Introduction

High fructose and saturated fatty acid-diet reported that increase level of plasma triglyceride and cholesterol in the rats.

High fructose and saturated fatty acid-diet have been reported to increase level of plasma triglyceride and cholesterol in the rats.

High-fructose and/or high-fat diet decrease in TG clearance so induce hypertriglyceridemia.¹ In the circulation, the triglycerides carried in chylomicrons are lipolyzed by lipoprotein lipase in the capillary bed of tissues using fatty acids for energy (i.e.skeletal muscle and heart) or storage (i.e.adipose tissue). The fatty acids storage will cause adipose tissue expansion and an increase in fatty acid release into the circulation,² and activate inflammatory pathways.^3,4

Free fatty acids could act as signal molecule activating nuclear factor kappa B (NFκB), an important transcription factor in the inflammatory response.^5,4 In this condition, pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and plasminogen activator inhibitor-1 (PAI-1) will increase, whereas the anti-inflammatory cytokines such as IL-10 will decrease.^6,7 Reduction of IL-10 expression will increase the severity of inflammation due to the function of this cytokine to inhibit the pro-inflammatory effect.^8,9

Interleukin-10 is a pleiotropic cytokine produced and secreted mainly by macrophages (M2).^10,11 This cytokine has immuno suppressive roles which inhibits the expression of inflammatory genes.¹² The mechanism of action for IL-10 is by inhibiting the activation of NFκB through the kB-inhibitor (IkB) which induces translocation p50 thereby inhibiting the translocation of the heterodimer p65/p50.¹³ Enhancement of IL-10 expression in an inflammatory state will reduce pro-inflammatory cytokines and reduce the negative effects of inflammation.¹¹

Some studies reported that short chain fatty acid (SCFA) can regulate the production of inflammatory mediators through macrophages.^14,15 The SCFA, mainly acetate, propionate, and butyrate, could be produced by colonic bacteria as a result of dietary fiber fermentation. Dietary fiber is an undigested carbohydrate that are resistant to gastrointestinal enzymes but could be fermented by colonic bacteria.¹⁶ Propionate acid plays an important role in inhibiting TNF-α and IL-6 secretion and increasing pro-inflammatory cytokines secretion (IL-4 and IL-10).¹⁷ Danuyanti et al.,¹⁸ reported that high fiber diet decrease TNF-α and IL-6 levels that may be related with suppression of toll-like receptor 4 (TLR4) and NFκB gene expression in hypertriglyceridemia rats. In this study we evaluate the benefits of diet with high fiber gene expression and levels of IL-10 cytokine in white adipose tissue in rats with high fat and fructose diet.

Material and Methods

This study was done after approvement from Ethical Committee of Integrated Research and Testing Laboratory, University of Gadjah Mada (Approval Number: 00065/04/LPPT//2017). Twenty-five (25) male Wistar rats, aged 8 weeks, body weight 180-200 g were divided into 5 groups: 1) normal control rats (N); 2) hyperlipidemia control rats (HL); 3) hyperlipidemia rats with fiber 1.04 g/rat/day (HL1); 4) hyperlipidemia rats with fiber 2.07 g/rat/day (HL2), and 5) hyperlipidemia rats with fiber 3.11 g/rat/day (HL3). The condition of hyperlipidemia was carried out using a diet high in fat and fructose for 7 weeks, and they were considered hyperlypidemia, if their plasma triglyceride levels were > 70.79 mg / dL.¹⁹ The blood samples used to analysis triglyceride levels were collected from the medial canthus sinus orbitalis from fasting rats during 10 hours. To get the plasma sample, the blood enters into EDTA tube, and then centrifuge 3000 rpm for 15 minutes. Diet high in fat and fructose were made by replacing fructose into sucrose, and trans-fat into corn starch in the same weight.^20,21

The rats were individually in cages and maintained under standard conditions (12:12-h light/dark cycle and 22-25°C room temperatures). Acclimation of the rats was done for 7 days using AIN-93M formulation with modification (L-cystine was substituted by DL-methionine and choline bitartrate by choline chloride) and water ad libitum.²² The normal diet composition consists of 61.94% corn starch, 14% casein, 10% sucrose, 4% corn oil, 5% cellulose, 3.5% mineral mixture, mixture of vitamin 1%, DL-methionine 0.3%, choline chloride 0.25%, and tetrabutilhydroquinone 0.008%. While the treatment diet for T1, T2, and T3 refers to a normal diet with substitution of corn starch using sweet potatoes and pumpkin with a total fiber of 6.88 g, 13.77 g and 20.65 g, respectively per 100 g of diet. The diet fiber content was examined by the Center for Food and Nutrition Studies, University of Gadjah Mada.

Before and after intervention, blood samples were collected from the medial canthus sinus orbitalis for serum IL-10 analysis using ELISA method (FineTest, Wuhan, China) and used as manufacturer’s protocol.²³ IL-10 were calculated based on a standard curve and the ELISA assay was performed duplicately. Range detection 31.25-2000pg/mL, the inter-assay: CV<10%, and the intra-assay: CV<8%. White the adipose tissue samples were collected from the retroperitoneal area for IL-10 gene expression analysis using quantitative PCR (qPCR).

The white adipose tissue samples were collected from the retroperitoneal area for IL-10 gene expression analysis by qPCR. Total RNA was extracted using TRIzol reagent (Invitrogen, USA), according to manufacturer’s protocol. Reverse transcription of 1 µg RNA was done based on Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The qPCR assay used SsoFast EvaGreen Supermix (Bio-rad, United Kingdom) with total reaction for qPCR was 10 µL . The conditions of qPCR were 95^oC for 10 minutes for early denaturation, followed by 40 cycles of 95^oC for 15 sec, 57^oC for 1 minute and 72^oC for 45 sec.

The conditions of qRT-PCR were 95^oC for 10 minutes for early denaturation, followed by 40 cycles of 95^oC for 15 sec, 57^oC for 1 minute and 72^oC for 45 sec. The results were normalized to β-actin housekeeping gene, and relative gene quantification was performed by using the 2⁻  method.²⁴ The specific primer sequences for IL-10 (purchased from Integrated DNA Technologies, Inc, Singapore) were Forward 5’-TTCCCTGGGAGAGAAGCTGA-3’, and Reverse  5’-ATGGCCTTGTAGACACCTTTGT-3’,²⁵ while it for β-actin gene were Forward 5’-ACGGTCAGGTCATCACTATCG-3`, and Reverse 5’-GGCATAGAGGTCTTTACGGATG-3’.²⁶

Statistical Analysis

All data were presented as the mean ± standard deviation (SD). Paired t-tests were used to evaluate the levels of serum IL-10 before and after intervention of high fiber diet. One way ANOVA was used to analyze the differences in IL-10 serum levels and the expression of IL-10 genes between the groups. Tukey’s honest significant difference (HSD) was post hoc tests. Differences were considered statistically significant at p<0.05.

Tukey’s honest significant difference (HSD) was post hoc tests. Differences were considered statistically significant at p<0.05.

Results

The levels of serum IL-10 before and after intervention of high fiber diet to hyperlipidemic rats are shown in Table 1. Before intervention, the levels of IL-10 in the N group were higher than the HL or HL with high-fiber diet groups. It showed that high trans-fat and fructose diet decrease IL-10 levels, which may induce inflammation. However, after administration of the high-fiber diet, the serum levels of IL-10 in hyperlipidemic rats increased significantly (p<0.05). In the present study, there was a decreased serum IL-10 levels in normal control.

Table 1. Mean of IL-10 level before and after high fiber diet.

Table 1: Analysis of serum IL-10 s using ELISA method before and after high fiber diets.

Note: Data is displayed as mean±standard deviation (SD); * p<0.05 is categorized as significant value; ^# p<0.05 is categorized as significant value; There were no significant differences in pre-test between groups p>0.05. Superscript ^a,b, and ^c indicate p<0.05 vs hyperlipidemia group according to One Way ANOVA test followed by Tukey HSD. P in row indicate the differences of serum IL-10 before and after high fiber diet in the same group. P in last row indicates the differences of plasma/serum IL-10 between group. ^{b, c} stated p<0.05 vs Hyperlipidemia group.

This study showed that the diet with 20.65% fiber could significantly increase IL-10 gene expression in HL3 (p<0.05). Although the diet containing 13.77% fiber can also increase IL-10 gene expression in HL2, it was not statically significant. It showed that high fiber diet could improve decrease inflammation through increase the gene expression and levels of IL-10. (Figure 1).

Figure 1: Expression of IL-10 on white adipose tissues of rats after administration of high fiber diet comparing those received normal diet.  Click here to View figure

Discussion

In this study, the rats consumption of a high-fat and fructose diet for 7 weeks, in the pre-test, had lower serum IL-10 levels compared to a normal diet (Table 1). This showed that the diets high in fat and fructose could induce a decrease in IL-10 anti-inflammatory cytokines. High fat diet is reported to induce systemic chronic low-grade inflammation.²⁷

High fat diet increases free fatty acid levels that may directly act on intestinal cells, and lead to elevated production of pro-inflammatory²⁷ or decrease anti-inflammatory cytokines. High plasma FFAs could upregulate the expression of TLRs in circulating macrophages, enabling macrophages to be activated (M1 phenotype).²⁷ M1 macrophages secrete TNF-α, IL-1 and MCP-1 to recruit monocytes to adipose tissue and increase the ratio of M1: M2 macrophages that cause a decrease in IL-10 cytokines. The decrease in M2 macrophages can reduce IL-10 levels because M2 macrophages in adipose tissue are important in secreting anti-inflammatory cytokines, especially IL-10, and are involved in maintaining homeostasis in white adipose tissue.²⁸ Therefore, high fat diet causes an increase in proinflammatory cytokines and a decrease in IL-10 levels (Kondo et al., 2018).^{29, 25, 26}

On the other hand, FFA molecules can act as signal transducer molecules that can bind to TLR 2 and TLR 4. Activation of TLR2 and TLR4 will trigger activation of NFkB which causes the release of proinflammatory cytokines and decreased IL-10 levels.^7,6 According to Zhu et al.,³⁰ FFA can increase regulation of TLR2 and TLR 4 gene expression when triglycerides (TG), total cholesterol (TC) or both increased.

In this study, both IL-10 gene expression and IL-10 levels in hyperlipidemia rats group were higher than those in normal control rats group. It may be an effect of adipose tissue compensatory. According to Juge-Aubry et al.,³¹ the adipose tissues always produces anti-inflammatory factors to limit pro-inflammatory effects. IL-10 is one anti-inflammatory factor that will be enhanced to limit TNF-α response during compensation. During acute inflammation, release of pro-inflammatory molecules such as IL-1 and TNF-α is followed release of local anti-inflammatory mediators such as IL-10 as act offset.³² Stoecklin et al.,³³ stated that increased expression of IL-10 mRNA was thought to be due to 3-5 h after an increase in TNF-α mRNA. The TNF-α may activate IL-10 gene expression in monocytes.

Administration high fiber diet for 6 weeks was able to increase significantly IL-10 levels in hyperlipidemia rats (HLT1, HLT2, HLT3), p<0.05. Fiber could not be digested by the small intestine enzymes but it would be fermented by colonic bacteria to produce short-chain fatty acids (SCFAs), especially butyrate, propionate and acetate. Butyric and propionate acids were reported to have anti-inflammatory activity.^34,35 Sasaki et al.,³⁶ reported that adequate amounts of fiber would increase the fermentation yield of SCFA, for instance, butyric acid. The study by Nastasi et al.,³⁷ showed that butyrate could suppress TNF-α, IL-6 via monocytes and macrophages and may increase the release of IL-10 anti-inflammatory cytokines.

In hyperlipidemia rats, the increase of IL-10  relate to elevate IL-10 gene expression. SCFAs have a possibility to modulate the expression of the IL-10 gene at the mRNA transcription stage. This mechanism accordances with research by Astakhova et al.,³⁸ found that SCFA could limit the activity of HDAC in the nucleus. The inhibition of HDAC activity will give access for transcription factors to the promoter and will provoke gene expression. Besides the effect of high fiber diet intervention, increased IL-10 serum levels in the intervention groups may also be supported by compensatory mechanisms after induction of high trans-fat and fructose. However, the increased IL-10 serum levels in the intervention groups between 2x until 3x compared to the hyperlipidemia rat group without intervention. Therefore, it showed that high-fiber diet could increase IL-10 serum levels.

This study suggested that a high-fiber diet may act as an anti-inflammatory agent, by increasing level and IL-10 gene expression in adipose tissue triggered by high-fat and fructose diets. Based on this study, high-fiber diet could be used to alternative therapeutic strategies in cases of hyperlipidemia and other cases involving inflammatory mechanisms such as obesity, diabetes mellitus and lipid-related cardiovascular disease. In this study, we did not perform the type of dietary fiber. The limitation of this study as we did not perform the analysis of dietary fiber type.

Conclusion

High fiber diet could improve inflammation through increase the gene expression and levels of IL-10 in white adipose tissue of hyperlipidemia rats model.

Acknowledgement

The authors give thanks to Dianandha Septiana Rubi, Budi Lestari and Sukamto for assistance with ELISA, qPCR, and statistical analyses. We are thankful to Klinik Bahasa staff for grammar assistance in writing the manuscript.

Funding

The research was funded by the Directorate General of Higher Education (DIKTI) and Baiturrahmah University Islamic Foundation.

Conflict of Interest

The authors declare there is no conflict of interest to disclose.

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