Suppression of Inflammation in Adipocyte-Macrophage Coculture by Passion Fruit Seed Extract: Insights into the p38 and NF-ҡB Pathway

Obesity, which is characterized by chronic low-grade inflammation, involves the infiltration of immune cells into adipose tissue, leading to the secretion of inflammatory cytokines and subsequent inflammation. Therefore, the aim of this study was to examine the potential of passion fruit seed extract (PSEE) in mitigating lipopolysaccharide (LPS)-induced inflammation in a coculture system comprising macrophages and adipocytes. PSEE demonstrated significant reductions in reactive oxygen species (ROS) and nitric oxide (NO) levels, primarily achieved through the downregulation of inducible nitric oxide synthase (iNOS) protein expression in LPS-induced adipocyte-macrophage cocultures. Furthermore, PSEE effectively suppressed the secretion of TNF-α and IL-1β by attenuating the gene expression of these cytokines, as well as other inflammation-related genes such as MMP-2, IL-6, and MCP-1. Notably, PSEE exhibited potent inhibitory effects on the p38 and NF-κB signaling pathways, thus alleviating inflammation in the LPS-induced adipocyte-macrophage cocultures. Additionally, PSEE led to a decrease in the expression of ACC, HSL, and FaSN, while aP2 and ATGL showed increased expression in LPS-induced cocultured macrophages and adipocytes. These findings suggest that passion fruit seed extract effectively combats inflammation by suppressing the p38 and NF-κB signaling pathways, resulting in reduced levels of proinflammatory cytokines, NO, and ROS production.


Introduction
Obesity, characterized by the excessive accumulation of fat, is a chronic low-grade systemic infammation that signifcantly increases the risk of various metabolic diseases, including cardiovascular disease, hypertension, type II diabetes, and other infammation-related disorders [1].Tis infammatory state is primarily attributed to chronic infammation within adipose tissue, which involves the infltration of macrophages and other infammatory cells, accompanied by the release of proinfammatory cytokines, all of which are closely associated with obesity [2].Te presence of macrophages in adipose tissue promotes the secretion of proinfammatory cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin 6 (IL-6), leading to the initiation of an infammatory response and the development of insulin resistance within adipose tissue [3].Consequently, reducing macrophage-induced infammation in adipose tissue could potentially mitigate the progression of obesity-related diseases.
Te coculture of adipocytes and macrophages is essential for studying various physiological and pathological processes [4].Te interaction between these two cell types plays a signifcant role in the development of infammation and metabolic disorders [4][5][6].In obesity, the cross-talk between adipocytes and macrophages can lead to the secretion of proinfammatory factors, contributing to chronic low-grade infammation.Tis interaction has been studied using in vitro coculture systems, which mimic the in vivo environment and help in understanding the complex cellular interactions and signaling pathways involved [7,8].Terefore, cocultured adipocytes and macrophages serve as a valuable tool for investigating the pathophysiology of various metabolic conditions and for the development of potential therapeutic interventions.
Various treatments for obesity, such as orlistat and lorcaserin, have been associated with certain limitations, including side efects.Orlistat can lead to side efects such as oily stools and increased defecation.Lorcaserin is associated with side efects such as serotonin syndrome [9].Passifora edulis Sims f. favicarpa, a variety of passion fruit known for its yellow or sour favor, has been extensively studied for its diverse range of biological activities in both in vitro and in vivo settings.Tese properties include antioxidant, antiinfammatory, antimicrobial, antihypertensive, hepatoprotective, and antidiabetic activities [10][11][12].In a previous study, our study reported that the seed extract of P. edulis is particularly rich in total phenolics, favonoids, carotenoids, and stilbenes, with a notable concentration of piceatannol [12].Moreover, passion fruit seed extract has demonstrated remarkable biological activity, including antioxidant and anti-infammatory properties, inhibition of pancreatic lipase and cholesterol esterase, as well as vasorelaxation in rat aortic rings [12,13].Despite the well-known anti-infammatory efects of piceatannol, no previous studies have explored these efects specifcally in relation to passion fruit seed extract.Terefore, the objective of this study was to investigate the inhibitory efects of passion fruit seed ethanolic extract (PSEE) on lipopolysaccharide (LPS)-induced infammatory responses in a coculture model consisting of 3T3-L1 adipocytes and RAW264.7 macrophages.

Preparation of Passion Fruit Seed Extract.
To obtain the passion fruit seed ethanolic extract (PSEE), the seeds were frst dried and then powdered.Te powdered seeds were mixed with 70% ethanol in a ratio of 1 : 10.Te mixture was allowed to extract at room temperature for 72 h.Subsequently, the extract solution was fltered using Whatman ® paper No. 1. Te solvent was then evaporated using an evaporator (Buchi, Switzerland), and the remaining extract was dried using a freeze dryer.Te resulting extract sample was stored in amber glass bottles at −20 °C for further analysis.Te phytochemical analysis of the extract was previously reported in our study [12].

Cell
Culture.In this study, murine 3T3-L1 preadipocytes (ATCC, USA) and murine macrophages RAW 264.7 (ATCC, USA) cells were cultured separately using diferent culture media.Te 3T3-L1 cells were cultured in Dulbecco's Modifed Eagle Medium (DMEM; Gibco, USA) high glucose (with a glucose content of 4.5 g/L).On the other hand, the RAW 264.7 cells were cultured in DMEM low glucose (with a glucose content of 1.0 g/L; Gibco, USA).Both culture media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 1% penicillinstreptomycin, and 1% L-glutamine (Gibco, USA).Both cell lines were incubated at a temperature of 37 °C, in a humidifed atmosphere with 5% CO 2 .

Cytotoxicity Test.
To assess cytotoxicity in the cocultured cells, the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed.Following the treatment protocol, the cocultured cells were incubated with MTT solution at a concentration of 500 μg/ mL for a duration of 2 h at 37 °C.After the incubation period, the absorbance of the cells was assessed at 570 nm using a microplate reader (SPECTROstar Nano, BGM Labtech, Germany).

Intracellular Reactive Oxygen Species (ROS) Production.
To analyze the production of reactive oxygen species (ROS) in the cocultured cells, the 2′-7′-dichlorofuorescin diacetate (DCFH 2 -DA) assay was conducted.After 24 h of LPS treatment, the cocultured medium was removed, and the cells were washed with PBS.Subsequently, 100 μL of 50 µM DCFH 2 -DA (Sigma, USA) was added to the cells and incubated for 1 h at a temperature of 37 °C.After the incubation period, the DCFH 2 -DA solution was removed, and the cells were washed with PBS.Te fuorescence intensity of the cells was then measured using a fuorescence microplate reader (Bio-tex, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
2.6.Nitric Oxide Assay.Te level of nitric oxide (NO) was determined using the Griess assay.For each treatment group, 100 μL of the cocultured medium was combined with 2 Advances in Pharmacological and Pharmaceutical Sciences 100 μL of Griess reagent (Sigma, USA).Te resulting mixture was then incubated for a duration of 10 min.After the incubation period, the NO level in the samples was detected at 540 nm using a microplate reader.Sodium nitrate was used as a standard for comparison in order to quantify the nitric oxide levels in the cocultured samples.

Determination of TNF-α and IL-1β
Level.Te TNF-α and IL-1β levels were determined using an ELISA assay (Merck Millipore, Burlington, Massachusetts, USA).To perform the assay, 100 μL of the cocultured medium was added to each well of a 96-well ELISA plate.Te plate was then incubated for 2.5 h at room temperature with gentle shaking.After the incubation, the plate was rinsed four times with 1X Wash Solution.Ten, the prepared Detection Antibody (100 μL) was applied to each well and incubated at room temperature for 60 min with gentle shaking.Following this, the prepared streptavidin solution (100 μL) was applied to each well and incubated at room temperature with gentle shaking for 45 min.Next, the TMB One-Step Substrate Reagent (100 μL) was incubated for 30 min.Finally, the reaction was stopped by adding the stop solution and measured at 450 nm using a microplate reader.

Real Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR).
Total RNA was extracted from the cocultured 3T3-L1 and RAW264.7 cells using the TRIzol reagent.Te concentration of the extracted RNA was measured using a Bio spectrometer (Eppendorf, Hamburg, Germany).Te extracted RNA was then converted into complementary DNA (cDNA) using a cDNA synthesis kit (Invitrogen, Waltham, Massachusetts, USA).Te cDNA template was mixed with the SensiFAST SYBR NO-ROX kit (Invitrogen ™ , USA) and quantifed using a real-time PCR detection system (Model FQD-96A, Bioer, China).Te quantifcation was performed using SYBR green dye to examine the mRNA expression levels of various genes, including ACC, aP2, FaSN, ATGL, HSL, LPL, leptin, resistin, Glut4, IsnR, adipoQ, and adiponectin receptors (adipoQ-R1 and adipoQ-R2).Additionally, the expressions of TNF-α, IL-1β, IL-6, MCP-1, MMP-2, and MMP-9 were also evaluated.Te housekeeping gene β-actin was used as a reference gene for normalization.Te gene expression analysis was performed using the 2 −ΔΔCt method, which allows for the comparison of relative gene expression levels between different samples.
2.9.Western Blot Analysis.Te cocultured cells were washed with PBS and subsequently lysed using RIPA bufer.Te lysis process was conducted for 30 min at a temperature of 4 °C.Afterward, the lysed cells were disrupted using a homogenizer and the resulting mixture was centrifuged at 14,000 rpm for 20 min.Te total protein concentration in the lysate was measured using Bradford's method.Ten, 75 μg of protein was loaded onto 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto PDVF membranes.Te membranes were blocked with 5% skim milk for 2 h and washed with 1X TBST for 1 h.Te membranes were then incubated with primary antibodies to ERK1/2 (Abcam, Cambridge, UK), pERK1/2 (Santa Cruz Biotechnology, CA, USA), p-p38 (Santa Cruz Biotechnology), p38 (Santa Cruz Biotechnology), nuclear factor-κB (NF-κB; Santa Cruz Biotechnology), and inducible nitric oxide synthase (iNOS; Santa Cruz Biotechnology) overnight at 4 °C.Te membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature.Te band of proteins was observed by the ECL detection reagent, followed by exposure to X-ray hyperflm, and the relative band density was measured by normalizing with β-actin using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis.
All data were reported as the mean ± standard error of the mean (SEM).Statistical comparisons were conducted using one-way analysis of variance (ANOVA), followed by a post-hoc LSD (Least Signifcant Diference) test using SPSS software.A p value less than 0.05 was considered statistically signifcant to indicate meaningful diferences between the groups.

Efects of PSEE against ROS and NO Production on LPS-Induced Infammation in Cocultured Adipocytes and
Macrophages.According to Figure 1(a), the production of ROS in the LPS group was signifcantly higher compared to the control group (p ≤ 0.05).Interestingly, treatment with PSEE at concentrations of 50 and 100 μg/mL resulted in a signifcant decrease in ROS production compared to the LPS group (Figure 1(a); p ≤ 0.05).As shown in Figures 1(b) and 1(c), the highest level of NO and iNOS protein expression was found in the LPS group compared to the control group (p ≤ 0.05).However, treatment with all concentrations of PSEE resulted in signifcantly lower NO levels and iNOS protein expression compared to the LPS group (p ≤ 0.05) (Figures 1(b) and 1(c)).Lastly, the efect of long-term treatment with PSEE on cell viability in cocultured adipocytes and macrophages was investigated (Figure 1(d)).Te results indicated that neither LPS nor the diferent concentrations of PSEE (10, 50, and 100 μg/mL) had a signifcant efect on cell viability, suggesting that PSEE treatment did not exhibit cytotoxicity in the cocultured cells (Figure 1(d)).Overall, these results demonstrated the potential of PSEE in reducing LPS-induced ROS and NO secretion without causing cytotoxicity in cocultured adipocytes and macrophages.Treatment with PSEE at concentrations of 50 and 100 μg/mL signifcantly downregulated the expression levels of MCP-1, IL-6, and MMP-2 compared to the LPS group (Figures 3(a)-3(c); p ≤ 0.05).However, the expression of MMP-9 was not signifcantly afected by the treatment with PSEE (Figure 3(d)).In addition, treatment with PSEE at concentrations of 50 and 100 μg/mL signifcantly upregulated the expression levels of adiponectin and adiponectin receptors (adipoQ-R1 and adipoQ-R2) (Figures 3(e) and 3(f )).

Efects of PSEE against the Secretion and Expression of TNF-α and IL-1β on LPS-Induced Infammation in
In contrast, PSEE (50 and 100 μg/mL) caused a signifcant decrease in leptin and resistin mRNA expression in the cocultured adipocytes and macrophages (Figures 3(g) and 3(h)).Tese fndings suggest that PSEE attenuated infammation in the cocultured adipocytes and macrophages by suppressing the expression of cytokines and adipokines.Advances in Pharmacological and Pharmaceutical Sciences lipoprotein lipase (LPL), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL)) were investigated in LPS-induced adipocytes and macrophages coculture.Te results demonstrated that PSEE at the concentrations of 50 and 100 μg/mL signifcantly increased the expression of ACC, HSL, and FaSN, while aP2, ATGL, and LPL exhibited a decrease in LPS-induced cocultured adipocytes compared to the LPS-alone condition (Figures 4(a)-4(f )).Moreover, treatment with PSEE at concentrations of 50 and 100 μg/mL led to a reduction in insulin resistance in the cocultured adipocytes with macrophages, as evidenced by a signifcant increase in the expression of Glut4 and insulin receptor genes (Figures 4(g) and 4(h)).

PSEE Inhibits Infammation through NF-κB and MAPK Pathway.
As shown in Figure 5, the NF-κB and p38 expression in their active forms were signifcantly upregulated following LPS treatment.However, in the presence of PSEE treatment, the phosphorylation of p38 and the expression of NF-κB p65 protein were signifcantly suppressed compared to the LPS group (Figures 5(a), 5(c) and 5(d); p ≤ 0.05).On the other hand, there was no signifcant diference in the expression levels of ERK1/2 among the experimental groups (Figures 5(a) and 5(b); p > 0.05).Tese results suggest that PSEE has the potential to signifcantly inhibit the activation of the infammatory response through the NF-κB and p38MAPK pathways.

Discussion
Obesity is characterized by chronic low-grade infammation, which occurs due to the infltration of infammatory cells into adipose tissue [18].Tis infltration leads to the transformation of monocytes into M1 macrophages, resulting in the secretion of proinfammatory cytokines such as TNF-α, IL-6, IL-1β, and MCP-1, thereby promoting adipose tissue infammation [19].Te interaction between adipocytes and macrophages plays a signifcant role in the development of chronic infammation in adipose tissue among obese individuals [18].Te objective of this study was to investigate the inhibitory efect of PSEE on LPS-induced infammation in a coculture of macrophages and adipocytes.Obesity is associated with increased production of free radicals, such as ROS and reactive nitrogen species, which contribute to the secretion and expression of proinfammatory cytokines [20,21].Elevated levels of NO are found in infamed adipocytes and macrophages, and increased NO production has been observed in obese individuals [22].ROS can activate signaling pathways, including NF-κB and MAPK, leading to the production of proinfammatory cytokines such as TNF-α and IL-1β, as well as the expression of iNOS [23,24].In this study, it was observed that treatment with PSEE at concentrations of 50 and 100 μg/mL signifcantly reduced the ROS levels in cocultured adipocytes and macrophages stimulated with LPS.Furthermore, the treatment with PSEE signifcantly      Advances in Pharmacological and Pharmaceutical Sciences inhibited the secretion of NO and the expression of iNOS protein in the cocultured cells stimulated with LPS.Tese fndings align with previous research, which consistently revealed that passion fruit seed extract and its constituents, including piceatannol and resveratrol, possess the capability to diminish ROS levels, suppress NO production, and mitigate iNOS protein expression across various cell types, including keratinocytes, adipocytes, and macrophages [15,16,25,26].In our previous report, we demonstrated that passion fruit seed extract, obtained through both ethanol and water extraction methods, led to a reduction in the levels of NO in LPS-induced RAW264.7 cells [12].Taken together, these experiments suggest that PSEE has anti-infammatory efects in cocultured adipocytes and macrophages by inhibiting ROS and NO production through the suppression of iNOS expression.Te interaction between adipocytes and macrophages plays a signifcant role in chronic infammation observed in obese individuals.Adipocytes contribute to the secretion of proinfammatory cytokines such as TNF-α, IL-1β, MMP-2, MMP-9, IL-6, and MCP-1, as well as adipokines like adiponectin, which further contribute to chronic infammation in adipose tissue of individuals with obesity [18].In obesity, MCP-1 is vital for macrophage infltration into adipose tissue, where both adipocytes and macrophages release MCP-1, intensifying macrophage recruitment [8,27].IL-6 is predominantly produced by infltrated adipose tissue macrophages, while elevated MMP-2 and MMP-9 levels in obesity contribute to adipose tissue remodeling, triggering proinfammatory cytokine secretion and amplifying the infammatory response [5,19].In this study, the treatment of cocultured adipocytes and macrophages with PSEE resulted in a signifcant reduction in the release and expression of TNF-α and IL-1β compared to the LPS-only group.Tis fnding was consistent with previous studies that demonstrated the ability of resveratrol and piceatannol to suppress the secretion of TNF-α and IL-1β [6,16].In addition, Li et al. [28] demonstrated that piceatannol exhibits antiinfammatory properties by reducing infammation through the inhibition of TNF-α production in cocultured adipocytes and macrophages, suppressing signaling pathways such as NF-κB and JNK pathways.Furthermore, the expressions of MMP-2, IL-6, and MCP-1 genes were signifcantly suppressed in response to PSEE.Tese results were consistent with previous studies reporting that piceatannol can reduce the production of MCP-1 and IL-6 in cocultures of adipocytes and macrophages [16,28].Additionally, resveratrol was shown to decrease the levels of IL-6 and MCP-1 in infamed adipocytes [29].Moreover, α-tocopherol that is found in passion fruit has been shown to inhibit the expression of the IL-6 gene in cocultures of adipocytes and macrophages [7].Furthermore, an imbalance among adiponectin (anti-infammatory), leptin, and resistin contributes to the development of moderate infammation associated with obesity and increased adipose tissue cell necrosis.Tis imbalance underscores the progression of insulin resistance and metabolic syndrome [2,30].Our fndings indicate that PSEE increases the expression of adiponectin and its receptor, while simultaneously decreasing the expression of leptin and resistin genes.Tis is the frst investigation that determines the properties of PSEE on adiponectin, leptin, and also resistin.Tese experimental fndings ofer compelling evidence of the anti-infammatory properties of PSEE, likely attributed to the presence of compounds like piceatannol and resveratrol.Te observed inhibition of proinfammatory cytokines and adipokines associated with infammation underscores the potential therapeutic signifcance of passion fruit seed extract in alleviating adipose tissue infammation.Infammation occurring within adipocytes is regulated by the activation of NF-κB and MAPK signaling pathways [17,19].In this study, we investigated the impact of PSEE on the expression of ERK1/2, p38MAPK, and NF-κB p65 proteins in cocultured adipocytes and macrophages stimulated with LPS.Te results demonstrated a signifcant reduction in the expression of NF-κB p65 and p38MAPK proteins upon treatment with PSEE.Tis fnding was consistent with a previous study that highlighted the inhibitory efect of resveratrol, a major component of passion fruit seed, on the phosphorylation of ERK1/2 and NF-κB p65 in cocultured adipocytes and macrophages [31].Te antiinfammatory efects of piceatannol, resveratrol, and α-tocopherol are thought to be mediated through the inhibition of the NF-κB pathway activation and/or SIRT1 activation [16].However, in contrast to the efect on NF-κB and p38 MAPK proteins, PSEE did not show any signifcant impact on the expression of ERK1/2 proteins in cocultured adipocytes and macrophages stimulated with LPS.Tese fndings suggest that the extract specifcally targets the NF-κB and p38 MAPK signaling pathways, while the ERK1/2 pathway remains unafected.Taken together, the results of this study indicate that the inhibition of NF-κB and p38 MAPK signaling pathways by the PSEE suggests its potential as a therapeutic agent for mitigating adipose tissue infammation.In addition to the MAPK and NF-κB pathways, the AMP-activated protein kinase (AMPK) and mTOR signaling pathways are linked to the development and regulation of obesity, making them potential targets for therapeutic intervention [14,32].Te efects of PSEE on the activation of the AMPK and mTOR signaling pathways should be evaluated for further study to understand the underlying mechanisms involved.
Lipolysis in adipocytes constitutes a pivotal metabolic pathway responsible for breaking down triglycerides into fatty acids and glycerol, thus generating energy.Furthermore, these lipolytic processes contribute to the reduction of fat content within adipose tissue [33,34].ACC, aP2, FaSN, LPL, HSL, and ATGL are the genes that experience upregulation in mature adipocytes, playing signifcant roles in the synthesis of triglycerides [35].In this study, it was observed that PSEE led to an increase in the expression of ACC, HSL, and FaSN genes, while concurrently resulting in a decrease in the expression of aP2, ATGL, and LPL genes in the infammation-co-cultured adipocytes and macrophages.Advances in Pharmacological and Pharmaceutical Sciences

Conclusion
Tis study demonstrated that the ethanolic extract of passion fruit seeds efectively reduces ROS and NO levels by inhibiting iNOS protein expression.Additionally, it exerts anti-infammatory efects in LPS-stimulated adipocytes by suppressing the activation of p38 and NF-κB signaling pathways, resulting in reduced production of proinfammatory cytokines including TNF-α, IL-1β, MMP-2, IL-6, and MCP-1, as well as decreased adipokine levels.Moreover, the ethanolic extract of passion fruit seeds increased the expression of lipogenic gene, including ACC, HSL, and FaSN.However, aP2, ATGL, and LPL were dramatically downregulated (Figure 6).Tese fndings contribute to the understanding of the potential therapeutic applications of passion fruit seed extract in mitigating adipose tissue infammation and adipogenesis-related disorders.However, further research is necessary to elucidate additional specifc mechanisms including AMPK and mTOR signaling pathways.In vivo studies are also required to confrm the efects of passion fruit seed extract against infammation in an obese animal model.

Figure 1 :
Figure 1: Inhibition of reactive oxygen species (ROS) and nitric oxide (NO) production by PSEE in lipopolysaccharide (LPS)-induced infammation in cocultured adipocytes and macrophages.Te 3T3-L1 preadipocytes were induced to undergo diferentiation into mature adipocytes and subsequently treated with PSEE (10, 50, and 100 μg/mL) for a duration of 12 days.Following the 12-day diferentiation phase, coculture was established with RAW264.7 macrophages and mature 3T3-L1 adipocytes.Infammation was induced by the addition of 1 μg/mL of LPS for a 24 h period.(a) Intracellular ROS production.(b) NO level.(c) Inducible nitric oxide synthase (iNOS) protein expression.(d) Cytotoxicity.All values are presented as the mean ± SEM from four independent experiments (n � 4).Statistical signifcance was determined using LSD post-hoc analysis, with signifcance accepted when p < 0.05 (# compared to the control group and * compared to the LPS group).

Figure 2 :
Figure 2: Inhibition of TNF-α and IL-1β secretion and gene expression by PSEE in lipopolysaccharide (LPS)-induced infammation in cocultured adipocytes and macrophages.Te 3T3-L1 preadipocytes were induced to diferentiate into mature adipocytes and treated with PSEE (10, 50, and 100 μg/mL) for a duration of 12 days.Following the 12-day diferentiation phase, coculture was established with RAW264.7 macrophages and mature 3T3-L1 adipocytes.Infammation was induced by the addition of 1 μg/mL of LPS for a 24 h period.(a) Secretion of TNF-α.(b) Secretion of IL-1β.(c) Gene expression of TNF-α.(d) Gene expression of IL-1β.All values are expressed as the mean ± SEM from four independent experiments (n � 4).* Signifcantly diferent from the LPS group; #signifcantly diferent from the control group.Statistical signifcance was measured using the LSD post-hoc test (p < 0.05).

Figure 5 :
Figure 5: Suppression of NF-κB and p38MAPK signaling pathways by PSEE in LPS-induced infammation in cocultured adipocytes and macrophages.(a) Western blot analysis of NF-κB, ERK1/2, and p38MAPK protein bands in response to PSEE treatment.(b) Relative expression of p-p38/p38 protein.(c) Relative expression of pERK/ERK protein.(d) Relative expression of NF-κB protein.All values are expressed as the mean ± SEM from four independent experiments (n � 4).Statistical signifcance was determined using LSD post-hoc analysis, with signifcance accepted when p < 0.05 (# compared to the control group and * compared to the LPS group).

Figure 6 :
Figure 6: Schematic summary of the inhibitory activity of PSEE in cocultured adipocytes and macrophages.