IMMUNO PROTECTIVE, ANTI-DIABETIC AND HISTOCHMICAL-ANTI OXIDATIVE ACTIVITIES OF L-CARNITINE, AND CALF THYMUS EXTRACT IN AGED MALE MICE.

Background: The mechanisms behind of immunosenescence have remained largely unknown in elderly. Some studies are referred the cause to that, L-carnitine is essential nutrient factor which it is important in transporting of long chain fatty acids to mitochondrial matrix, a process essential for fatty acid oxidation and energy release. The immunobiological properties of a new formulation of the lipid thymus calf extract (CYTOIMMUNE ®) Our results showed an improvement in RBCs count, Hb concentration and PCV after intraperitoneal administration of 200 mg/kg b.wt. of L-carnitine in aged male mice. This agree with the previous findings reporting that L-carnitine administration in male rats lead to increase in RBCs count, Hb concentration and PCV ( Qadir et al., 2009 ). (2014) stated that RBCs count and Hb content were increased in chicks fed diet supplemented by L-carnitine compared to those fed on control diet. This results may be due to an effect of L-carnitine on erythrocyte stability and survival. Therefore, by this mechanism PCV can be increased. Bommer (1999) demonstrated that L-carnitine improved anemia and also, L-carnitine improved erythropoietin efficiency that reported by Hurot et al. (2002) . In this study, L-carnitine treated groups were shown significantly decreased in neutrophil / lymphocyte ratio that may be attributed to increase level of lymphocyte production. This results are similar to results of Ohara et al. (2018) who reported that L-carnitine non oral administration was decreased N/L ratio than oral route. In addition, lymphocyte cells % were significantly increased for duckling fed diets supplemented with L- carnitine than control group that demonstrated by ( Awad et al., 2016) . Our results and a significant increase in CAT (catalase enzyme), in liver and kidney.These results clearly show the antioxidant and protective property of experimental preparations.

The thymus gland is the primary lymphoid organ that provide site of T cell production and activation, it represents a key organ of the immune system, (Haroun, 2018).With aging and after puberty, the thymus begins to shrink and involute and the production as well as development of T-cells reduce to a lower rate (Haroun, 2018 andJaneway et al., 2001), therefore, the thymus extracts have been shown to modulate the development, maturation and activation of T cells, stimulate production of interleukin-2 (IL-2), their effect on the specialization and migration of T-lymphocytes throughout the body. The thymus also releases hormones that regulate immune function (Colic et al., 2008). Pretreatment with the calf thymus extract increased the antioxidant enzymes in a dose-dependent manner (Ganie et al., 2014).
Natural thymic peptides have been isolated from calf thymus by mild acid extraction. Pharmaceutical containing natural peptides, thymalin was put into practice as immune corrector. Thymalin and thymogen were used in persons with chronic pathology and immune dysfunction. The previous results indicated that thymic peptides participate in the regulating mechanisms of inflammatory processes as cytokine antagonists (Morozov and khavinson, 1997). The researcher reported that thymalin stimulated T cells while acting with immunoglobulins (Wilson, 1999).Thymalin is a polypeptide complex extracted from the thymus. It is approved for medical application as an immunomodulator by USSR Ministry of Health Order No. 1008 of 10.11.1982,Registration No. 8.1008.8 (khavinson and Morozov, 2003. Barboza et al. (2000) have described the use of thymus extract in therapy of different diseases in few animal species (dogs, rabbits and pigs). The thymus extract acts upon the production, maturation and activation of T lymphocytes and macrophages, and stimulates the maturation of immature thymocytes (T6 cells) to T3 cells in the bone marrow.
Thymosin, soluble hormone-like peptides produced by the thymus gland, can mediate immune and non-immune physiological processes and have gained interest in recent years as therapeutics in inflammatory and autoimmune diseases (Severa et al., 2019). Thymalin and thymosin are the main components of thymus extract. After oral administration of crude thymus extract at dose 1 ml/chick significantly increased the total protein, globulin, albumin, T3, T4 and the body weight gain in chick of one-day-old. Thymus extract also increased total lymphocytic count (Abdel Fattah et al., 1999).
Further investigations are required to elucidate role of L-carnitine and calf thymus extract in various immune functions and hence the present study was designed to evaluate the effect of L-carnitine and calf thymus extract on certain immunological functions and performance characteristics in aged male mice.

Samples collection
Blood samples were collected from five randomly selected animals from each group. Blood samples were obtained from the retro-orbital sinus of the eye and each blood sample was divided into three portions. The first portion was collected in a small labeled dry and clean vial containing one drop of heparin as anticoagulant to determine the phagocytic activity and phagocytic index. The second portion of blood was collected in a small labeled dry and clean vial containing EDTA for hematological studies. The Third portion of blood was collected in plain clean dry and sterile centrifuge tubes and were allowed to coagulate and then centrifuged at 3000 rpm for 10 min. to separate sera for measurement of ALT , AST, immunoglobulins (Ig) and interleukin 2(IL2) .
Twenty-four hours after the last injection the mice were weighed and then the mice were sacrificed. Spleen from treated & untreated mice were removed & were weighed. Specimens from internal organs (liver, spleen and kidney) of all studied mice groups were taken for histological and immune histochemistery examinations.

Hematological examinations
RBCs& WBCs count, PCV and differential leukocytic count were measured.

Candida phagocytosis:
The Phagocytic activity of neutrophils performed according to the method described by Wilkinson (1981) as follow: In clean dry tube, the following aliquots were mixed; 100ul mice serum, 100ul heat killed Candida albicans (5x10 6 /ml) and 100ul of heparinized tested blood. The tube was mixed and then incubated at 37°c for 30 minutes, after which it was centrifuged at 1500 rpm for 5minutes.The supernatant was discarded leaving a droplet into which the sediment was re-suspended. Smear was prepared from the deposit, dried in air, fixed with methyl alcohol for about 3 min and stained with Giemsa stain for 30 min., the slides were washed well with water and dried in air, The slides were examined under oil immersion lens. Then one hundred neutrophils were examined and the number of neutrophils ingesting Candida was counted and expressed as percentage.
No of neutrophils ingesting Candida Total number of neutrophils Phagocytic index = Total no of ingested Candida/ Phagocytic activity % Body weight and immune-related organs relative weight: Just before killing at the end of experiment, final body weight of mice in all experimental groups was recorded. Upon being killed, the spleen was removed aseptically, weighed and their relative organ weights (ROW) were calculated according to For immunohistochemistry:-Specimens from the liver, and kidney, were fixed in 10% (v/v) formalin in phosphate buffered saline (PBS). They were processed for paraffin sections (4 µm in thickness). Immunostaining for CAT was performed using monoclonal antibody to human CAT (Santa Cruz Biotechnology, Inc. Texas, USA). Antigen retrieval was performed by heating at 98 °C in 10 mMtrisodium citrate, pH 6.0, with 0.05% (v/v) Tween-20 for 15 min. Endogenous peroxidases were quenched using 0.3% (v/v) H2O2 in PBS for 10 min. Sections were incubated with the monoclonal anti-CAT overnight at 4 °C. The CAT antibody was diluted in PBS at the ratio of 1:100 PBS. Normal mouse IgG (Santa Cruz Biotechnology, Inc. Texas, USA) diluted in the same way was used for negative control staining. After washing by PBS sections were incubated with biotinylated anti-mouse IgG binding protein (1:200) for 1 h, followed by incubation with avidin-biotin complex (1:50) (Santa Cruz Biotechnology) for 1 h. The immunoreaction products were visualized using a reaction mixture of 0.02% (w/v) 3, 3´-diaminobenzidine-4HCl and 0.05% (v/v) H2O2 in 10 mMTris-buffered saline, counterstained with hematoxylin, and then examined under a light microscope.

Statistical analysis:
Data obtained in this study was statistically analyzed for variance (ANOVA) with confidence limits set as 95 % (significance at p<0.05 probability level) described by Duncan (1955). The results were reported as mean ± standered error "SE", multiple range tests should be performed to compare among different groups of experiment. Statistical analysis was performed using SPSS statistical package.

Hematological analysis:
The mean values of the RBCs, Hb and PCV were significantly higher in group II , group III, respectively than control group. This values were non significantly higher in group IV compared to control one as shown in table 1.
1207 Table 1 illustrated that WBCs were significantly higher in group II compared to other groups. The mean values of neutrophils were significantly decreased in group III on comparison with other groups. Lymphocytes % were significantly higher in group III on comparison with other groups. In addition, Lymphocytes % were significantly higher in group II and group IV compared to control group as shown in figure 1. Moreover, neutrophil/lymphocyte (N/L) ratio was significantly lower in group III (0.32) compared to other groups and N/L ratio was significantly lower in group II (1.13) and group IV (1.58) in comparison with control group (3.11) . Monocytes % were significantly higher in group IV on comparison with other groups. Eosinophils were significantly lower in group IV, group III and group II, respectively on comparison with control group. There is no changes in basophils between groups.

Biochemical findings:
Levels of AST, ALT and glucose in control, L-carnitine at dose 200 mg/kg b.wt. , calf thymus extract at dose 0.5 mg/kg b.wt. and L-carnitine at dose 200 mg/kg b.wt. Plus calf thymus extract at dose 0.5 mg/kg b.wt. groups are shown in table 2 and figure 1. According to these data, it was determined that levels of AST and ALT were significantly decreased at group II, group III and group IV on comparison with control group. A significant decrease in glucose levels were observed in group IV (52.73 mg/dl), group II (55.93 mg/dl), and group III (62.60 mg/dl), respectively on comparison with control group (70.37 mg/dl).
Phagocytic function is the primary mechanism through the immune system eradicates most of pathogens and infections. Phagocytic activity and phagocytic index were illustrated in table 2 and figure 1. From this table we observed that there were significantly increased in phagocytic activity in group II (69%) , group III (62%) and group IV (55.25%) , respectively on comparison with control group ( 46.25%) and a significant increase in phagocytic index was observed in group II , group III and group IV on comparison with control group. Phagocytic activity and phagocytic index mainly higher in all treated groups than control one.

Total protein, albumin, globulin concentrations and A/G ratios are demonstrated in table 2.
There is no significant changes in total protein concentrations between all groups. However, the mean values of globulin were significantly increased in group II, group III and group IV, respectively when compared with control group (group I). A/G ratios and albumin concentrations were significantly lower in group II, group III and group IV on comparison with control group (group I).
Interleukin 2 are shown in table 2 and figure 2. The mean values of IL 2 were significantly increased in group II, group III and group IV, respectively on comparison with control group.
Relative spleen weight (RSW) was calculated and it was presented in table 2. There was significantly increased in RSW in group II, group IV and group III, respectively on comparison with control group.

Histological investigation and immunohisto-anti-oxidative activity:-
Renal histological examination of the treated group showed sever dilatation in the renal collecting tubules,Interstitial lymphocytic infiltrations, and glomerular expansion , expansion of Bowman's capsule and thickening of the blood vessel wall is indicated in the (L-carnitine 200mg/kg.b.wt.) and milled in thymus group as well as in mixed group with thymus and carnitine than normal tissue, (Figure3).
Liver histological sections of treated mice showed a normal histological structure of hepatic tissue that involved arrangement of hepatocyte in radiating manner around central veins, with mild dilatation of hepatic sinusoid and central vein, slight shrinkage of hepatocyte, that was present in high degree in (L-carnitine 200mg/kg.b.wt.) and milled in thymus group as well as in mixed group with thymus and carntine than normal tissue, (Figure 4). Spleen histological sections of treated mice don't show marked or observable internal changes than normal except that of enlarged area of white pulp (lymphocyte producing cells) especially in thymus group, then in mixed group and in L-carntine group respectively, (Figure 5).
With regards to the immune-histochemistery of anti-oxidative activity of catalase enzyme, which seems as qualitative and semi quantitative immune activity and appearas dark brownish color cells concentrated in perivascular area of liver tissue around central veins and portal vein of liver tissues of (L-carntine200 mg/kg.b.wt.) in high degree than control and other treated groups. Moreover, there are dark brown colors concentrated in renal collecting tubules of kidney tissues due to reaction of anti -oxidative investigations of treated extractions in aged mice was highly significant of L-carntine group, then in mixed group (combination between L-Carntine and thymus extract), then in thymus extract group respectively, (Figure 6&7).

Discussion:-
The investigation concerning the effect of L-carnitine on differnent tissues is rear, and further research is required for this purpose, (Boyacioglu et al., 2014). The beneficial effects of L-carnitine have been documented in rat models of renal ischemia reperfusion injury and renal disease (Mister et al., 2002 and Boyacioglu et al., 2014), because, Lcarnitine was known as an exogenous antioxidant agent in cells, due to its protective effects against lipid peroxidation and oxidative stress in the internal tissues, so can protect the tissues from damage by repairing oxidized membrane lipids, (Calò et al., 2006 and Ye et al., 2010). Therefore, it is reported to inhibit apoptosis. Additionally, supplementation with L-carnitine in aged rats improved the antioxidant status, (Boyacioglu et al., 2014).
Our results in aged male mice show that L-carnitine administration stimulated CAT activities in renal and liver tissues is highly significants,.Similar to that observed in our results, L-carnitine may play a major role in stability of antioxidant status in kidney, liver tissue (Boyaciogluet al., 2014).
The kidney plays the major role in carnitine biosynthesis, excretion, and acylation, kidney contains all enzymes needed to form carnitine from trimethyl lysine in activities exceeding that of the liver (Cibulka et al., 2006), and providing a protective effect against lipid peroxidation and oxidative stress (Ye et al., 2010), so our result in histology we observed that the presence of like, expansion of Bowman's capsule, dilatations in the tubule and interstitial infiltration. The antioxidant and free radical scavenger activities of L-carnitine have been proposed to have several mechanisms by inhibiting free radical generation, preventing the impairment of fatty acidbeta-oxidation in mitochondria and protecting tissues from damage by repairing oxidized membrane lipids (Calò et al., 2006).
In the old age, the thymus gland is atrophic and difficult to detect macroscopically, that is manifested with multiple organ dysfunction and increasing of Reactive oxygen species (ROS) or the free radicals that causing deterioration of the immune system, and increased incidence of infection,impaired responses to vaccines, autoimmunity, and cancer, thus increasing the rates of morbidity and mortality in elder age, (Schulz et al., 2015 and Haroun, 2018). The imbalance between oxidants like free oxygen radicals (reactive oxygen species, ROS) and antioxidants are considered as an important etiology of aging, (Desai et al., 2010) It seems that TFX may be a valuable therapeutic agent for the management of patients with chronic hepatitis B. (Skotnicki, 1989). It was concluded that the antibody-mediated chronic kidney graft rejection could be effectively inhibited or delayed with thymic hormones, (Gorski et al., 1985).
The damage to the immune response either by the aged adult mice and by induction of thymectomyin newborn mice, can be prevented by repeated injections of calf thymus extract which is very effective, while the extractsderived from other calf organs as kidney or muscle, are ineffective(Trainin and Linker-Israeli, 1967) due tothe existence of a diffusible product from thymus tissue in different strains and species, which, without being species specific, enables thymectomized animals to produce new lymphocytes and to develop immunologic competence (Law et al.,1964).It has also been reported that injection of calf thymus extract into thymectomized mice protects from the fatal effect of a viral infection, prevents the appearance of a wasting syndrome, and increases the number of lymphocytes in peripheral blood (De Sommer et al., 1963). Histologically, liver and spleen of these injected animals resembled those of neonatal mice (Czaplicki, &Błońska, 1989). Thymus calf extract treatment and neonatal thymus graft have similar corrective effects on age-related changes in mice (Basso et al., 2005).

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Our results showed an improvement in RBCs count, Hb concentration and PCV after intraperitoneal administration of 200 mg/kg b.wt. of L-carnitine in aged male mice. This agree with the previous findings reporting that L-carnitine administration in male rats lead to increase in RBCs count, Hb concentration and PCV (Qadir et al., 2009). Jameel (2014) stated that RBCs count and Hb content were increased in chicks fed diet supplemented by L-carnitine compared to those fed on control diet. This results may be due to an effect of L-carnitine on erythrocyte stability and survival. Therefore, by this mechanism PCV can be increased. Bommer (1999) demonstrated that L-carnitine improved anemia and also, L-carnitine improved erythropoietin efficiency that reported by Hurot et al. (2002).
In this study, L-carnitine treated groups were shown significantly decreased in neutrophil / lymphocyte ratio that may be attributed to increase level of lymphocyte production. This results are similar to results of Ohara et al.
(2018) who reported that L-carnitine non oral administration was decreased N/L ratio than oral route. In addition, lymphocyte cells % were significantly increased for duckling fed diets supplemented with L-carnitine than control group that demonstrated by (Awadet al., 2016).
In regardes to relative spleen weights were significantly increased than control, this refered to increase lymphocyte cell % (Boyacioglu et al., 2014), and due to decreased the total number of thymocytes and splenocytes by the effect of stress which is also accompanied by decreasing weight ratio of the spleen (Obmińiska& Szczypka, 2005).
Our findings revealed elevated in phagocytic activity and phagocytic index in group II (L-carnitine at dose 200 mg/kg b.wt.) in comparison to control group. These results agree with Thangasamy et al. (2008) who reported that L-carnitine at dose 300 mg/kg b.wt. for 7 successive days improved phagocytic activity and phagocytic index in aged rats. This results may be attributed to an increase in superoxide anion production in neutrophils of aged rats which lead to decrease in phagocytosis in control group. Furthermore, superoxide anion production decreased by Lcarnitine treatment. Therefore, by this mechanism phagocytic activity can be improved (Uysal et al., 2003).
The present study revealed that L-carnitine had a hepatoprotective effect that determined by decreased levels of hepatic marker enzymes as AST and ALT. This results are compatible with previous studies, L-carnitine improved liver function enzymes may be attributed to its antioxidant effects leading to protection of membrane permeability, prevention of the leakage of intracellular enzymes (Mousah et al., 2016).
Our results mentioned that L-carnitine reduced blood glucose level in aged male mice. These results may be due to its ability to stimulate fatty acid oxidation in mitochondria. This restores the intramitochondrial acyl-CoA/CoA ratio and stimulates pyruvate dehydrogenase activity and glucose oxidation. Our results showed improvement in RBCs count, Hb concentration and PCV after intraperitoneal administration of 0.5 mg/kg b.wt. of calf thymus extract in aged male mice. This agree with the previous findings reporting that calf thymus preparation (TFX-Polfa) was significantly increased in the hemoglobin concentration and erythrocyte counts ( Lewandowicz, 1990). There are significant increased in the blood picture (RBCs, Hb and PCV) in irradiated Wistar rats treated with thymic extract (Jovanovic et al., 2006).

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In this study, thymus extract treated groups were shown significantly decreased in neutrophil / lymphocyte ratio that may be attributed to increase level of lymphocyte production. This results are similar to results of Naik et al. (2005) who reported that there was a very significant increase in number of lymphocytes in calf thymus extract treated groups, when compared to the control group, which is one of the probable reason for overall immunopotentiation and this could be due to the fact that thymic hormones activates T-cell and activates lymphocyte production. Relative spleen weights were significantly increased, this refered to increase lymphocyte cell % (Abdel Fattah et  al., 1999).
In our study we documented a significant increased of phagocytic activity and phagocytic index. This agree with the previous findings reporting that thymic peptide mixtures (Thymosin fraction 5 thymulin) have been proved to stimulate the immune response and enhance phagocytosis (Jablonowska et al., 2005). The increase in phagocytic activity of phagocytes in calf thymus extract treated group could be due to the ability of the thymus extract to act as an immunomodulator by exerting control on cytokine production by peripheral blood mononuclear cells (Naik et al.,  2005).
The present study revealed that thymus extract had a hepatoprotective effect that determined by decreased levels of hepatic marker enzymes as AST and ALT. This results are compatible with previous studies, oral bovine thymus extracts in viral hepatitis induce broad-spectrum immune system enhancement presumably mediated by improved thymus gland activity and they induced accelerated decreases of liver enzymes (Pizzorno, 2016).
Our results mentioned that thymus extract reduced blood glucose level in aged male mice. This agree with the previous findings reporting that the efficacy of the use of thymalin in therapy of diabetes mellitus in children (Kurbanovetal, 1989). This is in contrast with few studies which stated thatthymosin f5 (thymic hormone) produces hyperglycemic effect (Sivas et al., 1982).The difference between the results of our study and of their studies may be due to the difference in active principle, dose and/ or route of administration of thymus extract protects suchanimals from the fatal effect of a viral infection, prevents theappearance of a wasting syndrome, and increases the number oflymphocytes in peripheral blood.
In this study, thymus extract significantly increased globulin and significantly decreased A/G ratio. This agree with the previous findings reporting that thymus extract significantly increased globulin (Naiket al., 2005).
In this study, thymus extract significantly increased interleukin 2 producing from T-helper cells. Our results are consistent with the hypothesis of (Morozov and Khavinson, 1997) who reported that thymalin was increased production of interferon and interleukin 2 by lymphocytes. In addition to report by (colic et al. 2008) who stated that thymus extract was increased production of interleukin 2. This results may be attributed to a positive effect of intraperitoneal administration of calf thymus extract on humoral and cellular immunity by increasing lymphocyte cells% (Naik et al., 2005).

Conclusion:-
From this study, it can be concluded that: a) thymic involution with age negatively impacts the immune system, so there are disturbance of hematological parameters in aged male mice, which can be improved by exogenous supplementation of L-carnitine or calf thymus extract or combination of them. b) With aging, there are declined in immune functions and intraperitoneal administration of L-carnitine and calf thymus extract or combination of them had an asignificant positive effect in enhancing immune response in aged male mice. C) The hepatoprotective effect of L-carnitine or calf thymus extract or combination between them in aged male mice by decreasing hepatic marker enzymes). Exogenous supplementation of L-carnitine or calf thymus extract or combination of them were decreased glucose level in aged male mice.D) The use of (thymus and L-carntine) preparations improve the histological architectures in the internal tissues (liver, kidney and spleen) and a significant increase in CAT (catalase enzyme), in liver and kidney.These results clearly show the antioxidant and protective property of experimental preparations. 1214