Ligng Yng, Cho Yng, Zhixiu Song, Min Wn, Hui Xi, Xin Yng,Dengfeng Xu, D Pn, Hechun Liu, Shokng Wng, Guiju Sun,*

a Key Laboratory of Environmental Medicine and Engineering of Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, China b Second Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210023, China

Keywords:Polyunsaturated fatty acid n-6 Fatty acids n-3 Fatty acids Postprandial metabolism Hypertriglyceridemia

A B S T R A C T Postprandial metabolism plays major roles in many pathological conditions. The n-6/n-3 polyunsaturated fatty acid (PUFA) ratio is closely related to various physiological disorders. This study aimed to investigate the effects of high fat meals with different n-6/n-3 PUFA ratios on postprandial metabolism in normal control(NC) and hypertriglyceridemia (HTG) rats. The postprandial response of triglyceride (TG) in HTG groups was higher than that in NC groups after different n-6/n-3 PUFA ratio meals. The HTG groups showed higher postprandial total cholesterol (TC) responses than NC groups after 1:1 and 20:1 ratio meals. The 5:1 n-6/n-3 PUFA ratio elicited lower postprandial responses of tumor necrosis factor α (TNF-α) than 1:1 and 10:1 ratios in HTG groups. The postprandial malondialdehyde (MDA) response was lower after a 5:1 n-6/n-3 PUFA ratio meal than 1:1 and 20:1 ratio meals in HTG groups. The 1:1 ratio resulted in a lower postprandial reactive oxygen species (ROS) level than 5:1 and 10:1 n-6/n-3 PUFA ratios in NC groups. The results showed that a low n-6/n-3 PUFA ratio improved postprandial dysmetabolism induced by a high fat meal in NC and HTG rats. A high n-6/n-3 PUFA ratio increased the difference in postprandial metabolism between NC and HTG rats.

1. Introduction

Postprandial metabolism plays an important role in the development of many pathological conditions [1]. The vascular system is often exposed to long-term postprandial hyperlipidemia and multiple atherogenic products during postprandial states [2]. Postprandial responses are affected by the quantity and composition of experimental meals, background diets, the characteristics of subjects, postprandial time and suitable markers [3,4]. Hypertriglyceridemia (HTG) is closely associated with postprandial hyperlipidemia and increased oxidative stress, inf lammation, and endothelial dysfunction [5,6].

n-6 andn-3 polyunsaturated fatty acids (PUFAs) have different physiological and metabolic characteristics and they interact with each other to regulate many important biological processes [7,8]. Moderate intake ofn-6 andn-3 PUFAs is beneficial to regulate various physiological responses such as lipid metabolism, inflammation,oxidative stress, platelet aggregation, and hemagglutination [9,10].Linoleic acid (18:2n-6; LA) andα-linolenic acid (18:3n-3; ALA)are the most commonn-6 andn-3 PUFAs. Compared with marinen-3 PUFAs, which include eicosapentaenoic (20:5n-3; EPA) and docosahexaenoic (22:5n-3; DHA), plantn-3 PUFA (ALA) is more abundant in many food sources [11,12]. Plant ALA and marine EPA and DHA have different physiological effects on postprandial lipid metabolism and oxidative stress in hyperlipidemic patients [13].Both plant and marinen-3 PUFAs attenuate inf lammation and induce characteristic pro-resolving lipid mediator metabolomes [14,15]. Our previous study showed that a highn-6/n-3 PUFA ratio with plantderivedn-3 fatty acids increases the difference in postprandial lipid responses between HTG and healthy subjects [16]. Another study showed that high fat diets with a lown-6/n-3 PUFA ratio improve lipid metabolism, inflammation, oxidative stress, and endothelial functions in rats using plant oils as then-3 fatty acid source, whereas a highn-6/n-3 PUFA ratio has adverse effects [10]. Furthermore, acute marinen-3 PUFA dietary supplementation improves postprandial lipid metabolism and inflammatory responses in rats with metabolic syndrome [17].

Despite these findings, few studies have reported the effect of high fat meals with differentn-6/n-3 PUFA ratios on postprandial lipid metabolism, inflammation, oxidative stress, and endothelial functions in HTG rats. Hence, the aim of the present study was to examine the effects of differentn-6/n-3 PUFA ratios on postprandial metabolism in normal control (NC) and HTG rats using plant sources ofn-3 fatty acids.

2. Materials and methods

2.1 Animals

Male Sprague-Dawley rats, which weighed 140-160 g, were purchased from Zhejiang Medical Experimental Animal Center(Hangzhou, China). The rats were fed in a single cage. The temperature was controlled at (22 ± 2) °C and the humidity was controlled at 50%-70% with 12 h light/12 h dark cycle in the feeding room. The rats were acclimatized for 1 week before experiments.

The rats were randomly divided into NC and HTG groups. The rats were fed a complete formula diet. The NC diet was prepared by referring to the AIN-76A experimental animal synthetic diet recommended by the American Society of Nutrition, which contained 230 g casein, 295 g corn starch, 310 g sucrose, 50 g cellulose, 70 g soybean oil, 30 g mixed mineral salts, 10 g mixed vitamins, 3 gDL-methionine, and 2 g choline chloride. HTG rats were fed high fat model feed. The high fat model diet consisted of 33.5% fat, 19.4%protein and 47.1% carbohydrate (% of energy), and the diet contained 215 g casein, 258 g corn starch, 265 g sucrose, 50 g cellulose, 117.5 g lard, 12.5 g cholesterol, 2 g bile salts, 30 g mixed mineral salts, 10 g mixed vitamins, 3 gDL-methionine, and 2 g choline chloride.

Before an experiment, no significant difference in body weight or blood lipids was found among the groups. Rats in each group were provided with the corresponding diet and had free access to water.The rats were weighed weekly and food intake was recorded every other day. When the HTG group had significantly higher triglycerides(TGs) than the NC group after 6 weeks, HTG rats were established.

NC and HTG rats were randomly divided into 4 subgroups according ton-6/n-3 PUFA ratios of 1:1, 5:1, 10:1 and 20:1,respectively, in liquid high fat meals, with 10 rats in each group.

2.2 Test meals

To minimize the effect on postprandial metabolism, postprandial metabolic meals were prepared as liquid meals. The liquid high fat meals consisted of water, blended oil, sucrose, casein, lactose,maltodextrin, and monoglycerides. The blended oil was butter, corn oil, linseed oil and olive oil. The liquid meals consisted of 50% fat,15% protein, and 35% carbohydrate (% of energy). The test meals contained 60 g blended oils, 40.5 g casein, 6.625 g lactose, 59.535 g sucrose, and 28.35 g maltodextrin per 500 mL. The formulas of the 4 test meals were similar except for differentn-6/n-3 PUFA ratios.The compositions of the test meals are shown in Table 1.

Table 1Compositions of the test meals.

2.3 Postprandial experimental procedure

The postprandial experiments were performed after establishment of the HTG rats. The rats were fasted overnight for 12 h with free access to water. Before the postprandial experiment, the liquid high fat meals were homogenized. After collecting a fasted blood sample, rats in each group orally received the homogeneous emulsion(2 g/100 g bw) in the fasted state. Subsequent blood samples were collected at 2, 4, and 6 h after meal consumption. Blood samples were collected sequentially from the tail vein using heparinized capillary tubes. The blood samples were centrifuged to separate serum for 10 min at 3 000 ×g, which was stored at -80 °C. Finally,all animals were anesthetized with sodium pentothal and sacrificed by decapitation.

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Southeast University (approval number: 20150516) on April 28, 2015. All animal care and experimental procedures complied with the principles of European Directive 2010/63/EU.

2.4 Serum biochemical analyses

The serum levels of TG, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), glucose (GLU), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA),reactive oxygen species (ROS) and von Willebrand factor (vWF)were measured. Enzymatic method was used to determine serum TG, TC and HDL-C levels. GLU was determined by oxidase endpoint method. Levels of IL-6 and TNF-α were measured using enzyme-linked immunosorbent assay (ELISA) kits purchased from Science Biotechnology (Yantai, China). Levels of MDA, ROS and vWF were measured using ELISA kits purchased from Jrdun BioTech(Shanghai, China). The determination method was carried out according to the instruction of these kits.

2.5 Statistical analysis

All data were processed by statistical software SPSS 17.0.Data were expressed as means ± standard deviations (SD). Oneway analysis of variance was used for significance test. Repeated measurement analysis of variance (ANOVA) was used to anaylze the time-varying repeatable measures.P＜ 0.05 indicated a statistically significant difference.

3. Results

3.1 Effects of different n-6/n-3 PUFA ratios on postprandial GLU and lipid responses in NC and HTG rats

3.1.1 Postprandial TG response

Except in the 1:1 (Fig. 1A) subgroup (P＞ 0.05), significant changes were observed in postprandial TG levels over time in 5:1(Fig. 1B), 10:1 (Fig. 1C), and 20:1 (Fig. 1D) subgroups (P＜ 0.01).As shown in Figs. 1A-D, the postprandial responses of TGs in HTG groups were higher than those in NC groups after the differentn-6/n-3 PUFA ratio meals (P＜ 0.01). There was a significant time effect on postprandial TG levels in both NC (Fig. 1E) and HTG (Fig. 1F)groups (bothP＜ 0.001). However, the postprandial responses of TGs were not significantly different among then-6/n-3 PUFA ratio treatments in NC (Fig. 1E) and HTG (Fig. 1F) groups (bothP＞ 0.05).

Fig. 1 The curve of TG concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. Effect of time, meal, meal × time:repeated measures ANOVA. Data presented as mean ± SD (n = 10). (A) 1:1 group, (B) 5:1 group, (C) 10:1 group, (D) 20:1 group, (E) NC group,(F) HTG group, the same below.

Fig. 2 The curve of TC concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. Effect of time, meal, meal × time:repeated measures ANOVA. Data presented as mean ± SD (n = 10).

3.1.2 Postprandial TC response

TC concentrations decreased within 2 h and then increased after differentn-6/n-3 PUFA ratio meals and there was a significant change in postprandial TC levels over time in all groups (P＜ 0.001)(Figs. 2A-D). The HTG groups showed higher postprandial TC responses than NC groups after 1:1 (Fig. 2A) and 20:1 (Fig. 2D)n-6/n-3 PUFA ratio meals (bothP＜ 0.05). Furthermore, the difference in postprandial responses of TC between NC and HTG groups tended to be broader after differentn-6/n-3 PUFA ratio meals along with the increase in postprandial time (Figs. 2A-D). However,the postprandial TC responses were not significantly different amongn-6/n-3 PUFA ratio subgroups in NC (Fig. 2E) and HTG (Fig. 2F)groups (bothP＞ 0.05).

3.1.3 Postprandial HDL-C response

For postprandial HDL-C responses, there was no meal effect(P＞ 0.05) or time × meal interaction (P＞ 0.05), but there was a significant time effect (P＜ 0.001) after differentn-6/n-3 PUFA ratio meals (Tables 2 and 3). As shown in Tables 2 and 3, postprandial HDL-C responses showed similar patterns over time with a decreasing trend from baseline.

Table 2Concentrations of HDL-C (mmol/L) at different time points before and after fat loading in NC rats with different n-6/n-3 PUFA ratios (mean ± SD, n = 10).

Table 3Concentrations of HDL-C (mmol/L) at different time points before and after fat loading in HTG rats with different n-6/n-3 PUFA ratios (mean ± SD, n = 10).

3.1.4 Postprandial GLU response

For postprandial GLU responses, there was no meal effect(P＞ 0.05) or time × meal interaction (P＞ 0.05) after differentn-6/n-3 PUFA ratio meals (Tables 4 and 5). As shown in Tables 4 and 5,a significant time effect was observed in postprandial GLU responses after differentn-6/n-3 PUFA ratio meals (P ＜0.001).Postprandial GLU levels peaked at 2 h, and then declined gradually (Tables 4 and 5).

Table 4Concentrations of GLU (mmol/L) at different time points before and after fat loading in NC rats with different n-6/n-3 PUFA ratios (mean ± SD, n = 10).

Table 5Concentrations of GLU (mmol/L) at different time points before and after fat loading in HTG rats with different n-6/n-3 PUFA ratios (mean ± SD, n = 10).

3.2 Effects of different n-6/n-3 PUFA ratios on postprandial inflammatory responses in NC and HTG rats

3.2.1 Postprandial IL-6 response

As shown in Tables 6 and 7, there was a significant time effect(P＜ 0.001) on postprandial IL-6 responses and postprandial IL-6 levels showed an increasing trend after differentn-6/n-3 PUFA ratio meals (Tables 6 and 7). The postprandial responses of IL-6 exhibited no meal effects or time × meals interaction (P＞ 0.05) after differentn-6/n-3 PUFA ratio meals (Tables 6 and 7). However, the postprandial IL-6 concentrations at 6 h in HTG groups were significantly lower than those in NC groups after 5:1 and 10:1n-6/n-3 PUFA ratio meals(P＜ 0.05) (Tables 6 and 7).

Table 6Concentrations of IL-6 (ng/L) at different time points before and after fat loading in NC rats with different n-6/n-3 PUFA ratios (mean ± SD).

Table 7Concentrations of IL-6 (ng/L) at different time points before and after fat loading in HTG rats with different n-6/n-3 PUFA ratios (mean ± SD).

3.2.2 Postprandial TNF-α response

The postprandial responses of TNF-α in HTG groups were higher than those in NC groups after the 1:1 (Fig. 3A) and 10:1 (Fig. 3C)n-6/n-3 PUFA ratio meals (bothP＜ 0.01). The postprandial TNF-α levels showed a decreasing trend over time after the 5:1n-6/n-3 PUFA ratio meal in both NC and HTG groups (Fig. 3B), but exhibited an increasing trend after the 20:1n-6/n-3 PUFA ratio meal (Fig. 3D)in both NC and HTG groups. The postprandial responses of TNF-α

were not significantly different amongn-6/n-3 PUFA ratio treatments in NC groups (P＞ 0.05) (Fig. 3E), but the 5:1n-6/n-3 PUFA ratio meal caused lower postprandial TNF-α responses than 1:1 and 10:1n-6/n-3 PUFA ratio meals in HTG groups (P＜ 0.05) (Fig. 3F).

3.3 Effects of different n-6/n-3 PUFA ratios on postprandial oxidative stress responses in NC and HTG rats

3.3.1 Postprandial MDA response

Althouth the postprandial responses of MDA were not significantly different between NC and HTG groups after differentn-6/n-3 PUFA ratio meals (Figs. 4A-D) (P＞ 0.05), the 5:1 (Fig. 4B)n-6/n-3 PUFA ratio meal tended to reduce the postprandial MDA response in both NC and HTG groups. Conversely, 1:1 (Fig. 4A) and 20:1 (Fig. 4D)n-6/n-3 PUFA ratio meals caused an increasing trend of postprandial MDA responses in both NC and HTG groups. Furthermore, the postprandial MDA levels at 4 h in the HTG group were higher than those in the NC group after the 10:1n-6/n-3 PUFA ratio meal(P＜ 0.05) (Fig. 4C). The 5:1 and 10:1n-6/n-3 PUFA ratio meals caused lower postprandial responses of MDA than the 1:1n-6/n-3 PUFA ratio meal in NC groups (P＜ 0.05) (Fig. 4E). Additionally, the 1:1 and 20:1n-6/n-3 PUFA ratio meals caused higher postprandial responses of MDA than the 5:1 and 10:1n-6/n-3 PUFA ratio meals in HTG groups (P＜ 0.05) (Fig. 4F).

Fig. 3 The curve of TNF-α concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. a-c Mean values with differentletters were significantly different (P ＜ 0.05). Effect of time, meal, meal × time: repeated measures ANOVA. *Significantly different between groups at the same time point, P ＜ 0.05. Data presented as mean ± SD (n = 10).

Fig. 4 The curve of MDA concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. a, bMean values with differentletters were significantly different (P ＜ 0.05). Effect of time, meal, meal × time: repeated measures ANOVA. *Significantly different between groups at the same time point, P ＜ 0.05. Data presented as mean ± SD (n = 10).

3.3.2 Postprandial ROS response

As shown in Figs. 5A-D, postprandial ROS levels were increased significantly in both NC and HTG groups after differentn-6/n-3 PUFA ratio meals (P＜ 0.001). The postprandial responses of ROS were not significantly different between NC and HTG groups after differentn-6/n-3 PUFA ratio meals (P＞ 0.05)(Figs. 5A-D). However, the postprandial ROS concentration at 6 h in the HTG group was significantly higher than that in the NC group after the 1:1n-6/n-3 PUFA ratio meal (P＜ 0.05) (Fig. 5A). Additionally,the 20:1n-6/n-3 PUFA ratio meal caused higher postprandial ROS level at 4 h in the HTG group than that in the NC group (P＜ 0.05)(Fig. 5D). Conversely, the HTG group showed lower postprandial ROS levels at 6 h than the NC group after the 5:1 PUFA ratio meal(P＜ 0.05) (Fig. 5B).

The postprandial responses of ROS were significantly different amongn-6/n-3 PUFA ratio subgroups of the NC group (P＜ 0.01)(Fig. 5E), but not significantly different amongn-6/n-3 PUFA ratio meals in HTG groups (P＞ 0.05) (Fig. 5F). The postprandial response of ROS after the 1:1 PUFA ratio meal was lower than that after 5:1 and 10:1n-6/n-3 PUFA ratio meals in NC groups (P＜ 0.05)(Fig. 5E). Additionally, the difference in postprandial response of ROS amongn-6/n-3 PUFA ratio subgroups tended to widen in both NC (Fig. 5E)and HTG (Fig. 5F) groups along with the increase in postprandial time.

Fig. 5 The curve of ROS concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. a, bMean values with differentletters were significantly different (P ＜ 0.05). Effect of time, meal, meal × time: repeated measures ANOVA. *Significantly different between groups at the same time point, P ＜ 0.05. Data presented as mean ± SD (n = 10).

Fig. 6 The curve of vWF concentrations over 6 h after fat loading with different n-6/n-3 PUFA ratios in NC and HTG groups. Effect of time, meal, meal × time:repeated measures ANOVA. *Significantly different between groups at the same time point, P ＜ 0.05. Data presented as mean ± SD (n = 10).

3.4 Effects of different n-6/n-3 PUFA ratios on postprandial endothelial functions in NC and HTG rats

3.4.1 Postprandial vWF response

The postprandial responses of vWF were not significantly different between NC and HTG groups after 1:1 (Fig. 6A), 10:1(Fig. 6C) and 20:1 (Fig. 6D)n-6/n-3 PUFA ratio meals (allP＞ 0.05)except for the 5:1 (Fig. 6B) subgroup (P＜ 0.05). However, the postprandial concentrations changed significantly over time in both NC and HTG groups after 1:1 (Fig. 6A), 10:1 (Fig. 6C), and 20:1 (Fig. 6D)n-6/n-3 PUFA ratio meals (P＜ 0.05) except for the 5:1 (Fig. 6B)n-6/n-3 PUFA ratio (P＞ 0.05). The postprandial vWF concentrations at 6 h in HTG groups were significantly higher than in NC groups after the 1:1n-6/n-3 PUFA ratio meal (Fig. 6A) (P＜ 0.05).

The postprandial responses of vWF were not significantly different amongn-6/n-3 PUFA ratio subgroups in both NC (Fig. 6E)and HTG (Fig. 6F) groups (bothP＞ 0.05). A decreasing tendency was observed in postprandial vWF levels from baseline after the 1:1n-6/n-3 PUFA ratio meal (Fig. 6F), whereas the 10:1n-6/n-3 PUFA ratio meal caused a slight upward trend in postprandial vWF concentrations in HTG groups (Fig. 6F). Additionally, the postprandial vWF concentrations increased, peaked at 4 h, and then decreased below the baseline after the 20:1n-6/n-3 PUFA ratio meal in HTG groups (Fig. 6F).

4. Discussion

HTG is a common dyslipidemia. Postprandial lipemia is closely related to inflammation, oxidative stress, and endothelial dysfunction [18].Although some studies have been carried out [19,20], the definitions of HTG vary [21] and there is no widely agreed definition of postprandial lipemia [19,22]. However, some HTG animal models may provide additional insights into HTG metabolism [23-25].Reducing the ratio ofn-6 ton-3 fatty acids is preferable to reduce the risk of many chronic diseases [7]. Thus, we established an animal model of fasting and postprandial HTG in rats fed a high fat diet and to evaluate the effects of differentn-6/n-3 PUFA ratios on postprandial metabolism in HTG rats using plant sources ofn-3 fatty acids. Considering the different effects of saturated fatty acid (SFA),monounsaturated fatty acid (MUFA), and PUFA on postprandial metabolism [26], the SFA:MUFA:PUFA ratio in meals was fixed in this study.

Triglyceride-rich lipoprotein (TRL) increases after meals and a delay in its clearance leads to postprandial hyperlipidemia [27]. As expected, our study showed that postprandial TG levels increased in both HTG and NC groups. Furthermore, the HTG groups had higher postprandial TG responses than NC groups after differentn-6/n-3 PUFA ratio meals. However, our results indicated no difference in the postprandial TG response after differentn-6/n-3 PUFA ratio meals in both HTG and NC groups. A possible reason was that the high intake ofn-6 PUFA, which is similar to high intake ofn-3 PUFA,reduced TG in hyperlipidemia [28,29]. Althoughn-3 PUFA (ALA)has a stronger anti-hyperlipidemic effect thann-6 PUFA (LA) [30],the postprandial time was too short to distinguish the postprandial TG response after different fatty acid challenges [31]. Our results were consistent with the hypothesis that the fasting TG concentration is an important factor that affects the postprandial TG response [32].Postprandial TGs suggest impaired TG clearance more conveniently and effectively compared with fasting TG levels [33]. The increase of the postprandial TG concentration is a potential driving force for the decrease of HDL-C. HDL-C usually decreases after a high fat diet and is inversely correlated with TG [34]. As expected, our results showed that HDL-C decreased, reached the mimimum value at 2 h, and then slightly increased after high fat meals. Blood GLU in each group increased, reached a peak at 2 h, and then decreased after the meals. However, the postprandial HDL-C and GLU responses were unaffected by differentn-6/n-3 PUFA ratios and no significant difference in postprandial HDL-C and GLU responses was found between HTG and NC groups after differentn-6/n-3 PUFA ratio meals. In support of our results, a previous study has shown that postprandial GLU and TG concentrations did not differ between high fat meals rich in LAs (n-6 PUFA) and linolenic acids (n-3 PUFA) in male rats [35]. Our results also showed that the postprandial TC levels decreased and then slowly increased after the different meals. This may be due to the slow absorption and synthesis of cholesterol [36].Interestingly, our study indicated that the difference in postprandial TC concentrations between HTG and NC groups tended to widen as the postprandial time increased after differentn-6/n-3 PUFA ratio meals. Additionally, the HTG groups showed higher postprandial TC responses than NC groups after 1:1 and 20:1n-6/n-3 PUFA ratio meals. Thus, our study indicated that postprandial TC responses in HTG and NC rats were different from postprandial TG and HDL-C responses after differentn-6/n-3 PUFA ratio meals. This finding was consistent with other studies in which plant-derivedn-3 PUFA (ALA)had a significant effect on TC, and marinen-3 PUFAs (EPA and DHA) improved the TG and HDL-C status [37,38]. We speculated that HTG increased the postprandial TG response and a very high or lown-6/n-3 PUFA ratio increased the difference in the postprtandial TC response between HTG and NC rats. Alternatively, we aslo hypothesized that the beneficial effects of a lown-6/n-3 PUFA ratio on lipid levels may require a longer time to be better observed [37].

Nutrition-mediated postprandial inflammation is characterized by increased circulating IL-6 and TNF-α levels [39].n-6/n-3 PUFA ratios may be important determinants of the postprandial inflammation response [40].n-3 PUFAs have an anti-inflammatory effect andn-6 PUFAs have a pro-inflammatory effect. Thus, maintaining a propern-6/n-3 PUFA ratio is important to reduce inflammation [41]. Our study revealed a significant increase in postprandial IL-6 levels after the high fat meal in both HTG and NC groups, which was consistent with previous studies [42]. However, no difference in the postprandial IL-6 response was found between HTG and NC groups after differentn-6/n-3 PUFA ratio meals. Interestingly, our results showed that the 5:1 and 10:1n-6/n-3 PUFA ratios tended to decrease the postprandial IL-6 levels at 6 h in HTG groups compared with those in NC groups. Our study indicated that an appropriately lown-6/n-3 PUFA ratio may be partly beneficial to ameliorate HTGinduced elevation of postprandial IL-6. The increase in postprandial TNF-α levels may result from a higher percentage of TNF-α-producing monocytes in circulation after a fatty meal [43]. Our results showed that postprandial TNF-α concentrations tended to decrease after 5:1n-6/n-3 PUFA ratio meals in both NC and HTG groups. Furthermore, the 5:1 subgroup had a lower postprandial TNF-α response than the 1:1 and 10:1 subgroups of the HTG groups. Conversely, our results indicated that the postprandial TNF-α responses in HTG groups were higher than those in NC groups after 1:1 and 10:1n-6/n-3 PUFA ratio meals. The results also suggested that an excessively lown-6/n-3 ratio (1:1) was detrimental to postprandial inflammation. Wei et al. [44] reported that a lown-6/n-3 PUFA ratio decreased the concentration of serum TNF-α and IL-6. We speculate that an appropriately lown-6/n-3 PUFA ratio meal inhibits postprandial inflammation compared with very low or highn-6/n-3 PUFA ratios.

Oxidative stress induced by postprandial lipemia may adversely affect multiple physiological processes [18]. A diet rich inn-3 PUFA may reduce postprandial oxidative stress [45]. Supplementation ofn-3 PUFAs in a diet significantly enhances the antioxidant effect because of their strong free radical-scavenging ability [46,47].MDA is widely regarded as a biomarker of lipid peroxidation [48].Our resutls showed that the 5:1 and 10:1n-6/n-3 PUFA ratio meals caused lower postprandial MDA responses than 1:1 and 20:1n-6/n-3 PUFA ratio meals in HTG groups. Furthermore, the 5:1 and 10:1n-6/n-3 PUFA ratios caused a lower postprandial MDA response than the 1:1n-6/n-3 PUFA ratio in NC groups. Moreover, 1:1, 10:1 and 20:1n-6/n-3 PUFA ratio meals tended to elevate postprandial MDA responses, whereas the 5:1n-6/n-3 PUFA ratio meals induced a slight decline in the postprandial MDA level in HTG groups. Thus,our results indicated that the lown-6/n-3 PUFA ratio of 5:1 improved postprandial MDA response in HTG groups. There is an imbalance between the production and elimination of ROS by the antioxidant system in the postprandial state [49,50]. In the present study,postprandial ROS levels were increased after differentn-6/n-3 PUFA ratio meals in both HTG and NC groups. Furthermore, the difference in the postprandial ROS response amongn-6/n-3 PUFA ratio meals tended to increase along with the postprandial time in both NC and HTG groups. Our results showed that the 1:1n-6/n-3 PUFA ratio meal induced a lower postprandial ROS response than the 5:1 and 10:1n-6/n-3 PUFA ratio meals in NC groups. An interesting finding was that the HTG group showed a higher postprandial ROS level at 6 h than the NC group after the 1:1n-6/n-3 PUFA ratio meal, whereas the 5:1n-6/n-3 PUFA ratio induced a lower postprandial ROS level at 6 h in the HTG group than in the NC group. Our study indicated that 5:1 was the optimaln-6/n-3 PUFA ratio for the postprandial ROS response in the HTG group, whereas 1:1 was the optimaln-6/n-3 PUFA ratio in the NC group. Therefore, it appeared that lown-6/n-3 PUFA ratios were more effective in improving postpranpranal ROS. This observation was in agreement with the findings of Wei et al. [51] who showed that a highn-6/n-3 PUFA ratio reduced the antioxidant capacity. Conversely, high intake ofn-3 PUFAs increase the antioxidant capacity by reducing the level of arachidonic acid(AA) and its products [52], thereby decreasing the level of endothelial ROS [53] and scavenging superoxide [54].

Postprandial lipemia causes endothelial dysfunction via transient increases of oxidant stress, proinflammatory cytokines, and soluble adhesion molecules [55,56]. vWF is a marker of endothelial dysfunction, and elevated plasma vWF levels may be an acute phase reactant [57]. To date, few studies have examined the effects of differentn-6/n-3 PUFA ratios on postprandial vWF responses.Although our results showed that the postprandial responses of vWF were not significantly different amongn-6/n-3 PUFA ratio subgroups in both NC and HTG groups, the 1:1n-6/n-3 PUFA ratio tended to reduce the postprandial vWF levels, whereas the 10:1 and 20:1n-6/n-3 PUFA ratios caused an increasing trend of the postprandial vWF levels in both HTG and NC groups. However, the postprandial vWF response did not show a marked change over time after the 5:1n-6/n-3 PUFA ratio meal and the postprandial vWF response in the HTG group was higher than that in the NC group after 5:1n-6/n-3 PUFA ratio meals. Furthermore, the 20:1n-6/n-3 PUFA ratio meal caused a higher postprandial vWF concentration at 4 h in the HTG group than the NC groups. Our results suggested that then-6/n-3 PUFA ratio influenced transient changes in postprandial vWF levels.The transient postprandial endothelial dysfunction may be mediated by increased postprandial TG and inflammatory responses in HTG subjects [58]. The results were consistent with the hypothesis that a lown-6/n-3 PUFA ratio improves postprandial endothelial functions.Conversely, a highn-6/n-3 PUFA ratio causes postprandial endothelial dysfunction. This was consistent with our previous findings of reduced serum levels of vWF in rats fed high fat diets with a lown-6/n-3 PUFA ratio [10].

The present study demonstrated that high fat meals increased postprandial lipid metabolism, inflammation, and oxidative stress.A highn-6/n-3 PUFA ratio enhanced the difference in postprandial metabolism after high fat meals between NC and HTG rats. A propern-6/n-3 PUFA ratio improved postprandial metabolism in HTG groups. Furthermore, our results were in accordance with the hypothesis that excessive intake ofn-6 PUFAs orn-3 PUFAs is closely related to the pathogenesis of many chronic diseases [59].However, further study is necessary to determine the underlying metabolic pathways.

This study had some limitations. Most small animal models are resistant to the development of atherosclerosis unless provided with a high fat and cholesterol diet or genetic manipulation [60].Additionally, rats are naturally deficient in cholesteryl ester transfer protein (CETP) activity [61]. Compared with anti-atherosclerosis rats, there is evidence that people with atherosclerotic susceptibility have significantly higher mean CETP activity [62]. Humans were different from rodents in postprandial metabolism [63]. Thus, the results observed in rats might not be directly extrapolated to humans.It is important to point out that other oxidative markers provide additional information about postprandial oxidative stress caused by diets with differentn-6/n-3 PUFA ratios and some oxidative stress markers such as C-reactive protein (CRP) might require more time to change. Additionally, a relationship between dietaryn-6/n-3 PUFA ratios and the dose effect was not proven in this study. Despite the above limitations of this study, to our knowledge, this was the first study to investigate the effects of differentn-6/n-3 PUFA ratios on postprandial lipid metabolism, inflammation, oxidative stress, and endothelial functions in normal and hypertriglyceridemic rats using plant sources ofn-3 fatty acids.

5. Conclusion

This study demonstrated that high fat meals elevated postprandial lipid metabolism, inflammation, and oxidative stress, which were influenced by dietaryn-6/n-3 PUFA ratios in both NC and HTG rats. Our results indicated that a lown-6/n-3 PUFA ratio of 5:1 may provide beneficial effects on postprandial dysmetabolism induced by a high-fat diet. Conversely, a highn-6/n-3 PUFA ratio of 20:1 increased the difference in postprandial metabolism between NC and HTG rats. However, the adverse effects of an excessively lown-6/n-3 PUFA ratio of 1:1 on postprandial metabolism should be a concern.Further studies are required to elucidate the mechanisms related to the regulation of postprandial HTG metabolism by the dietaryn-6/n-3 PUFA ratio. Our study also suggested that the difference in postprandial metabolism between normal and HTG rats may increase over postprandial time. Further research can be conducted to study the effects of postprandial time after multiple meals on postprandial metabolism. Additionally, the results observed in rats might not be directly applicable to humans and whether such changes occur in humans remains to be determined.

Declaration of conflicting interest

The authors declare no conflict of interest. The authors alone are

responsible for the content and writing of this article.

Acknowledgements

This work was supported by National Key Research and Development Plan (2016YFD0400604) and National Natural Science Foundation of China (82073551).