Trivalent Chromium Promotes Healing of Experimental Colitis in Mice by Suppression of Inflammation and Oxidative Stress

Ulcerative colitis (UC) has reactive oxygen species (ROS) and immunologic pathways implicated in its pathogenesis. The search for new therapeutic protocols in managing UC is tailored in suppressing or preventing these pathways. The influence of trivalent chromium (Cr ³⁺), an essential mineral on experimental colitis was investigated. Mice were grouped into 3; group 1 (control) received clean drinking water while groups 2 and 3 received 10 and 100 ppm Cr ³⁺ respectively for 12 weeks through drinking water. After Cr ³⁺ administration, 5 animals per group were sacrificed on day 0. Thereafter, experimental colitis was induced intra-rectally using acetic acid (4%, 0.3mL) and 5 mice per group were subsequently sacrificed on days 3, 7 and 14. Blood and colonic tissues were obtained and processed appropriately. Blood Cr ³⁺ level, haematological variables, gross and microscopic colitis scores, colonic myeloperoxidase (MPO), Superoxide Dismutase (SOD) and malondialdehyde (MDA) levels were determined using standard methods. Colon cytokine mRNA genes were quantified using real-time PCR. There was a significant decrease in colon gross and histology scores on days 3 and 7 in chromium treated compared with control. The MPO and MDA in chromium groups reduced significantly compared with control while SOD activities increased significantly in Cr ³⁺ groups compared with control. Total RNA increased in chromium groups compared with control on day 3 post-colitis. There was up-regulation of IL-10, down-regulation of TNF- α and IFN- λ in chromium administered groups compared with control. Chromium enhanced healing of colitis by suppression of ROS, inflammation and promotion of antioxidant activities.

• Colitis
• Oxidative Stress
• Trivalent Chromium
• Inflammation
• Mice

1. Introduction

Inflammatory bowel disease (IBD) comprises of both ulcerative colitis (UC) and Crohn’s disease (CD) which is often characterized by extensive and severe intestinal inflammation resulting from deranged immune system in response to increasing loads of commensal micro-biota [1] . Although, the pathophysiology is still vague, reactive oxygen species ( ROS ) and immunologic processes are key theories that have been proposed as the possible mechanism of its pathologic nature [2] [3] [4] [5] . This has driven the search for therapeutic strategies that can be focused or targeted to the needs of individual patients. Conditions such as the patient’s genetic components, disease states, and environmental influences have all played vital roles in the treatment and prognosis of the disease [5] [6] . Nevertheless, increase in antioxidant activities are also known to provide relieve for these conditions [7] [8] .

Chromium, specifically trivalent (Cr³⁺) is an essential mineral that human requires in trace amounts, although its mechanism of actions and the quantity that is beneficial to health have no clear-cut definitions [9] . It exists mainly in two forms, trivalent which are the more stable bio-available forms and present in food substances; hexavalent, the toxic form available from industrial pollutions. Chromium in this study is limited to trivalent form and has been reported to promote actions of insulin [10] [11] , aid metabolism of fats, carbohydrates and protein [12] [13] , among other beneficial effects.

The non-specificity of available treatment in the clinical management of ulcerative colitis and the increasing use of trivalent chromium as dietary supplement have increased our curiosity towards evaluating the probable activities of oral chromium on normal colon and experimental colitis. This study aim was to investigate the effect of chromium exposure basically on the reactive oxygen species and inflammatory pathways in both normal and experimental colitis in mice.

2. Materials and Methods

2.1. Ethical Considerations

This study was carried out in strict adherence to the recommendations in the Guide for the Care and Use of Laboratory Animals released by US National Institute of Health [14] . The protocol was approved by the Committee of Animal Care and Use of Maebashi Institute of Technology, Japan (No. 15-009).

2.2. Animals and Treatment Protocols

Sixty male, slc:ddY mice ( 25.3 ± 2.1 g, 5 weeks old) were purchased from SLC Incorporation, Japan, kept in the animal room at the Department of Biotechnology, Maebashi Institute of Technology and were used for the experiments. The animals were acclimatized for 2 weeks with access to standard feeds and water ad libitum. They were grouped into 3: Control (n = 20) were allowed free access to clean drinking water and groups 2 and 3, were administered oral 10 ppm, (n = 20) and 100 ppm (n = 20) trivalent chromium respectively through their drinking water for 12 weeks.

2.3. Chemicals and Drugs

Chromium was obtained from Koshin Chemicals, Japan. Hexadecyltrimethylammonium Bromide, o-Dianisidine Dihydrochloride, and Sodium nitrite were procured from Tokyo Chemical Industry, Co., Limited, Japan. All Other chemicals were of highest purity and analytical grade. RNA retraction and reverse transcription kits were obtained from Qiagen, Japan. Real-time PCR kits were obtained from Brilliant Agilent^®, UK; Primers for standards and RT-PCR assays all from qStandard^®, London.

2.4. Acute Colitis Induction, Stool Consistency and Gross Assessment of Colonic Damage

After Cr³⁺ exposure, mice (n = 5 per group) were sacrificed (day 0) for gross, histology, haematological variables, biochemical parameters and gene expression studies. Thereafter, experimental colitis was induced using the modified method by Choudhary et al. (2001) [15] . Briefly, 0.1 mL of 4% acetic acid was introduced using a 3 mm soft paediatric catheter which was advanced 3 cm from anal opening under light ketamine anaesthesia (0.01 mL/g) for 60 seconds in 24 h fasted mice. Mice were maintained in a head-down position for 30 seconds following introduction of acetic acid to prevent immediate extrusion of the solution (Control had normal saline of the same proportion instilled). Stool consistency was scored starting from 24 h post induction and daily for 7 days after colitis induction using the method described by Fukuda et al. [16] where 0 = normal stool, 1 = loose stool without visible blood, 2 = loose stool with visible blood, 3 = bloody diarrhea. The mice were anaesthetized with xylaxine (0.0005 mL/g) and ketamine (0.015 mL/g) cocktails. Thereafter, laparotomy was conducted and the distal 8 cm of the colon was resected for gross colitis scores, histology, biochemical assessment and cytokine mRNA gene expression on day 0 (before colitis induction) and days 3, 7 and 14 after induction of colitis in the mice. The gross scoring method described by Morris et al. [17] where, no damage = 0, localized hyperemia with no ulcers = 1, linear ulcers with no significant inflammation = 2, linear ulcer with inflammation at one site = 3, more site of ulcers and inflammation, the size of ulcer <1 cm =4 and multiple inflammations and ulcers, the size of ulcer >1 cm =5 was adopted.

2.5. Histology and Histomorphometry Assessment of Colon Tissues

All sections for light microscopy were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin (H&E). Accuscope TS view, China was used to capture images and to evaluate morphological changes and measurement of histomorphometry variables.

2.6. Determination of Blood Parameters

Blood samples (1 mL) were analyzed using an automated machine following blood sample collection from cardiac puncture into a 5 mL heparinized bottle. Each of the samples collected were ran sequentially using KX-21 haematological analyzer by Symex Kobe^® Japan.

2.7. Lipid Peroxidation Determination

This was assessed by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method described by Varshney and Kale [18] . The method described the value of malondialdehyde (MDA) in the tissue homogenates during lipid peroxidation with thiobarbituric acid (TBA) forming a pinkish MDA-TBA complex that was read spectrophotometrically at 532 nm.

2.8. Determination of Antioxidant Status in Colonic Tissues

Catalase activity was determined according to the method described by Sinha [19] . The procedure described the reduction of dichromate in the presence of acetic acid to chromic acetate when heated in the presence of H₂O₂, with the formation of perchromic acid as an unstable intermediate. The resultant chromic acetate was determined spectrophotometrically at 530 nm. Superoxide Dismutase (SOD) method described by Misra and Fridovich [20] was adopted using o.5 mL of tissue homogenates which was added to 2.5 ml of 0.05 M carbonate buffer at pH 10.2 to equilibrate in glass cuvette. The reaction commenced with the addition of 0.3 mL freshly prepared 0.3 mM epinephrine. Increase in absorbance at 480 nm was observed every 30 s for 1 min and 1 unit of SOD activity was given as amount of SOD expected to cause 50% inhibition of the oxidation of adrenaline.

2.9. Determination of Total Nitrite

Total tissue nitrite determination was done using the method described by Ignarro et al. [21] . The assay relies on a diazotization reaction that was originally described by Griess [22] , which uses sulfanilamide and N-1-napthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions.

2.10. Determination of Myeloperoxidase Activity

The method described by Kim et al. [23] was adopted using tissue homogenates.

2.11. RNA Extraction and Quantification of Absolute Real-Time PCR

Total RNA was obtained with RNeasy Mini Kit from Qiagen, Japan and the protocol was followed strictly. Briefly, 30 mg of sample were stored in RNAlater RNA stabilization reagent, (Qiagen^®) pending homogenization. Tissue was thawed on ice and subsequently homogenized after lysing with appropriate lysing buffer at 4˚C. The lysate was centrifuged for 3 minutes at maximum speed and supernatant was carefully pipetted. Ethanol (70%) was added to the pipetted supernatant, volume for volume and mixed well. The mixture (700 μL) was added to 2 mL RNeasy spin column and centrifuged for 15 seconds at >8000 x g. The flow-through was discarded and another 700 μL wipeout buffer (RW1) added to the spin column, this was centrifuged at >8000 x g for 15 sec.

The flow-through was also discarded and 500 μL RPE buffer added to wash out membrane-bound RNA and also centrifuged for 15 seconds at >8000 x g. This was repeated for 2 minutes. The spin-column is now placed in a new collection tube and spin for 1 minute to dry the membrane. Then 50 μL of RNase-free water was added to the column directly and centrifuged for 1 minute at >8000 x g to elute the RNA. The eluted fraction was quantified using 1 μL of total RNA sample on Nanodrop^® 2000 spectrophotometer at 260/280nm. Total RNA was further expressed on 2% Agarose gel electrophoresis. The eluted RNA was then stored at −80˚C pending reverse transcription, thereafter 150 ng/μL RNA was reverse transcribed into complementary DNA.

QuantiTect Reverse Transcription Kit from Qiagen^® was used and all reactions set-up was on ice. Briefly, genomic DNA elimination reaction was done using gDNA wipeout buffer, template RNA and RNase-free water mixture incubated for 2 minutes at 42˚C, then returned immediately on ice. The reverse- transcriptase master mix was constituted with 1 μL Quantiscript reverse transcriptase, 4 μL Quantiscript RT Buffer and 1 μL RT Primer mix all added to the entire purified genomic DNA reaction described above and quickly returns on ice. This was incubated for 15 minutes at 42˚C. Incubation was repeated for 3 minutes at 95˚C to inactivate Quantiscript Reverse Transcriptase. The reverse-transcription reaction was then placed on ice and immediately proceeds for real time PCR. Prior to use in PCR, the cDNA was diluted 10-folds (1:10) by tRNA (10 μg/mL), to reduce contamination if any.

Thereafter, Brilliant III Ultra-Fast SYBR^® Green QPCR Master Mix obtained from Agilent Technologies, UK. Standard assays, primers and cytokine assays were all gotten from qStandards^®, UK with the following document number for standards, mmu_Actb_001; mmu_Gapdh_001; mmu_Rpl13_002; mmu_Ifng_001, mmu_Il1a_001; mmu_Il6_001; mmu_Il10-001 and mmu_Tnf_001 were used. Applied Biosystems^® 7500 Fast Real-Time PCR System was used to amplify the eight mRNA genes, three were reference genes (B-Actin, Gapdh and Rpl13) and five cytokines of interest (IL-1α, IL-6, IL-10, TNF-α and IFN-λ) (Table 1).

The protocols were strictly followed. Briefly, a total volume of 20μL was used for the PCRs. 4 μL diluted cDNA, 10 μL Brilliant III SYBR green mix, 1 μL forward primer, 1 μL reverse primer and 4 μL Samples were first denatured at 95˚C for 3 min, this was followed by amplification for 40 cycles; denaturation, 95˚C

Table 1

Oligonucleotide primers used for real time quantitative PCR

Primer Sequence
Genes β-actin GAPDH RPL13 IFN-λ IL-1α IL-6 IL-10 TNF-α	Forward 5’-caccatgaagatcaagatcattgct-3’ 5’-catttcctggtatgacaatgaatacg-3’ 5’-tgattggcgtttgagattggc-3’ 5’-cggcacagtcattgaaagcc-3’ 5’-cgcttgagtcggcaaagaaat-3’ 5’-gccttcttgggactgatgct-3’ 5’-gccgggaagacaataactgc-3’ 5’-gtctactgaacttcggggtgat-3’	Reverse 5’-taaaacgcagctcagtaacagt-3’ 5’-ggatagggcctctcttgctc-3’ 5’-gcttcagtatcatgccattccg-3’ 5’-tgtcaccatccttttgccagt-3’ 5’-tggcagaactgtagtcttcgt-3’ 5’-gccattgcacaactcttttctca-3’ 5’-ttggcaacccaagtaaccct-3’ 5’-tgagaagatgatctgagtgtgagg-3’

for 5 sec, annealing and extension was at 60˚C for 27 seconds and ramping was done between 65˚C and 95˚C rising by 1˚C per step. Polymerase chain reaction amplification was performed in triplicate. The standard curves for each gene were generated using the specific standard fragments from qStandards^®. The absolute copy numbers were generated by the PCR machine and the amounts determined by normalization against the three reference genes were subsequently analyzed.

The RNA and PCR products were subjected to electrophoresis on 1.5 % Agarose gels with 1 × running buffer Tris-Acetic acid-EDTA (TAE × 1), at 50 V for 90 min and visualized by means of ethidium bromide stain under ultra violet light before they were transferred for digital gel photographs with Quantum LAS 4000.

2.12. Statistical Analysis

Data were recorded as mean ± S.E.M and analyzed using descriptive statistics one way ANOVA followed by a post-hoc test (Newman-Keul’s comparison test) with GraphPad Prism version 5 (GraphPad software, San Diego, CA). Differences were considered significant at P < 0.05.

3. Results

3.1. Effect of Oral Chromium on Stool Consistency in Acetic Acid-Induced Colitis

The stool consistency scores were significantly low in the chromium groups by days 3, 4 and 5 following colitis induction compared with control group (Figure 1).

3.2. Effects of Oral Chromium on Colon Macro and Micro Architecture (MAG X100), Pre- and Post-Induction of Colitis

The colon histoarchitecture varied with significant increase in crypt heights in the chromium groups, 10 ppm (163.1 ± 10.59 μm) and 100 ppm (165.71 ± 12.26 μm) compared to control (132.13 ± 8.24 μm). Mucosa width increased signify-

References

1. Abraham, C. and Medzhitov, R. (2011) Interactions between the Host Innate Immune System and Microbes in Inflammatory Bowel Disease. Gastroenterology, 140, 1729-1737. https://doi.org/10.1053/j.gastro.2011.02.012

2. Keshavarzian, A., Morgan, G., Sedghi, S., Gordon, J.H. and Doria, M. (1990) Role of Reactive Oxygen Metabolites in Experimental Colitis. Gut, 31, 786-790.https://doi.org/10.1136/gut.31.7.786

3. Grisham, M.B. (1994) Oxidant and Free Radicals in Inflammatory Bowel Disease. Lancet, 344, 859-861. https://doi.org/10.1016/S0140-6736(94)92831-2

4. Millar, A.D., Rampton, D.S., Chander, C.L., Claxson, A.W., Blades. S., Coumbe, A. et al. (1996) Evaluating the Antioxidant Potential of New Treatments for Inflammatory Bowel Disease Using a Rat Model of Colitis. Gut, 39, 407-415.https://doi.org/10.1136/gut.39.3.407

5. Fiocchi, C. (1998) Inflammatory Bowel Disease: Etiology and Pathogenesis. Gastroenterology, 115, 182-205. https://doi.org/10.1016/S0016-5085(98)70381-6

6. Podolsky, D.K. (2002) Inflammatory Bowel Disease. New England Journal of Medicine, 347, 417-429. https://doi.org/10.1056/NEJMra020831

7. Nosal’ova, V., Cerna, S. and Bauer, V. (2000) Effect of N-Acetylcysteine on Colitis Induced by Acetic Acid in Rats. General Pharmacology, 35, 77-81.https://doi.org/10.1016/S0306-3623(01)00094-5

8. Kanodia, L., Borgohain, M. and Das, S. (2011) Effect of Fruit Extract of Fragaria vesca Leave on Experimentally Induced Inflammatory Bowel Disease in Albino Rats. Indian Journal of Pharmacology, 43, 118-121.https://doi.org/10.4103/0253-7613.75660

9. National Institute of Health (NIH) (2013) Chromium Dietary Supplement Facts Sheet. National Institute of Health, Bethesda, 1-7.

10. Mertz W. ,et al. (1993)Chromium in Human Nutrition: A Review Journal of Nutrition 123, 626-633.

11. Mertz, W. (1998) Interaction of Chromium with Insulin: A Progress Report. Nutritional Review, 56, 174-177. https://doi.org/10.1111/j.1753-4887.1998.tb06132.x

12. Stoecker, B.J. (2001) Chromium. In: Bowman, B. and Russell, R., Eds., Present Knowledge in Nutrition, 8th Edition, ILSI Press, Washington DC, 366-372.

13. Vincent, J.B. (2003) The Potential Value and Toxicity of Chromium Picolinate as a Nutritional Supplement, Weight Loss Agent and Muscle Development Agent. Sports Medicine, 33, 213-230. https://doi.org/10.2165/00007256-200333030-00004

14. National Institute of Health Publication (1996) Guide for the Care and Use of Laboratory Animals. Revised No. 85-23.

15. Choudhary, J., Blackstock, W., Creasy, D. and Cottrell, J. (2001) Matching Peptide Mass Spectra to EST and Genomic DNA Databases. Trends Biotechnology, 19, S17-S22.

16. Fukuda, R., Hirota, K., Fan, F., et al. (2002) Insulin-Like Growth Factor 1 Induces Hypoxia-Inducible Factor 1-Mediated Vascular Endothelial Growth Factor Expression, Which Is Dependent on MAP Kinase and Phosphatidylinositol 3-Kinase Signaling in Colon Cancer Cell. The Journal of Biological Chemistry, 277, 38205-38211. https://doi.org/10.1074/jbc.M203781200

17. Morris, G.P., Beck, P.L., Herrigge, M.S., et al. (1989) Hapten Induced Model of Chronic Inflammation and Ulceration in the Rat Colon. Gastroenterology, 96, 795-803.

18. Varshney, R. and Kale, R.K. (1990) Effect of Calmodulin Antagonists on Radiation Induced Lipid Peroxidation in Microsomes. International Journal of Biology, 158, 733-741. https://doi.org/10.1080/09553009014552121

19. Sinha K.A. ,et al. (1972)Colorimetric Assay of Catalase Analytical Biochemistry 47, 389-394.

20. Misra, H.P. and Fridovich, I. (1972) The Role of Superoxide Anion in the Autoxidation of Epinephrine and a Simple Assay for Superoxide Dismutase. Journal of Biological Chemistry, 25, 3170-3175.

21. Ignarro, L.J., Buga, G.M., Wood, K.S., et al. (1987) Endothelium-Derived Relaxing Factor Produced and Released from Artery and Vein Is Nitric Oxide. Proceedings of National Academy of Sciences, 84, 9265-9269. https://doi.org/10.1073/pnas.84.24.9265

22. Griess, P. (1879) Bemerkungen zu der abhandlung der H.H., Weselsky und Benedikt. “Ueber einige azoverbindungen.” Chemische Berichte, 12, 426-428. https://doi.org/10.1002/cber.187901201117

23. Kim, J.J., Shajib, M.S., Manocha, M.M., et al. (2012) Investigating Intestinal Inflammation in DSS-Induced Model of IBD. Journal of Visual Experiments, 60, e3678. https://doi.org/10.3791/3678

24. Elson, C.O., Sartor, R.B., Tennyson, G.S. and Riddell, H. (1995) Experimental Models of Inflammatory Bowels Disease. Gastroenterology, 109, 1344.

25. Smyth, S.S., McEver, R.P., Weyrich, A.S., et al. (2009) Platelet Colloquium Participants. Platelet Functions beyond Hemostasis. Journal of Thrombosis Haemostasis, 7, 1759-1766. https://doi.org/10.1111/j.1538-7836.2009.03586.x

26. Morrell, C.N., Aggrey, A.A., Chapman, L.M. and Modjeski, K.L. (2014) Emerging Roles for Platelets as Immune and Inflammatory Cells. Blood, 123, 2759-2767. https://doi.org/10.1182/blood-2013-11-462432

27. Lih-Brody, L. Powell, S.R., Collier, K.P., et al. (1996) Increased Oxidative Stress and Decreased Antioxidant Defenses in Mucosa of Inflammatory Bowel Disease. Digestive Diseases and Sciences, 41, 2078-2086. https://doi.org/10.1007/BF02093613

28. Thomson, A., Hemphill, D. and Jeejeebhoy, K.N. (1998) Oxidative Stress and Antioxidants in Intestinal Disease. Digestive Diseases and Sciences, 16, 152-158. https://doi.org/10.1159/000016859

29. Bhattacharyya, A., Chattopadhyay, R., Mitra, S. and Crowe, S.E. (2014) Oxidative Stress: An Essential Factor in the Pathogenesis of Gastrointestinal Mucosal Diseases. Physiological Reviews, 94, 329-354. https://doi.org/10.1152/physrev.00040.2012

30. Iles, K.E., Dickinson, D.A., Watanabe, N., et al. (2002) AP-1 Activation through Endogenous H2O2 Generation by Alveolar Macrophages. Free Radical Biology and Medicine, 32, 1304-1313.

31. Pechova, A. and Pavlata, L. (2007) Chromium as an Essential Nutrient: A Review. Veterinarni Medicina, 52, 1-18.

32. Okada, S., Tsukada, H. and Ohba, H. (1983) Enhancement of Ribonucleic Acid Synthesis by Chromium (III) in Regeneratin Rat Liver. Journal of Inorganic Biochemistry, 19, 95-103.

33. Deisenroth, C. and Zhang, Y. (2010) Ribosome Bio-genesis Surveillance: Probing the Ribosomal Protein Mdm2-p53 Pathway Oncogene. Epub, 29, 204253-204260. https://doi.org/10.1038/onc.2010.189

34. Steffen, K.K., McCormick, M.A., Pham, K.M., et al. (2012) Ribosome Deficiency Protects against ER Stress in Saccharomyces cerevisiae. Genetics, 191, 107-118. https://doi.org/10.1534/genetics.111.136549

35. Wan, W., Lim, J.K., Lionakis, M.S., et al. (2011) Genetic Deletion of Chemokine Receptor Ccr6 Decreases Atherogenesis in ApoE-Deficient Mice. Circulatory Research, 109, 374-381. https://doi.org/10.1161/CIRCRESAHA.111.242578

36. Wier, E.M., Neighoff, J., Sun, X., et al. (2012) Identification of an N-Terminal Truncation of the NF-Kappa B p65 Subunit That Specifically Modulates Ribosomal Protein S3-Dependent NF-kappaB Gene Expression. The Journal of Biological Chemistry, 287, 430. https://doi.org/10.1074/jbc.M112.388694

37. Yang, H.J., Youn, H., Seong, K.M., et al. (2013) Phosphorylation of Ribosomal Protein S3 and Antiapoptotic TRAF2 Protein Mediates Radio Resistance in Non-Small Cell Lung Cancer Cells. Journal of Biological Chemistry, 288, 2965-2975. https://doi.org/10.1074/jbc.M112.385989

38. Tsirogianni, A.K., Moutsopoulos, N.M. and Moutsopoulos, H.M. (2006) Wound Healing: Immunological Aspects. Injury, 375, S5-S12.

39. WanYong, H., Huynh, K.Y., Swee, K.Y., et al. (2009) Traditional Practice, Bioactivities and Commercialization Potential of Elephantopus scaber Linn. Journal of Medicinal Plant Research, 3, 1212-1221.

40. Cheville, N.F. (1999) Introduction to Veterinary Pathology. 2nd Edition, Iowa State University Press, Ames, 118-119.

41. Vitarbo, E.A., Chatzipanteli, K., Kinoshita, K., et al. (2004) TNF Expression and Protein Levels Following Fluid Percussion Injury in Rats: The Effect of Injury Severity and Brain Temperature. Neurosurgery, 55, 416-425. https://doi.org/10.1227/01.NEU.0000130036.52521.2C

42. Seno, H., Miyoshi, H., Brown, S.L., et al. (2009) Efficient Colonic Mucosal Wound Repair Requires Trem2 Signaling. Proceedings of National Academy of Sciences, 106, 256-261. https://doi.org/10.1073/pnas.0803343106

43. Goresky, T., Dirisina, R., Sinh, P., et al. (2012) p53 Mediates TNF-Induced Epithelial Cell Apoptosis in IBD. American Journal of Pathology, 181, 1306-1315.

44. Fan, L., Young, P.R., Barone, F.C., et al. (1996) Experimental Brain Injury Induces Differential Expression of Tumor Necrosis Factor-Alpha mRNA in the CNS. Brain Research. Molecular Brain Research, 36, 287-291.

45. Knoblach, S.M., Fan, L. and Faden, A.I. (1999) Early Neuronal Expression of Tumor Necrosis Factor after Experimental Brain Injury Contributes to Neurological Impairment. Journal of Neuroimmunology, 95, 115-125.