What is the effect of reduced glutathione on the antioxidant properties of tilapia?

 With the deepening of nutritional research on small peptides, there is now a more comprehensive understanding of the metabolic characteristics and nutritional effects of small peptides, and more and more studies have shown that the addition of small peptides to animal feeds can effectively avoid the competition between free amino acids, increase the utilization rate of proteins and micronutrients in the feeds, promote the growth of animals, improve the feed remuneration, and enhance the immunity and anti-stress ability of animals, so the application of small peptides in animal production is also attracting more and more attention. Therefore, the application of small peptides in animal production has received more and more attention.  Reduced glutathione is a biologically active peptide composed of p-glutamic acid, cysteine and glycine (p-Glu-Cys-Gly), which is widely distributed in natural organisms, and is abundant in the liver, muscle and blood of animals, playing an important role in the metabolic activities of tissues and organs.  Many studies have shown that the epithelium of the small intestine has a transporter system that transports exogenous reduced glutathione to the intracellular compartment, and that exogenous reduced glutathione can be directly absorbed by the small intestine into the blood circulation [1-5].  

 

Reduced glutathione has been widely used in human medicine as a drug for the treatment of various diseases and as a functional food and nutritional enhancer in the food processing industry.   Studies have shown that the gastrointestinal tract of fish can also absorb and utilize bioactive peptides [6-7].  In this experiment, glutathione reduction was added to the diet of tilapia, and the growth of fish was observed. The serum level of insulin-like growth factor-I (IGF-I) and the activity of antioxidant enzymes in muscle tissues of fish were measured to investigate the effects of glutathione reduction on the growth of fish and antioxidant properties and its possible mechanisms, so as to provide the theoretical basis for the use of glutathione reduction as an aquatic feed additive.

 

1 Materials and Methods

1 . 1 Test animals and drugs

Oreochromis niloticus × O. aureus, provided by Guangzhou Huadu District Hengtai Agricultural Development Co.  Reduced Reduced glutathione, purity > 98% (mass ratio), relative molecular mass 307 . 33 (product of AMRESCO).

 

1 . 2 Grouping and feeding management of test animals

A total of 360 tilapia with the same body length, body mass and body condition were randomly divided into 4 groups with 3 replicates of 30 tilapia in each group.   Group I was the control group and fed with basal diet.   Groups II, III and M were treated with 100, 200 and 400 mg/kg of reduced glutathione in the basal diet, respectively.  The basal diet was "999 Tilapia Medium Adult Feed" from Guangdong Zhengda Kangdi Company (main ingredients: crude protein ≥ 28% (lysine > 1%), crude ash < 14%, water ≤ 13%, crude fiber ≤ 14%, inorganic salt 0.5 % ~ 1.5 %), and the main ingredient of "999 Tilapia Medium Adult Feed" was "999 Tilapia Medium Adult Feed" from Guangdong Zhengda Kangdi Company. 0.5 % ~ 1.5 %, Calcium 0.5 %, Lysine 0.5 %. 0.5 % ~ 1.5 %, calcium 0.882 %, phosphorus 0.5 % ~ 1.5 %. 882 %, phosphorus 0.682 %). 682 %).  The addition of reduced glutathione and pelleting were carried out in the Aquatic Research Laboratory of the Institute of Animal Husbandry, Guangdong Academy of Agricultural Sciences, Guangdong Province, China: 100, 200 and 400 mg/kg of reduced glutathione were added to the basic feed after crushing, and the pellets with a particle size of 2 mm were made after mixing evenly, then blown-drying to dry the dry data, and then storing them in a refrigerator at -4 ℃.  The feeding experiments were conducted at Hengtai Agricultural Economy Co., Ltd. in Huadu District, Guangzhou City, China, in an aquarium of 60 cm × 60 cm × 80 cm. The aquarium was fed twice a day with saturation feeding at 9:00 a.m. and 5:00 p.m., and the residual feed was sucked up after 1 h. The water was changed every day, and the aquarium was inflated for 18 h every day.  The water temperature was 25~30 ℃, PH7.1~7.5 ℃, and the water was aerated for 18 hours a day to increase oxygen. 1 ~ 7 . 3 .   The test water temperature was 25~30 ℃, pH7.1~7.3. The test fish were domesticated on the basal diet for 15 d, and the test period was 60 d. The test fish were domesticated on the basal diet for 15 d.

 

1 . 3 Measurement indicators and methods

1 . 3 . 1 Body mass Fish were weighed at d 0, 20, 40 and 60 of the experiment. Relative body mass growth rate (RSGR) was calculated at the end of the experiment.

RSGR = (final body mass - initial body mass)/initial body mass × 100% .

 

1 . 3 . 2 Serum levels of insulin-like growth factor - I (IGF-I)    

On the 40th and 60th day of the experiment, after weighing the body mass each time, 2~3 mL of blood was collected from 5 fish of close to the average body mass and length in each replicate, and the serum was prepared and stored at -20 ℃.  The serum was prepared and stored at -20 ℃. The serum IGF-I level was determined by radioimmunoassay (RIA).  The principle and procedure are described in the instruction manual of the kit (purchased from Tianjin Jiuding Medical Biotechnology Co., Ltd.).

 

1 . 3 . 3 Antioxidant enzyme activity  

On the 20th, 40th and 60th days of the experiment, after weighing each fish, four fish of close to the average body mass were randomly selected from each replicate, and the muscle tissue samples were collected and stored at -70 ℃ for further use.  The total antioxidant capacity (T-AOC), glutathione peroxidase (GPP) and total superoxide dismutase (T-SOD) were determined according to the methods described in the kit.  The kits were purchased from Nanjing Jianjian Bioengineering Institute.

 

1 . 4 Data statistics and processing

All data were analyzed by SPSS (10 . All data were analyzed by analysis of variance (ANOVA) using SPSS (10.0) statistical processing software, and multiple comparisons were performed using the least significant difference (LSD) method.  The data were expressed as mean ± standard error.

 

2 Results and analysis

2 . 1 Effect of reduced glutathione on fish growth.

At the beginning of the experiment, there was no significant difference in the body mass of the fish in each group.  At d 20, the body mass of treatment groups II and M was significantly greater than that of the control group (P < 0.05) (Figure 1A). At the end of the experiment, the RSM of each treatment group was significantly higher than that of the control group (P < 0.05) (Figure 1A).  At the end of the experiment, the RSGR of all treatment groups was greater than that of the control group, and the RSGR of groups II and M were 95% and 52% higher than that of group I, respectively (P < 0.05) (Figure 1B). The differences were significant (P < 0.05) (Figure 1B).

 

2 . 2 Effect of reduced glutathione on serum IGF-I levels in fish

As shown in Fig. 2, at the 40th and 60th d of the experiment, treatment group II was significantly lower than the control group and other treatment groups (P < 0.05), while treatment group M was significantly higher than the control group and other treatment groups (P < 0.05). The results showed that at 40 d and 60 d of the experiment, treatment group II was significantly lower than the control and other treatment groups (P < 0.05), while treatment group M was significantly higher than the control and other treatment groups (P < 0.05), with 53% and 45% higher than the control, respectively. M was significantly higher than the control and other treatment groups (P < 0.05), 53% and 45% higher than the control group, respectively. Treatment group III was higher than the control group at d 40 but lower than the control group at d 60, but the differences were not significant (P > 0.05).

 

2.3 Effect of reduced glutathione on antioxidant enzyme activities in fish muscle tissue

T-AOC activity (Figure 3A): The T-AOC activity in muscle tissue of all groups showed an increasing and decreasing trend during the test period.  Comparing the time points of the experimental period, on the 20th day, the T-AOC activity of all treatment groups with reduced glutathione was significantly higher than that of the control group (P < 0.05); on the 40th day, the T-AOC activity of all groups increased significantly, and that of the treatment group IV increased by 2.7-fold, which was significantly higher than that of the control group and the other treatment groups (P < 0.05); on the 60th day, the T-AOC activity of all groups decreased again, but the T-AOC activity of all groups decreased significantly, and the T-AOC activity of all groups decreased significantly, and that of all groups decreased significantly. At 60 d, the T-AOC activity of all groups decreased significantly, but all treatment groups were still higher than the control group, and the difference between treatment group " and the control group was significant (P < 0.05).

T-SOD activity (Figure 3B): Comparing the time points during the experiment, there was no significant difference in T-SOD activity in muscle tissue of all groups on the 20th day; on the 40th day, the activity of all groups increased exponentially, and the activity of "treatment group" and "treatment group IV" was higher than that of the control group by 24% and 40%, which was significant (P < 0.05); on the 60th day, the activity of T-SOD in all groups, except for treatment group IV, still showed an increasing trend, and the activity of "treatment group" was significantly higher than that of the control group (P < 0.05). - By the 60th day, except for treatment group IV, the T SOD activity of all other groups still showed an increasing trend, and the treatment group was significantly higher than the control group (P < 0.05).

 

Reduced glutathione-PX activity (Fig. 3 C): There was no significant difference in reduced glutathione-PX activity in muscle tissues of fish from all groups at all time points during the comparison test (P > 0.05). (P > 0.05).  On the 20th day, the activity of all treatment groups with reduced glutathione was lower than that of the control group; on the 40th day, the activity of all treatment groups was higher than that of the control group, although the activity of all treatment groups declined as that of the control group; and on the 60th day, the activity of all groups increased again and was lower than that of the control group in all treatment groups.

 

3 Discussion

In fish, antioxidants mainly include antioxidant enzymes (e.g., glutathione-PX, SOD, CAT, etc.) and non-enzymatic antioxidants (e.g., vitamin E, vitamin C, glutathione-R, etc.).  The antioxidant enzymes SOD, CAT and reduced glutathione-PX are important components of the body's antioxidant defense system, and the measurement of these three enzymes is often used to indirectly reflect the dynamics of free radical reactions and tissue damage in the body [9-11].  Lash et al. [12] showed that oral administration of reduced glutathione to mice increased the levels of reduced glutathione in the jejunum, colon and stomach. Reduced glutathione in the gastrointestinal tract and in the epithelial cells plays an important role in the defense of the gastrointestinal tract against toxicants and peroxides.  In the present experiment, the addition of reduced glutathione to the diet significantly increased the T-AOC and T-SOD activities of fish muscle tissues, which were significantly higher than those of the control group (P < 0.05). This was significantly higher than that of the control group (P < 0.05).  This may be attributed to the fact that the exogenous glutathione reduces the damage of H2 O2 to the cells by altering the metabolism of glutathione reduced/glutathione S (GSSH) in the fish, thus relieving the pressure of SOD and enhancing the antioxidant capacity of the organism [13-15].  On the other hand, there was no significant change in the activity of reduced glutathione-Px in each treatment group compared with the control group. The authors speculated that the exogenous reduced glutathione increased the level of reduced glutathione in the serum of fish, and reduced glutathione could directly scavenge the excess radicals, which resulted in less stress in the body, and thus did not induce the production of too much reduced glutathione-Px.

 

Studies on weaned piglets [16] and yellow-feathered broiler chickens [17] have shown that the addition of reduced glutathione to feed can promote the growth of animals by increasing the serum level of IGF-I. In this experiment, the body mass of fish in the 100 mg/kg and 400 mg/kg groups was greater than that in the control group.  In this experiment, the body mass of fish in all treatment groups was greater than that of the control group, and the RS-GR of fish in the 100 mg/kg and 400 mg/kg groups was significantly greater than that of the control group.  Meanwhile, the serum IGF-I level was significantly higher in the 400 mg/kg group than in the control group.  In fish, the serum levels of growth hormone (GH) and IGF-I reflect the growth and metabolism of fish.   IGF-I has the function of promoting cell and tissue metabolism, cell mitosis, cartilage and bone growth, etc. IGF-I is the main factor mediating the growth-promoting effect of GH.  Chen et al [18] reported that exogenous injection of recombinant IGFs could promote the growth of tilapia, and the serum levels of IGF-I in the fish in the present study also showed a close correlation with their body mass.  Meanwhile, Ho- bor et al. [19] found that IGF-I could not effectively form and maintain its inherent disulfide bonds under the redox condition in vivo, and with the increase of GSSG/reduced glutathione concentration, the number of mismatched disulfide bonds increased, which might lead to the accelerated clearance of IGF-I in the organism. In this experiment, due to the addition of exogenous reduced glutathione, the content of reduced glutathione in the fish increased, thus reducing the chance of free radicals and abnormally matched disulfide bonds appearing in the fish, and lowering the clearance rate of IGF-I, so the IGF-I level in the serum of the treated group was elevated.

 

Therefore, it can be concluded that the addition of reduced glutathione to the diet may have a positive effect on the growth performance of fish by improving the antioxidant capacity of the muscle tissue and increasing the serum level of IGF- I in fish.

 

References:

［1］ LINDER M, BURLET G D, SUDAKA P, et al. Transport of glutathione by intestinal brush border membrance vesicles ［J］.  Biochem Biophy Res Comm, 1984, 123: 929-936.

［2］ HAGEN T M , JONES D P. Transepithelial transport of glutathione in vascularly perfused small intestine of rat ［J］.  Am J Physiol. 1987, 252(1):607-647.

［3］ HAGEN T M, WIERZBICKA G T, BOWMAN B B, et al. Fate of dietary glutathione: disposition in the gastrointesti- nal tract［J］.  Am J Physiol, 1990, 259(4):530-613.

［4］ VINCENZINI M T , FAVILLI F , IANTOMASI T , et al.

Glutathione - mediated transport across intestinal brush - border membranes [J].  Biochim Biophys Acta, 1980, 542 (2): 107- 114.

［5］ VINCENZINI M T , FAVILLI F , IANTOMASI T , et al. Intestinal uptake and transmembrane transport systems of intact reduced glutathione: Characteristics and possible biological role ［J］. Intestinal uptake and transmembrane transport systems of intact reduced glutathione: Characteristics and possible biological role ［J］.  Biochim Biophys Acta, 1982, 611(3):13-23.

［6］ MCLEAN E, DONALDSON E M. Abosorption of bioactive proteins by the gastrointestinal tract of fish: A review［J］.  J Aquat Anim Health, 1990, 2:1-11.

［7］ SIRE M F, VERNIER J M. Intestinal absorption of pro- tein in teleost fish［J］.   Comp Biochem Physiol , 1992, 103A(4):771-781.

［8］ Zhu L P, Chen X Q. Common experimental methods in immunology［M］. Common experimental methods in immunology [M].  Beijing: People's Army Medical Press, 2000, 192-194.

［9］ RUDNEVA I I. Blood antioxidant system of black sea e- lasmobranch and teleosts［J］.    Comp Biochem Physiol, 1997, 118C(2):255-260 .

[10] MOURENTE G, TOCHER D R, DIAZ E, et al. Relation- ships between antioxidants , antioxidant enzyme activities and lipid peroxidation products during early development in Dentex dentex eggs and larvas[J].  Aquaculture, 1999, 179:309-324 .

［11］ TRENZADO C, HIDALGO M C, GARCIA-GALLEGO M, et al. Antioxidant enzymes and lipid peroxidation in stur- geon Acipenser naccarii and trout oncorhynchus mykiss: A comparative study[J].  Aquaculture, 2006, 254:758-767 .

［12］ LASH L H, HAGEN T M, JONES D P, et al. Exogenous glutathione protects intestinal epithelial cells from oxidative injury［J］.  Pro Natl Acad Sci USA, 1986, 83: 4641-4645.

［13］ MARACINE M, SEGNER H. Cytotoxicity of metals in iso- lated fish cells: Importance of the cellular glutathione sta- tus［J］.  Comp Biochem Physiol, 1998, 120A:83-88 .

［14］ PENA-LLOPIS S, PENA J B, SANCHO E, et al. Gluta- thione-dependent resistance of the European eel Anguilla anguilla to the herbicide molinate［ J］.   Chemosphere, 2001, 45:671-681 .

[15] PENA-LLOPIS S, FERRANDO M D, PENA J B. Fish tol- erance to organophosphate-induced oxidative stress is de- pendent on the glutathione metabolism and enhanced by N- acetylcysteine[J].  Aquatic Toxicology, 2003, 65:337-360.

［16］ Liu Pingxiang.  Growth-promoting effect of glutathione on weaned piglets and its mechanism ［D］.  Guangzhou: South China Agricultural University, College of Animal Science, 2002.

［17］ WEI Jianfu, LIU Li, FU Weilong, et al.  Effects of carnosine and glutathione on growth and hormone levels in broiler chickens［J］.  Chemistry of life, 2004, 24:66-68.

［18］ CHEN Jyh-yih , CHEN Jian-chyi , CHANG Chi-yao , et al. Expression of recombinant tilapia insulin-like growth factor-I and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides[J].  Aquaculture, 2000, 181: 347-360.

[19] HOBOR S , LJUNG J L , UHLEN M , et al. Insulin-like growth factor I and " are unable to form and maintain their native disulfides under in vivo redox conditions[J].  febs l, 1999, 443:271-276 .

 

Can reduced glutathione protect the myocardium in patients with carbon monoxide poisoning?

 Acute carbon monoxide poisoning (ACMP), commonly known as gas poisoning, is the main cause of acute poisoning deaths, in China, the incidence of ACMP and the death rate of all kinds of acute poisoning of the first [1] O ACMP refers to a certain amount of inhalation of carbon monoxide at a certain time of acute hypoxic disease caused by cerebral hypoxia is the most prominent symptom O Clinical patients with ACMP often focus on the recovery of neurological dysfunction caused by cerebral hypoxia, often ignoring the damage to the myocardium O Recent studies [2] found that moderate and severe ACMP patients are often combined with significant myocardial damage O And cardiac damage is often hidden Clinical attention to ACMP patients is often focused on the recovery of neurological dysfunction caused by brain tissue hypoxia, often ignoring the damage to the myocardium O Recent studies [2] found that patients with moderate and severe ACMP often combined with obvious myocardial damage O While the cardiac damage is often masked, heart failure and severe cardiac arrhythmia often occur, which can lead to deterioration of the patient's condition and death of the crusher O Therefore, for patients with ACMP, protection of the myocardial function is an important link in the treatment of ACMP O From January 2003 to December 2009, the authors used the following methods to evaluate the effectiveness of the treatment of ACMP patients. From January 2003 to December 2009, the author used reduced glutathione (GSH) to treat myocardial damage caused by ACMP, and observed the changes of creatine kinase isoenzyme (CK-MB) and troponin I (cTnI) before and after the treatment, which are reported as follows.

 

1 Objects and Methods

1.1 Subject of the study    

Ninety-three patients with acute moderate or severe ACMP who were admitted to our hospital from January 2003 to December 2009, all of them had a clear history of ACMP and positive COHb qualitative test (using the boiling method and healthy human venous blood as the control) O were randomly divided into 52 cases in the treatment group and 41 cases in the control group: male in the treatment group, male in the control group, male in the treatment group, male in the control group, male in the control group, male in the treatment group, male in the control group, male in the treatment group, male in the control group.

In the control group, there were 20 males and 21 females with a median age of 35.43 (18-79) years. O Degree of poisoning: 38 moderate and 14 severe cases in the treatment group, 25 and 16 cases in the control group respectively. O Excluding those who died during treatment and those who had suffered from cardiomyositis, cardiac failure, shock, serious arrhythmia and other recent history of poisoning. O Screening criteria: no cardiac, hepatic, renal or thyroid diseases before poisoning, diagnosis and grading were established according to the "Diagnostic standards for occupational acute carbon monoxide poisoning (GBZ23-2002)" of the Ministry of Health. O Differences between the two groups in terms of gender, age, and severity were not statistically significant (p<0.05).

 

1.2 Treatment   

Both groups were treated with conventional therapy: (1) high-flow oxygen inhalation by normal-pressure nasal cannula; (2) hyperbaric oxygen therapy; (3) dehydration and cranial pressure reduction therapy; (4) energy synthesis therapy; (5) prevention of infections and symptomatic treatment O In the treatment group, on the basis of the conventional treatment, GSH was added 1,800 mg/times per day, intravenously once a day, and the course of treatment was 7 d. The treatment group was treated with GSH for 7 days.

 

1.3 Measurement indicators    

CTnI was measured by ELISA (BeCkman, Germany), and serum CTnI ≥0.40 μg/L was considered as myocardial injury. CK-MB was measured by enzyme rate method (Olympus Vistro 250, Japan), and the reference range of normal CK-MB value was ≤25 U/L. All patients were admitted to the hospital and at the end of treatment. All patients had 3 mL of venous blood collected at the time of admission and at the end of the treatment, and CTnI and CK-MB were measured separately after serum separation.

1.4 Statistical processing SPSS13.0 statistical software was used to complete the data analysis, the measurement data were expressed by x ± s, t-test was performed, and the rate of comparison was performed by the "2-test," and the difference of P < 0.05 was regarded as statistically significant.

 

2 Results

There was no statistically significant difference in serum CTnI levels between the two groups before treatment (P > 0.05), and there was a statistically significant difference in serum CTnI levels between the two groups before and after treatment (both P < 0.01). At the end of the treatment course, serum CTnI levels of the treatment group were significantly lower than those of the control group, and the difference was statistically significant (P < 0.05), as shown in Table 1.

There was no statistically significant difference between the serum CK-MB levels of the two groups before treatment (P > 0.05), and the difference between the serum CK-MB levels of the two groups before and after treatment was statistically significant (both P < 0.01). At the end of the treatment course, the serum CK-MB levels of the treatment group were significantly lower than those of the control group, and the difference was statistically significant (P < 0.05), as shown in Table 2.

 

3 Discussion

ACMP is one of the most common life and occupational poisoning, has become an important cause of morbidity and mortality Carbon monoxide inhalation, 85% and the blood of red blood cells hemoglobin binding, the formation of stable COHb, impede the hemoglobin oxygen and oxygen release, the body of all tissues can be a certain degree of damage, but because of the brain tissue is the most sensitive to oxygen deprivation, it is the first to be damaged The presence of COHb in the blood makes the brain, heart and other systems of vascular endothelial cells to reduce energy synthesis, impaired cell metabolism, resulting in intracellular acidosis, increased permeability of cell membranes, endothelin and other vasoconstrictors released into the blood. The presence of COHb in the blood reduces the energy synthesis of vascular endothelial cells of brain, heart and other systems, impaired cell metabolism, resulting in intracellular acidosis, increased permeability of cell membranes, increased release of vasoconstrictive substances such as endothelin into the bloodstream, which in turn causes structural damage to cardiac myocytes. In addition, disorders of neurofluidic regulation manifested by increased levels of catecholamines and epinephrine, which further cause coronary artery spasm and constriction, elevation of blood pressure, accelerated heart rate, resulting in myocardial ischemia, and the heart rate increased. The incidence of myocardial damage caused by ACMP was reported to be 13.58%~32.00%, and the incidence and severity of myocardial damage increased significantly with the aggravation of toxicity. In addition, patients with myocardial damage had no obvious subjective symptoms, or they were covered up by the symptoms of central nervous system damage, which were often neglected and delayed in treatment. In addition, most patients with myocardial damage do not have obvious subjective symptoms, or are masked by symptoms of central nervous system damage, which are often neglected by the clinic and delayed diagnosis.

 

GSH participates in triple completion acid cycle, promotes bile acid metabolism, and can activate a variety of enzymes, thus promoting sugar, fat and protein metabolism GSH can bind with free radicals in the body through Ryukyl radicals to accelerate the excretion of free radicals, and can reduce tissue damage and promote the repair of patients suffering from systemic or localized hypoxemia caused by intoxication. Through the reaction of transmethylation and trans-propylation of amino acids, GSH also protects the synthesis of the liver, and detoxification, CK-MB and troponin I/T (CTnI/T) are specific and sensitive markers for myocardial injury. CTnI/T is the most sensitive and specific serum biochemical marker for myocardial injury. CTnI and CTnT have the same sensitivity, but the specificity of CTnI is higher than that of CTnT, so CTnI is the most sensitive and specific marker for myocardial injury. Therefore, CTnI is the most sensitive and specific as an indicator of myocardial damage.

 

In the course of ACMP treatment, after normobaric and hyperbaric oxygen therapy, with the improvement of microcirculation and the recovery of tissue oxygenation, there is hypoxia/reoxygenation (A/R) injury in the tissue, which may cause permanent damage to the myocardium if no active and effective measures are taken. GSH can reduce the A/R injury of cardiomyocytes by inhibiting the expression of inflammatory factors and other pathways. The main mechanism of A/R injury is calcium overload, The main mechanisms of A/R injury are calcium overload, oxygen radicals, and leukocyte aggregation, etc. However, GSH can inhibit the release of calcium ions by binding to the luciferase site of the small molecule chloride channel protein, leading to structural changes in the protein, and inhibiting the opening of the channel. When [Ca2+] rises to a certain concentration, the Ca2+-Mg2+-ATPase enzyme is activated, hydrolyzes the ATP to provide energy, and pumps the Ca2+ out of the cell or uptake it from the endoplasmic reticulum and mitochondria, thus reducing the cytosolic Ca2+ concentration. At the same time, GSH can directly eliminate oxygen radicals, hydrogen peroxide and hydroxyl radicals, and can also react with myeloperoxidase-derived oxidants produced by leukocytes during oxidative stress, such as hypochlorous acid and chloramine, preventing them from participating in the oxygen radical generation reaction; and GSH can also inhibit the activities of NADPH and cytochrome P450 reductase to reduce the free radicals produced by the NADPH oxidase system. oxidase system, thus reducing the cytotoxicity of this system in A/R injury.[4] Hu Tao et al.[5] used high dose of vitamin C, and Xiao Li et al.[6] used edaravone oxygen radical scavenger in the treatment of ACMP with good results.

 

In this study, the levels of CTnI and CK-MB were significantly lower in the treatment and control groups before and after treatment, indicating that myocardial damage in patients with moderate-to-severe ACMP is easy to occur. The levels of cTnI and CK-MB in the treatment group with the addition of GSH were significantly lower than those in the control group (P < 0.05), indicating that GSH has a certain auxiliary therapeutic effect on the elevation of cTnI and CK-MB in the patients with ACMP 9. In summary, GSH has a protective effect on cardiomyocytes of ACMP patients, and in clinical practice, cardiac damage caused by ACMP 9 should be emphasized and early intervention is recommended.

 

References.

[1] Rapheal J C. Acute carbon monoxide poisoning [J].  Rev Prat , 2008 ,58(8) :849-854.

[2] Henry C R , Satran D , Lindgren B , et al. Myocardial injury and long term mortality following moderate to severe carbon monoxide poisoning [J]. jama , 2006 ,295(4) :398-402.

[3] ZHANG Jianguo,ZHANG Huiru,SHI Xueying,et al. Dynamic changes of serum enzyme activity in patients with acute carbon monoxide poisoning [J].  Chinese Journal of Labor Health and Occupational Diseases , 2003 ,21 (1) :51-53.

[4] Scholz R W , Reddy P V , Wynn M K , et al. Glutathione dependent factors and inhibition of rat liver microsomal lipid peroxidation [J].  Free Radic Biol Med , 1997 ,23 (5) :815 - 828.

[5] HU Tao ,YIN Wen ,LI Xiaoyi ,et al. Protective effect of high-dose vitamin C on myocardium of patients with acute carbon monoxide poisoning [J].   China Emergency Medicine , 2009 ,29(7): 637-639.

[6] XIAO Li,WANG Dajun. Therapeutic effect of ganglioside and edaravone combined with hyperbaric oxygen in the treatment of acute carbon monoxide poisoning [J]. Shandong Medicine , 2010 ,50(31): 102-103.

 

What is the effect of reduced glutathione on the growth of turbot?

 Scophthalmus max imus, commonly known as turbot, is native to the northeastern coast of the Atlantic Ocean. It has a fast growth rate, adapts to low water temperature, accepts feeds easily and has a high conversion rate [1], which makes it particularly suitable for factory farming along the northern coast of China [2]. With the expansion of industrial scale and the change of culture mode, the factory farming mode has also brought some impacts on turbot culture while increasing the production. The deterioration of the water environment caused by high-density culture[3] , the oxidative rancidity of feeds due to improper preservation during processing, storage and transportation[4] , diseases, and operational stresses caused by transportation and other management aspects[5] can cause oxidative stress to turbot, resulting in damage to its growth and development, and even its death.

 

Studies have shown that nutritional regulation can be used to eliminate or alleviate the stress caused by stress in aquatic animals, and non-nutritive immune enhancers (immunoglycans, antioxidants, etc.) can promote their growth and stress resistance[3] .

 

Glutathione (usually referred to as reduced glutathione) is an active peptide formed by the peptide bonding of glutamic acid, cysteine and glycine, which plays an important role in a wide range of cellular activities[6] . Reduced glutathione is a non-protein, low molecular weight thiol that is a non-enzymatic antioxidant, and it performs a variety of functions such as scavenging of oxygen radicals, detoxification, maintenance of DNA biosynthesis, and cellular immunity through the transfer of electrons and protons[7] . The most important function is to maintain the dynamic balance between oxidation and antioxidant[8] .

 

Currently, studies on the application of reduced glutathione in aquafeeds have shown that the addition of appropriate amounts of reduced glutathione to feeds can significantly improve the performance of Oreo-chromis niloticus GIFT[9] , rainbow trout Oncorhy nchus mykiss[10] , shrimp Litopenaeus v. annamei[11] , O. niloticus × O. aureus[12] , brown toothfish Paralichthys oliveaceus[13] , and other species of flounder, including O. niloticus × O. aureus[14] . annamei)[11] , Oncorhynchus mykiss (Oncorhynchus mykiss)[10] , Litopenaeus v. annamei (Litopenaeus v. annamei)[11] , O. niloticus × O. aureus (O. niloticus × O. aureus)[12] , and Paralichthys oliveaceus (Paralichthys oliveaceus)[13] , whereas the addition of reduced glutathione to the diets of grass carp Ctenopharyngodon idella (Ctenopharyngodon idella) did not have any significant effect on growth and bait coefficient. In grass carp (Ctenophary ngodon idella), the addition of reduced glutathione to the diet had no significant effect on growth and bait coefficient, but significantly increased the total antioxidant capacity of the liver and decreased the levels of reactive oxygen species (ROSs) in the liver and serum[14] , and the addition of reduced glutathione to the diet of the abalone H aliotis discus hannailna (H aliotis discus hannailna) did not have any significant effect on the rate of increase in mass, but it did have an improvement in the antioxidant system[15] .

The author chose turbot as the test object, and added different levels of reduced glutathione to the basic feed to study its effect on the growth and development and antioxidant capacity of turbot, and to determine the optimal amount of reduced glutathione in turbot feed, so as to provide a theoretical basis for the application of reduced glutathione in turbot feed and the healthy aquaculture of turbot.

 

1 Materials and Methods

1 .1 Test feed

Fishmeal and soybean meal were used as the main protein sources, high-gluten wheat flour as the main sugar source, and fish oil and soy lecithin as the main fat source to formulate nitrogen- and energy-equivalent basic feeds. The formula and nutritional composition of the basal feed are shown in Table 1. Reduced glutathione 0, 100, 200, 400 and 600 mg/kg were added to the basal diets to formulate five kinds of experimental diets.

The raw materials are crushed and passed through a 60-mesh sieve, then expanded and mixed step by step according to the test ratios, and then extruded into pellets with a particle size of 2 mm by a pelletizer, then naturally air-dried to a moisture of about 10% and put into self-sealing bags, and then placed in a -20 ℃ refrigerator for freezing and storing.

 

1 .2 Feeding and management

Turbot juveniles were purchased from Dalian Tianzheng Industrial Co., Ltd. and fed with basic feed for two weeks, and then randomly grouped into individuals without disease or trauma and with an initial mass of (23.08± 0.09) g after they had adapted to the feed and aquaculture environment.

Fifteen 60 cm × 45 cm × 40 cm aquariums (actual water consumption: 90 L) were used, each aquarium was used as one culture unit, and five treatments were set up with three replicates of each treatment, each replicate was stocked with 14 fishes, and the culture experiment lasted for 8 weeks. The aquariums were fed twice a day (8:00 and 18:00) under natural light, and the residual bait was collected after 30 min of feeding. The water temperature ranged from 14 to 18.5 ℃, and the dissolved oxygen was >6 mg/L. The water was changed once/d, and the water volume ranged from 33.3% to 50%.

 

1 .3 Sample Collection

Before the end of the experiment, turbot was fasted for 24 h, and each tank was weighed and counted separately. Five fish were randomly taken from each aquarium, weighed and measured, and the livers were collected, weighed and frozen in liquid nitrogen at -80 ℃. The mass gain rate and specific growth rate were calculated according to the following formula:

Mass increase rate/% = (m2 - m1)/m1 × 100%

Specific growth rate/% - d-1 = (lnm2 - lnm1 ) / t × 100%

where m1 is the initial body mass (g), m2 is the final body mass (g), and t is the incubation time (d).

 

1 .4 Sample Determination

The collected liver tissues were mechanically homogenized by adding pre-cooled 0.86% physiological saline as homogenizing medium (m/V=1/9) in an ice-water bath, and then centrifuged at 4 ℃ and 3000 r/min for 10 min, and the supernatant was extracted for the determination of the indexes.

For the determination of total protein, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Kaumas Brilliant Blue Protein Quantification Test Kit was used; for the determination of total antioxidant capacity, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Total Antioxidant Capacity Test Kit was used, which utilized the colorimetric measurement of antioxidant substances by reacting with Fe2+ and pheophytin; for the determination of malondialdehyde, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Malondialdehyde Test Kit was used, which utilized the colorimetric measurement of the red product formed by condensation of malondialdehyde and thiobarbituric acid. The determination of malondialdehyde was done by colorimetric reaction between malondialdehyde and thiobarbituric acid; the determination of superoxide dismutase was done by superoxide dismutase test kit of Nanjing Jianjian Institute of Biological Engineering; the determination of reduced glutathione was done by micro enzyme assay using micro reduced glutathione test kit of Nanjing Jianjian Institute of Biological Engineering; the determination of glutathione peroxidase and glutathione reductase was done by glutathione peroxidase of Nanjing Jianjian Institute of Biological Engineering; the determination of glutathione peroxidase was done by glutathione peroxidase of Nanjing Jianjian Institute of Biological Engineering. Glutathione peroxidase and glutathione reductase were determined using a glutathione peroxidase kit from Nanjing Jianjian Institute of Biological Engineering, using the reaction of dithiodinitrobenzoic acid with sulfhydryl compounds for colorimetric determination; glutathione sulfotransferase was determined using a glutathione sulfotransferase kit from Nanjing Jianjian Institute of Biological Engineering, using glutathione sulfotransferase catalyzed by the binding of reduced glutathione with 1-chloro 2,4-dinitrobenzene substrate for colorimetric determination.

 

1 .5 Statistical analysis

The experimental data were expressed as mean ± standard deviation. The data were analyzed by one-way ANOVA using SPSS 21.0 software. Duncan's multiple comparisons were used to test for differences between groups if the differences were significant (P < 0.05).

 

2 Results

2 .1 Effect of reduced glutathione on the growth of turbot (Scophthalmus maximus)

The addition of reduced glutathione to the feed increased the mass gain rate and specific growth rate of turbot to different degrees, and the mass gain rate and specific growth rate of turbot showed a tendency of increasing and then decreasing with the increase of the added amount of reduced glutathione (Table 2). In the experimental group with 200 mg/kg of reduced glutathione, the mass gain rate and specific growth rate of turbot were significantly higher than those of the other groups (P < 0.05), while the differences among the other groups were not significant (P > 0.05). The predictive model was established by linear regression analysis, and the regression equation showed that the maximum specific growth rate of turbot reached 1.67% when the dietary reduced glutathione was added at 189.70 mg/kg (Figure 1).

 

2.2 Effect of reduced glutathione on malondialdehyde content, total antioxidant capacity and superoxide dismutase activity in turbot liver

The addition of reduced glutathione did not significantly affect the malondialdehyde content, total antioxidant capacity and superoxide dismutase activity in the liver of turbot (P＞0.05) (Figs. 2-4), and the malondialdehyde content in the liver of turbot showed a tendency of decreasing and then increasing with the increase of the added amount of reduced glutathione, among which the control group had the highest malondialdehyde content and the test group had the lowest malondialdehyde content when the added amount of reduced glutathione was 200 mg/kg. The control group had the highest malondialdehyde content and the test group with 200 mg/kg reduced glutathione had the lowest. The total antioxidant capacity and superoxide dismutase activity in the liver of turbot showed a tendency of increasing and then decreasing with the increase of reduced glutathione, with the highest levels in the control group and the lowest level in the test group at 200 mg/kg of reduced glutathione, which were (1.99± 0.12) and (99.32± 3.09) U/mg, respectively.

 

2.3 Effects of reduced glutathione on reduced glutathione content and glutathione peroxidase activity in liver of turbot (Scophthalmus maximus)

With the increase in the amount of reduced glutathione added to the feed, the reduced glutathione content and glutathione peroxidase activity in the liver of turbot showed a tendency to increase and then decrease (Figs. 5 and 6). Reduced glutathione content in the liver of turbot was highest when 200 mg/kg of reduced glutathione was added to the feed, and the liver content of reduced glutathione was significantly higher in the groups with 200 and 400 mg/kg of reduced glutathione than in the control group (P < 0.05). The glutathione peroxidase activity in the liver of turbot was highest at 200 mg/kg of reduced glutathione, but the difference between the experimental group and the control group was not significant (P＞0.05).

 

2.4 Effect of reduced glutathione on glutathione sulfotransferase and glutathione reductase activities in turbot liver

The activities of glutathione sulfotransferase and glutathione reductase in the liver of turbot showed a decreasing and then increasing trend with the increase of reduced glutathione in the feed (Figs. 7 and 8). When 200 mg/kg of reduced glutathione was added to the diet, the activities of glutathione sulfotransferase and glutathione reductase in the liver of turbot were the lowest, which were (38.08± 5.68)U/mg and (6.87± 0.87)U/g, respectively, and were significantly lower than those in the control group (P<0.05).

 

3 Discussion

3.1 Effects of reduced glutathione on the growth of turbot (Scophthalmus maximus)

Studies have shown that the addition of reduced glutathione to feed can have a positive effect on the growth performance of aquatic animals, and can promote the growth of aquatic animals[16] , and the mechanism of action is the result of the coordination of multiple systems. Liu Xiaohua et al[11] showed that the addition of a certain amount of reduced glutathione to feed can improve the quality increase rate and feed efficiency of shrimp Vannamei, and pointed out that reduced glutathione can destroy the disulfide bond of the growth inhibitory molecules through the intermediate metabolite cysteamine, to release the growth inhibitory hormone control on growth hormone, and to promote growth hormone in the existing level, so as to promote the growth of the organism. Zhao Hongxia et al.[17] showed that the addition of reduced glutathione to feed can promote the growth of grass carp by regulating the level of growth hormone and increasing the level of insulin-like growth factor I. Zhou Tingting et al.[18] showed that the addition of reduced glutathione to feed can increase the level of insulin-like growth factor I in the growth hormone of grass carp. Zhou Tingting et al.[9] showed that the addition of reduced glutathione to feed could promote protein synthesis and increase the intake of Jifu tilapia. These findings have been confirmed in experiments with rainbow trout[10] , brown turbot[13] , Pelteoba grus fulv idraco[18] and Onirofus tilapia[19] .

 

In this experiment, different doses of reduced glutathione were added to the basic feed formula, and the results showed that the addition of reduced glutathione to turbot feed could significantly improve the growth performance of turbot, and the rate of increase in mass and specific growth rate of turbot in the experimental group with the addition of reduced glutathione was significantly higher than that of the control group with no reduced glutathione, and reached a significant level at the addition rate of 200 mg/kg. The significant level was reached at 200 mg/kg, which is consistent with the results of the above experiments. In addition, it has been shown that reduced glutathione in the intestinal lumen of animals can protect the intestinal mucosa by scavenging peroxides[20] , and in a study by Venurini[21] , it was shown that reduced glutathione can enhance the feeding response of Hydra attenuate. Thus, the growth-promoting effect of reduced glutathione on turbot is the result of a series of mechanisms, and the specific growth mechanism needs to be further investigated.

 

3.2 Effect of dietary reduced glutathione on the antioxidant capacity of turbot (Scophthalmus maximus)

In practice, there are many factors that cause oxidative stress to organisms, resulting in the production of a large number of reactive oxygen radicals (RORs), which lead to the production of reactive oxygen species (RORs) exceeding their decomposition rate and causing damage to organisms and even to their growth and development. Lipid peroxidation leads to the production of secondary products, such as malondialdehyde, which is an important indicator of oxidative stress damage caused by reactive oxygen species in different marine organisms[22] . In this experiment, malondialdehyde decreased and then increased with the addition of reduced glutathione to the diets, and the levels of malondialdehyde in the reduced glutathione group were lower than those in the control group, and the lowest level was found in the reduced glutathione group at 200 mg/kg, but did not reach a significant level. The exogenous addition of reduced glutathione reduced the oxidative damage of cells to a certain extent.

 

In order to maintain the balance between oxidation and reduction reactions, organisms have a whole set of antioxidant system. The antioxidant system continuously scavenges free radicals, regulates the level of reactive oxygen species in the organism, and at the same time participates in a variety of biochemical reactions to effectively regulate the oxidative stress of the organism, thus ensuring the stability of the internal and external cellular environment of the organism and its normal physiological functions. With the increase of reduced glutathione in the feed, the malondialdehyde content in the liver of turbot was lower than that of the control group, and the increase of total antioxidant capacity and superoxide dismutase activity also proved that the moderate addition of reduced glutathione in the feed could improve the antioxidant capacity of turbot to a certain extent.

 

The changes in glutathione peroxidase activity after the addition of reduced glutathione showed the same trend as that of reduced glutathione, total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity, but did not reach a significant level. The decrease of glutathione reductase activity in this experiment also proved that the dynamic balance of reduced glutathione/oxidized glutathione could be regulated by the addition of exogenous reduced glutathione, but not by the endogenous oxidized glutathione. It is not necessary to convert endogenous oxidized glutathione to reduced glutathione catalyzed by glutathione reductase to increase the reduced glutathione content in the body. This result is similar to that of brown turbot[13] , but different from that of rainbow trout[10] and jiffy tilapia[23] , so the mechanism of glutathione reductase in turbot needs to be further investigated.

 

Glutathione sulfotransferase exists in large quantities in hepatocytes, and when hepatocytes are damaged, glutathione sulfotransferase will be rapidly released into the blood, so glutathione sulfotransferase can also be used as a sensitive indicator of liver injury[24] . In this experiment, malondialdehyde showed a decreasing and then increasing trend with the increase of reduced glutathione, with the highest in the control group and the lowest in the experimental group with 200 mg/kg of reduced glutathione, and the glutathione sulfotransferase showed an opposite trend to malondialdehyde. It was further proved that the moderate addition of reduced glutathione could reduce the oxidative damage in the cells. In addition, when the glutathione peroxidase of turbot showed a decreasing trend, the glutathione sulfotransferase showed a corresponding increasing trend. This result is similar to that of brown turbot[13] and rainbow trout[25] . The results suggest that glutathione sulfotransferase can also be used as an antioxidant protection mechanism when the activity of glutathione peroxidase is low.

 

3.3 Optimal levels of reduced glutathione in turbot feeds

It has been shown that reduced glutathione, although an important scavenger of free radicals in the body, can be a pro-oxidant at concentrations of up to 1 mmol/L in living organisms, causing DNA damage[26] . In addition, excessive accumulation of reduced glutathione as a precursor of oxidants can be toxic[27] , and some compounds can be converted to cytotoxic, genotoxic, or mutagenic metabolites by combining with reduced glutathione[28] .

 

The present study showed that the addition of reduced glutathione to feed increased the accumulation of reduced glutathione in the liver to a certain extent, which was significantly different from that of the control group, which was consistent with the results of Wang Fangqian et al[13] . This result was consistent with that of Wang Fangqian et al[13] . However, He Fen et al[29-30] showed that the addition of reduced glutathione to feed did not have a significant effect on the liver reduced glutathione content, which may be related to the different types of test subjects, and the exact mechanism needs to be further investigated.

 

It was also found that the growth performance and antioxidant capacity of turbot showed a tendency of increasing and then decreasing with the increase of reduced glutathione content, and the malondialdehyde content in the liver showed a tendency of increasing after the addition of 200 mg/kg, which indicated that the antioxidant capacity of the organism declined and a certain degree of oxidative damage appeared when the added amount of reduced glutathione was higher than a certain level. The results were similar to those of the shrimp Penaeus vannamei[11] , brown turbot[13] and Pelteobagrus fulvidraco[18] . The results showed that only when the amount of reduced glutathione was added in the appropriate range, it had the effect of promoting growth and antioxidant capacity; if it was added in excess, it would lead to the excessive accumulation of reduced glutathione in turbot, resulting in oxidative damage, which could have toxic effects on fish. The optimal level of reduced glutathione in aquatic feeds varies according to the target species. The optimal level of reduced glutathione in feeds for rainbow trout is 200 mg/kg[10] , in feeds for shrimp, Penaeus vannamei, the optimal level is 174.13 mg/kg[11] , in feeds for brown flounder, the optimal level is 368.92 mg/kg[13] , in feeds for yellow croaker, the optimal level is 368.92 mg/kg[14] , in feeds for catfish, Pelteobagrus fulvidraco, and in feeds for catfish, Pelteobagrus fulvidraco, the optimal level is 368.92 mg/kg[15] . 13]; the optimum level of reduced glutathione in Pelteobagrus fulvidraco feed is 357.69 mg/kg[18]; the optimum level of reduced glutathione in Jiffy tilapia juvenile feed is 355.13 mg/kg[23]; in the present experiment, the specific growth rate was used as the evaluation index, and a prediction model was established by linear regression analysis, through which the optimum level of reduced glutathione in turbot feed was determined to be 189.13 mg/kg[24]. The optimal level of reduced glutathione in turbot feed was 189.70 mg/kg[23] .

 

4 CONCLUSIONS

Addition of appropriate amount of reduced glutathione to turbot feed can promote the growth of turbot, improve the ability of turbot to resist oxidative stress, remove oxygen radicals in the body, and alleviate the oxidative damage to turbot in the process of aquaculture, and the optimal amount of reduced glutathione added to the feed is 189.70 mg/kg.

 

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