ما هو تركيز الكلوريد داخل الخلايا في الفئران التائية أو الخلايا التوتية

p = 0.005, in gonad-intact males.

of the gonad-intact control with

ص < 0.04, in castrated males.

3.4. The DCA Effect on RNA Expression of NKCC1 in Thymus

Slc12a2 RNA expression in the thymus after normalization with Glpdh gene in analyzed rats groups is shown in Table 4.

ص < 0.0001, DCA-treated gonad-intact males compared with their control.

ص = 0.015, DCA-treated castrated male compared with their control.

Expression difference in the Slc12a2 و Glpdh genes between the DCA-treated and the control groups is considered as the ∆CT value. ال Slc12a2 RNA levels in experimental groups after normalization with the Glpdh gene are shown in Figure 5. A significant difference was found between the ∆CT values of the gonad-intact male control and DCA-treated groups (p < 0.0001). In the castrated male group, the expression of the Slc12a2 و Glpdh genes between the DCA-treated and the control groups was also significant (p = 0.015). The difference between the ΔCT of target and reference genes as expressed by the ΔΔCT is shown in Table 4. The RNA expression level ( ) in the gonad-intact DCA-treated male was 0.103-fold change lower than in the control. This means the 90% downregulation of expression as the expression level is decreased by 90% to the level of 10% under control conditions. Also, there was a significant Slc12a2 gene expression change in the castrated male DCA-treated group as compared with the control: its expression level was found to be decreased by 76% (Table 4). However, we found no statistical significance in the test RNA expression when comparing the control and the DCA-treated groups in both gonad-intact and castrated female rats (p > 0.05).

The correlation of thymus weight with ∆CT in the controls and in the DCA-treated gonad-intact and castrated groups of both genders was insignificant (p > 0.05) no significant differences comparing correlation coefficients of the control with the DCA-treated, respectively, groups were found (p > 0.05 Table 3).

4. Discussion

DCA has been employed as an investigational medicine for indication in the chronic treatment of conditions related to pathologically increased cell proliferation such as cancer and pulmonary hypertension [1, 10, 14]. The DCA principal site of action is to inhibit the PDHK, keeping the PDC in the unphosphorylated catalytically active form [5]. PDHK has been suggested as a specific target in proliferating cancer cells [13]. PDH is present in all tissues. Also, the DCA effect is related with a decrease of the blood lactate acid level [12]. Many aspects of the contribution of the DCA pharmacological mechanisms and their relationship with gender-related pathophysiological processes have not been investigated. PDH activity was higher in young female than in young male rats and decreased after ovariectomy but not after orchidectomy [29]. Estradiol treatment increased the expression of several PDH subunits [30]. Eight out of 11 genes evaluated for the PDC had higher expression levels in adult male hearts compared to females [31].

The rat thymus could be a valuable model for investigating the impact of a medicinal product on thymocyte proliferation. The study tested the hypothesis that the influence of DCA on thymus weight might be gender-related. It was based on the recent published data in which DCA has been shown to decrease thymus weight in gonad-intact male rats [20]. To exclude the influence of gonadal hormones on thymus involution, castrated male and female rats were studied also. We expected that changes might occur in HCs because they represent the stage of thymic epithelial cell differentiation [32, 33] and participate in removing the matured or apoptotic thymocytes [34, 35]. Both thymocytes and thymic epithelial cells possess androgen receptors [36].

A comparison of rat thymus weight of gonad-intact and castrated rats is related with a significant increase of thymus weight in castrated rats of both genders. Castration induces a thymus hyperplasia which is related with increased rat thymocyte proliferation [37]. Thymic involution is a complex process related with numerous molecular mechanisms, ageing, and gonad hormones included [38]. Androgens induce a decline of thymus weight in NZB mice, which results in the gender dimorphism of thymus weight between males and females [39]. In castrated female rats at the age of one month, the number of thymocytes and recent thymic emigrants in the peripheral blood was increased after a month as compared with the control animals [40]. The surgical and chemical castration of 12-month-old male rats caused regeneration of the atrophied thymus [41]. The Sprague–Dawley rat castration enhances thymic weight whilst gender hormones reduce the castration-induced thymus hypertrophy [42].

The study results show a decrease of thymus weight after four weeks of DCA treatment only in the gonad-intact males and females and no DCA treatment impact on thymus weight in the age-matched castrated DCA-treated male and female groups. These data indicate that the DCA acts on thymus weight synergistically with gonad hormones of both genders in gonad-intact males and females.

No relationship was determined between the thymus weight and the rat body weight in the tested DCA-treated groups of both genders despite the fact that castrated DCA-treated rat groups showed a significant body weight decrease no rat body weight change was noted in the DCA-treated gonad-intact rat groups of both genders. A significant body weight decrease in DCA-treated castrated rats may indicate a change in the gonad-hormone-dependent DCA metabolism or a possible increased toxicity in the conditions of the reduced gonad hormone levels.

The Effect of Castration and DCA Treatment on the HCs in Rat Thymus. The study data indicate that the HCs appeared to be low in male and female thymus of gonad-intact control rat groups, and no gender-related difference was determined. These data confirm the literature data that HCs are poorly expressed in the thymus of rodents [43]. Tanaka et al. described HCs in spontaneous thymoma of 10-week-old Sprague–Dawley rats, and they were evaluated as medullar differentiation areas [44]. Rat castration is related with the increased HCs in female and male thymus [18].

The DCA treatment caused a significant increase of the HCs in the DCA-treated gonad-intact males as compared with their control, and no such increase in the DCA-treated gonad-intact females was found. There was gender-related difference in the HCs when comparing DCA-treated gonad-intact males and females: males showed significantly higher HCs, and no gender-related differences were found in the castrated DCA-treated groups when comparing the castrated control and castrated DCA-treated groups of both genders. The findings of DCA effect on thymus weight, on correlation between thymus weight and HCs may indicate that under the influence of DCA treatment the thymic epithelial cells undergo gender-related changes which could depend on the sex hormones.

In the thymus microenvironment, thymic epithelial cells are an important component supporting thymocyte development [45] which may be under the sex hormone control. When gonad-intact DCA-treated thymus diminishes in weight or increases in castrated animals, the increase in the HCs may have different pathophysiological mechanisms which can be related to the apoptotic thymocyte removal. Both thymic epithelial cells and thymocytes possess functional androgen receptors and can respond to testosterone [36]. The gonad-hormone-related mechanism of thymopoiesis is not clear by using androgen-receptor knockout mice it was shown that thymic epithelial cells but not thymocytes or fibroblasts contribute to the determination of thymic cellularity [46]. The thymic epithelial cells in gonad-intact aged and in castrated male mice express similar sets of genes, and they are not altered after castration [47]. The mechanisms of the synergistic effect of DCA and testosterone on gonad-intact male thymus remain to be elucidated.

The study indicates some thymic epithelial cells to transform into HCs after reduction of gonad hormones by castration of both gender rats. When castrated male and female animals were treated with DCA, we noticed the further tendency of increase in HCs in the thymus of both sexes, and it was more pronounced in males. This supports the findings of Gray et al. that the thymic stroma is a dynamic population of thymocytes, and it may play a more direct role in the selection of thymocytes [48]. The study results suggest that different mechanisms are responsible for changes in thymopoiesis and thymus epithelial cells transformation into HCs under the DCA influence in gonad-intact and castrated animals. The DCA influences the thymopoiesis suppressing thymocyte proliferation, and this effect was gender-related and more pronounced in gonad-intact male rats in contrast, in castrated rats after DCA treatment such DCA effect was not expressed.

The DCA Effect on RNA Expression of NKCC1 in Thymus. The existence of NKCC1 in rat thymocytes was the background to check the influence of DCA on its activity. The NKCC1 functional activity in rat thymocytes was found to be related with the chloride anion influx sensitive to the NKCC inhibitor furosemide, indicating that part of the chloride influx is mediated by NKCC1 [22, 23, 49].

The study shows that the DCA could target the NKCC1 protein where a gender-related difference of the DCA effect on the NKCC1 gene expression in rat thymus was determined. A significant difference was found between the ∆CT values (expression difference in the Slc12a2 و Glpdh genes) in the DCA-treated gonad-intact and control males as well as in the castrated male groups. ال Slc12a2 gene RNA expression level (the value) in the gonad-intact DCA-treated males was decreased by 90% under control conditions and significantly decreased (by 76%) in the castrated males. No changes in the tested NKCC1 RNA gene expression level were determined in the studied gonad-intact and castrated female DCA-treated groups as compared with the control of both gonad-intact and castrated female rats.

There are two NKCC isoforms: NKCC1 and NKCC2 the NKCC1 is distributed in various tissue types, among them in the thymus [24, 25] of different species [50]. NKCC1 mRNA levels were higher in males than in females on the day of birth, and the total NKCC1 protein levels were higher in the embryonic male than in female hypothalamus [51].

The DCA increases the reactive oxygen species generation [52]. The DCA produces time- and concentration-dependent increase in the superoxide anion and nitric oxide production in zebrafish [53]. The DCA effect on NKCC1 might be related with the oxidative stress because the NKCC activity in different cell types is regulated by oxidation and nitration, and the NKCC activity may depend on the levels of free oxidative radicals or nitric oxide donors free oxidative radicals and protein tyrosine nitration can affect the NKCC structure and its function the NKCC1 activity in endothelial cells was inhibited by the oxidant tertbutylhydroperoxide exposition [54, 55].

Young adult female mice have a lower oxidative stress and a higher pyruvate dehydrogenase complex activity as compared with young adult males, indicating that females may be better protected against the ROS damage. The depletion of steroids by ovariectomy potentially enhanced oxidative damage, whereas orchidectomy did not modify the oxidative stress parameters in mice [29]. The female rats showed a lower production of hydrogen peroxide in cardiac mitochondria as compared to males [56]. Increased formation of ROS inhibits the rat thymocyte proliferation [57].

The DCA treatment was reported to significantly reduce the thymus weight, and such changes are in concert with the DCA-induced increase of thymocyte percentage in the G₂-M cycle phase and the reduced percentage of the G₀-G₁ phase [20]. The DCA-induced G₂-M phase arrest in multiple myeloma cell lines induced by DCA was found also, where the described DCA effect supposed to be related with the oxidative stress caused by the DCA [17, 58]. Additionally, the increased reactive oxygen species generation in relationship with DCA treatment appears with a concomitant cellular shift from glycolysis to oxidative metabolism, resulting in an increased apoptosis [59]. The DCA causes Treg induction and Th17 suppression in the T-cell differentiation process which is dependent on the reactive oxygen species [60].

The induction of NKCC1 RNA expression suppression indicates that DCA may be important in regulating the intracellular chloride thymocyte concentration. The impact on the intracellular chloride concentration would have the antiproliferative effect [61, 62]. The NKCC1 plays an important role in cancer cell proliferation, apoptosis, invasion [62–64], has a potential role in cancer progression of tumors with a high NKCC1 expression, and is recognized as a cancer therapeutic target [26].

DCA acts as inhibitor of glutathione S-transferase-zeta1 (GSTζ). Elimination of DCA including glutathione- (GSH-) dependent oxygenation to glyoxylic acid mainly depends on GSTζ-catalyzed dechlorination by mitochondrial or cytosolic enzymes [65–67]. Regarding cytosolic GSTζ، ال

for GSH obtained in female rats is 2.5–3.2-fold higher than males the higher of GSH is associated with lower access or binding to GSH [67]. It has been reported that DCA metabolism could be gender- and sex hormone dependent in rats: activity of glycolate oxidase in gonad-intact females and gonadectomized male rats was significantly lower than in gonad-intact males ones (glycolate oxidase is involved in the conversion of DCA metabolite glycolic acid to oxalate in rat liver) testosterone increases and estrogens decrease activity of glycolate oxidase in male rats [68]. The preclinical studies of DCA metabolism were conducted only in male rats [69–74]. Data on DCA metabolism in males cannot be directly extrapolated to females. The scientific guidelines for the preclinical safety evaluation of pharmaceuticals require both genders to be used [75] the principles of Good Clinical Practice require that the available nonclinical information on an investigational product should be adequate to support the proposed clinical research [76] e.g., gender-related investigational medicines effects remain to be explored firstly by preclinical studies.

5. الاستنتاجات

(i) The DCA treatment decreases the thymus weight of gonad-intact rats of both genders, but such impact is absent in castrated rat groups, indicating the synergistic pharmacological mechanism of DCA and gonad hormones. (ii) The different mechanisms are responsible for changes in thymus epithelial cells transformation into Hassall’s corpuscles under the DCA effect in gonad-intact and castrated animals. (iii) The DCA treatment decreases the NKCC1 gene RNA expression in the gonad-intact and castrated males, and no such effect was determined in females. (iv) The investigation of the DCA treatment efficacy should be evaluated with regard to gender and the exposition of gonad hormones.

توافر البيانات

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

تضارب المصالح

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

شكر وتقدير

The present study was partially funded by research fund of the Lithuanian University of Health Sciences and by the Research Council of Lithuania.

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حقوق النشر

Copyright © 2019 Jūratė Stanevičiūtė et al. هذا مقال مفتوح الوصول يتم توزيعه بموجب ترخيص Creative Commons Attribution License ، والذي يسمح بالاستخدام غير المقيد والتوزيع والاستنساخ في أي وسيط ، بشرط الاستشهاد بالعمل الأصلي بشكل صحيح.

آلية التأثيرات السمية المناعية لكلوريد ثلاثي بوتيل القصدير على الخلايا التوتية الفئران

كلوريد ثلاثي بوتيل القصدير ، مركب معروف من القصدير العضوي ، هو مادة سامة بيئية منتشرة. لا تزال التأثيرات السمية المناعية لكلوريد ثلاثي بوتيل القصدير على نظام الثدييات وآليتها غير واضحة. تم تصميم هذه الدراسة لاستكشاف طريقة عمل موت الخلايا المبرمج الناجم عن ثلاثي بوتيل القصدير ومسارات موت الخلايا المبرمج الموازية الأخرى في الخلايا التوتية الفئران. تمت ملاحظة الاستجابة المبكرة في الإجهاد التأكسدي متبوعًا بإزالة الاستقطاب من غشاء الميتوكوندريا وتنشيط كاسباس 3. المعالجة المسبقة مع ن- أسيتيل سيستئين وبوثيونين سلفوكسيمين مثبط بشكل فعال الحمض النووي الناجم عن موت الخلايا المبرمج الناجم عن ثلاثي بوتيل القصدير ورفع مستوى G الفرعي₁ السكان ، على التوالي. تمنع المعالجة المسبقة لمثبطات Caspase موت الخلايا المبرمج الناجم عن ثلاثي بوتيل القصدير. تشير اللطخة الغربية والقياس الخلوي للتدفق إلى عدم وجود إزاحة للعامل المحفز لموت الخلايا المبرمج والنوكلاز الداخلي G في الجزء النووي من الميتوكوندريا. يتم رفع مستويات الكالسيوم 2+ داخل الخلايا بشكل ملحوظ بواسطة كلوريد ثلاثي بوتيل القصدير. توضح هذه النتائج بوضوح مسار موت الخلايا المبرمج المعتمد على الكاسبيز وتدعم دور الإجهاد التأكسدي ، وإزالة الاستقطاب من غشاء الميتوكوندريا ، وتنشيط كاسباس 3 ، والكالسيوم أثناء استماتة كلوريد ثلاثي بوتيل القصدير (TBTC) الناجم عن موت الخلايا المبرمج الغدة الصعترية.

هذه معاينة لمحتوى الاشتراك ، والوصول عبر مؤسستك.

شاهد الفيديو: تكاثر الفيروس داخل الخلية عن طريق القاء الحمض النووى Dna داخل الخلية (كانون الثاني 2023).

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