Food & Function REVIEW Cite this: Food Funct. , 2021, 12 , 30 Received 28th July 2020, Accepted 30th October 2020 DOI: 10.1039/d0fo01976c rsc.li/food-function The e ff ects of low-ratio n-6/n-3 PUFA on biomarkers of in fl ammation: a systematic review and meta-analysis † Yali Wei, a Yan Meng, b Na Li, c Qian Wang b and Liyong Chen * a,b Objective : The purpose of the systematic review and meta-analysis was to determine if low-ratio n-6/n-3 long-chain polyunsaturated fatty acid (PUFA) supplementation a ff ects serum in fl ammation markers based on the current studies. Methods : PubMed, Embase and The Cochrane library databases were systemati- cally searched to fi nd randomized controlled trials (RCTs) on the e ff ect of low-ratio n-6/n-3 PUFA inter- vention on in fl ammation markers up to July 2020. Data were pooled using standardized mean di ff erence (SMD) and 95% con fi dence intervals (95% CI), with P value ≦ 0.05 as statistical signi fi cance. Results : Thirty-one RCTs were included in the meta-analysis. The analysis indicated that increasing low-ratio n-6/ n-3 PUFA supplementation decreased the level of tumor necrosis factor- α (TNF- α ) (SMD = − 0.270; 95% CI: − 0.433, − 0.106; P = 0.001) and interleukin 6 (IL-6) (SMD = − 0.153; 95% CI: − 0.260, − 0.045; P = 0.005). There were no signi fi cant e ff ects on C-reactive protein (CRP) (SMD = − 0.027; 95% CI: − 0.189: 0.135; P = 0.741). Subgroup analysis indicated that there was a signi fi cant reduction in TNF- α serum con- centration in subjects from Asia (SMD: − 0.367; 95% CI: − 0.579, − 0.155; P = 0.001) and in subjects with diseases (SMD: − 0.281; 95% CI: − 0.436, − 0.127; P < 0.001). In the subgroup of the n-6/n-3 ratio ≦ 5, low- ratio n-6/n-3 PUFA supplementation could decrease the level of TNF- α (SMD: − 0.335; 95% CI: − 0.552, − 0.119; P = 0.002). Serum IL-6 decreased signi fi cantly in patients from the Europe subgroup (SMD: − 0.451; 95% CI: − 0.688, − 0.214; P < 0.001), but not in Asia (SMD: − 0.034; 95% CI: − 0.226, 0.157; P = 0.724), North America (SMD: − 0.115; 95% CI: − 0.274, 0.044; P = 0.157) and Oceania (SMD: 0.142; 95% CI: − 0.557, 0.842; P = 0.690). Conclusion : Low-ratio n-6/n-3 PUFA supplementation could decrease signi fi - cantly the concentration of serum TNF- α and IL-6, but not decrease CRP concentration. Introduction The inflammatory response is an important part of the human immune system. The human body resists external stimulation by producing massive inflammatory cells and secreting inflammatory factors. Inflammatory mediators play a dominant and antagonistic role in the homeostasis of organisms. However, strong inflammatory responses impair normal physiological activities. Chronic inflammation is con- sidered an underlying pathological condition. 1 Inflammation is a key factor in the pathogenesis of many chronic diseases. 2 Inflammatory cells secrete excessive inflammatory mediators, such as TNF- α , IL-6 and CRP involved in the inflammatory response. When inflammatory mediators are present for a long time, they can cause a variety of chronic diseases and even cancer. 3,4 As important members of PUFA, omega-6 fatty acid ( ω -6 fatty acid or n-6 fatty acid) and omega-3 fatty acid ( ω -3 fatty acid or n-3 fatty acid) have attracted much attention in recent years. Among omega-3 fatty acids, there are α -linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-6 fatty acids include linoleic acid (LA), γ -linolenic acid (GLA) and arachidonic acid. In recent years, many scholars have studied the e ff ects of omega-3 and omega- 6 fatty acids on inflammatory factors. Many studies have docu- mented that EPA and DHA are anti-inflammatory and promote inflammation regression. 5 – 7 Some studies have found no e ff ects of omega-3 fatty acid on inflammatory markers. 8 A † Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0fo01976c a Department of Toxicology and Nutrition, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, China. E-mail: chenle73@sina.com, weiyali0811@163.com; Tel: (+86)15168867157 b Department of Nutrition, Shandong Provincial Hospital A ffi liated to Shandong University, Jinan, China. E-mail: mengsandy@aliyun.com, wangqian.0821@163.com c Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences/Shandong Provincial Food for Special Medical Purpose Engineering Technology Research Center/Key Laboratory of Agro-Products Processing Technology of Shandong Province/Key Laboratory of Novel Food Resources Processing, Ministry of Agriculture and Rural A ff airs, Jinan, China. E-mail: 2803729865@qq.com 30 | Food Funct. , 2021, 12 , 30 – 40 This journal is © The Royal Society of Chemistry 2021 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online View Journal | View Issue higher ratio of n-6/n-3 PUFA increases the risk of obesity, further increasing the levels of inflammatory factors. 9 A pre- vious RCT indicated that supplementation with a low n-6/n-3 PUFA ratio had no e ff ect on inflammatory markers. 10 There is no consensus on the e ff ects of supplementation with omega-3 and omega-6 fatty acids on inflammatory markers. There is a competitive inhibition relationship between derivatives of omega-6 and omega-3 fatty acids. The mecha- nism of interactions is unclear between omega-3 and omega- 6 fatty acids in the human body. It is more practical to research the e ff ects of the ratio of omega-6 to omega-3 fatty acids on inflammation. The purpose of this study was to investigate whether supplementation with a low n-6/n-3 PUFA ratio was beneficial to reduce the level of inflammatory markers. Methods Search strategy Three databases (PubMed, Embase and the Cochrane Library) were exhaustively retrieved to find relevant articles using the following term: “ n-6/n-3 fatty acid ratio ” OR “ n-6/n-3 PUFA ” OR “ omega-3 fatty acid ” OR “ n-3 PUFA ” OR “ n-6 PUFA ” OR “ omega-6 fatty acid ” OR “ Docosahexaenoic Acid ” OR “ DHA ” OR “ eicosapentaenoic acid ” OR “ EPA ” OR “ α -linolenic acid ” OR “ fish oil ” paired with the following: inflammation OR inflammatory OR “ inflammatory factors ” OR “ C reactive protein ” OR “ C-reactive protein ” OR “ high-sensitivity C-reactive protein ” OR hs-CRP OR interleukin-6 OR “ interleukin 6 ” OR IL-6 OR “ tumor necrosis factor- ” OR “ tumor necrosis factor ” OR TNF- α . References from experimental papers and review were searched to find the missing target studies. Inclusion and exclusion criteria The inclusion and exclusion criteria of selected articles were as follows: (1) the study was a randomized controlled or random- ized crossover population trial; (2) the subjects were adults, excluding infants and children; (3) in terms of intervention type, oral feeding was included, whereas enteral nutrition and intravenous input were excluded; (4) the n-6/n-3 ratio was pro- vided in the article, or the ratio could be obtained by calcu- lation; (5) at least one of three inflammatory factors TNF- α , IL-6 and CRP, the mean and standard deviation were provided in the article; (6) non-original studies, repetitive articles and non-English articles were excluded. Data extraction and quality assessment Data extraction was carried out by two researchers indepen- dently according to the inclusion criteria. If there was any con- troversy, the original literature was checked again, and the third researcher would jointly negotiate to solve the problem when no consensus could be reached. The basic information was extracted, including the author, year, region, population and basic information about the subjects (age, sex, body mass index (BMI)). For trails, the following information was extracted in detail: the number of participants in the experi- mental group and control group, whether the participants were healthy and what diseases they su ff ered from, interven- tion duration and n-6/n-3 PUFA ratio. Cochrane Collaboration ’ s tool was used to evaluate the quality of the included articles. The content of assessment included seven aspects: random sequence generation, allo- cation concealment, blinding, observation bias, loss to follow- up, selective reporting and other bias. Statistical analysis Stata (version 12.0; Stata Corporation, College Station, TX) and RevMan software (version 5.1; Cochrane Collaboration, Oxford, United Kingdom) were used to evaluate the e ff ects of low-ratio n-6/n-3 PUFA intervention on the indicators of three inflamma- tory factors. SMD and 95% CI were used to compare e ff ect sizes between trials due to various measurements applied in the included studies. Specifically, the result was considered significant at P ≤ 0.05. I 2 statistics were conducted to evaluate the degree of heterogeneity. I 2 > 50% suggested significant het- erogeneity between the selected studies, and the random- e ff ects model was used for data analysis. Otherwise, the fixed- e ff ects model was used. Sensitivity analyses were carried to identify sources of heterogeneity. 11 In this study, subgroup analyses were conducted according to the di ff erent classifi- cations of region, physical condition and n-6/n-3 PUFA ratio. Meta-regression was used to investigate whether there was any significant linear relationship between e ff ects and duration or ratio. Egger ’ s test and Begg ’ s funnel plots were used to assess potential publication bias. 12 Results Search results and study characteristics A flow chart of the retrieval strategy is shown in Fig. 1. After searching three databases and removing repeated papers, 10,171 papers were found. There were 3527 potentially relevant articles after excluding non-human studies and non-RCT, and 1305 articles assessed for eligibility by screening of the title and abstract. After the full text was read and a quality assess- ment conducted, 31 studies finally met the inclusion and exclusion criteria. The detailed features of the included studies are presented in Table 1. Selected articles were published from 2003 to 2019: five articles from the USA; 13 – 17 nine articles from Canada, 18 – 20 Iran 21 – 23 and Greece 24 – 26 (three articles each); 12 articles from Spain, 27,28 Denmark, 29,30 Germany, 31,32 UK, 33,34 Australia 35,36 and China 37,38 (two articles each); five articles from Japan, 39 New Zealand, 40 Norway, 41 Italy 42 and Croatia 43 (one article each). The 31 included trials included a total of 1450 partici- pants ranging in age from 18 to 76. Some subjects were healthy, and some su ff ered from diseases. The types of disease included metabolic syndrome, hyperlipidemia, rheumatoid arthritis, coronary artery disease, gestational diabetes, polycys- tic ovary syndrome, type 2 diabetes, hemodialysis, dyslipide- Food & Function Review This journal is © The Royal Society of Chemistry 2021 Food Funct. , 2021, 12 , 30 – 40 | 31 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online mia, obesity, ischemic stroke, hypertriacylglycerolemia and coronary heart disease. Duration ranged from 35 days to 24 weeks. There were two comparisons in the paper by Chiang, Y. L. et al. 13 and Baril-Gravel, L. et al ., 18 respectively. Kaul, N. et al. , 20 Wallace, Fiona A. et al. 34 and Zhang, J. et al. 37 each had three randomized, parallel controlled trials. Four groups of RCTs involving 123 subjective were carried out simul- taneously in the paper by Zhou, Q. et al. 38 The quality-assessment results are presented in Table 2. All 31 articles used a randomized controlled approach, most using single- or double-blind studies and concealed sup- plement allocation. Observation bias and loss to follow-up bias were not found in most studies. No other sources of bias were identified in most studies. E ff ects of low-ratio n-6/n-3 PUFA on inflammatory biomarkers A total of 14 included articles with 24 e ff ect values investigated the e ff ect of low-ratio n-6/n-3 PUFA supplementation on TNF- α . As shown in Fig. 2, the experimental group with low-ratio n-6/n-3 PUFA supplementation could significantly reduce the serum concentration of TNF- α compared with the control group with placebo (SMD = − 0.270; 95% CI: − 0.433, − 0.106; P = 0.001), with a lower heterogeneity ( I 2 = 37.6%). Results for IL-6 were reported in 15 eligible studies, which resulted in a total of 24 comparisons (Fig. 3). Overall, the meta-analysis showed that serum IL-6 concentration in the experimental group was significantly lower than that in the control group, with statistical significance (SMD = − 0.153; 95% CI: − 0.260, Fig. 1 Flow diagram of the literature search and selection. Review Food & Function 32 | Food Funct. , 2021, 12 , 30 – 40 This journal is © The Royal Society of Chemistry 2021 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online − 0.045; P = 0.005). In addition, IL-6 did not reveal any signifi- cant heterogeneity among the trials ( I 2 = 0.0%). However, 24 trails with 32 comparisons showed that low-ratio n-6/n-3 PUFA supplementation did not a ff ect the level of CRP (SMD = − 0.027; 95% CI: − 0.189, 0.135; P = 0.741). The tests for hetero- geneity were highly significant ( I 2 = 63.9%) (Fig. 4). Sensitivity analysis and subgroup analysis A sensitivity analysis was performed to identify the sources of high heterogeneity. As shown in the ESI, † the sensitivity ana- lysis did not find that the removal of any study could change the e ff ect of low-n-6/n-3 PUFA supplementation on CRP. A sub- group analysis was carried out by stratifying region, duration, health status and n-6/n-3 PUFA ratio to investigate further the e ff ects of low-ratio n-6/n-3 PUFA supplementation on the three inflammatory markers (Table 3). A subgroup analysis of the TNF- α by region classification showed significant di ff erences in the studies from Asia and no di ff erences from other continents. In Asia, low-ratio n-6/n-3 PUFA supplementation significantly reduces the level of serum TNF- α (SMD: − 0.367; 95% CI: − 0.579, − 0.155; P = 0.001). However, in Europe, North America, and Oceania, the serum TNF- α concentration did not show a significant reduction (SMD: − 0.035, 95% CI: − 0.283, 0.214; P = 0.784; SMD: − 0.394, 95% CI: − 0.842, 0.054; P = 0.085; SMD: − 0.175, 95% CI: − 0.875, 0.524; P = 0.623). The included studies stratified by health status indicated that low-ratio n-6/n-3 PUFA supplemen- tation led to lower serum levels of TNF- α in participants who su ff ered from disease (SMD: − 0.281; 95% CI: − 0.436, − 0.127; P < 0.001), with statistically significance. However, supplemen- tation did not reduce TNF- α levels in healthy subjects (SMD: − 0.230; 95% CI: − 0.614, 0.147; P = 0.232). A stratified analysis was conducted according to whether the ratio of n-6/n-3 PUFA supplementation was >5. The results showed that when the ratio of n-6/n-3 PUFA was ≦ 5, the di ff erence between the control group and the experimental group was statistically sig- nificant (SMD: − 0.335; 95% CI: − 0.552, − 0.119; P = 0.002). When the ratio was >5, there was no statistical significance (SMD: − 0.137; 95% CI: − 0.357, 0.084; P = 0.224) (Table 3). The studies stratified by region indicated that the pooled e ff ect showed a significant reduction in the IL-6 level in Europe (SMD: − 0.451; 95% CI: − 0.688, − 0.214; P < 0.001), not Table 1 Characteristics of the included studies Study Year Country Design Population Participants Duration n-6/n-3 Indicators Baril-Gravel 2014 Canada RCCT 114/114 MetS 4 W 1.6/46.7;2.4/46.7 IL-6, CRP Capo 2014 Spain RCT 9/6 Healthy 8 W 1.079/8.39 TNF- α , IL-6 Chiang 2012 USA RCCT 25/25 HLP 4 W 4.7/9.4; 5.1/9.4 TNF- α , IL-6, CRP Cornish 2018 Canada RCT 11/12 Healthy 12 W 6.49/9.16 TNF- α , IL-6 Damsgaard 2008 Denmark RPT 14/17 Healthy 8 W 1.51/7.33 IL-6, CRP Dawczynski 2018 Germany RCCT 25/25 RA 10 W 0.92/5.66 CRP Dawczynski 2013 Germany RCT 17/24; 17/14 HLP 10 W 4.1/7.21;1.91/7.21 CRP Agh 2017 Iran RCT 24/21 CAD 8 W 94.88/145.45 CRP Hallund 2010 Denmark RPT 23/22 Healthy 8 W 0.16/3.33 IL-6, CRP Han 2012 USA RCrT 18/18 High LDL 35 D 2.73/7.81 CRP Jamilian 2016 Iran RCT 27/27 GDM 6 W 18.54/123.5 CRP Kalgaonkar 2011 USA RCT 17/14 PCOS 6 W 4.61/22.06 TNF- α , IL-6, CRP Kaul 2008 Canada RCT 22/22 Healthy 12 W 0.05/46;0.3/46;3/46 TNF- α , CRP Kondo 2014 Japan RCrT 23/23 DM2 4 W 2.2/6.4 CRP Kontogianni 2013 Greece RCrT 37/37 Healthy 6 W 1.4/8.3 TNF- α , CRP Kooshki 2011 Iran RCT 17/17 HD 10 W 4.65/128.57 TNF- α , IL-6, CRP Martorell 2014 Spain RCT 9/6 Healthy 8 W 1.079/8.39 CRP Minihane 2005 UK RPT 15/14 Healthy 6 W 9/16 CRP Munro 2012 Australia RCT 18/14 Obesity 4 W 4.33/8.47 TNF- α , IL-6, CRP Murphy 2007 Australia RCT 38/36 Overweight 24 W 3.97/6.72 CRP Paschos 2007 Greece RPT 18/17 Dyslipidemic 12 W 0.27/148.8 TNF- α Poppitt 2009 New Zealand RCT 47/48 Ischemic stroke 12 W 0.28/8.97 CRP Rallidis 2003 Greece RPT 50/26 Dyslipidemic 12 W 1.3/13.2 IL-6, CRP Seierstad 2005 Norway RPT 20/19 CHD 6 W 0.1/1.64 TNF- α , IL-6, CRP Sofi 2013 Italy RCrT 20/20 Healthy 10 W 0.44/1.27 TNF- α , IL-6 Stupin 2018 Croatia RCT 20/16 Healthy 3 W 2.63/7.29 CRP Lee 2014 USA RPT 21/16 DM2 8 W 1.3/10.2 CRP Vargas 2011 USA RCT 17/17 PCOS 6 W 0.01/7.27 CRP Wallace 2007 UK RCT 8/8 Healthy 12 W 3.03/8.13;5.53/8.13;3.6/8.13 TNF- α , IL-6 Zhang 2012 China RCT 32/33; 32/33; 29/ 33 Hypertriacylglycerolemia 8 W 4.6/15.1; 5.4/15.1; 7.3/15.1 TNF- α , IL-6, CRP Zhou 2019 China RCT 23/24; 25/24; 25/ 24; 26/24 HLP 12 W 6.98/14.93; 4.53/14.93; 3.5/ 14.93; 2.03/14.93 TNF- α , IL-6 Abbreviations: CAD: coronary artery disease; CHD: coronary heart disease; CRP: c-reactive protein; D: day; DM2: type 2 diabetes mellitus; GDM: gestational diabetes; HD: hemodialysis; HLP: hypercholesterolemia; IL-6: interleukin-6; MetS: metabolic syndrome; PCOS: polycystic ovary syndrome; RA: rheumatoid arthritis; RCCT: randomized controlled cross trial; RCrT: randomized crossover trial; RCT: randomized controlled trial; RPT: randomized parallel trial. Food & Function Review This journal is © The Royal Society of Chemistry 2021 Food Funct. , 2021, 12 , 30 – 40 | 33 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online Table 2 Quality assessment of the included studies Study Random sequence generation Allocation concealment Blinding Observation bias Loss to follow-up Selective reporting Other bias Baril-Gravel Yes Unclear Unclear No Yes No No Capo Yes Yes Unclear Unclear Yes No No Chiang Yes Unclear Yes No Yes No No Cornish Yes Yes Yes No Yes No No Damsgaard Yes Yes Yes No Yes No No Dawczynski Yes Yes Yes No Yes No No Dawczynski Yes Yes Yes No Yes No No Agh Yes Yes Yes No Yes No No Hallund Yes Yes Yes No Yes No No Han Yes Yes Yes No Unclear Unclear Unclear Jamilian Yes Yes Yes No Yes No No Kalgaonkar Yes Yes Yes No Yes No No Kaul Yes Yes Yes No Yes No No Kondo Yes No No Unclear Yes No Unclear Kontogianni Yes Yes Yes Unclear Yes No Unclear Kooshki Yes Yes Yes No Unclear Unclear Unclear Martorell Yes Yes Unclear Unclear Yes No No Minihane Yes Yes Yes Unclear Unclear Unclear Unclear Munro Yes Yes Yes No Yes No No Murphy Yes Yes Yes No Yes No No Paschos Yes Unclear Yes Yes Unclear Unclear Unclear Poppitt Yes Yes Yes No Yes No No Rallidis, Yes No No Yes No No Yes Seierstad Yes Yes Yes No Yes No No Sofi Yes Unclear Unclear Unclear Yes No No Stupin Yes Yes Yes No Unclear Unclear No Lee Yes Unclear Yes No Yes No No Vargas Yes Unclear Yes Unclear Unclear Unclear Unclear Wallace Yes Yes Yes No Yes Yes No No Zhang Yes Yes Yes No Yes No No Zhou Yes Yes Yes No Yes No No Fig. 2 Forest plot of the e ff ect of di ff erent n-6/n-3 PUFA ratios on TNF- α Review Food & Function 34 | Food Funct. , 2021, 12 , 30 – 40 This journal is © The Royal Society of Chemistry 2021 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online in Asia, North America and Oceania (SMD: − 0.034, 95% CI: − 0.226, 0.157, P = 0.724; SMD: − 0.115, 95% CI: − 0.274, 0.044, P = 0.157; SMD: 0.142, 95% CI: − 0.557, 0.842, P = 0.690). A sub- group analysis showed that the e ff ect of low-ratio n-6/n-3 PUFA on the reduction in IL-6 level in the healthy population (SMD: − 0.394; 95% CI: − 0.715, − 0.073; P = 0.016) was more obvious Fig. 3 Forest plot of the e ff ect of di ff erent n-6/n-3 PUFA ratios on IL-6. Fig. 4 Forest plot of the e ff ect of di ff erent n-6/n-3 PUFA ratios on CRP. Food & Function Review This journal is © The Royal Society of Chemistry 2021 Food Funct. , 2021, 12 , 30 – 40 | 35 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online than in patients (SMD: − 0.111; 95% CI: − 0.228, − 0.005; P = 0.060). As for the stratification of duration, the extraction results showed that there was a significant di ff erence between the experimental group with low-ratio n-6/n-3 PUFA and the control group with placebo when the duration was ≦ 8 weeks (SMD:-0.135; 95% CI: − 0.264, − 0.007; P = 0.039). There was no significant di ff erence between the two groups when the dur- ation was >8 weeks (SMD: − 0.194; 95% CI: − 0.404, 0.016; P = 0.071) (Table 3). The subgroup analysis of CRP was not statisti- cally significant (Table 3). Meta-regression and publication bias The duration of low-ratio n-6/n-3 PUFA supplementation ranged from 35 days to 24 weeks in the included studies. We conducted a meta-regression analysis and found that there was no linear relationship between TNF- α concentration and dur- ation. Similarly, IL-6 was not linearly related to duration. The same results were found in the meta-regression of ratio and inflammation markers (Table 4). Begg ’ s test and Egger ’ s test found no publication bias in TNF- α (PBegg = 0.785, PEgger = 0.729), IL-6 (PBegg = 0.333, PEgger = 0.307) and CRP (PBegg = 0.466, PEgger = 0.337) (ESI † ). Discussion To the best of our knowledge, this article is the first meta-ana- lysis to investigate the e ff ects of low-ratio n-6/n-3 PUFA sup- plementation on inflammatory factors. A total of 31 RCTs involving 1450 participants were included in this meta-ana- lysis. The results showed that low-ratio n-6/n-3 PUFA sup- plementation significantly reduced serum TNF- α and IL-6 concentrations. The subgroup analysis indicated that the e ff ect of low-ratio n-6/n-3 PUFA on the reduction in TNF- α level in Asian countries was more obvious than in other countries. Low-ratio n-6/n-3 PUFA supplementation significantly decreased serum IL-6 concentration in Europe, but not in other regions. Eating habits vary from region to region, not only in terms of nutri- ents, but also in terms of dietary patterns that a ff ect changes in markers of inflammation. The e ff ects of dietary habits on markers of inflammation are also inconsistent. 44 Meta-ana- lyses have also found that supplementation with omega-3 fatty acids has di ff erent e ff ects on blood sugar between Asians and Europeans, which may be due not only to dietary habits, but also to ethnic and environmental di ff erences. 45 Similarly, the Table 3 Pooled e ff ects on TNF- α , IL-6, and CRP in various subgroups Variables Subgroups Number SMD (95% CI) P TNF- α Region Europe 8 − 0.035 ( − 0.283, 0.214) 0.784 Asia 8 − 0.367 ( − 0.579, − 0.155) 0.001 North America 7 − 0.394 ( − 0.842, 0.054) 0.085 Oceania 1 − 0.175 ( − 0.875, 0.524) 0.623 Participants Health 10 − 0.230 ( − 0.614, 0.147) 0.232 Diseases 14 − 0.281 ( − 0.436, − 0.127) <0.001 Duration ≦ 8 weeks 10 − 0.194 ( − 0.374, − 0.015) 0.034 >8 weeks 14 − 0.320 ( − 0.608, − 0.032) 0.029 Ratio >5 8 − 0.137 ( − 0.357, 0.084) 0.224 ≦ 5 16 − 0.335 ( − 0.552, − 0.119) 0.002 IL-6 Region Europe 9 − 0.451 ( − 0.688, − 0.214) <0.001 Asia 8 − 0.034 ( − 0.226, 0.157) 0.724 North America 6 − 0.115 ( − 0.274, 0.044) 0.157 Oceania 1 0.142 ( − 0.557, 0.842) 0.690 Participants Health 8 − 0.394 ( − 0.715, − 0.073) 0.016 Diseases 16 − 0.111 ( − 0.228, 0.005) 0.060 Duration ≦ 8 weeks 13 − 0.135 ( − 0.264, − 0.007) 0.039 >8 weeks 11 − 0.194 ( − 0.404, 0.016) 0.071 Ratio >5 8 − 0.204 ( − 0.421, 0.013) 0.065 ≦ 5 16 − 0.128 ( − 0.284, 0.027) 0.106 CRP Region Europe 12 0.077 ( − 0.137, 0.290) 0.482 Asia 7 − 0.460 ( − 1.022, 0.102) 0.109 North America 11 0.108 ( − 0.088, 0.304) 0.280 Oceania 2 − 0.172 ( − 0.793, 0.449) 0.588 Participants Health 9 0.228 ( − 0.114, 0.571) 0.191 Diseases 23 − 0.112 ( − 0.291, 0.067) 0.220 Duration ≦ 8 weeks 22 − 0.143 ( − 0.344, 0.058) 0.164 >8 weeks 10 0.210 ( − 0.050, 0.471) 0.114 Ratio >5 15 0.064 ( − 0.081, 0.209) 0.388 ≦ 5 17 − 0.144 ( − 0.444, 0.156) 0.347 Table 4 Results of meta-regression of TNF- α and IL-6 b SE t P 95% CI TNF- α Duration − 0.030 0.282 − 1.07 0.295 ( − 0.089, 0.028) Ratio 0.000 0.000 − 0.56 0.584 ( − 0.001, − 0.000) IL-6 Duration − 0.013 0.017 − 0.82 0.423 ( − 0.048, 0.021) Ratio 0.002 0.005 0.31 0.761 (0.009, 0.012) Review Food & Function 36 | Food Funct. , 2021, 12 , 30 – 40 This journal is © The Royal Society of Chemistry 2021 Published on 24 November 2020. Downloaded on 5/18/2023 3:18:19 PM. View Article Online e ff ect of low-ratio n-6/n-3 PUFA supplementation on serum inflammatory markers may also vary from region to region. In the subgroup analysis, participants were divided into a healthy group and a disease group according to their physical health status. A diet with low-ratio n-6/n-3 PUFA supplemen- tation significantly reduced TNF- α levels in sick individuals but not in healthy individuals. Studies have shown that sup- plementation with DHA and EPA significantly reduced CRP concentrations, especially in subjects with dyslipidemia and higher baseline CRP concentrations. 46 We speculated that in the patients, the inflammatory factor level was higher, and the e ff ect of low-ratio n-6/n-3 PUFA supplementation was more obvious. The pooled e ff ect of serum TNF- α concentration stra- tified by health status could further illustrate the e ff ect of low- ratio n-6/n-3 PUFA supplementation on inflammatory markers. Therefore, in an inflammatory state, the interaction between omega-3 and omega-6 is complex. High levels of n-6 fatty acids could counteract the anti-inflammatory e ff ects of n-3 fatty acids. 47 Omega-6 and omega-3 fatty acids compete for the bio- logical synthase, causing di ff erent physiological e ff ects on the body. There is a balance between n-6 and n-3 PUFA. 48,49 With respect to IL-6, there was a statistically significant decrease in healthy individuals, but not in patients. A meta-analysis showed that supplementation of omega-3 fatty acids alone could not reduce inflammation levels in patients with renal disease. 50 Omega-3 fatty acids reduced CRP levels but did not reduce IL-6 levels in patients undergoing dialysis. 51 The anti- inflammatory e ff ects on colorectal cancer were also di ff erent at di ff erent doses and durations. 52 It takes more than 1 g d − 1 of omega-3 fatty acids to reduce inflammation in patients with heart failure. 53 The e ff ects of low n-6/n-3 PUFA supplemen- tation on inflammatory factors need to be refined further for di ff erent diseases, as well as for di ff erent doses of intake. On the other hand, it may also be shown that diets with a low- ratio n-6/n-3 PUFA can help reduce the levels of inflammatory factor and prevent inflammation-related diseases in healthy men. The studies stratified by duration indicated that TNF- α was significantly reduced regardless of whether the duration was longer than or less than 8 weeks, whereas the change in CRP level was not statistically significant. There was a significant decrease in serum IL-6 levels within 8 weeks of low-ratio n-6/ n-3 PUFA supplementation, although there was no significant decrease when the duration was beyond 8 weeks. Molecular biology studies have shown that n-3 PUFA, n-6 PUFA and their derivatives can target transcription factors to regulate gene expression and participate in the process of inflammation regression by modifying cell-membrane composition. 54 An RCT showed that supplementation with a low ratio of n-6/n-3 PUFA at 26 weeks altered gene expression and reduced the expression of inflammation-related genes, which suggests that long-term supplementation of low-ratio n-6/n-3 PUFA could reduce the incidence of inflammation. 55 Regarding the e ff ect of IL-6 stratified by duration, further analysis revealed that most of the studies of >8 weeks ’ duration came from Asian. The pooled e ff ect of a significant reduction in the ≦ 8 weeks subgroup may be due to confounding factors caused by regional di ff erences. Subgroup analysis showed that there was a significant decrease in TNF- α when the ratio of n-6/n-3 PUFA was no higher than 5. Some derivatives of n-6 fatty acids, such as endogenous cannabinoids, target NF- κ B to participant in the inflammation response. The concentration of endocanna- binoids is influenced by a dietary intake of omega-6 and omega-3 fatty acids. Diets with a high omega-6 fatty acid content can lead to an overactive endocannabinoid system. Diets with a high omega-6/omega-3 ratio lead to an increase in endocannabinoid signaling and related agents, leading to an inflammatory state. 56 It is particularly important to find an appropriate ratio of n-6/n-3 PUFA. The results in this paper suggest that a ratio of no higher than 5 is more conducive to reducing the level of inflammatory markers. A number of RCTs are needed to further explore the optimal ratio of n-6/n-3 PUFA. Conclusion Previous studies have focused on the e ff ects of EPA and DHA on inflammation, or on the e ff ects of single n-6 or n-3 PUFA on inflammatory markers. This paper is a novel study on the e ff ect of the ratio of n-6/n-3 PUFA on inflammatory markers. The type of fatty acids in the diet is complex, and it is rarely a single intake of n-6 or n-3 PUFA. The study of the e ff ect of the n-6/n-3 PUFA ratio on inflam- matory biomarkers has practical significance for inflam- mation-related diseases. In conclusion, this meta-analysis pro- vides evidence that low-ratio n-6/n-3 PUFA supplementation has obvious e ff ects on lowering TNF- α and IL-6 levels. Con fl icts of interest There are no conflicts of interest to declare. Acknowledgements This study was funded in full by the Major project of Shandong Province, China; grant number 2018YYSP020 (To Liyong Chen). References 1 M. E. Kotas and R. Medzhitov, Homeostasis, inflammation, and disease susceptibility, Cell , 2015, 160 (5), 816 – 827, DOI: 10.1016/j.cell.2015.02.010. 2 R. 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