ABSTRACT

The study aimed to investigate the effect of methionine deficiency on conventional nutrient composition, NO and NOS activity in the liver and kidney, and expression of genes related to the NF-κB signal pathway. One hundred 1-day-old broilers were divided into control and methionine deficiency group, and provided with a standard diet. The methionine-deficient group was fed with a methionine-deficient diet for 42 days. Moisture content, protein content, and crude lipid content were determined by freeze drying, the Kjeldahl method, and the ether extraction method. The concentration of NO was measured using the nitrate reductase method, and the activity of NOS was measured using colorimetry. mRNA expression of related genes in the NF-κB signal pathway was detected by reverse transcription-polymerase chain reaction (RT-PCR). The results showed the contents of crude protein and crude lipid in the liver and kidney of the methionine-deficient group were significantly higher than those in the control group (p<0.05 or p<0.01). There was no significant change in dry matter (p>0.05). Methionine deficiency resulted in significantly increased NO concentration and total nitric oxide synthase (TNOS) activities in the liver and kidney (p<0.01). In contrast, cNOS and inducible iNOS activities significantly decreased (p<0.01). The expression of NF-κB, TNF-α, IFN-γ, IL-1, and IL-6 mRNA, which are related to the NF-κB signal pathway of the methionine-deficient group, were significantly higher than those in the control group (p<0.05 or p<0.01). It is concluded that long-term feeding diets lacking methionine will lead to changes in nutrient composition and inflammatory injury of the liver and kidney.

Keywords:
Kidney; liver; methionine deficiency; NO; NOS

INTRODUCTION

Methionine is known as the first limiting amino acid and is also an essential functional amino acid for poultry. In addition to participating in the body’s protein synthesis, methionine is also related to cell proliferation, differentiation, apoptosis, and oxidative stress, as well as the regulation of intestinal immune function and the elimination of free radicals (Wu et al., 2017Wu P, Tang L, Jiang W. The relationship between dietary methionine and growth, digestion, absorption, and antioxidant status in intestinal and hepatopancreatic tissues of sub-adult grass carp (Ctenopharyngodon idella). Journal of Animal Science and Biotechnology 2017;8(4):970-83. https://doi.org/10.1186/s40104-017-0194-0
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https://doi.org/10.1556/004.2018.006... ; Séité et al., 2018Séité S, Mourier A, Camougrand N, et al. Dietary methionine deficiency affects oxidative status, mitochondrial integrity, and mitophagy in the livers of rainbow trout (Oncorhynchus mykiss). Scientific Reports 2018;8(1):10151-64. https://doi.org/10.1038/s41598-018-28559-8
https://doi.org/10.1038/s41598-018-28559... ). Methionine side chains are located on the surface of proteins and act as endogenous antioxidants in proteins (Levine et al., 1996Levine RL, Mosoni L, Berlett BS, et al. Methionine residues as endogenous antioxidants in proteins. Proceedings of the National Academy of Sciences of the United States of America 1996;93(26):15036-40. https://doi.org/10.1073/pnas.93.26.15036
https://doi.org/10.1073/pnas.93.26.15036... ). Methionine, which is exposed on the cell surface, could protect other amino acids and act as an antioxidant to prevent cell damage (Salmon et al., 2016Salmon AB, Kim G, Liu C, et al. Effects of transgenic methionine sulfoxide reductase A (MsrA) expression on lifespan and age-dependent changes in metabolic function in mice. Redox Biology 2016;10:251-256. https://doi.org/10.1016/j.redox.2016.10.012
https://doi.org/10.1016/j.redox.2016.10.... ). Methionine can react with reactive oxygen species and produce two types of methionine sulfoxide isomers: S-methionine sulfoxide and R-methionine sulfoxide (Moskovitz, 2005Moskovitz J. Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation and prevention of aging-related diseases. Biochimica et Biophysica Acta 2005;1703(2):213-9. https://doi.org/10.1016/j.bbapap.2004.09.003
https://doi.org/10.1016/j.bbapap.2004.09... ). These two isomers can be reduced to methionine under the action of methionine sulfoxide reductase. The decrease of methionine sulfoxide reductase activity is closely related to oxidative stress diseases, and its overexpression is beneficial to protect the body from oxidative stress damage and maintain the stability of protein spatial conformation (Cabreiro et al., 2006Cabreiro F, Picot CR, Friguet B, et al. Methionine sulfoxide reductases: relevance to agingand protection against oxidative stress. Annals of the New York Academy of Sciences 2006;1067:37-44. https://doi.org/10.1196/annals.1354.006
https://doi.org/10.1196/annals.1354.006... ).

The content of nutrients in the body is closely related to life activities. Conventional nutrients include water, crude protein, fat, and dry matter. Among them, water is an important part of the animal body, being the carrier of various nutrients and metabolites, and also an indispensable substance for temperature regulation (Häussinger, 1996Häussinger D. The role of cellular hydration in the regulation of cell function. Biochemical Journal 1996;313(3):697-710. https://doi.org/10.1042/bj3130697
https://doi.org/10.1042/bj3130697... ; Montain el al., 1999Montain SJ, Latzka WA, Sawka MN. Fluid replacement recommendations for training in hot weather. Military Medicine 1999;164(7) 502-8. https://doi.org/10.1093/milmed/164.7.502
https://doi.org/10.1093/milmed/164.7.502... ). Protein is essential for repairing body tissues and can replace the thermogenic effects of carbohydrates and fats. When the heat supply is insufficient, proteins can decompose in the body and oxidize to release heat energy (Hayamizu, 2017Hayamizu K. Amino acids and energy metabolism: an overview. Sustained Energy for Enhanced Human Functions and Activity 2017:339-49. https://doi.org/10.1016/B978-0-12-805413-0.00021-1
https://doi.org/10.1016/B978-0-12-805413... ). Excess protein can be stored in the liver, blood, and muscles or be converted into fat by deamination; it will be re-decomposed for heat supply when nutrients are insufficient. Fat is the main source of animal heat energy, the best form of chemical energy storage in the body and an important component of animal tissues. Fat is used as the solvent of liposoluble vitamins in feed to ensure the digestion, absorption, and utilization of liposoluble vitamins by animals (Wei et al., 2020Wei L, Surma M, Yang Y, et al. ROCK2 inhibition enhances the thermogenic program in white and brown fat tissue in mice. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 2020;34(1):474-93. https://doi.org/10.1096/fj.201901174RR
https://doi.org/10.1096/fj.201901174RR... ; Vanqa, 2022Vanqa N, Mshayisa VV, Basitere M. Proximate, physicochemical, techno-functional and antioxidant properties of three edible insect (Gonimbrasia belina, Hermetia illucens and Macrotermes subhylanus) flours. Foods 2022;11(7):976. https://doi.org/10.3390/foods11070976
https://doi.org/10.3390/foods11070976... ).

Oxidative stress is a state in which the organism produces more free radicals, or the ability to eliminate them decreases, and the imbalance of oxidation and antioxidant system leads to body injury. A free radical is an atom or group of atoms created in this process that can exist independently and contain unpaired electrons (Laederach et al., 2007Laederach A, Shcherbakova I, Jonikas MA, et al. Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding. Proceedings of the National Academy of Sciences USA 2007;104(17):7045-50. https://doi.org/10.1073/pnas.0608765104
https://doi.org/10.1073/pnas.0608765104... ). Active nitrogen radicals are free radicals that cause oxidative stress, including nitric oxide (NO), nitrogen dioxide (NO₂), and nitrite peroxide (ONOO-), which are called active nitrogen intermediates (MacMicking et al., 1997MacMicking J, Xie Q, Nathan C. Nitric oxide and macrophage function. Annual Review of Immunology 1997;15(1):323-50. https://doi.org/10.1146/annurev.immunol.15.1.323
https://doi.org/10.1146/annurev.immunol.... ). NO is produced by cytokines in the pathological state of the body, which are new messenger molecules that can affect transcription factors and serve as cytotoxic molecules with dual protective and toxic effects. While NO is involved in physiological processes, its catalytic production of nitric oxide synthase (NOS) also plays an important role in many disease processes (Ohshima & Bartsch, 1994Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 1994;305(2):253-64. https://doi.org/10.1016/0027-5107(94)90245-3
https://doi.org/10.1016/0027-5107(94)902... ; Abd-Ellatef et al., 2017Abd-Ellatef GF, Ahmed OM, Abdel-Reheim ES, et al. Ulva lactuca polysaccharides preventWistar rat breast carcinogenesis through the augmentation of apoptosis, enhancement of antioxidant defense system, and suppression of inflammation. Breast Cancer: Targets and Therapy 2017;9:67-83. https://doi.org/10.2147/BCTT.S125165
https://doi.org/10.2147/BCTT.S125165... ). Active nitrogen intermediates are directly related to NOS. Therefore, the amount of NO is closely related to the activity of NOS, and they also have a certain impact on the oxidative stress state of the body. At present, the expression characteristics of NOS in cerebral ischemia, cerebral oedema, and colitis are widely discussed in important studies (Krieglstein et al., 2001Krieglstein CF, Cerwinka WH, Laroux FS, et al. Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide andnitric oxide. The Journal of Experimental Medicine 2001;194(9):1207-18. https://doi.org/10.1084/jem.194.9.1207
https://doi.org/10.1084/jem.194.9.1207... ; Zhou et al., 2010Zhou L, Li F, Xu HB, et al. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nature Medicine 2010;16(12):1439-43. https://doi.org/10.1038/nm.2245
https://doi.org/10.1038/nm.2245... ), but the influence of methionine deficiency on its activity is rarely studied. As a common signaling pathway, the NF-κB signaling pathway controls many genes related to inflammation and plays an important regulatory role in inflammatory response and apoptosis. The function of the cell during inflammation depends on its signaling response to activation with neighboring cells and a combination of hormones, which particularly has an effect on cytokines through specific receptors (Alharbi et al., 2021Alharbi KS, Fuloria NK, Fuloria S, et al. Nuclear factor-kappa B and its role in inflammatory lung disease. Chemico-Biological Interactions 2021;345:109568. https://doi.org/10.1016/j.cbi.2021.109568
https://doi.org/10.1016/j.cbi.2021.10956... ; Chaithongyot et al., 2021Chaithongyot S, Jantaree P, Sokolova O, et al. NF-kappaB in gastric cancer development and therapy. Biomedicines 2021;9(8):870-91. https://doi.org/10.3390/biomedicines9080870
https://doi.org/10.3390/biomedicines9080... ; Zinatizadeh et al., 2021Zinatizadeh MR, Schock B, Chalbatani GM, et al. The Nuclear factor kappa B (NF-κB) signaling in cancer development and immune diseases. Genes & Diseases 2021;8(3):287-97. https://doi.org/10.1016/j.gendis.2020.06.005
https://doi.org/10.1016/j.gendis.2020.06... ).

The liver is the main organ of metabolism and synthesis in the body. It plays a vital role in immunity, heat production, and the metabolism of fat, sugar, protein, and other substances (Hoekstra et al., 2013Hoekstra LT, de Graaf W, Nibourg GA, et al. Physiological and biochemical basis of clinical liver function tests: a review. Annals of Surgery 2013;257(1):27-36. https://doi.org/10.1097/SLA.0b013e31825d5d47
https://doi.org/10.1097/SLA.0b013e31825d... ). The kidney is the main place for the excretion of biological metabolites, which can filter blood impurities and maintain the metabolic balance of the body (Kim et al., 2019Kim AD, Lake BB, Chen S, et al. Cellular recruitment by podocyte-derived pro-migratory factors in assembly of the human renal filter. IScience 2019;20:402-14. https://doi.org/10.1016/j.isci.2019.09.029
https://doi.org/10.1016/j.isci.2019.09.0... ). As the main organs of body metabolism, they are closely related to the oxygen content in the body, thus affecting the free radicals produced by the oxidative metabolism. At present, the immunological effects of the liver have been deeply studied, and the links between antigen-presenting cells, KC cells, and NK cells in the liver and liver disease have been systematically explained (Gao et al., 2009Gao B, Radaeva S, Park O. Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases. Journal of Leukocyte Biology 2009;86(3):513-28. https://doi.org/10.1189/JLB.0309135
https://doi.org/10.1189/JLB.0309135... ). At the same time, the research direction on kidney disease mainly focuses on the pathogenesis and treatment of acute kidney injury, chronic kidney disease, and glomerular disease (Liu, 2011Liu Y. Cellular and molecular mechanisms of renal fibrosis. Nature Reviews Nephrology, 2011;7(12):684-96. https://doi.org/10.1038/nrneph.2011.149
https://doi.org/10.1038/nrneph.2011.149... ). The relationship between NO content and NOS activity in liver and kidney functions remains to be studied for broiler chickens in a methionine-deficient state.

Therefore, this study used broiler chickens to explore the effects of changes in conventional nutrients, NO content, NOS activity, and gene expression related to the NF-κB signaling pathway in the liver and kidney during methionine deficiency. This study aimed to investigate the role of methionine in liver and kidney metabolism, oxidative stress, and inflammatory damage and to provide a theoretical basis for the preparation of feed for the breeding industry.

MATERIALS AND METHODS

Experimental animals and groups

100 1-day-old AA broiler chickens (39±3g) were purchased from Chengdu Wenjiang Zhengda Breeding Co., LTD. The broilers were randomly divided into two groups (control and methionine deficiency groups), with 50 birds in each group. Each group was raised in an experimental cage, and the management method was consistent with conventional brooding. The experiment lasted for 42 days, as shown in Table 1.

Both the control group and methionine deficiency diets were prepared from corn, soybeans, and wheat purchased from Jining, Shandong, and the feed formula was based on (Wu et al., 2018Wu BY, Zhu M, Ruan T, et al. Oxidative stress, apoptosis and abnormal expression of apoptotic protein and gene and cell cycle arrest in the cecal tonsil of broilers induces by dietary methionine deficiency. Research in Veterinary Science 2018;121:65-75. https://doi.org/10.1016/j.rvsc.2018.10.009
https://doi.org/10.1016/j.rvsc.2018.10.0... ). The rest of the protein content, energy, vitamin, and trace element additions are added based on the nutritional standards of NRC (1994) for chickens. The control group was feed with the conventional diet, and the methionine deficiency groups were fed with a methionine-deficient diet.

Sample preparation

At 14, 28, and 42 days, 15 chickens were randomly selected in the C group and MD group. They were killed, liver and kidney tissues were taken out, isolated in a sterile environment, and washed with normal saline. Part of the liver and kidney tissue was used for nutrient determination, and part was made into 10% liver and kidney tissue homogenate, which was used to detect NO and NOS indicators. The other tissue was stored in liquid nitrogen and used for mRNA detection.

Detection of the conventional nutrient content

The dry matter, crude protein, and crude fat contents of liver and kidney tissue samples were determined. The freeze-drying method determined the moisture content (GB/T6435-1986). The content of crude protein was determined by the Kjeldahl method (GB/T6432-1994). The ether extraction method (GB/T6433-1994) determined the crude fat content.

Determination of NO concentration: nitrate reductase method

NO is chemically active, and metabolism in the body quickly changes it into NO^2- and NO^3-, and NO^2- is further converted to NO^3-. In this method, NO^3- was specifically reduced to NO^2- by nitrate reductase, and its concentration was determined by color. The NO concentration was determined according to the manufacturer’s instructions (Nitric Oxide assay kit, A012-1, purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China), as shown in Table 2.

The experimental results were calculated according to the formula of NO concentration, NO content (μmol/L) = (measured OD value - blank OD value) / (Standard OD value - blank OD value) × Standard concentration (100 μmol/L) × dilution ratio.

Determination of NOS activity: colorimetric method

NOS catalyzes the reaction of L-Arg with oxygen to produce NO, which then forms colored compounds with nucleophilic substances. The absorbance was measured at 530nm wavelength, and the NOS activity could be calculated according to the absorbance. There are two main types of NOS: cNOS and iNOS. The method was conducted according to the Nitric Oxide Synthase (NOS) typed assay kit instructions (A014-1, purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the protocols are shown in Table 3.

The subsequent steps were measuring the corresponding absorption value, using the standard product concentration as the horizontal coordinate and the corresponding OD value as the longitudinal coordinate, drawing the linear regression curve of the standard product, and finally calculating the concentration value of each sample according to the curve equation.

Detection of NF-κB signaling pathway-related gene mRNA expression

The extraction of total RNA was carried out according to the manufacturer’s instructions. After the concentration and purity were determined, the RNA was reverse-transcribed into cDNA using the PrimeScript RTreagent kit (RR037A, Takara) with gDNA Eraser. The gene sequence was obtained from GenBank, and the GAPDH gene was selected as the internal reference gene. The primer sequence was designed using Primer Express 6.0 software and synthesized by Shanghai Bioengineering Co., LTD. The primers are shown in Table 4. The qRT-PCR reactions (25 µl total volume) included 12.5 µl of SYBRR Premix Ex TaqTM II (Takara Bio, Japan), 1µl of forward and 1 µl of reverse primers, 8.5 µl of RNase-free water (Tiangen Biotech, Co., Ltd., Beijing, China), and 1 µl of cDNA. A C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA) was used to perform qRT-PCR. The thermal cycling conditions were as follows: 95ºC for 3 min, followed by 44 cycles of 95ºC for 10 s, Tm of the specific primer pair for 30 s, and 95ºC for 10 s, followed by 72ºC for 10 s. Melting curve analysis displayed only one peak for each PCR product. β-actin was used as the internal reference housekeeping gene. Expression fold changes were calculated using the 2^-ΔΔCT method.

Statistical analysis

The data obtained in this experiment were all collected using the SPSS 20.0 software and analyzed using the sample T-test. The results were expressed as mean ± standard deviation, p<0.05 meant significant difference, and p<0.01 meant extremely significant difference.

RESULT

Content of routine nutrients in the liver and kidney

As shown in Table 5, the crude protein of the MD group was significantly or extremely significantly lower than that of the C group (p<0.05 or p<0.01), and the fat content in the liver and kidney of the MD group was extremely significantly higher than that of the C group (p<0.01). At the same time, there was no significant change in dry matter.

Results were expressed as mean ± standard deviation, ^* indicated a significant difference compared with the C group (p<0.05), ^** indicated an extremely significant difference compared with the C group (p<0.01). The following tables are represented in the same way.

Changes in NO concentration in the liver and kidney

Compared with the C group, NO concentration in the liver and kidneys of the MD group increased. The concentration of NO in the liver increased significantly at 28 days (p<0.05) and was extremely significant at 42 days (p<0.01). The concentration of NO in the kidney increased extremely significantly at 42 days (p<0.01) (Figure 1).

Results were expressed as mean ± standard deviation, and ^* indicated a significant difference compared with the C group (p<0.05). ^** indicated an extremely significant difference compared with the C group (p<0.01), the following figures are the same.

Changes in TNOS activity in the liver and kidney

As shown in Figure 2, TNOS activity in the liver and kidney of the MD group was overall increased when compared with the C group. The TNOS activity in the liver increased significantly at 14 days (p<0.05), and was extremely significant at 28 days and 42 days (p<0.01). The TNOS activity in the kidney increased significantly at 28 days and 42 days (p<0.05).

Changes in cNOS activity in the liver and kidney

cNOS activity in the liver and kidneys of the MD group decreased when compared with the C group. The cNOS activity in the liver decreased extremely significantly at 28 days and 42 days (p<0.01). The cNOS activity in the kidney decreased significantly at 28 days (p<0.05) and extremely significantly at 42 days (p<0.01), which is shown in Figure 3.

Changes in iNOS activity in the liver and kidney

As shown in Figure 4, compared with the C group, iNOS activity in the liver decreased significantly at 28 days (p<0.05), and extremely significant at 42 days (p<0.01). The iNOS activity in the kidney decreased extremely significantly at 28 days and 42 days (p<0.01).

Gene expression associated with the NF-κB signaling pathway in the liver and kidney

The mRNA expressions of NF-κB signaling pathway-related genes in the liver and kidney (such as NF-κB, TNF-α, IFN-γ, IL-1, and IL-6) in the MD group were significantly or extremely significantly higher than those of the C group (p< 0.05 or p<0.01) (Table 6).

DISCUSSION

This study determined the dry matter, crude fat and crude protein, NO concentration, TNOS activity (including iNOS and cNOS), and gene expression associated with the NF-κB signaling pathway in the liver and kidney. The results showed that the content of crude fat and crude protein, NO concentration, and TNOS activity all increased in the liver and kidney when broilers were treated with a methionine deficiency diet. Meanwhile, the mRNA expressions of NF-κB signaling pathway-related genes such as NF-κB, TNF-α, IFN-γ, IL-1, and IL-6 were all significantly or extremely significantly higher than that of the control group. These results indicate that metabolic disorders occurred in the liver and kidney, with varying degrees of injuries.

In detecting the conventional nutrient content, we found that the crude fat and protein contents in the liver and kidney of the met-deficient group were significantly higher than those of the control group. It was speculated that the methionine deficiency changed the intensity of fat metabolism in the liver and kidney. Methionine can provide methyl through its metabolite s-adenosine methionine, which can work as a methyl donor to promote the synthesis of carnitine, betaine, and choline (Wang et al., 2021Wang C, Ma C, Gong L, et al. Preventive and therapeutic role of betaine in liver disease: A review on molecular mechanisms[J]. European Journal of Pharmacology 2021;912:174604. https://doi.org/10.1016/j.ejphar.2021.174604
https://doi.org/10.1016/j.ejphar.2021.17... ). These substances can promote the synthesis of apolipoprotein, accelerate the outward transport of fat, promote the oxidation of fat, and reduce the activity of fat synthase, thus reducing fat accumulation (James et al., 2002James SJ, Melnyk S, Pogribna M, et al. Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology. The Journal of Nutrition 2002;132(8):2361S-2366S. https://doi.org/10.1093/jn/132.8.2361S
https://doi.org/10.1093/jn/132.8.2361S... ). Methionine can also regulate the concentration of related enzymes in fat metabolism by up-regulating the expression levels of lipolysis-related genes and down-regulating the expression levels of fat synthesis-related genes to regulate the lipid metabolic response (Wang et al., 2022). On the contrary, methionine deficiency can significantly down-regulate the expression of lipolysis enzyme, up-regulate the expression of fatty acid synthase, promote fat synthesis, and reduce lipolysis (Aissa et al., 2014Aissa AF, Tryndyak V, De Conti A, et al. Effect of methionine-deficient and methionine-supplemented diets on the hepatic one-carbon and lipid metabolism in mice. Molecular Nutrition & Food Research 2014;58(7):1502-12. https://doi.org/10.1002/mnfr.201300726
https://doi.org/10.1002/mnfr.201300726... ). In addition, methionine deficiency can induce the synthesis of homocysteine (Tang et al., 2010Tang B, Mustafa A, Gupta S, et al. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine ?-synthase. Nutrition 2010;26(11-12):1170-5. https://doi.org/10.1016/j.nut.2009.10.006
https://doi.org/10.1016/j.nut.2009.10.00... ) and increase the level of homocysteine, leading to oxidative stress of the endoplasmic reticulum and promoting the conversion of glucose into lipid substances. Elevated homocysteine levels also affect vascular permeability and inhibit fat transfer in the liver and kidney, thus leading to fat accumulation (Hansrani & Stansby, 2008Hansrani M, Stansby G. The use of an in vivo model to study the effects of hyperhomocysteinaemia on vascular function. Journal of Surgical Research 2008;145(1):13-8. https://doi.org/10.1016/j.jss.2007.02.055
https://doi.org/10.1016/j.jss.2007.02.05... ). Moreover, methionine is one of the raw materials for protein synthesis (Asante et al., 2019Asante I, Chui D, Pei H, et al. Alterations in folate-dependent one-carbon metabolism as colon cell transition from normal to cancerous. The Journal of Nutritional Biochemistry 2019;69:1-9. https://doi.org/10.1016/j.jnutbio.2019.02.008
https://doi.org/10.1016/j.jnutbio.2019.0... ), which can initiate protein translation and stabilize protein structure (Aledo, 2019Aledo JC. Methionine in proteins: the cinderella of the proteinogenic amino acids. Protein Science 2019;28(10):1785-96. https://doi.org/10.1002/pro.3698
https://doi.org/10.1002/pro.3698... ). Therefore, a lack of methionine can inhibit mRNA translation and lead to protein synthesis obstruction, thus affecting protein stability and reducing protein levels in the liver and kidney.

NOS is an important enzyme that regulates NO synthesis, and the stronger its activity, the faster the rate of NO synthesis (Carmignani et al., 2000Carmignani M, Volpe AR, Boscolo P, et al. Catcholamine and nitric oxide systems as targets of chronic lead exposure in inducing selective functional impairment. Life Sciences 2000;68(4):401-15. https://doi.org/10.1016/s0024-3205(00)00954-1
https://doi.org/10.1016/s0024-3205(00)00... ). NO is a cytokine with dual effects under physiological and pathological conditions in the body, which is unstable and easily oxidized. In small amounts, it can serve as an antioxidant and anti-apoptotic substance (Nagahawatta et al., 2022Nagahawatta DP, Liyanage NM, Jayawardhana H, et al. Anti-fine dust effect of fucoidan extracted from ecklonia maxima leaves in macrophages via inhibiting inflammator signaling pathways. Marine Drugs 2022;20(7):413. https://doi.org/10.3390/md20070413
https://doi.org/10.3390/md20070413... ). On the contrary, if the synthesis of NO significantly increases beyond the range of controllable levels in the body, it will lead to injuries to the body. It has been shown that in pathological conditions, cNOS level would increase, iNOS would be activated and expressed in large quantities, and then continuously catalyze the production of a large number of NO, resulting in the production of various active nitrogen groups (RNS) (McCall et al., 2009McCall DO, McGartland CP, McKinley MC, et al. Dietary intake of fruits and vegetables improves microvascular function in hypertensive subjects in a dose-dependent manner. Circulation 2009;119(16):2153-2160. https://doi.org/10.1161/CIRCULATIONAHA.108.83129
https://doi.org/10.1161/CIRCULATIONAHA.1... ; Ascherio et al., 2010Ascherio A, Munger KL, Simon KC. Vitamin D and multiple sclerosis. The Lancet Neurology 2010;9(6):599-612. https://doi.org/10.1016/S1474-4422(10)70086-7
https://doi.org/10.1016/S1474-4422(10)70... ). Therefore, due to a lack of methionine, the body does not clear excess free radicals promptly, which stimulates oxidative stress and disrupts the balance of the body, ultimately leading to an increase in the activity of NOS and an acceleration of NO synthesis. The long-term high NO content in the liver and kidney has a significant toxic effect on these two metabolic organs. These results suggest that adding an appropriate amount of methionine to control NOS activity and NO synthesis can protect the liver and kidney. In the studies of NOS or its related disease, it has been pointed out that only a very small amount of NOS expression is expressed in normal liver and kidney tissues, while in some diseases the cNOS and iNOS expression are increased, which are then related to the occurrence of cancer tissues (Gardner et al., 1998Gardner CR, Heck DE, Yang CS, et al. Role of nitric oxide in acetarninophen-induced hepatotoxicity in the rat. Hepatology 1998;27(3):748-54. https://doi.org/10.1002/hep.510270316
https://doi.org/10.1002/hep.510270316... Marchetti et al., 2005Marchetti B, Serra PA, Tirolo C, et al. Glucocorticoid receptor-nitric oxide crosstalk and vulnerability to experimental parkinsonism: pivotal role for glia-neuron interactions. Brain Research Reviews 2005;48(2):302-21. https://doi.org/10.1016/j.brainresrev.2004.12.030
https://doi.org/10.1016/j.brainresrev.20... ; Cook, 2006Cook S. Coronary artery disease, nitric oxide and oxidative stress: The "Yin-Yang" effect-A Chinese concept for a worldwide pandemic. Swiss Medical Weekly 2006;136(7-8):103-13. https://doi.org/10.4414/smw.2006.11068
https://doi.org/10.4414/smw.2006.11068... ; Kawanishi et al., 2006Kawanishi S, Hiraku Y, Pinlaor S, et al. Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis. Biological Chemistry 2006;387(4):365-72. https://doi.org/10.1515/BC.2006.049
https://doi.org/10.1515/BC.2006.049... ). In the present study, we found that the activities of cNOS and iNOS in both the liver and kidney show a decreasing trend, which is contrary to the conclusion above. According to the pertinent studies (Chang et al., 2004Chang K, Lee SJ, Cheong I, et al. Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of I?B. Experimental & Molecular Medicine 2004;36(4):311-24. https://doi.org/10.1038/emm.2004.42
https://doi.org/10.1038/emm.2004.42... ; Vo et al., 2005Vo PA, Lad B, Tomlinson JA, et al. Autoregulatory role of endothelium-derived NO on lipopolysaccharide-induced vascular iNOS expression and function. The Journal of Biological Chemistry 2005;280(8):7236-43. https://doi.org/10.1074/jbc.M411317200
https://doi.org/10.1074/jbc.M411317200... ), under high concentrations of NO, the binding activity of NF-κB and DNA is inhibited, thus down-regulating the expression of iNOS, which prevents the occurrence of inflammation to a certain extent. In our study, when methionine was lacking, the concentration of NO increased significantly, and the level of NF-κB and the activity of iNOS decreased. It is speculated that the organism may try to maintain the homeostasis of its internal environment and resist the inflammatory response, which leads to a high concentration of NO in the body. The level of NF-κB is also adjusted by negative feedback, and the expression of iNOS is inhibited. In addition, (Scicinski et al., 2015Scicinski J, Oronsky B, Ning S, et al. NO to cancer: The complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001. Redox Biology 2015;6:1-8. https://doi.org/10.1016/j.redox.2015.07.002
https://doi.org/10.1016/j.redox.2015.07.... ) found that cNOS played a small role in the synthesis of NO, and the expression of cNOS could protect the body under normal circumstances. However, in this study, the synthesis of cNOS was lower than the normal value, and the activity of cNOS is related to Ca²⁺, so it is speculated that methionine deficiency may have caused the decrease in the activity of Ca²⁺ and calmodulin (CAM), thus reducing the activity of cNOS. If cNOS is at a low level for a long time, its protective function in the body will be reduced, and tissues or organs will be injured.

NF-κB signaling pathway plays an important role in inflammatory response, regulating the transcription process of a variety of cellular inflammatory factors such as NF-κB, TNF-α, IFN-γ, IL-1, IL-6 genes. An abnormal NF-κB signaling pathway will bring adverse effects on the body. The inflammatory factors in the methionine-deficient group were higher than those in the control group, suggesting that methionine could regulate the NF-κB signaling pathway. Methionine can regulate the expression balance of NF-κB/IκBα through its metabolite s-adenosine methionine (SAM). In normal cells, NF-κB binds to its inhibitor IκBα protein in an inactive state. When stimulated by external signals, IκBα is degraded, activating the expression of NF-κB, thereby promoting the release of inflammatory factors, and activating the expression of IκBα gene synthesis, inhibiting NF-κB activity (Irrera et al., 2020Irrera N, Bitto A, Vaccaro M, et al. PDRN, a bioactive natural compound, ameliorates imiquimod-induced psoriasis through NF-?B pathway inhibition and Wnt/?-Catenin signaling modulation. International Journal of Molecular Sciences 2020;21(4):1215-29. https://doi.org/10.3390/ijms21041215
https://doi.org/10.3390/ijms21041215... ). Methionine deficiency can lead to an imbalance of NF-κB/IκBα, resulting in an abnormal NF-κB signaling pathway and liver cell damage. Methionine also regulates the expression of inflammatory cytokines through SAM, which, as an important methyl donor, is an important source of methylated DNA (Martínez et al., 2017Martínez Y, Li X, Liu G, et al. The role of methionine on metabolism, oxidative stress, and diseases. Amino Acids, 2017;49(12):2091-8. https://doi.org/10.1007/s00726-017-2494-2
https://doi.org/10.1007/s00726-017-2494-... ). SAM can increase the methylation degree of inflammatory factors such as IL-6 and TNF-α and inhibit the expression of inflammatory factor genes, thus inhibiting the inflammatory response (Shen et al., 2017Shen J, Wu S, Guo W, et al. Epigenetic regulation of pro-inflammatory cytokine genes in lipopolysaccharide-stimulated peripheral blood mononuclear cells from broilers. Immunobiology 2017;222(2):308-15. https://doi.org/10.1016/j.imbio.2016.09.009
https://doi.org/10.1016/j.imbio.2016.09.... ). Lack of methionine reduces SAM levels and leads to reduced methylation degree of inflammatory factor genes, thus promoting the expression of inflammatory factors, aggravating inflammatory response, inducing cell damage, destroying tissues and organs of the body, and seriously affecting body performance.

In conclusion, methionine deficiency can hurt liver and kidney function in broilers. The results in this study demonstrated that, in a state of methionine deficiency, crude protein and crude fat contents, NO concentration, TNOS, iNOS and cNOS activities in the liver and kidney were all changed through the NF-κB signaling pathway.

ACKNOWLEDGEMENTS

The authors would like to thank their co-workers at China West Normal University for their assistance in performing the experiment and the analysis.

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