Effects of crude thymus gland extract on some physiological and biochemical parameters in local rabbits

Ghorbat Saleh Ali,Amad M.Saleh Jubrail,Zeliha Selamoglu,Betul Ozdemir and Malika Kassem Najeeb

Abstract—In the present study,36 local rabbits of different ages were divided into two groups:18 infants and 18 adult rabbits of both sexes were used.Each group of rabbits were subdivided randomly into two subgroups (control and treated).Treated groups,received intraperitoneally calf thymus extract obtained from abattoir and homogenized with 0.15M NaCl,centrifuged and the supernatant was Millipore filtrated and stored at -20°C and used in a dose of 0.25 mL/kg body weight daily for two weeks.Control groups received similar doses of normal saline intraperitoneally.Data obtained from each treated group were compared to those of the control group.Thirty days after the last injection,animals were anesthetized and blood samples were collected for biochemical,hematological investigations.Animals treated with crude thymus extract showed significant decreasesin RBC,Hb and PCV.Blood indices(MCV and MCHC)alsoshowed significant decreases in treated adults.Leukocyte count and the percentage of lymphocytes showed highly significant decrease in both treated groups,crude extract seems to induce immunosuppressive action through anti proliferative properties.Biochemical parameters showed high significant decrease of serum albumin level in the treated adult group but no changes were observed in the infant group,while total protein markedly decreased only in treated adult,which could be explained on the base of decreased negative acute protein and increased catabolism of proteins in the liver.Changes in the lipid profile showed significant increase in triglyceride,VLDL,and LDL level,while HDL level was decreased in both treated groups.Changes in the HDL composition which is expected and decrease of antioxidant lead to increase in free radicals which induced LDL and VLDL oxidation and initiation of atherosclerosis.In all treated groups highly significant increase of AST,ALT and ALP activation were obvious as a result of liver cell damages.

Keywords—Blood,Rabbit,Serum,Thymus extract

I.BACKGROUND

The thymus,a primary lymphoid organ is located in the anterosuperior mediastinum and development of T cell is important immunological function of the tissue.The thymus is a bilobed structure organ is divided by numerous septa into multiple lobules.Each lobule is consisted of two morphologically distinctive areas;outer,more cellular;cortex and an inner,less cellular;medulla.It is actually an epithelial organ in which its epithelial cells provide a meshwork containing thymocytes and smaller numbers of other lymphoid cells [1],[2].Thymus function particularly susceptible to various negative influences ranging from age-related involution to atrophy as a consequence of a series of hormones,including sex and steroids hormone,malnutrition and infection.So,capacity of T-lymphocyte differentiation,declines with a change in the composition of T-lymphocyte subsets produced [3].

II.MATERIALS AND METHODS

This study was performed on different ages of local domestic rabbits of both sexes.Infants of about two months ages weighing about 528.9±9.6g and adult rabbits their ages are between 8-12 months weighing about 1671.1±30.6g were used.The study design was approved by the Duhok General Directorate of Animal Ethical Committee of Iraq.Animals were kept in the animal house of College of Medicine at University of Duhok.All animals were kept in individual cages during the whole experimental period,kept under similar conditions at 18-24°C in a 12-hours dark-light cycle and maintained with free access to food diet (barely and vegetable) and drinking water.Preparation of Calf Thymus Extract Fresh Calf thymus extract was obtained from adult caw,homogenized in 0.15 M NaCl,pH 5.7 (tissue:solvent=3:1)at 0–4°C.The homogenate was then centrifuged at 5000 rpm in refrigerated centrifuge at 0°C for 30 minutes.The clear supernatant obtained,microfiltrated through 45μm filter paper using vacuum pump before injection.Extracts were used immediately after preparation or stored at– 20°C for further experiments[4].

III.EXPERIMENTAL DESIGN

Thirty six rabbits of different ages(18 infants and 18 adults)were used in this study.They were divided into four groups according to the‘a(chǎn)ge(Infants or adults):

First group:Control Infant (CI) group:nine infant rabbits were treated with intraperitoneal injection normal saline in dose 0.25 mL/kg of body weight,daily for 14 days,considered as a control group for Infants.

Second group:Treated Infant (TI)group:nine infant rabbits were treated with intraperitoneal injection thymus extract in dose 0.25 mL/kg of body weight,daily for 14 days.

Third group:Control Adult (CA) group:nine adult rabbits were treated with intraperitoneal injection normal saline in dose by 0.25 mL/kg of body weight,daily for 14 days,considered as a control group for adults.

Fourth group:Treated Adult (TA) group:nine adult rabbits were treated with intraperitoneal injection thymus extract in dose 0.25 mL/kg of body weight,daily for 14 days.

Ⅳ.SAMPLES COLLECTION

Blood samples were collected by cardiac puncture from anesthetized rabbit’s overnight fasted animals.The blood sample which is collected in gel tube centrifuged for 10 minutes at 3000 rpm,serum was stored in (-20°C) for biochemical analysis.Anticoagulant tube was used for investigation of some hematological parameters.

The results obtained were expressed as mean ± standard error of mean of 36 rabbits,statistical significant differences determined by unpaired comparison t-test using Statistics Package for the Social Sciences (SPSS) software package,version 16.Changes were considered statistically significant whenPvalue was found from t-table less than(0.05).

V.RESULTS

Effects of the Crude Thymus Extract on some Hematological Parameters

Table 1 summarizes the effect of crude thymus extract injection on some erythrocyte parameters in infant and adult rabbits.The results revealed RBC counts significantly (P<0.05) decreased in treated adult group while high significant(P<0.01) decrease in treated infant group.Hb concentration and PCV value showed highly significant (P<0.001)reduction in both treated groups.On the other hand in treated infant group,blood indices exhibited statistically nonsignificant changes however MCV and MCHC significant showed reduction in treated adults.Data shows the effect of crude thymus extract injection on leukocyte count.In both treated groups,total leukocyte count were significantly (P<0.001)reduced.

Table 2 shows the effect of the extract on some biochemical data.The values of total protein,albumin,and globulin in treated infant group showed no significant difference in response to the extract administration.However in treated adults;total protein concentration decreased significantly (P<0.05) and albumin concentration declined with high significance (P<0.01),while globulin concentration did not significantly changed.Almost all treated infant and adult rabbits enzymes AST,ALT and ALP concentrations showed highly significantly increased (P<0.01 to 0.001) (Figures 4.7-4.9).

Impact of Thymus Extract on Lipid Profiles Parameters

Table 3 shows hows serum total cholesterol and triglyceride,HDL,LDL and VLDL levels in control and treated infant and adult rabbits.In treated infant group,the triglyceride,VLDL and LDL concentrations significantly (P<0.05)increased,while cholesterol concentration highly significantly increased (P<0.01).HDL concentration significantly (P<0.05) declined.In treated adult rabbits,triglyceride,VLDL and LDL and cholesterol concentrations were significantly (P<0.05 to 0.01) increased.In contrast HDL concentration showed high significant(P<0.01)decreased.

Table 1.Effects of thymus extract injection on erythrocyte parameters(*statistically significant among groups)

Table 2.Effects of crude thymus extract on some liver function parameters AST,ALT,ALP,total serum protein,serum globulin,and albümin(*statistically significant among groups)

Table 3.Effects of thymus extract on lipid profile parameters(*statistically significant among groups)

Ⅵ.DISCUSSION

Effect of Crude Thymus Extract on Erythrocytes Parameters

Effect of the crude calf thymus extract after 30 days from the last injection in infant rabbits caused significant decline in the total number of erythrocytes,Hb value and PCV percentage.In treated adult rabbits,high significant reduction was observed Hb value,PCV percentage and significant reduction in RBC count,MCH and MCHC were observed.During injection of thymus extract,may increase activity of the phagocytic cells such as neutrophils and macrophages.The mechanism involved in the interpretation of this reduction in erythrocyte indices induced by the crude thymus extract is not completely clear,however it could be explained on the base of inhibition of erythropoietin according to other researches have which reported that increased secretion proinflammatory cytokines in the site injection,such as TNF and IL-1 inhibit erythropoiesis in vivo and in vitro through disrupted erythroid development via the inhibition of this precursor in bone marrow[5].Phatho-Physiological stimuli by this mechanism may contribute to the decrease in erythropoiesis following impaired ability of the erythroid progenitors to respond to erythropoietin [6] and suppression of erythropoietin gene expression [7] that reduction in erythropoietin availability results in a selective heamolysis of youngest erythrocyte and shortened erythrocytes life span[8].

The action of the other proinflammatory cytokine,IL-6 is less effective than the above factors,it may exert its effects on erythropoiesis through regulation of iron metabolism [9],which can up regulate the production of hepcidin in the liver [10].Hepcidin may decrease the responsiveness of the bone marrow erythroid progenitor to erythropoietin[11].High levels of hepcidin lead to decrease iron in the plasma and absorbed iron is sequestered for storage in the reticuloendothelial system through exerting double inhibitory effect;on ferroportin-1 receptor on the macrophages;inhibition of iron release and on the enterocyte receptor;inhibiting intestinal absorption of iron(The net results may be diminished quantity of iron available for erythropoiesis which may down regulate the hemoglobin concentration [11,12].In contrast to our findings,in vivo study by Balleariet al.[13] showed the effect of bovine thymic extract administration on the hemopoiesis of healthy elderly were caused significant increase in the number of blastocyte formation unit-erythrocyte and statistically increased hemoglobin concentration and erythrocyte counts.

The results of the present study showed that crude thymus extract caused decline in the total and percentage of lymphocytes in both treated inflants and adults.This reduction may be related to the high molecular weight (more than 60 KDa)of the crude thymus extract components.Similar results were previously demonstrated to possess both immunosuppressive activities on T-lymphocytes by lymphocyte-inhibiting-factor and antiproliferative properties through inhibitory action on DNA synthesis in T-lymphocytes [14].Different results were obtained by administration of thymic hormones separately,from these studies;thymosin fraction5,which is a low molecular weight of the extract content,induced twofoldenhance-ment of T-lymphocytes during in vitro incubation of murine thymocytes by Zatz and Goldstein [15],another study by Peng et al.[16],showed that bovine thymic extract induced lymphocyte differentiation (thymopoiesis) in embryonic stem cells to CD3+and CD4+/CD8+lymphocytes.

Effect of Crude Thymus Extract on Liver Function

The activity of serum aminotransferases (ALT,AST) and ALP were found to be elevated significantly in rabbits 30 days after last dose of the crude calf thymus extract injection.

Injection of the crude thymus extract intraperitoneally under normal circumstances,pass through the intestinal mesentery and subsequently into the liver blood stream induces activation of Kupffer cells increasing phagocytic action are critical for the rapid clearance of external material also facilitate generation of inflammatory response leading to recruitment of inflammatory cells such as neutrophils,monocytes,NK,T and B lymphocytes to the liver and producing reactive oxygen species,increases peroxidation phospholipids on the plasma membrane and changes permeability of cell membrane and that can promote damage of cells in the liver tissue and release and raise the level of ALT and AST enzyme may resemble study by Janeway and Medhitov [17].

Effect of Crude Thymus Extract on Lipid Profiles

The present study showed in treated inflants,triglyceride,VLDL and LDL concentrations significantly increased and cholesterol concentration showed high significant increase,while HDL significantly declined.In adult treated triglyceride,VLDL and LDL concentrations showed highly significant increased and cholesterol only significantly increased;in contrast HDL concentration showed high significant decrease.The inflammatory response of the effect thymus extract on lipid profile in treated adult group was more prominent than treated neonatal group.

The injection of thymus extract seems to increase phagocytosis leading to increase free radicals in adults and inflants,might be associated with acute phase response mediated by several multifunctional cytokines secretion,including TNF-α,IL-1 β and IL-6,and characteristically involves stimulation of specific plasma proteins production,such as serum amyloid protein [18] and reduction of apoprotein A that have essential role in the HDL composition [19].Amyloid protein replaces the apoprotein A reducing the atherogenic potential of these lipoproteins that causes decline in antioxidant action by phospholipid oxidation in LDL lipoprotein,leading to modification in the activity of LDL [20],LDL and VLDL oxidation which is an important mechanism for the initiation of atherosclerosis[21].This oxidation promotes the recruitment and retention of monocytes.LDL and VLDL particles are protected tissue against invasion of free radical and oxidation by antioxidants carrier,primarily alpha-tocopherol (6 molecules vitamin E/LDL particle),with minor amounts of gamma-tocopherol,carotenoids[22].

In inflants,injection of crude thymus extract may induce some acute phase proteins production such as amyloid in the liver,significantly lower than in adults.Changes of HDL concentration is lesser in infant than in adult so the inflammatory response is less which decreases transport of antioxidant materials to the damage tissue.

Elevation of Cholesterol in treated rabbit may be related to pro inflammatory cytokines that increases in response to cholesterol synthesis in hepatic cell and decrease hepatic cholesterol catabolism by inhibiting cholesterol 7alphahydroxylase,the key enzyme in bile acid synthesis.Also decrease HDL cholesterol levels and induce alteration in the composition of HDL,lead to decreased reverse cholesterol transport [23].

Unlike our study G?kkus?u and Oz [24] demonstrated that thymosin F5 might normalize the changes in lipid levels and decrease lipid peroxide and elevate glutathione in the plasma,liver and aorta tissues of atherosclerotic in rabbits with high doses of cholesterol in diet.While in thymectomized adult rats caused hypercholesterolemic animals and supplementation with thymic peptide limited free radical damages to the hepatic cells,according to these findings,it was implied that thymic peptide possesses antioxidant activity.Another study by Armutcu et al.[25].showed that in rats fed with fructose lead to hepatic steatosis and a defect in the free radical defense system treatment by Tα1 may decrease biochemical liver function and caused morphologic changes.

Ⅶ.CONCLUSION

The present study indicates that crude thymus extract is toxic and induces inflammatory response predominantly in adult than in infant.

Authors’Contribution:

Study concept and design:G.S.A.and M.K.N.Analysis and interpretation of data:Z.S.,B.O.and G.S.A.Drafting of the manuscript:Z.S.,B.O.and G.S.A.Statistical analysis:

A.M.S.J.and M.K.N.Administrative,technical,and material support:A.M.S.J.and M.K.N.

Ethics:

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Acknowledgements:

The authors would like to acknowledge and thank Department of Biology,Collage of Science,University of Duhok,Iraq for his continued encouragement and support of this project.

Citation:Ali GS,Jubrail AM,Selamoglu Z,Ozdemir B,Najeeb MK.Effects of crude thymus gland extract on some physiological and biochemical parameters in local rabbits.TMR Clin Res.2021;4(3):12.doi:10.53388/TMRCR2021082 5003

Executive editor:Si-Yu Bian.

Submitted:10 May 2021,Accepted:24 August 2021,

Online:25 August 2021.

? 2021 By Authors.Published by TMR Publishing Group Limited.This is an open access article under the CC-BY license(http://creativecommons.org/licenses/BY/4.0/)