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To high-fat feed, we added the same amounts of various fatty acids to the following groups: high fat base group (HFD group), 2% caprylic acid feed (C8:0 group), 2% palmitic acid feed (C16:0 group) and 2% eicosapentaenoic acid feed (EPA group). The proportion of energy from fat in the HFD group was 37.11%, whereas that in the other groups was 39.68%. The feed, as previously described[13], was provided by Beijing Huafukang Company (license No.: SCXK Beijing 2014-0008).
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The experimental animals included 56 male 6-week-old C57BL/6J mice, which were purchased from SPF (Beijing) biotechnology Co, Ltd. The mice were randomly divided into four groups according to body weight. High fat feed was provided for 1 week and then replaced by intervention feed. The experiment was completed at 12 weeks. During the experiment, fasting weight was measured once per month (drinking water was not limited during fasting the night before measurements). The mouse bedding and drinking water were replaced every 2–3 days, and the remaining amount and added amount of mouse feed were recorded. The daily average feed consumption of each mice was calculated. All experimental procedures were performed in strict accordance with the regulations of the Ethics Committee of the International Association for the Study of Pain (Zimmermann, 1983) and National Guidelines for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006). This research was approved by the Animal Care and Use Committee of the Chinese PLA General Hospital.
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The RAW246.7 cell line was obtained from the American Type Culture Collection, and the cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin (Invitrogen), 0.1 mg/mL streptomycin (HyClone) and 1 mm sodium pyruvate at 37 ℃ under humidified air and 5% CO2. The cell count was adjusted to 4 × 105 – 8 × 105/mL (measured with a hemocytometer: cell number/mL = 100 cells per 100 cells/400 × 10,000 dilution factor), were harvested for subsequent assays.
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Various fatty acids were diluted with 95% ethanol solution and serum-free medium (containing 20 mg/mL BSA); 200 mmol/L was added to culture wells, and 0.3 mmol/L with cAMP was added to culture wells.
Experimental groups and treatments: RAW 264.7 cells were seeded in 24-well plates at approximately 2 × 105 per well and randomly divided into five groups (n = 6): normal group, LPS group (final LPS concentration 50 ng/mL), LPS+C8:0 group (LPS final concentration 50 ng/mL and C8:0 final concentration 200 mmol/L), LPS+EPA group (LPS final concentration 50 ng/mL and EPA final concentration 200 mmol/L) and LPS+cAMP group (LPS final concentration 50 ng/mL and cAMP final concentration is 0.3 mmol/L). After 24 h, the cells were collected and isolated to extract RNA and proteins for analysis after being washed three times with refrigerated PBS.
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The levels of serum lipid were determined using commercial kits. TC and triglycerides (TG) (Wako, Osaka, Japan), and HDL-C and LDL-C (Abcam, Cambridge, UK) were measured with commercial kits, and the ratio of HDL-C to LDL-C was subsequently calculated.
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After the addition of fatty acids, the cells were continuously cultured for 24 h, and cell lysates were collected from each group. After 4 ℃, 3,000 r/min centrifugation for 10 min, the supernatant was collected. TNF-α, IL-1β, IL-6, interleukin-10 (IL-10) and MCP-1 were determined in cells and serum according to the manufacturer’s instructions for the ELISA kit (R&D Systems, Minneapolis, MN, USA).
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Total RNA from RAW cells and aorta samples (approximately 50 mg) was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed with a reverse transcription system kit (NEB, M-MLV kit). The cycling conditions were 25 ℃ for 5 min, 50 ℃ for 15 min, 85 ℃ for 5 min and 4 ℃ for 10 min for cDNA, then 50 ℃ for 2 min, 95 ℃ for 10 min, 95 ℃ for 30 s and 60 ℃ for 30 s. After 40 cycles, dissolution curves were analyzed, and only the sample with a single dissolution peak was collected. With normalization to the β-actin gene, the relative expression levels were calculated with the △Ct method, and the experiments were repeated in triplicate. The primers used in the current study are listed in Table 1.
Indicators Primer Sequence (5‘−3’) Size (bp) β-actin Forward GGCCGAGGAGCAAGAATGG 118 Reverse CATGCACTCTGCGATACGCT ACBA1 Forward GCTTGTTGGCCTCAGTTAAGG 135 Reverse GTAGCTCAGGCGTACAGAGAT JAK2 Forward AGTGGCGGCATGATTTTGTT 181 Reverse GCTCGAACGCACTTTGGTAA STAT3 Forward GTAGAGCCATACACCAAGCAGCAG 123 Reverse AATGTCGGGGTAGAGGTAGACAAGT p65 Forward CACCGGATTGAAGAGAAGCG 194 Reverse AAGTTGATGGTGCTGAGGGA Note. ABCA1, ATP-binding cassette transporter A1; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; p65, pNF-κBp65. Table 1. RT-PCR primer list
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The cells were extracted with RIPA buffer (CST). Immunoblotting for STAT3 (1:1,000), JAK2 (1:1,000), p-STAT3 (1:1,000), p-JAK2 (1:1,000), p65 (1:1,000), β-actin (1:1,000) and ABCA1 (1:200) was performed with established procedures. Bands were visualized with a chemiluminescence detection system (Vilber Lourmat, France) according to the manufacturer’s instructions.
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On the basis of a preliminary experiment, G*Power 3.1.9.2 software (Heinrich-Heine University, Germany) was used to calculate the minimum sample size required for the detection of a significant difference (P < 0.05). All data in this report are presented as the mean ± standard derivation, and significance was defined as P < 0.05 (two-tailed). A one-way analysis of variance was performed for data analysis. The Tukey-Kramer multiple comparison was used to determine the statistical significance of differences among various groups. SPSS 26.0 (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses.
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At 4, 8 and 12 weeks during the intervention, the fasting body weights in the C8:0 group and EPA group were found to be significantly lower than those in the HFD group and C16:0 group (P < 0.05) (Figure 1). During the study, no significant difference in average feed intake was observed (P > 0.05) (Figure 2).
Figure 1. Effects of different fatty acids (C8:0, C16:0 and EPA) on fasting body weight of C57BL/6J mice. C8:0, caprylic acid; C16:0, palmitic acid; EPA, eicosapentaenoic acid; HFD, high-fat diet. Data in the figure are expressed as the mean ± SD with 14 samples in each group (n = 14). aP < 0.05, versus high-fat group; bP < 0.05, versus C16:0 group; cP < 0.05, versus EPA group
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After 12 weeks of intervention, half the mice (n = 7) were randomly selected from each group and intraperitoneally injected with LPS. The expression levels of lipid metabolism indexes, inflammatory factors and mRNA of ABCA1 and JAK2/STAT3, pathway related factors in arteries were measured.
At the end of the intervention, compared with that in the HFD group, the serum TC in the C8:0 group was significantly lower (P < 0.05). The levels of serum TC and LDL-C were significantly lower in the EPA group than the HFD group, whereas the LDL-C levels were significantly higher, and the HDL-C/LDL-C ratio was clearly higher, than that in the C16:0 group (P < 0.05) (Table 2). Our data showed that C8:0 decreased the level of TC. EPA decreased the TC and LDL-C, and up-regulated the HDL-C/LDL-C ratio (P > 0.05).
Indicators −LPS +LPS C8:0 C16:0 EPA HFD C8:0 C16:0 EPA HFD TC (mmol/L) 3.27 ± 0.14a 3.49 ± 0.29 3.15 ± 0.38a 3.67 ± 0.16 3.47 ± 0.23a 3.95 ± 0.40 2.86 ± 0.48a,b 4.26 ± 0.80 TG (mmol/L) 0.67 ± 0.11 0.62 ± 0.21 0.71 ± 0.24 0.93 ± 0.35 0.71 ± 0.19 0.66 ± 0.11 0.63 ± 0.27 0.57 ± 0.09 HDL-C (mmol/L) 1.39 ± 0.13 1.19 ± 0.25 1.28 ± 0.16 1.40 ± 0.16 1.47 ± 0.13 1.44 ± 0.08 1.38 ± 0.18 1.29 ± 0.11 LDL-C (mmol/L) 0.56 ± 0.07c 0.55 ± 0.01c 0.45 ± 0.03a,b 0.55 ± 0.03c 0.44 ± 0.04a 0.52 ± 0.07 0.39 ± 0.07a,b 0.54 ± 0.08 HDL-C/LDL-C 2.52 ± 0.45 2.17 ± 0.46 2.82 ± 0.42b 2.55 ± 0.27 3.32 ± 0.25a 2.83 ± 0.44 3.69 ± 0.75a,b 2.45 ± 0.56 Note. C8:0, caprylic acid; C16:0, palmitic acid; EPA, eicosapentaenoic acid; HFD, high-fat diet; LPS, lipopolysaccharide; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; aP < 0.05, versus HFD group; bP < 0.05, versus C16:0 group; cP < 0.05, versus EPA group. Table 2. Effects of different fatty acids on blood lipids in C57BL/6j mice without or with LPS injection (
$\bar x$ ± s, n = 7)At the end of the intervention, after injection of LPS, the serum TC and LDL-C levels were significantly lower, and the HDL-C/LDL-C ratio was clearly higher, in the C8:0 group than the HFD group (P < 0.05). Moreover, the serum TC and LDL-C levels were significantly lower, and the HDL-C/LDL-C ratio was clearly higher, in the EPA group than the C16:0 and HFD groups (P < 0.05) (Table 2). Our study indicated that both C8:0 and EPA decreased the TC and LDL-C levels and increased the HDL-C/LDL-C ratio after LPS treatment.
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At the end of the intervention, the levels of IL-1β and TNF-α in the HFD group (+LPS) were significantly higher, and those of IL-10 were significantly lower, than those in the HFD group (-LPS) (P < 0.05). The levels of TNF-α and MCP-1 clearly increased in the C16:0 group (+LPS), thus indicating that LPS increased the serum inflammation in the mice (P < 0.05) (Table 3).
Indicators −LPS +LPS C8:0 C16:0 EPA HFD C8:0 C16:0 EPA HFD IL-1β 4.89 ± 0.53 4.81 ± 1.37 4.67 ± 0.82 4.65 ± 0.48* 4.81 ± 0.91 5.33 ± 1.08 5.10 ± 0.68 5.43 ± 0.46 IL-6 110.78 ± 17.19 117.18 ± 24.29 114.56 ± 19.85 113.53 ± 15.99 119.89 ± 13.51 127.49 ± 30.40 119.46 ± 16.80 119.42 ± 22.97 IL-10 3.29 ± 1.09 2.42 ± 1.09 2.62 ± 0.60 2.57 ± 0.54* 4.85 ± 1.06a,b,c 2.50 ± 1.00 2.66 ± 0.90 1.89 ± 0.28 MCP-1 44.75 ± 10.54 51.93 ± 6.02* 44.08 ± 9.04 52.54 ± 6.04 48.51 ± 10.38b 61.65 ± 3.60 51.70 ± 4.97b 58.86 ± 6.47 TNF-α 4.00 ± 0.69 4.73 ± 0.92* 3.87 ± 0.97 4.78 ± 0.80* 4.05 ± 1.09a,b 6.23 ± 1.01 4.26 ± 0.70a,b 6.91 ± 0.86 Note. C8:0, caprylic acid; C16:0, palmitic acid; EPA, eicosapentaenoic acid; HFD, high-fat diet; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; MCP-1, monocyte chemoattractant protein-1; *P < 0.05, versus LPS group; aP < 0.05, versus HFD group; bP < 0.05, versus C16:0 group; cP < 0.05, versus EPA group. Table 3. Effects of different fatty acids on serum inflammatory factors in C57BL/6j mice (
$\bar x$ ± s, n = 7)After intraperitoneal injection of LPS, the levels of MCP-1 in the C8:0 group were significantly lower than those in the C16:0 group, and the level of TNF-α was significantly lower than those in the HFD group and the C16:0 group (P < 0.05). Compared with those in the other three groups, the IL-10 levels in the C8:0 group were significantly higher, whereas the TNF-α levels in the EPA group were significantly lower than those in the HFD group and C16:0 group. In addition, the MCP-1 levels in the EPA group were significantly lower than those in the C16:0 group (P < 0.05). Our data suggested that C8:0 significantly inhibited LPS-stimulated serum inflammation in mice (Table 3).
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By the end of the intervention, the mRNA levels of JAK2/STAT3 significantly increased; those of NF-κB and TNF-α dramatically decreased in each group after LPS injection; and those of ABCA1 also significantly increased in the C8:0 and EPA groups (P < 0.05) (Table 4). Compared with those in the other three groups, the expression levels of ABCA1 in the C8:0 group were significantly higher, whereas those of TNF-α and NF-κB in the C8:0 group and EPA group were significantly lower than those in the HFD group and C16:0 group. JAK2 in the C8:0 group was significantly higher than that in the C16:0 group, and STAT3 in the C8:0 group was significantly higher than that in the HFD group (P < 0.05).
Indicators −LPS +LPS C8:0 C16:0 EPA HFD C8:0 C16:0 EPA HFD ABCA1 0.17 ± 0.04a,b,c,* 0.09 ± 0.02 0.11 ± 0.01* 0.09 ± 0.02 0.22 ± 0.02a,b,c 0.07 ± 0.03 0.14 ± 0.01b 0.10 ± 0.05 JAK2 0.21 ± 0.03b,* 0.14 ± 0.04* 0.18 ± 0.05 0.16 ± 0.04* 0.57 ± 0.23a,b,c 0.29 ± 0.15 0.17 ± 0.02 0.26 ± 0.10 STAT3 0.23 ± 0.05a,* 0.17 ± 0.06* 0.19 ± 0.03* 0.15 ± 0.03* 0.93 ± 0.30a,b,c 0.26 ± 0.05 0.34 ± 0.15 0.38 ± 0.16 TNF-α 0.08 ± 0.01a,b,* 0.15 ± 0.04* 0.08 ± 0.02a,b,* 0.17 ± 0.06* 0.15 ± 0.06a,b 0.27 ± 0.08 0.14 ± 0.06a,b 0.26 ± 0.07 NF-κB 0.08 ± 0.03a,b,* 0.13 ± 0.03* 0.09 ± 0.02a,b 0.15 ± 0.03* 0.12 ± 0.02a 0.18 ± 0.04 0.10 ± 0.04a 0.24 ± 0.08 Note. C8:0, caprylic acid; C16:0, palmitic acid; EPA, eicosapentaenoic acid; HFD, high-fat diet; LPS, lipopolysaccharide; ABCA1, ATP-binding cassette transporter A1; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor α; NF-κB, nuclear factor kappa B; *P < 0.05, versus +LPS (comparison in the same group ±LPS); aP < 0.05, versus HFD group; bP < 0.05, versus C16:0 group;
cP < 0.05, versus EPA group.Table 4. Effects of different fatty acids on the mRNA expression of ABCA1/JAK2/STAT3 pathway-related molecules in the aorta in C57BL/6j mice (
$\bar x$ ± s, n = 7)After intraperitoneal injection with LPS, the expression levels of ABCA1 and JAK2/STAT3 were significantly higher in the C8:0 group than the HFD, C16:0 and EPA groups, the mRNA levels of NF-κB, TNF-α were lower than those in the HFD group (P < 0.05), and the mRNA levels of TNF-α were also lower than those in the C16:0 group (P < 0.05). Meanwhile, compared with that in the C16:0 group, the expression of ABCA1 mRNA in the EPA group was significantly higher, and the expression of TNF-α mRNA was inhibited. Moreover, compared with that in the HFD group, the expression of NF-κB and TNF-α mRNA in the EPA group was inhibited (P < 0.05) (Table 4). Our data revealed that C8:0 up-regulated ABCA1 and resulted in downstream inhibition of the mRNA levels of NF-κB and TNF-α.
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After LPS treatment, the levels of cytokines in RAW264.7 cells significantly increased, and consequently the levels of TNF-α, MCP-1, IL-6 and IL-1β were significantly higher than those in the normal group, whereas those of IL-10 were significantly lower (P < 0.05). The levels of TNF-α, MCP-1, IL-6 and IL-1β in the LPS+C8:0 group, LPS+EPA group and LPS+cAMP group were significantly lower than those in the LPS group, but the levels of IL-10 were higher (P < 0.05). The levels of TNF-α, IL-6 and MCP-1 were significantly lower in the LPS+C8:0 group and LPS+EPA group than the LPS+cAMP group (P < 0.05) (Figure 3). Compared with those in the LPS+EPA group, the levels of IL-1β were higher, and the levels of IL-6 were lower, in the LPS+C8:0 group. Our findings indicated that C8:0 inhibited the expression of inflammatory factors in RAW264.7 cells stimulated by LPS.
Figure 3. Effects of C8:0 and EPA on inflammatory factors of RAW264.7 cells stimulated by LPS. TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; MCP-1, monocyte chemoattractant protein-1; NC, normal contrast; LPS, lipopolysaccharide; C8:0; caprylic acid; EPA, eicosapentaenoic acid; cAMP, cyclic adenosine monophosphate. Data in the figure are expressed as the mean ± SD with 6 samples in each group (n = 6). *P < 0.05, versus NC group; aP < 0.05, versus LPS group; bP < 0.05, versus LPS+EPA group; cP < 0.05, versus LPS+cAMP group
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Compared with that in the normal group, the expression level of ABCA1 mRNA in RAW264.7 cells treated with LPS was clearly lower, and the expression of JAK2, STAT3 and p65 mRNA was higher (P < 0.05). Moreover, the expression of ABCA1 mRNA in the LPS+C8:0 group was clearly higher than that in the LPS and LPS+EPA group and the expression of JAK2 and p65 mRNA was significantly lower than that in the LPS group (P < 0.05). Furthermore, the mRNA expression level of STAT3 in the LPS+C8:0 group was significantly higher than that in the LPS+EPA group (P < 0.05). However, the mRNA expression of ABCA1, JAK2, STAT3 and p65 in the LPS+C8:0 group was no different from that in the LPS+cAMP group (P > 0.05). Compared with that in the LPS+cAMP group, the mRNA expression of ABCA1 and STAT3 was lower, and the expression of p65 was higher, in the LPS+EPA group (Figure 4). Thus, C8:0 up-regulated the ABCA1 and STAT3 pathway in RAW264.7 cells stimulated by LPS and decreased the mRNA expression of p65.
Figure 4. Effects of C8:0 and EPA on JAK2/STAT3 mRNA expression in RAW264.7 cells stimulated by LPS. NC, normal contrast; LPS, lipopolysaccharide; C8:0, caprylic acid; EPA, eicosapentaenoic acid; cAMP, cyclic adenosine monophosphate; ABCA1, ATP-binding cassette transporter A1; JAK2, Janus Kinase 2; STAT3, signal transducer and activator of transcription 3; p65, pNF-κBP65. Data in the figure are expressed as the mean ± SD with 6 samples in each group (n = 6). *P < 0.05, versus NC group; aP < 0.05, versus LPS group; bP < 0.05, versus LPS+EPA group; cP < 0.05, versus LPS+cAMP group
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Compared with that in the normal group, the expression of ABCA1, p-JAK2 and p-STAT3 protein was significantly lower after LPS treatment, whereas the expression of p65 protein was significantly higher (P < 0.05). Meanwhile, in the LPS+C8:0 group and LPS+EPA group, compared with the LPS group and LPS+cAMP group, the protein expression levels of ABCA1, p-JAK2 and p-STAT3 were significantly higher (P < 0.05). Compared with that in the LPS group, p65 protein expression was significantly lower in the LPS+C8:0 group and LPS+EPA group (P < 0.05). Therefore, C8:0 up-regulated ABCA1 and the p-JAK2/p-STAT3 pathway in RAW264.7 cells stimulated by LPS and decreased the expression of p65 inflammatory factor protein (Figure 5).
Figure 5. Effects of C8:0 and EPA on JAK2/STAT3 protein expression in RAW264.7 cells stimulated by LPS. NC, normal contrast; LPS, lipopolysaccharide; C8:0, caprylic acid; EPA, eicosapentaenoic acid; cAMP, cyclic adenosine monophosphate; ABCA1, ATP-binding cassette transporter A1; JAK2, Janus Kinase 2; STAT3, signal transducer and activator of transcription 3; p65, pNF-κBP65. Data in the figure are expressed as the mean ± SD with 4 samples in each group (n = 4). *P < 0.05, versus NC group; aP < 0.05, versus LPS group; bP < 0.05, versus LPS+EPA group; cP < 0.05, versus LPS+cAMP group
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Feed Composition
Experimental Animals
Cell Culture
Preparation of Fatty Acids[13]
Serum Lipid Level Measurements
Inflammatory Factor Detection
RNA Extraction
Western Blot Analysis
Statistical Analysis
Effects of Different Fatty Acids on Feed Intake and Body Weight in C57BL/6J Mice
Effects of Different Fatty Acids on Blood Lipids in C57BL/6J Mice
Effects of Different Fatty Acids on Serum Inflammatory Factors in C57BL/6J Mice
Effects of Different Fatty Acids on the mRNA Expression of ABCA1 and JAK2/STAT3 Pathway-Related Molecules in C57BL/6j Mouse Aorta
Effects of Caprylic Acid on Inflammatory Factors in RAW264.7 Cells Stimulated by LPS
Effects of Caprylic Acid on JAK2/STAT3 mRNA Expression in RAW264.7 Cells Stimulated by LPS
Effects of Caprylic Acid on the Expression of JAK2/STAT3 Pathway Proteins in RAW264.7 Cells Stimulated by LPS
21166Supplementary Materials.pdf |