الفهرس 
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Abstract
Thyroid hormones (THs) are indispensable regulators of cellular activities, including the immune system. The relationship between hypothyroidism (HPO) and oxidative stress (OS) is probably based on the lower activity of the internal antioxidant system, which does not supply sufficient cell protection against free radical accumulation, thereby resulting in oxidative damage. Thyroid-stimulating hormone (TSH) when stimulated in HPO causes hyperplasia, neovascularization, and histopathological alterations in thyrocytes.
Being predictable that HPO may have an impact on the liver given that THs are involved in the metabolism of all the cells in the body. Abnormal liver functions were observed in 15% to 76% of patients with thyroid problems. HPO has been associated with the elevation of cytokines involved in the development of hepatic necrosis, increased hepatocyte death, and vascular permeability.
HPO can impact pulmonary functioning, resulting in a restrictive pattern of lung damage. Low thyroxin (T4) levels in hypothyroid people can lead to a loss of inspiratory muscle strength and hypoventilation. HPO can cause a variety of respiratory symptoms, ranging from mild dyspnea to forceful respiratory failure; in addition, dynamic lung functions are significantly reduced in HPO.
Carbimazole (CBZ) is an antithyroid drug, similar to methimazole (MMI). It acts as a prodrug because it is converted to MMI after administration. Eltroxin (ELT) or levothyroxine is a standard drug for HPO treatment. It significantly ameliorates the changes in THs levels.
Flavonoids have antioxidant, anti-inflammatory, anti-bacterial, anti-mutagenic, and anti-cancer properties. Hesperidin (HSP) is a natural antioxidant that has a broad scope of pharmacological actions. HSP modulates the antioxidant index, and anti-apoptosis, and indirectly activates the nuclear factor-kappa B (NF-kB) signaling pathway to modulate inflammation. HSP was shown to be nontoxic, fast-digested, had no accumulative effects, and did not elicit any allergic reactions in experiments on male and female rats.
The present study aimed to evaluate the potential protective effect of eltroxin and hesperidin against liver, thyroid and lung injury induced by carbimazole in adult male albino rats. Oxidative stress (OS), apoptosis, inflammation, histopathological, ultrastructural, immunohistochemical, and biochemical changes were examined concerning nuclear factor erythroid 2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), and nuclear factor-kappa B.
Thirty-two adult male albino rats were equally divided into four groups. group I: control non-treated rats. group II: rats received CBZ only (20 mg/kg/day) dissolved in distilled water for one week to induce HPO. group III: Rats were given HSP (200 mg/kg/day) dissolved in 1 % carboxymethylcellulose, then followed by a dose of CBZ (20 mg/kg/day) dissolved in distilled water. group IV: Rats were given ELT (0.045 mg/kg/day), followed by a dose of CBZ (20 mg/kg/day) dissolved in distilled water. All treatments were given by oral gavage for 12 weeks. All rats were sacrificed after 90 days of the experiment.
THs levels were increased in the hypothyroid group treated with HSP and the hypothyroid group treated with ELT. There was a significant improvement in the levels of TSH in the hypothyroid group treated with HSP compared to the hypothyroid group treated with ELT compared to the CBZ-treated group. Total protein and albumin were significantly higher in the hypothyroid group treated with HSP compared to the CBZ-treated group. Liver enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), activity was significantly boosted as compared to the CBZ-treated group.
Administration of HSP and ELT separately with CBZ significantly lowered the levels of cholesterol (CHOL), triglycerides (TRIGLY), LDL, and VLDL compared to CBZ group. Furthermore, the administration of HSP and ELT separately in the presence of CBZ significantly increased HDL levels compared to CBZ group. The cardiovascular risk indices 1 and 2 were significantly lower in groups III and IV. The antiatherogenic index (AAI) was significantly decreased in the hypothyroid group treated with ELT as compared to the hypothyroid group treated with HSP.
Groups III and IV showed significant upregulation of Nrf2 expression and HO-1 activity compared to the CBZ-treated group. The current study demonstrated a significant decrement in lipid peroxidation levels and a significant increase in the antioxidant parameters in groups III and IV compared to the CBZ-treated group in thyroid, liver, and lung tissues.
Tumor necrosis factor-alpha (TNF-α), IL-17, and COX-2 of thyroid tissues were significantly decreased in groups III and IV compared to the CBZ-treated group. IL-10 level of thyroid tissues was markedly increased in groups III and IV compared to the CBZ-treated group.
TNF-α and IL-1β levels of liver tissues were markedly reduced in groups III and IV compared to the CBZ-treated group. IL-37 level markedly increased in groups III and IV compared to the CBZ-treated group. The liver of the CBZ-treated group showed a significant increase in alpha-smooth muscle actin (α-SMA) expression. Interestingly, administration of HSP or ELT with CBZ restored the α-SMA index like the control level. PCNA was significantly higher in the hypothyroid group treated with HSP than in ELT-treated group.
Significant increases appeared in IL-6, and TNF- α of lung tissues in CBZ-treated group when compared to the control group. In comparison with the CBZ-treated group, HSP and ELT significantly reduced IL-6 and TNF-α levels in the hypothyroid group treated with HSP than in ELT-treated group.
Histopathological assessment of the thyroid tissues in the CBZ-treated group demonstrated loss of normal architecture of the follicles with absent or scanty amounts of colloids. Most of the follicular cells were enlarged and vacuolated. The examination by the electron microscope showed that the cytoplasm was filled with dilated rER containing an amorphous substance and shrunken heterochromatic nuclei with irregular nuclear membrane. Additionally, congested blood capillaries and electron-dense mitochondria were observed.
The thyroid tissues in the hypothyroid group treated with HSP revealed a normal architecture and the restoration of most follicles filled with acidophilic colloids; the follicular cells presented with oval to rounded nuclei. Normal endoplasmic reticulum and lumens filled with a moderately dense colloid was observed. In the hypothyroid group treated with ELT, the thyroid tissues showed a slight improvement in the majority of the follicles.
The mean area of Tg immunostaining in the thyroid sections was significantly lower in the CBZ-treated group compared to those in groups III and IV. NF-kB and PCNA expression was significantly increased in the CBZ-treated group and markedly decreased following HSP co-treatment. Mild NF-κB and PCNA expression were observed in the hypothyroid group treated with ELT compared to that in the CBZ-treated group.
Histological investigation of liver sections from the CBZ-treated group demonstrated hepatocytes with marked cytoplasmic vacuolation. Moreover, pyknotic or karyolitic nuclei, focal necrosis, hepatocyte disarray, and dilated congested sinusoids were also detected. The examination by the electron microscope revealed that hepatocytes exhibited unclear contours of the plasmalemma and remnants of cytoplasmic organelles. Dense clusters of inflammatory cells, multiple pleomorphic mitochondria, numerous lipid droplets, and an accumulation of glycogen surrounded by a large number of mitochondria were observed.
On the other hand, the liver sections of the hypothyroid group treated with HSP showed the nearly normal structure of hepatocytes with vesicular nuclei. The examination by the electron microscope demonstrated that hepatocytes of the hypothyroid group treated with HSP demonstrated marked recovery of the hepatocyte nuclei along with intact clear nuclear and normal mitochondrial features. Kupfer cells had normal structures and normal nuclei. The liver of the hypothyroid group treated with ELT showed moderate improvement, except for cytoplasmic vacuolation of a few hepatocytes and slightly dilated blood sinusoids. The examination by the electron microscope showed hepatocytes with nearly normal mitochondria, glycogen granules, and slightly normal nuclei with intact nuclear envelopes.
A significant decrease in the area percentage of the iNOS and NF-κB immunoreactions was noted in the sections of the hypothyroid group treated with HSP and the hypothyroid group treated with ELT compared to the CBZ group. Bcl-2 immunostaining was significantly increased in the hypothyroid group treated with HSP, while insignificantly increased in the hypothyroid group treated with ELT compared to the CBZ-treated group.
Histological investigation of lung sections from the CBZ-treated group demonstrated marked hyperplasia of a dilated bronchiole, collapsed alveoli, and other compensatory effects. Thickening of the interalveolar septa, significant augmentation of the inflammatory mononuclear cells in peribronchial and perivascular areas were also found. Alveolar hemorrhage, and significant cellular architectural alterations involving dilatation of congested blood vessels and the appearance of fat cells were also noted. The examination by the electron microscope revealed a Type I-containing nucleus with heterochromatin clumps, deposition of collagen fibers, and some vacuoles. Degenerated P2 with an irregular, shrunken pyknotic nucleus, lamellar bodies with a marked depletion of the surfactant material, absence of microvilli, severe cytoplasmic vacuolation, numerous collagen fibers, and bulged vacuolated mitochondria with destroyed cristae were also detected.
The hypothyroid group treated with HSP showed a nearly normal appearance of the bronchioalveolar unit parenchyma, with clear alveoli that were lined with Type I and Type II pneumocytes (p1 and p2) containing normal nuclei, bronchioles, thin intra alveolar septa, and few infiltrations of inflammatory cells. Electron microscopic examination revealed normal alveolar P1 and P2 cells with rounded nuclei and peripheral heterochromatin, numerous lamellar bodies with a concentric lamella of secretory material, normal mitochondria, and intact microvilli.
The hypothyroid group treated with ELT showed mild thickened interalveolar septa, bronchioles, and mild focal thickening of interstitial connective tissue with a few infiltrations of inflammatory cells. The examination by the electron microscope revealed that the P1 had an elongated nucleus, and the adjacent P2 contained a slightly normal nucleus. Slightly normal mitochondria, a moderate number of lamellar bodies, and short microvilli on the surface were also observed.
The immunohistochemical expression for iNOS, NF-κB, and nuclear proliferative marker Ki-67 in the lung tissue was significantly reduced in groups III and IV as compared to the CBZ-treated group.
The findings of this study indicated that HPO should be treated to reduce the potential for thyroid, liver, and lung impairment. The biochemical, histopathological, immunohistochemical, and ultrastructural endpoints of the current investigation highlight that HSP at a high dose (200 mg/kg) could effectively protect against thyroid, liver, and lung damage from the severe damage provoked by CBZ by acting on different levels.
The protective effect of HSP may be attributed to the activation of the Nrf2/HO-1 pathway, suppression of NF-κB pathways, regulation of apoptotic and proliferative markers, and its anti-hyperlipidemic, antioxidant, and anti-inflammatory potential. The findings of this study can stimulate further research on the protective role of HSP in other HPO-induced diseases. It is worth noting that the ELT dose was used more judiciously, but HSP can be used at a high dose with restricted side effects and a non-accumulative nature.