The effects of low-ratio n-6/n-3 PUFA on biomarkers of inflammation: a systematic review and meta-analysis

Abstract

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 affects serum inflammation markers based on the current studies. Methods: PubMed, Embase and The Cochrane library databases were systematically searched to find randomized controlled trials (RCTs) on the effect of low-ratio n-6/n-3 PUFA intervention on inflammation markers up to July 2020. Data were pooled using standardized mean difference (SMD) and 95% confidence intervals (95% CI), with P value ≦ 0.05 as statistical significance. 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 significant effects on C-reactive protein (CRP) (SMD = −0.027; 95% CI: −0.189: 0.135; P = 0.741). Subgroup analysis indicated that there was a significant reduction in TNF-α serum concentration 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 significantly 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 significantly the concentration of serum TNF-α and IL-6, but not decrease CRP concentration.

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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 considered 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 effects of omega-3 and omega-6 fatty acids on inflammatory factors. Many studies have documented that EPA and DHA are anti-inflammatory and promote inflammation regression.^5–7 Some studies have found no effects of omega-3 fatty acid on inflammatory markers.⁸ A higher ratio of n-6/n-3 PUFA increases the risk of obesity, further increasing the levels of inflammatory factors.⁹ A previous RCT indicated that supplementation with a low n-6/n-3 PUFA ratio had no effect on inflammatory markers.¹⁰ There is no consensus on the effects 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 mechanism of interactions is unclear between omega-3 and omega-6 fatty acids in the human body. It is more practical to research the effects 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 randomized 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 provided in the article, or the ratio could be obtained by calculation; (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 independently according to the inclusion criteria. If there was any controversy, 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 experimental group and control group, whether the participants were healthy and what diseases they suffered from, intervention 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, allocation 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 effects of low-ratio n-6/n-3 PUFA intervention on the indicators of three inflammatory factors. SMD and 95% CI were used to compare effect sizes between trials due to various measurements applied in the included studies. Specifically, the result was considered significant at P ≤ 0.05. I² statistics were conducted to evaluate the degree of heterogeneity. I² > 50% suggested significant heterogeneity between the selected studies, and the random-effects model was used for data analysis. Otherwise, the fixed-effects model was used. Sensitivity analyses were carried to identify sources of heterogeneity.¹¹ In this study, subgroup analyses were conducted according to the different classifications 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 effects 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 assessment conducted, 31 studies finally met the inclusion and exclusion criteria.

Fig. 1 Flow diagram of the literature search and selection.

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 Italy⁴² and Croatia⁴³ (one article each). The 31 included trials included a total of 1450 participants ranging in age from 18 to 76. Some subjects were healthy, and some suffered from diseases. The types of disease included metabolic syndrome, hyperlipidemia, rheumatoid arthritis, coronary artery disease, gestational diabetes, polycystic ovary syndrome, type 2 diabetes, hemodialysis, dyslipidemia, 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 simultaneously in the paper by Zhou, Q. et al.³⁸

Table 1 Characteristics of the included studies

Study	Year	Country	Design	Population	Participants	Duration	n-6/n-3	Indicators
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.
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

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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 supplement 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.

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

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Effects of low-ratio n-6/n-3 PUFA on inflammatory biomarkers

A total of 14 included articles with 24 effect values investigated the effect 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 (I² = 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, −0.045; P = 0.005). In addition, IL-6 did not reveal any significant heterogeneity among the trials (I² = 0.0%). However, 24 trails with 32 comparisons showed that low-ratio n-6/n-3 PUFA supplementation did not affect the level of CRP (SMD = −0.027; 95% CI: −0.189, 0.135; P = 0.741). The tests for heterogeneity were highly significant (I² = 63.9%) (Fig. 4).

Fig. 2 Forest plot of the effect of different n-6/n-3 PUFA ratios on TNF-α.

Fig. 3 Forest plot of the effect of different n-6/n-3 PUFA ratios on IL-6.

Fig. 4 Forest plot of the effect of different n-6/n-3 PUFA ratios on CRP.

Sensitivity analysis and subgroup analysis

A sensitivity analysis was performed to identify the sources of high heterogeneity. As shown in the ESI,† the sensitivity analysis did not find that the removal of any study could change the effect of low-n-6/n-3 PUFA supplementation on CRP. A subgroup analysis was carried out by stratifying region, duration, health status and n-6/n-3 PUFA ratio to investigate further the effects of low-ratio n-6/n-3 PUFA supplementation on the three inflammatory markers (Table 3).

Table 3 Pooled effects 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

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A subgroup analysis of the TNF-α by region classification showed significant differences in the studies from Asia and no differences 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 supplementation led to lower serum levels of TNF-α in participants who suffered from disease (SMD: −0.281; 95% CI: −0.436, −0.127; P < 0.001), with statistically significance. However, supplementation 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 difference between the control group and the experimental group was statistically significant (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 effect showed a significant reduction in the IL-6 level in Europe (SMD: −0.451; 95% CI: −0.688, −0.214; P < 0.001), not 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 subgroup analysis showed that the effect 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 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 difference 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 difference between the two groups when the duration was >8 weeks (SMD: −0.194; 95% CI: −0.404, 0.016; P = 0.071) (Table 3). The subgroup analysis of CRP was not statistically 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 duration. 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).

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)

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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-analysis to investigate the effects of low-ratio n-6/n-3 PUFA supplementation on inflammatory factors. A total of 31 RCTs involving 1450 participants were included in this meta-analysis. The results showed that low-ratio n-6/n-3 PUFA supplementation significantly reduced serum TNF-α and IL-6 concentrations.

The subgroup analysis indicated that the effect 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 nutrients, but also in terms of dietary patterns that affect changes in markers of inflammation. The effects of dietary habits on markers of inflammation are also inconsistent.⁴⁴ Meta-analyses have also found that supplementation with omega-3 fatty acids has different effects on blood sugar between Asians and Europeans, which may be due not only to dietary habits, but also to ethnic and environmental differences.⁴⁵ Similarly, the effect 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 supplementation significantly reduced TNF-α levels in sick individuals but not in healthy individuals. Studies have shown that supplementation 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 effect of low-ratio n-6/n-3 PUFA supplementation was more obvious. The pooled effect of serum TNF-α concentration stratified by health status could further illustrate the effect 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 effects of n-3 fatty acids.⁴⁷ Omega-6 and omega-3 fatty acids compete for the biological synthase, causing different physiological effects 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 effects on colorectal cancer were also different at different 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 effects of low n-6/n-3 PUFA supplementation on inflammatory factors need to be refined further for different diseases, as well as for different 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 effect of IL-6 stratified by duration, further analysis revealed that most of the studies of >8 weeks’ duration came from Asian. The pooled effect of a significant reduction in the ≦8 weeks subgroup may be due to confounding factors caused by regional differences. 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-κB to participant in the inflammation response. The concentration of endocannabinoids 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 effects of EPA and DHA on inflammation, or on the effects of single n-6 or n-3 PUFA on inflammatory markers. This paper is a novel study on the effect 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 effect of the n-6/n-3 PUFA ratio on inflammatory biomarkers has practical significance for inflammation-related diseases. In conclusion, this meta-analysis provides evidence that low-ratio n-6/n-3 PUFA supplementation has obvious effects on lowering TNF-α and IL-6 levels.

Conflicts 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).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0fo01976c

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