

Cell Counting Kit (CCK-8) Assay
BV2 cells were seeded in 6-well plates (3 × 10 5 cells/well), pre-treated with DSK for 4 h, and then stimulated with LPS 250 ng/mL for 24 h. The neuroprotective effect of DSK was evaluated by assessing the cell viability of SH-SY5Y cells treated with the LPS-conditioned BV2 medium for 24 h. Unstimulated BV2 medium was used as control. Cell viability was performed using the CCK-8 kit (Sigma-Aldrich) according to the manufacturer's instructions. The absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Benchmark, Biorad). The treatments were replicated three times in six independent experiments, and cell viability was calculated by normalizing the values to the control's mean [12].

Preparation of Cell Lysate
BV2 cells were seeded in 6-well plates (3 × 10 5 cells/well), pre-treated with DSK for 4 h, and then stimulated with LPS 250 ng/mL for 24 h. Then, the conditioned BV2 medium was collected and centrifuged (1000× g for 10 min, 37 • C). The pellet was discarded, and the supernatant was stored at −80 • C for cytokines dosages or used immediately to treat SH-SY5Y cells. Cell lysates were prepared using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl 1% sodium deoxycolate, 1% Tryton X-100, 2 mM PMSF) (Sigma-Aldrich) and the insoluble pellet was separated by centrifugation (12,000 × g for 30 min, 4 • C). The total protein concentration in the supernatant was measured using the Bradford colorimetric method (Sigma-Aldrich) [10].

Supernatant Protein Precipitation
TCA (25 µL) was added to 50 µL of medium and incubated at 4 • C for 10 min. This was followed by centrifugation at 14,000 rpm for 5 min. The supernatant was then removed, the pellet was washed three times by resuspending in cold acetone, and centrifuging again at 14,000 rpm for 5 min. At this point the microtubes were placed in a heated chamber, favoring the evaporation of the acetone and the drying of the pellets. Finally, the pellet was resuspended in loading buffer 4x and the samples were stored at −20 • C.

Statistical Analysis
Data are expressed as the mean ± SEM of three-six experiments and assessed by oneway or two-way ANOVA, followed by a Tukey post hoc test if significance was detected. For each test, a value of p < 0.05 was considered significant. The software GraphPad Prism (version 5.0, San Diego, CA, USA) was used in all statistical analysis.

DSK Reduced Cytotoxicity Induced by LPS in Microglia Cells
To evaluate the anti-inflammatory activity of DSK, we stimulated BV-2 microglial cells with LPS 250 ng/mL for 24 h. After the inflammatory stimulus, microglial cells assume an inflammatory phenotype. Under basal conditions, none of the tested concentrations (1, 10, 100, and 1000 µg/mL) of DSK altered cell viability after 24 h of treatment. The inflammatory stimulus reduces cell viability by approximately 30% compared to the control group. The pre-treatment with DSK (10 to 1000 µg/mL) of LPS-stimulated cells was able to counteract the cytotoxic effect of the inflammatory stimulus ( Figure 1). chamber, favoring the evaporation of the acetone and the drying of the pellets. the pellet was resuspended in loading buffer 4x and the samples were stored at −2

Statistical Analysis
Data are expressed as the mean ± SEM of three-six experiments and assessed way or two-way ANOVA, followed by a Tukey post hoc test if significance was de For each test, a value of p < 0.05 was considered significant. The software GraphPad (version 5.0, San Diego, CA, USA) was used in all statistical analysis.

DSK Reduced Cytotoxicity Induced by LPS in Microglia Cells
To evaluate the anti-inflammatory activity of DSK, we stimulated BV-2 mi cells with LPS 250 ng/mL for 24 h. After the inflammatory stimulus, microglial c sume an inflammatory phenotype. Under basal conditions, none of the tested con tions (1, 10, 100, and 1000 µg/mL) of DSK altered cell viability after 24 h of treatme inflammatory stimulus reduces cell viability by approximately 30% compared to t trol group. The pre-treatment with DSK (10 to 1000 µg/mL) of LPS-stimulated ce able to counteract the cytotoxic effect of the inflammatory stimulus ( Figure 1).

Activation of NF-κB Pathway Was Reduced by DSK Treatment in LPS-Stimulated BV2
It is well established that deacetylation by HDAC is not only directed to histone proteins, but also to enzymes and transcription factors. In fact, the deacetylation of specific lysine residues of transcription factors, such as NF-κB, can modulate their activity [14]. Activation of NF-κB p65 started with the degradation of IkB ⍺ proteins. With the degradation of IkB⍺, the p-NF-kBp65 complex is freed to move into the nucleus where it can trigger the expression of target genes resulting in an inflammatory response [15]. LPS increased the activity of this transcription factor by reducing (−53%) the levels of IKB⍺, the NF-κB inhibitory cytoplasmic protein. DSK 10 (-28%), 100 (−13%), and 1000 (+0.1%) µg/mL was able to counteract in a dose-dependent manner this effect reporting the value similar to the CTRL group, thus reducing the pro-inflammatory effect of NF-κB ( Figure 3).

DSK Modulates Levels of Pro-and Anti-Inflammatory Cytokines Released from LPS-Stimulated BV2 Cells
NF-κB is a transcription factor that primarily regulates transcription of genes encoding for inflammatory factors, such as cytokines. Among these, the most peculiar for microglial activation is represented by IL-1β [16]. Further to the regulation of pro-inflammatory cytokines expression, NF-κB regulates transcription of cytokines with anti-inflamma-

Activation of NF-κB Pathway Was Reduced by DSK Treatment in LPS-Stimulated BV2
It is well established that deacetylation by HDAC is not only directed to histone proteins, but also to enzymes and transcription factors. In fact, the deacetylation of specific lysine residues of transcription factors, such as NF-κB, can modulate their activity [14]. Activation of NF-κB p65 started with the degradation of IkB α proteins. With the degradation of IkBα, the p-NF-κBp65 complex is freed to move into the nucleus where it can trigger the expression of target genes resulting in an inflammatory response [15]. LPS increased the activity of this transcription factor by reducing (−53%) the levels of IKBα, the NF-κB inhibitory cytoplasmic protein. DSK 10 (−28%), 100 (−13%), and 1000 (+0.1%) µg/mL was able to counteract in a dose-dependent manner this effect reporting the value similar to the CTRL group, thus reducing the pro-inflammatory effect of NF-κB ( Figure 3). flammatory activity of LPS-stimulated isolated microglial cells [12]. As shown in Figure  2, LPS increased HDAC-1 expression (+65%), while pretreatment with DSK 10 (−13%), 100 (−4%), and 1000 (−6%) µg/mL was able to reduce HDAC1 expression to levels that are comparable to those of CTRL.

Activation of NF-κB Pathway Was Reduced by DSK Treatment in LPS-Stimulated BV2
It is well established that deacetylation by HDAC is not only directed to histone proteins, but also to enzymes and transcription factors. In fact, the deacetylation of specific lysine residues of transcription factors, such as NF-κB, can modulate their activity [14]. Activation of NF-κB p65 started with the degradation of IkB ⍺ proteins. With the degradation of IkB⍺, the p-NF-kBp65 complex is freed to move into the nucleus where it can trigger the expression of target genes resulting in an inflammatory response [15]. LPS increased the activity of this transcription factor by reducing (−53%) the levels of IKB⍺, the NF-κB inhibitory cytoplasmic protein. DSK 10 (-28%), 100 (−13%), and 1000 (+0.1%) µg/mL was able to counteract in a dose-dependent manner this effect reporting the value similar to the CTRL group, thus reducing the pro-inflammatory effect of NF-κB ( Figure 3).

DSK Modulates Levels of Pro-and Anti-Inflammatory Cytokines Released from LPS-Stimulated BV2 Cells
NF-κB is a transcription factor that primarily regulates transcription of genes encoding for inflammatory factors, such as cytokines. Among these, the most peculiar for microglial activation is represented by IL-1β [16]. Further to the regulation of pro-inflammatory cytokines expression, NF-κB regulates transcription of cytokines with anti-inflammatory activity, reducing them in case of inflammation. Among these, one of the best known

DSK Modulates Levels of Pro-and Anti-Inflammatory Cytokines Released from LPS-Stimulated BV2 Cells
NF-κB is a transcription factor that primarily regulates transcription of genes encoding for inflammatory factors, such as cytokines. Among these, the most peculiar for microglial activation is represented by IL-1β [16]. Further to the regulation of pro-inflammatory cytokines expression, NF-κB regulates transcription of cytokines with anti-inflammatory activity, reducing them in case of inflammation. Among these, one of the best known is IL-10 [17]. In the in vitro model of neuroinflammation, we observed that cells treated with LPS were characterized by a strong increase in the level of the pro-inflammatory cytokine IL-1β (+61%) and by a reduction of the anti-inflammatory cytokine IL-10 (−37%). DSK inhibited the release of IL-1β at each tested concentration (Figure 4A), counteracting a proinflammatory response. In addition, the concentration of 100 µg/mL significantly increased IL-10 release (+39%) (Figure 4B), promoting an anti-inflammatory response. 6 with LPS were characterized by a strong increase in the level of the pro-inflammatory cytokine IL-1β (+61%) and by a reduction of the anti-inflammatory cytokine IL-10 (−37%). DSK inhibited the release of IL-1β at each tested concentration (Figure 4A), counteracting a proinflammatory response. In addition, the concentration of 100 µg/mL significantly increased IL-10 release (+39%) (Figure 4B), promoting an anti-inflammatory response.
Nutraceuticals 2022, 2,6 with LPS were characterized by a strong increase in the level of the pro-inflammatory cytokine IL-1β (+61%) and by a reduction of the anti-inflammatory cytokine IL-10 (−37%). DSK inhibited the release of IL-1β at each tested concentration (Figure 4A), counteracting a proinflammatory response. In addition, the concentration of 100 µg/mL significantly increased IL-10 release (+39%) (Figure 4B), promoting an anti-inflammatory response.

Discussion
Despite the degree of malnutrition existing on the planet, according to the World Health Organization (WHO), obesity represents one of the main public health problems in the world. It certainly represents an actual socio-economic problem [18]. The co-presence of systemic inflammation and obesity was recognized some time ago, with early reported evidence suggesting that obese people have elevated circulating pro-inflammatory cytokines compared to control groups [19]. Thus, a fat-rich diet leads to infiltration and

Discussion
Despite the degree of malnutrition existing on the planet, according to the World Health Organization (WHO), obesity represents one of the main public health problems in the world. It certainly represents an actual socio-economic problem [18]. The copresence of systemic inflammation and obesity was recognized some time ago, with early reported evidence suggesting that obese people have elevated circulating pro-inflammatory cytokines compared to control groups [19]. Thus, a fat-rich diet leads to infiltration and activation of microglia with activation of inflammatory signaling [20]. Importantly, central inflammation may contribute to insulin resistance, promoting weight gain, and sustaining obesity [21]. Microglia are the main CNS immune cells and protect the homeostasis of the CNS [22]. Hence, if an unbalanced diet can induce an alteration in microglial activity, this can result in an alteration at the neuronal level, which can lead to a possible onset of degenerative diseases [23]. Thus, finding a product that can control both body weight and central inflammation could help prevent damage at the CNS level in overweight and obese patients. Complementary and alternative treatments for weight loss include medicinal plants, and their active components, that have been extensively used since ancient times [24].
The anti-obesity effect of polyphenols from commonly used herbal preparations has been documented [25]. Indeed, several clinical studies reported that the consumption of green tea [26] caffeine [27], cathechins [28], and epigallocatechin gallate (EGCG) [29] may improve weight maintenance by preventing or limiting weight regain. Moreover, several polyphenols could play a role in preventing the development of neuroinflammation [30]. For these reasons, our aim was to investigate the possible anti-inflammatory activity of DSK, a food supplement containing a combination of polyphenol-based herbal extracts, used to promote the metabolism of carbohydrates and lipids for the control of body weight and sense of hunger, in an in vitro model of neuroinflammation. Increasing evidence suggest that complex conditions with a multifactorial origin, such as obesity, benefits of a multitarget approach. In this contest, we want to emphasize and valorize the synergism of different plant extracts that can work together to reduce inflammation as a potential adjuvant in the control of obesity and related disorders.
LPS-stimulated BV2 cells have been widely used as a model of neuroinflammation. The stimulation with LPS increased the activation of several neuroinflammatory markers, such as HDAC-1, IKBα, and cytokines [12]. DSK was able to reduce the activation of all these pro-inflammatory markers. HDAC-1 is an epigenetic factor involved in inflammatory processes [31]. Recently, direct regulation of HDAC-1 has also been seen in modulating several transcription factors, including NF-κB [14,31]. Inhibition of HDAC-1 is, therefore, an important step towards reducing chronic inflammatory processes. Known inhibitors, such as Vorinostat, and others in the experimental phase, such as LG325, have been shown to reduce activation of the pro-inflammatory microglial phenotype in animal models in which microgliosis is certainly a key factor (e.g., neuropathic pain) [14,[32][33][34][35]. In fact, HDAC-1 inhibition also leads to a reduction in the expression of genes involved in the transcription of the NF-κB factor and its entire pathway. NF-κB is undoubtedly an important marker of microglial activation both in the early stages of the inflammatory process and in the more advanced phases [36]. IKB is an inhibitory protein that, when present in large quantities, can block the nuclear translation of NF-κB. However, its degradation leads to the phosphorylation of the p65 subunit of NF-κB complex and its nuclear translation, thus, causing an increase in the expression of pro-inflammatory factors [15]. The receptors to which LPS, which is the stimulus we used to reproduce a model of chronic inflammation, binds mainly at the level of BV2 on TLR4 receptors, which are directly linked to the activation of the NF-κB pathway [30]. Thus, both through the epigenetic modulation of HDAC1 and TLR4, microglial cells lead to an increase in the degradation of IKB-alpha and simultaneously to an increase in the transcription of pro-inflammatory proteins. Among these, IL1ß is certainly one of the most characteristic of microglial cells. This cytokine is released by the inflammatory microglia and causes an alteration in normal activity in the CNS [12,37]. Cytotoxic factors from activated microglial cells cause damage to neurons and the use of polyphenols could potentially ameliorate this neurotoxic effect [38]. DSK was not only able to reduce pro-inflammatory markers, but it also increased the expression of IL-10, an anti-inflammatory cytokine able to restore the physiological function of microglial cells. An important role for IL-10 has been observed in obesity. Indeed, in high-fat dietinduced obesity in mice, IL-10 expression-inducing gut bacteria can suppress obesity [39]. Moreover, the reduction of IL-10 expression in serum was detected in obese children and in high-fat diet rats [40]. Thus, the increase of IL-10 expression could represent a protective approach for metabolic syndrome. Neuroinflammation plays a key role in the pathogenesis of neurological disorders in obese patients [41]. Currently, the interaction between glial cells and neurons is an increasingly interesting field for the development of new therapies [42][43][44]. By treating SH-SY5Y neuronal cells with the LPS-conditioned medium of DSK-pretreated BV-2 cells, we showed that the anti-inflammatory effect of DSK can prevent the induction of neurotoxicity caused by microglia hyperactivation.

Conclusions
In conclusion, in this work we wanted to investigate the possible anti-inflammatory activity of a DSK supplement, which is mainly used for weight control. The key role of microglia in producing inflammatory factors that may impair normal neuronal activity in overweight people has recently been highlighted. In an in vitro model of neuroinflammation, we have seen that DSK is able to reduce the main markers linked to microglial activation, and this may be a good starting point for further investigation of its activity in preclinical and clinical studies of neuroinflammatory-related pathologies.