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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 51  |  Issue : 3  |  Page : 91-97

Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats


Department of Surgery, Division of Pediatric Surgery, National Defense Medical Center, School of Medicine, Tri-Service General Hospital, Taipei, Taiwan

Date of Submission03-Sep-2017
Date of Decision11-Oct-2017
Date of Acceptance16-Nov-2018
Date of Web Publication21-Jun-2018

Correspondence Address:
Dr. Guan-Yeu Diau
325, Chenggung Rd., Section 2, Neihu District, Taipei City 11490
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/fjs.fjs_148_17

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  Abstract 

Background: Dietary adjuvant management for the cholestatic liver disease before/after surgery is an important clinical issue. This study investigated the possibility for the dietary supplementation of docosahexaenoic acid (DHA) to treat cholestasis liver through the bile duct ligation (BDL) rat model.
Materials and Methods: Thirty-six male Wistar rats were divided into four groups (N: no BDL; BL, 1P, 5P: received BDL) and consumed either a regular diet (N, BL) or of DHA-enriched diet (1P: at 1% and 5P: at 5% weight percentage) for 4 weeks. The liver fatty acids (FAs) profiles, serum aspartate transaminase (AST), alanine aminotransferase (ALT), total bilirubin, alkaline phosphatase, interleukin-2 (IL-2), interferon γ (INF-γ), and pathological examination with H and E, Masson, CD56 (natural killer cell), CD68 (macrophage) were examined.
Results: The DHA dietary supplement increased liver DHA after BDL. Liver DHA N 8.09 ± 0.60% and BL 8.41 ± 0.55% were the lowest than the supplemented groups 1P 12.57 ± 1.16%, 5P 18.36 ± 2.00% (P = 0.000). However, liver arachidonic acid (20:4n-6) 1P 23.13 ± 2.19% was the highest than N 18.86 ± 4.31%, BL 17.13 ± 3.07%, 5P 18.78 ± 1.76% (P = 0.001). The serum AST (U/L) in N 147.4 ± 28.2 and 1P 155.9 ± 35.1 were lower than B 317.1 ± 195.8, 5P 326.9 ± 141. 8 (P = 0.006). The serum alkaline phosphatase (U/L) showed the same trend N 49.8 ± 5.4, 1P 67.6 ± 21.1 were lower than the BL 172.2 ± 108.1, 5P 171.1 ± 149.1 (P = 0.017). Pathological examination with H and E, Masson revealed the fibrosis was prominent in BL, 5P. However, there were no significant differences in serum ALT, total bilirubin, IL-2, INF-γ, and immunohistochemical stain for the CD56, CD68.
Conclusions: The results suggested that optimal dietary supplementing of DHA (1P) had less destruction and liver enzymes released after the BDL. However, higher enriched DHA (5P) could not benefit from this dietary treatment. The body weight did not increase even with this enriched high FAs diet after BDL for 4 weeks.

Keywords: Bile duct ligation, cholestasis liver disease, docosahexaenoic acid, hepatic injury


How to cite this article:
Diau GY, Kuo SM, Lin CW. Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats. Formos J Surg 2018;51:91-7

How to cite this URL:
Diau GY, Kuo SM, Lin CW. Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats. Formos J Surg [serial online] 2018 [cited 2018 Nov 18];51:91-7. Available from: http://www.e-fjs.org/text.asp?2018/51/3/91/234878


  Introduction Top


There is an unmet, compelling need for a safe and effective dietary adjuvant for the patient suffered from the cholestasis liver disease. Dietary adjuvant treatment for the cholestatic liver disease is still a major challenge for surgeons. However, the effective immunomodulation diets contribute to the pre- and post-operative care are still limited at the present time. Docosahexaenoic acid (DHA) (22:6n-3) is the most unsaturated fatty acid (FA) in mammalian tissue, and it is found at a particularly high concentration in the sperm,[1],[2],[3] cerebral cortex [4],[5] and retina.[6] It concentrated mainly in the serine and ethanolamine phosphoglycerides, especially at the sn-2 position.[1],[7] There is a large portion of FA metabolism occurred in the liver. The DHA tissue contents in the liver might affect its function and outcome by cholestasis.

To investigate the impaction of liver damage by cholestasis, we used the bile duct ligation (BDL) rat model to simulate the damage. For the detrimental effects by cholestasis, a considerable amount of oxygen radicals generated. Recent studies provided evidence that the oxygen free radicals can cause cellular damage, such as the biliary atresia in pediatric patients.[8] DHA reported to have the ability to reduce these free radicals. Its definite mechanisms are still elusive. The reported mechanisms involved in the affecting cytokine production,[9] leukotriene level,[10] cellular populations,[11] function of the immune cells,[12],[13],[14] increase anti-oxidative enzymes,[13],[14],[15],[16] protective effects for apoptosis,[17],[18],[19] replenish the lost cell membrane,[8],[20] decrease blood viscosity,[21],[22] and influence on the inflammatory gene expression,[23],[24],[25] etc.

Modulation of the immune system might be one of the crucial roles for the successfully treatment of cholestasis liver disease. The DHA reported to have the ability of immunomodulation. This might be one of the mechanisms that attenuate the detrimental effects after the liver destruction. Literatures reported DHA could downregulation of the proliferation of CD4 cells,[9],[26],[27] inhibit the lymphocyte function,[28],[29] decrease CD8 expression,[26],[27] modulate mitogen-activated protein kinase,[30] decrease natural killer (NK) cell function,[31],[32] affect the immunoglobulin status,[33] decreased cytokine production and inflammatory markers,[32],[34],[35] inhibit antigen-presenting function of lymphocyte,[36] and modulate gene expression,[34] etc. This study examined the immune reactions through the pathological examination of the liver tissue (H and E, Masson, CD56, CD68). Furthermore, the cytokines affected by the DHA administration after the cholestasis, for example, interleukin-2 (IL-2) production is still controversial. Most literature supported the DHA reduced the amount of IL-2 in the serum;[37],[38] however, some shown no obvious effects at all.[28],[29] Based on our understanding, the changes after the DHA supplement in cholestasis were still not examined up to now. The serum concentration of IL-2 and interferon γ (INF-γ) was checked through enzyme-linked immunosorbent assay (ELISA).

The single cell DHA were used instead of the traditional DHA manufactured from the fish oil. Long chain polyunsaturated (LCP) FAs are abundant in the deep sea fish. Fish oil was also a well-known source for the essential n-3 FA DHA production. It might have the possibility of contamination by various chemicals and heavy mental from deep-sea large fish sources oil. Recently, single cell oil from cultured microalgae (Crypthecodinium cohni) has been used as an alternative source of DHA in North America.[40],[41],[42]

We proposed that supplementing dietary DHA could decrease the rat liver damage by the BDL and provided enough energy supply to this mishap situation.


  Materials and Methods Top


Experimental animals and diet

All institutional and national guidelines for the care and use of laboratory animals were followed and compliance with the ethical standard. Experiments evaluated and passed the ethical standard (issued # Institutional Animal Care and Use Committee [IACUC]-09-107) by Animal Experimentation of IACUC and performed in accordance with the procedures outlined in the guidelines for IACUC, National Defense Medical Center. Male Wistar rats (250 g) divided into four groups (N, BL, 1P, 5P): N and BL (n = 18) consumed a regular diet based on a purified rodent diet (AIN 93G, Dyets, PA, USA) and 1P and 5P (n = 18) consumed a supplemented single cell DHA powder (DHASCO ®, Martek Bioscience, Columbia, MD, USA) at various strength for 4 weeks after BDL (BL, 1P, 5P received BDL). 1P: 1% DHA powder + 99% rodent regular diet powder. 5P: 5% DHA powder + 95% rodent regular diet powder. The purified rodent diet was grounded into powder. Then, it could mix with the DHA powder without any difficulty. This single-cell source DHA is chosen because they are used in the manufacturing of many North American products. The rats gave food and water ad libitum, and food consumption and body weight monitored throughout the experimental period.

Bile duct ligation

The bile duct ligation (BDL) surgery performed according to the method described by Gross.[43] Male Wistar rats (250 g) anesthetized with Zoletil (5–7 mg/kg) injection intramuscularly. The common bile duct identified and double ligated using 5-O silk thread and divided in the middle between those two ligatures to ensure the complete transaction of the bile duct. Two days after surgery, the presence of bilirubin in the urine turned it a deep yellow color, indicated successful ligation. Details described as cited.[43]

Histopathological examination

The liver evaluated for the histopathologic examination at the end of the experiment. Sections approximately 4 mm thick were stained with hematoxylin and eosin (H and E) staining for routine histology.

The Masson's trichrome staining for collagen to evaluate the fibortic change of the liver.

The immunohistochemical staining was performed for CD56 (NK cells) and CD68 (macrophage) in some occasions to evaluate the possible cellular response by our dietary manipulation.

Lipid extraction and fatty acids analyses

Total lipids will be extracted from the liver use the method of Bligh and Dyer [44] modified for the water content of the tissue to ensure a single-phase solvent mixture. FA methyl ester analyzed using a (gas chromatography-17A, Shimadzu Co, Tokyo, Japan) with a BPX 70 capillary column (60 m × 0.32 mm I. D. × 0.25 μm film; SGE, Austin, TX, USA) and H2 as carrier gas. Details were described as cited.[45]

Serum liver enzyme profiles analyses

The serum level of aspartate transaminase (AST), alanine aminotransferase (ALT), total bilirubin and alkaline phosphatase examined at the end of this study. Serum concentration of these parameter checked by the clinical, pathological laboratory in the Tri-Service General Hospital.

Cytokines analyses

IL-2 and IFN-γ serum levels examined at the end of this study. Serum concentrations of these cytokines were checked by colorimetric sandwich ELISA. (R and D Systems Inc., Minneapolis, MN, USA). Details were described as cited.[34]

Statistical analyses

All results were expressed as means and standard deviations. Student's t-test and one-way ANOVA used for statistical analysis. Duncan's multiple-range test employed to indicate the statistical significance at P < 0.05.


  Results Top


Characteristics of the animals were presented in [Table 1]. There were no significant differences in any parameter associated within groups statistically at the beginning of the experiment. However, the body weight and weight gain of the unreceived BDL (N) is significant increased than the received BDL groups (BL, 1P, 5P). This suggested that BDL could stress the rat health and decrease the body weight gain. 1P and 5P group did not increase the body weight compared with BL group. This revealed that even with enriched high-energy DHA dietary manipulation did not affect the body weight gain after BDL. This phenomena contrast to our previous study (in preparation, not published yet) through the burn rat model that higher enriched DHA diet increased the weight gain.
Table 1: Characteristics of Wistar rats

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FA compositions of the diet (RD) and fortified formula (1P, 5P) details were listed in [Table 2]. Dietary 20:4n-6 were 0.87 ± 0.00%, 0.79 ± 0.28%, and 0.81 ± 0.18% of total FAs, respectively. The 22:6n-3 were 2.52 ± 0.24%, 14.52 ± 0.02%, and 27.54 ± 0.24% separately.
Table 2: Fatty acids composition of the various diets

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The DHA dietary supplementation increased liver DHA after a 4-week feeding in 1P, 5P [Table 3]. N 8.09 ± 0.60%, BL 8.41 ± 0.55% were the lowest than the supplemented groups 1P 12.57 ± 1.16%, 5P 18.36 ± 2.00%. However, the 20:4n-6 concentrations revealed the 1P was the highest than N, BL, 5P. This results contrast to the general theory that the DHA competes with the enzyme system to reduce the ascorbic acid (AA) level in 1P.
Table 3: Liver fatty acids composition in various groups

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The serum AST (U/L) N 147.4 ± 28.2 and 1P 155.9 ± 35.1 were lower than the 5P 326.9 ± 141. 8, BL 317.1 ± 195.8. The serum alkaline phosphatase (U/L) showed the same trend N 49.8 ± 5.4 and 1P 67.6 ± 21.1 were lower than the 5P 171.1 ± 149.1, BL 172.2 ± 108.1 [Table 4]. This suggested that only relative optimal concentration (1P) decrease some of the liver enzymes release after BDL. However, there were no significant differences in serum ALT, bilirubin, IL-2, INF-γ.
Table 4: Serum biochemistry data of Wistar rats

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Pathological examination with H and E, Masson stain of the liver tissue revealed the fibrosis was prominent in BL, 5P [Figure 1]. However, there were no significant differences for the CD56, CD68.
Figure 1: Liver pathology H and E, Masson

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Ethical approval

The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the institute. Informed written consent was obtained from all patients prior to their enrollment in this study.


  Discussion Top


The result revealed the enriched DHA diet increased DHA tissue level in the liver after BDL. However, the tissue level of 20:4n-6 (arachidonic acid [AA]) increased a lot in the most effective 1P. There were no significant differences between the N, BL, and 5P. This trend was not universally compatible with most of literatures and traditional theory.[42] The previous knowledge was that n-3 FAs bearing its biological function by competing with the n-6 FAs through the same enzyme system. If n-3 FAs administration increased, the n-6 FAs should decrease. It is well known that AA (20:4n-6, AA) is oxygenated and further transformed into a variety of products, for example, Plasma prostaglandin E2, thromboxane A4, leukotriene A4, which mediate or modulate the inflammatory cascades. Our data revealed that 1P had the highest tissue AA unexpectedly. Usually, the serum AA rose, especially in the situation of liver cirrhosis, for example, cystic fibrosis,[46] hepatitis B.[47] The AA also exhibited an inverse relationship to serum bilirubin.[48] As a result, the AA in 1P should not so high theoretically. One of the possible explanations is that the DHA supplementation increased the lipid peroxidation. In human, the LCP-supplemented formulae improved LCP status in severe cholestasis infant, but might also enhance the lipid peroxidation.[41] This peroxidation resulted in the overproduction of FAs in both the AA and DHA. The 5P could possibly attenuate the AA more than 1P. This explained why there was the highest tissue AA content in 1P. Based on the result of this experiment, another tentative speculation was that AA might have a more crucial impact on the BDL rat. However, the total n-6/n-3 ratio was consisted with the traditional enzyme system competition theory. As a result, this unusual increased AA might imply that AA stands for more important role than other FAs only in the optimal dosing (1P) situation.

The impaired cholestasis liver might benefit from the DHA predominately at 1P. However, a lot of metabolism of the FAs, for example, lipogenesis, β-oxidation, and esterification was performed at the liver. We do not know if the administration of the DHA might cause more burdens under this situation. Furthermore, some of the liver enzyme profile, for example, the bilirubin and AST were not affected by the dietary manipulation. The destructive parameters liver enzymes were not universally showed beneficial effect. The question that less or increase burden of the live was not answered in this preliminary study. The FAs compositions between N and BL revealed that BDL only has effect on increase 18:0, total n-3 FAs, decrease total n-9 FAs, total n-6/n3 ratio. Most of the FAs composition did not have significant differences after BDL. This implied that the liver function was not so bad after a 4-week BDL. However, the body weight loss after the BDL was significant. One of the possibilities was that there might be a huge FAs pooling in the body. The cholestasis liver might take the FAs back from other body components.

The body weight gain did not increase in the DHA supplemented group. The FAs are efficient energy resources. It could produce more energy than the carbohydrate and protein per weight. The results of this experiment suggested that even with higher dietary enrichment of DHA (5P), it could only partially compensate the basic energy requirement after BDL. Lots of the metabolism of FAs were accomplished in the liver. One of the possibilities of the body weight failed to gain might be due to the liver function severely compromised by the BDL. Furthermore, the disease state by cholesteric liver might also decrease the foot intake. The daily food intake was not recorded in this experiment. This possibility could not be answered this time. It was also possible these rodents did not like the diets we prepared. The taste of DHA powder might be not so delicious for them. This powder was directly packed into capsules and sold by stores for human product. This result was quite different from the literatures and our previous study using rat model in burn injury, which revealed that the higher concentration dietary DHA would increase the body weight gain significantly. This suggested that there were differences among the various species and models of diseases.

The serum immunological and inflammatory parameters, for example, serum IL-2, INF-γ did not show significant differences statistically. The previous literature revealed DHA impact on the immune system were controversial.[26],[27],[29],[31],[32],[33] Up to now, the controversies existed and varied with different researchers. Furthermore, the histobiochemical stain for the NK cells and macrophage, which stand for a very important inflammation response after the BDL, did not revealed significant difference among various groups. Further studies would be advisable and necessary to search the possibility mechanisms about the changes after BDL.

The safety issue regarding the high dietary DHA supplementation is also a major clinical concern.[40],[41] Immunomodulation might also be possible when the toxicity of the DHA reached especially in 5P. However, there were no observable side effects, for example, bleeding tendency in those rats. It was not clear that the failure of body weight gain came from BDL or dietary manipulation is this survey. Even with the relative lower toxicity of DHA, the administration of dietary DHA in the cholestasis liver patient should be more cautiously and need more strong clinical trials to prove its safety and effectiveness.

Another rare clinical situation is the cholestasis liver occurred in the neonate, for example, biliary atresia.[8] Unlike AA, the DHA is an essential FA which is also vulnerable to limitations in the supply of precursor from food.[45] It is not very clear to what extent would the compromised liver function in the progression of biliary atresia. However, under the assumption that the metabolism of the liver should not as good as the normal one, dietary administration these essential FAs seemed reasonable. This is especially important for the pediatric group who need the FAs to build up body mass, especially for the important organs, e.g. brain, retina. Further study is necessary to make this approach more practical. Treatment for the biliary atresia is still mostly relying on surgery. The baby suffered from biliary atresia might also mimic the situation of BDL. However, the ligature of bile duct and anesthesia was extreme difficult, especially in the very tiny unweaning breast milk-fed rat. Furthermore, the dietary manipulation for breastfeeding through the mother rat was also very difficult. The artificially fortified mother rat milk and route of feeding are very hard to achieve. This is the future target for our team to struggle on.


  Conclusions Top


The results of this experiment concluded that only optimal dietary supplementing of DHA (1P) could reduce the tissue destruction, decrease serum AST, alkaline phosphatase after BDL. However, the higher enriched DHA (5P) did not have this beneficial effect at all. The dietary administration of various enriched DHA did not have any effect on the body weight gain after BDL for 4-weeks. Finally, the DHA enriched dietary did not have any observable side effect after BDL.

Acknowledgements

The authors thank the input and help by Dr. Shyi-Jou, Chen, Chief of Department of Pediatics, Tri-Service General Hospital, Taipei, Taiwan, ROC.

Financial support and sponsorship

This work was supported by Ministry of National Defense Medical Affairs Bureau grant DOD100-I-02, Taiwan, ROC.

Conflicts of interest

There are no conflicts of interest.

 
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