Alpha‐linolenic acid given as an anti‐inflammatory agent in a mouse model of colonic inflammation

Abstract This study examined the relationship between the high‐fat, high‐sugar diet (HFHSD) and trinitrobenzene sulfonic acid (TNBS) induced mouse colitis, the therapeutic effect of alpha‐linolenic acid (ALA) on mouse colitis, and the relationship between HFHSD and hyperlipidemia. We also examined the possible underlying mechanisms behind their interactions. Female BABL/c mice were fed with HFHSD for the 9 weeks. At the same time, ALA treatment (150 or 300 mg/kg) was administered on a daily basis. At the end of the 9 weeks, experimental colitis was induced by the intra‐colonic administration of TNBS. Body weight, spleen weight, disease activity index (DAI), histological changes, T‐cell‐related cytokine level, and lipid profiles were measured after treatment. TNBS induced severe clinical manifestations of colitis and histological damage. Low‐ALA (150 mg/kg) administration profoundly ameliorated TNBS‐induced clinical manifestations, body weight loss, spleen weight loss, and histological damage. On the contrary, the high‐ALA (300 mg/kg) administration did not ameliorate colitis and even exacerbated the symptoms. HFHSD consumption assisted TNBS in changing IL‐12, IFN‐γ, IL‐2, and IL‐17A in the liver. As expected, these changes were recovered through low‐ALA. In addition, HFHSD had a significant impact on the total cholesterol (TC), high‐density lipoprotein cholesterol (HDL‐C), and triglyceride (TG), which related to the increased risk of hyperlipidemia. In summation, HFHSD exacerbated the TNBS‐induced colitis via the Th1/Th17 pathway. The Low‐ALA (150 mg/kg) exhibited protective effects against the TNBS‐induced colitis via the Th1/Th2/Th17 pathway.

Although a clear pathology of IBD has not been established, it is generally accepted that the course and development of IBD in the genetically susceptible host depend on the interaction of the immune system, intestinal microbiota, and environmental risk factors (Kaser et al., 2009). Numerous environmental factors such as diet, smoking, appendectomy, nonsteroidal anti-inflammatory drugs, and antibiotics have been studied in relation to IBD (Molodecky & Kaplan, 2010). According to epidemiological investigations throughout the past decade, the incidence of IBD has doubled or tripled in several Asian countries. It is largely due to the increased consumption of the high-fat, high-sugar Western food, which contains high saturated fatty acid and n-6 polyunsaturated fatty acids (n-6 PUFAs) and low in n-3 polyunsaturated fatty acids (n-3 PUFAs; Manzel et al., 2014;Ng, 2015). The ratio of n-6 to n-3 PUFAs in the typical Western diet approaches 10:1 or even 25:1 (Palmquist, 2009;Poudyal, Panchal, Diwan, & Brown, 2011), whereas some researchers suggested ratio is 1:1 (Simopoulos, 2002).
Some encouraging studies showing that n-3 PUFA-rich diets exempted IBD in the clinic (Papadia et al., 2010) and animal models (Ibrahim et al., 2012;Monk et al., 2012). One study found that the transgenic mice rich in endogenous n-3 PUFAs were protected from colitis (Hudert et al., 2006). However, the efficacy of n-3 PUFAs as a form of complementary and alternative medicine (CAM) of IBD is still ambiguous, especially dose-dependent actions of n-3 PUFAs on IBD efficacy have not been well described (Calder, 2013). For instance, Hillary suggested to establishing a tolerable upper limit for DHA, regarding to the fact that dietary high-dose-DHA-rich fish oil diet exacerbated mouse colitis (Woodworth et al., 2010). In addition, despite plant-derived ALA is the major n-3 fatty acid consumed in most human diets. Most of the related studies focused on marinederived EPA/DHA supplements or EPA/DHA-rich fish oil (Calder, 2013). The remission efficacy of ALA-rich flaxseed oil for IBD has been well documented, but the effectiveness of purified n-3 PUFAs pathway primary fatty acid ALA is still less clear (Cohen, Moore, & Ward, 2005;Hassan et al., 2010;Monk et al., 2012). Therefore, it is worth investigating the effect of modifying n-6/n-3 ratio to provide reference to clinic practice. High-fat, high-sugar diet (HFHSD) also leads to hyperlipidemia and insulin resistance (IR), which may lead to coronary heart disease (CHD) (Munshi, Joshi, & Rane, 2014).
More interestingly, the n-3 PUFAs reduce the risk of this kind of diseases partly by improving the blood lipid profile (Poudyal et al., 2011). Given its potentially serious health effects, we are also interested in examining the risk of hyperlipidemia in the mouse colitis model.
In the present study, we investigate the effects of HFHSD and ALA supplement to trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis by a dietary intervention strategy, which is consistent with the current popular Western food. These experiments suggest that consuming a low dose of ALA daily could significantly prevent the incidence of colitis by the capacity of ALA and its metabolites to adjust the expression of T helper (Th) cell-related cytokines in the colon. However, over-increasing n-3/n-6 PUFAs ratio did not protect mice from TNBS-induced colitis and could even exacerbate the symptoms.

| Animals
All animal studies were performed in accordance with protocols ap- with free access to food and tap water. The mice were allowed to acclimate to these conditions for at least 7 days before inclusion in experiments.

| Induction of colitis and experimental design
Unless indicated the mice were fed with purified 45% HFHSD (Trophic Animal Feed High-tech Co., Ltd), and in which all nutritional requirements were met or exceeded (Table 1). In the meantime, the mice were gavaged with a 0.1 ml vehicle of ALA (Sigma-Aldrich) solution daily for 9 weeks. For induction of colitis, the food was withdrawn overnight for 12 hr, and then, prior to the hapten, 0.1 ml of TNBS (2 mg in 50% ethanol; Sigma-Aldrich) was administered intrarectally via a catheter 3 days before sacrifice. Control mice received 50% ethanol. The mice were divided into four groups (n = 9). ALA was dispersed in 0.25% Tween-20 just before using to prevent oxidation. The four treatment groups were as follows: (a) vehicle/EtOH, (b) vehicle/TNBS, (c) ALA (150 mg kg −1 day −1 )/TNBS, and (d) ALA (300 mg kg −1 day −1 )/TNBS. Three days after TNBS administration, the mice were euthanized.
Following the TNBS challenge, the mice were examined for clinical colitis by measuring daily weight, daily hem-occult positivity, and the presence of gross blood and stool consistency, and the disease activity index (DAI) was calculated according to the method used in our laboratory (Wen et al., 2015).

| Histopathological analysis and histological scoring
The mice were sacrificed by cervical dislocation at the end of the experiment. A specimen from the distal third of the colon was fixed overnight with 4% phosphate-buffered (pH 7.2) paraformaldehyde, then embedded in paraffin and cut longitudinally at 5 μm thickness, and stained with hematoxylin and eosin (H&E; Wen et al., 2016). Three sections per slide were scored on a blind basis, and histological analysis was performed on a blind basis as standard protocols.

| Tissue lipid profile analysis
Total cholesterol, HDL cholesterol, LDL cholesterol, and triglyceride in the colon and in the liver were measured using the ELISA Kit (Beijing Beihua Clinical Reagent Co., Ltd).

| Statistical analysis
All results are expressed as mean values with their standard deviation for each group. Significant differences were carried out with Origin 2017 (OriginLab), and were established by one-way ANOVA with post hoc Fisher's least significant difference (LSD) test for comparative analysis with vehicle/TNBS group unless otherwise indicated. p < .05 was regarded as statistically significant.

| The effect of ALA on the clinical index of TNBS-induced colitis mice
In this paper, body weight, spleen weight, and DAI were regarded as a clinical index of TNBS-induced colitis mice (Figure 1). Mice body weight was tracked for 4 weeks until the sacrifice. The body weight of the vehicle/EtOH increased slightly from 22.83 ± 2.05 g (5 weeks) to 22.96 ± 4.05 g (9 weeks) with daily consumption of HFHSD.
Compared to the vehicle/TNBS group, the low-ALA (150 mg/kg)/ TNBS group had significantly higher body weight ( Figure 1a). The vehicle/TNBS group had significantly higher spleen weight than the other three groups (Figure 1b), which meant there was a more active immune response. The DAI was determined by scoring changes in the body weight, hem-occult positivity, or gross blood and stool consistency (Cooper, Murthy, Shah, & Sedergran, 1993). In our experiment, the vehicle/EtOH group and low-ALA (150 mg/kg)/TNBS group had significantly lower the DAI than the vehicle/TNBS group, but the high-ALA treatment did not reduce the DAI in the TNBS-induced colitis (Figure 1c).

| The effect of ALA and HFHSD on histopathological damage of TNBS-induced colitis mice
Trinitrobenzene sulfonic acid is a very strong chemical for inducing mouse colitis. The histopathological image of vehicle/TNBS mouse indicated that the overall surface epithelium was almost destroyed by the TNBS, and two obvious regions of inflammatory cell infiltration appeared in mouse colon (Figure 2b). Low-ALA-treated mice had

| The effect of ALA and HFHSD on clinical index of TNBS-induced colitis mice
In this study, Th1 cell-related cytokines IL-12 and IFN-γ were found significantly lower in the vehicle/EtOH group and low-ALA/TNBS group compared to vehicle/TNBS group (Figure 4a

| D ISCUSS I ON
As an important environmental factor in the IBD pathogenesis, daily diet, especially HFHSD Western food, has been studied extensively in both clinical and animal research. It has been widely accepted that the unbalanced n-3 and n-6 PUFAs in HFHSD contribute significantly to IBD pathogenesis, and our data support this. In the present study, mice were fed with HFHSD and were supplemented with ALA to balance the n-3 and n-6 PUFAs. We found that consuming a low dose of ALA daily could significantly remit TNBS-induced colitis in mice via the Th1/Th2/Th17 pathway. However, over-increasing n-3/n-6 PUFAs ratio did not protect mice from TNBS-induced colitis and could even exacerbate the symptoms. A tolerable upper limit for ALA intake is needed to establish, particularly in the context of chronic inflammatory conditions such as IBD.
In the recent decades, the morbidity of IBD increased in Asian population largely due to consumption of the Western diet, that leading to an imbalance in the ratio of n-6/n-3 PUFAs, in an incli- Trinitrobenzene sulfonic acid-induced colitis in the female BALB/c mice was applied to study the function of ALA in this experiment. The observation that the last 4 weeks body weight of HFHSDfed mice without colitis (vehicle/EtOH group) showed no substantial increase (Figure 1a) was likely due to the BALB/c mouse is a very stable mouse strain. However, we found that the hepatic TC and hepatic HDL-C of the HFHSD-fed vehicle/EtOH mice were significantly higher than in the normal diet-fed vehicle/EtOH mice (Figure 3a,c).
These results suggest that the HFHSD increases the risk of hyperlipidemia, even if the body weight does not change much.
As mentioned previously, increased in the lipid profile is a characteristic of hyperlipidemia. In our study, no significant differences were found in the colonic lipid profiles (Figure 3e Values are expressed as mean ± standard deviation, (n = 9). Significant difference between HFHSD-fed groups was analyzed by comparing to the vehicle/TNBS group, *p < .05, **p < .01. Besides, significant difference between different diet-fed groups was analyzed by comparing between the same intrarectal administration groups, † p < .05 still do not indicate whether ALA has benefit for hyperlipidemia.
Further relative study could display without the interference of experimental colitis.
High-fat, high-sugar diet also has a significant contribution in the mouse colitis. The histology score (Figure 2g) was significantly higher in the HFHSD-fed vehicle/TNBS mice than in the normal diet vehicle/TNBS mice. In addition, infiltration of inflammatory cells was found in the HFHSD-fed vehicle/TNBS mouse colon (Figure 2a), but not in the normal diet vehicle/EtOH mice (Figure 2e), indicating that HFHSD causes low-grade inflammation.
Alpha-linolenic acid, EPA, and DHA are grouped together as well-studied n-3 PUFAs. Evidence suggests that human as well as rodents among other mammals, do not efficiently convert medium-chain ALA to long-chain EPA or DHA, most studies focused on EPA and DHA (Poudyal et al., 2011). However, we still keep interesting in ALA due to following reasons. First, studies proved that ALA-rich oil could be a beneficial functional food on IBD (Cohen et al., 2005;Hassan et al., 2010;Poudyal, Panchal, Ward, & Brown, 2013). Secondly, the dietary intake of ALA is much higher than EPA and DHA among people who do not regularly consume oily fish and consume ALA-rich flaxseed oil daily (Baker et al., 2016). Thirdly, unlike men do not efficiently convert ALA to EPA and DHA, women possess a higher capacity for ALA conversion (Baker et al., 2016). In young women, estimated net fractional ALA inter-conversion was EPA 21%, DPA 6%, and DHA 9% (Burdge & Wootton, 2002). In addition, model animals, like rats and mice, can also convert ALA to EPA and DHA (Scott & Bazan, 1989;Sinclair, Attar-Bashi, & Li, 2002). Lastly, it also suggests that ALA has extra physiological responses, not relying on its metabolism to DHA and EPA. For instance, as mentioned before ALA competes with LA for the same metabolic pathway and so will result in the reduction of the AA content in the tissues, which might be important for immunoregulation (Baker et al., 2016;Calder, 2013). TGF-β, IL-6, IL-2, and IL-1 can drive naive CD4 cells to become IL-17-producing Th17 cells via signals through RORγt. Finally, in the presence of IL-2 and high concentration of TGF-β, naive CD4 cells can develop into IL-10 and TGF-β production Treg-β cells (Vojdani & Lambert, 2011). A great deal of research has been done on the role of mucosal Th1/Th2 balance in IBD. More recently, researchers have also focused on the Th17 and Treg (Leppkes et al., 2009;Round & Mazmanian, 2010). There are growing evidences that Th17-related cytokine IL-17 has a highly pathogenic role in the IBD pathogenesis, while Treg-related cytokine IL-10 is an immunosuppressive cytokine that protects humans and mice from IBD (Geuking et al., 2011;Leppkes et al., 2009;Round & Mazmanian, 2010 (Wen et al., 2016).
In order to understand the overall effect of ALA on Th cells, we studied seven cytokines related Th1, Th2, Th17, and Treg cells in our experiment. Diets in the current study mimicked 2 g/day (low-ALA) and 4 g/day (high-ALA) human ALA consumption. Our results suggest that low-ALA has protective effects against TNBS-induced colitis via the Th1/Th2/Th17 pathway. The decrease of IL-17 and the increase of TGF-β were observed in the high-ALA (300 mg/kg) group, whereas IL-2 was not significantly changed in this group. Considering that the balance of the Th17/Treg will shift to Treg cells by signals from IL-2 and high concentration of TGF-β, high-dose ALA might disrupt Th17 pathway in the TNBS-induced colitis. Furthermore, Treg pathway was not influenced by high-dose ALA supplement, because no significant difference of IL-10 was observed.
Taking together, these results support the need to establish a tolerable upper limit for ALA intake, particularly in the context of chronic inflammatory conditions such as IBD. Considering the conversion of ALA to DHA is more on experimental animals than in humans (Baker et al., 2016). It still needs further investigation in the clinic.

ACK N OWLED G M ENTS
This study was funded by National Natural Science Fund (81660730) Jianling Wang (Lanzhou University) for their technical assistance.
We also thank Core Facility of School of Life Sciences, Lanzhou University for experiment facilities. We also thank LetPub (www. letpub.com) for its linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study was approved by the Animal Care and Ethics committee of the Lanzhou University.