Both Matricaria chamomilla and Metformin Extract Improved the Function and Histological Structure of Thyroid Gland in Polycystic Ovary Syndrome Rats through Antioxidant Mechanism

There is increasing proof that polycystic ovary syndrome (PCOS) is associated with the increased frequency of thyroid disturbances. Chamomile (Matricaria chamomilla L.) herb and metformin showed therapeutic efficacy against polycystic ovary syndrome (PCOS). This study aimed to investigate the possible therapeutic effect of both chamomile flower extract and metformin against thyroid damage associated with PCOS in rats. The PCOS model was developed in rats by injecting estradiol valerate, and it was confirmed to be associated with thyroid hypofunction biochemically and pathologically. Treatment of PCOS rats with both chamomile extract and metformin resulted in an improvement in serum level of thyroid hormones (TSH, p < 0.01; T3 and T4, p < 0.05) and the disappearance of most thyroid gland pathological changes demonstrated by light and electron microscopes. They also reduced the level of serum estrogen (p < 0.01). Both chamomile extract and metformin decreased MDA (p < 0.05) and increased GPx and CAT (p < 0.01). Only chamomile extract increased GSH (p < 0.01). Both treatments reduced the apoptotic death of thyroid cells as noted by the reduction of caspase-3 immunoexpression (p < 0.01). In conclusion, both Matricaria chamomilla extract and metformin ameliorated hypothyroidism associated with PCOS through an antioxidant and antiapoptotic mechanism.


Introduction
Subclinical hypothyroidism (SCH), which may be interpreted as an increased thyroid-stimulating hormone (TSH) level together with normal thyroxine (T4) level and absence of obvious symptoms of hypothyroidism is often more prevalent than overt hypothyroidism [1]. Despite the fact that SCH is

Study Groups
Four groups of rats were used (n = 6). Group 1: control, rats in this group were injected with 0.2 mL of corn oil. Group 2: PCOS, rats in this group were left without treatment. Group 3: M. chamomilla, rats in this group were orally administered M. chamomilla flower extract (75 mg/kg) daily for 30 days after the establishment of the model (Farideh et al. 2010). Group 4: metformin, rats in this group were orally administered metformin (50 mg/100 g body weight) daily for 30 days after the establishment of the model (Elia 2006).

Assessment of Percent Body Weight (% BW) Increase
Rats BW was assessed at the beginning of the experiment (initial BW) and at the end of 12 weeks (final BW). The % BW increase was calculated by the following equation: % BW increase = ((Initial BW−Final BW)/Initial BW) × 100

Sampling
At the end of the experiment blood samples were gathered by heart puncture and the serum was then separated and kept frozen at −80 • C for determination of thyroid function markers and oxidative stress/antioxidant measures. The ovaries and left thyroid lobes were then dissected and kept in 10% neutral buffered formalin for assessment of PCOS induction, thyroid gland histopathological alterations and thyroid gland immunohistochemical expressions. The right thyroid lobes were collected and kept 1 h in 2.5% glutaraldehyde, postfixed for 30 min in 1% osmium tetroxide [19].

Assessment of Thyroid Gland Weight (Thy W)
Thy W in g was determined for each rat.

Assessment of PCOS Induction
Haematoxylin and eosin (H & E) stained sections of the ovary were examined for the presence of multiple cysts in the PCOS rats (n = 18) compared to the control rats (n = 6).

Assessment of Histopathological Alteration Using Light and Electron Microscope
For the light microscopic examination, the formalin-fixed thyroid glands were embedded in paraffin wax, serially sectioned at 3-5 mm and stained with H & E. In addition, the glutaraldehyde-fixed thyroid glands were embedded in Epon, cut at 0.5-1 mm, stained with toluidine blue.
For the electron microscope examination, ultrathin sections of the thyroid gland were stained with uranyl acetate and lead citrate.

Statistical Study
Results were represented as mean ± SE. Data were examined using ANOVA followed by the Tukey HSD test via the online calculator, astatsa.com/One-Way_Anova_with_TukeyHSD/. The p-value was fixed at 0.05.

Volatile Constituents of M. Chamomilla Extract
SPE-GC/MS analysis of M. chamomilla extract revealed the presence of many volatile compounds. The high concentrations compounds were bisabolol oxide, pentacosane, tetracosane, and tricosane (Table 1, Figure 1 1,2).  (2) A bar chart showed the % of volatile constituents present in M. chamomilla extract. (2) A bar chart showed the % of volatile constituents present in M. chamomilla extract.

Proof of PCOS Induction
In estradiol valerate injected rats (PCOS), ovaries sections stained with H & E showed marked histological features of PCOS such as the presence of many cysts and degenerated follicles compared to the control rats' sections ( Figure 2a,b). In estradiol valerate injected rats (PCOS), ovaries sections stained with H & E showed marked histological features of PCOS such as the presence of many cysts and degenerated follicles compared to the control rats' sections ( Figure 2a,b).

Effect of M. Chamomilla Extract and Metformin on Body and Thyroid Gland Weight
Induction of PCOS, as well as administration of either M. chamomilla or metformin, resulted in no changes in rats' body weight mass (Table 2).
On the other hand, the induction of PCOS significantly (p < 0.01) increased rats' thyroid gland weight relative to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in non-significant (p = 0.879) changes in rats' thyroid gland weight relative to the PCOS rats. However, the administration of metformin to PCOS rats significantly (p < 0.05) decreased rats' thyroid gland weight relative to the PCOS rats ( Table 2). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group.

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Dysfunction
The induction of PCOS significantly (p < 0.01) increased serum TSH concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.01) decrease in serum TSH concentration compared to the PCOS rats ( Table 3).
Induction of PCOS significantly (p < 0.05) decreased serum T3 and T4 hormone concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.05) increase in serum T3 and T4 hormone concentration relative to the PCOS rats (Table 3).

Effect of M. Chamomilla Extract and Metformin on Body and Thyroid Gland Weight
Induction of PCOS, as well as administration of either M. chamomilla or metformin, resulted in no changes in rats' body weight mass ( Table 2). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group.
On the other hand, the induction of PCOS significantly (p < 0.01) increased rats' thyroid gland weight relative to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in non-significant (p = 0.879) changes in rats' thyroid gland weight relative to the PCOS rats. However, the administration of metformin to PCOS rats significantly (p < 0.05) decreased rats' thyroid gland weight relative to the PCOS rats ( Table 2).

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Dysfunction
The induction of PCOS significantly (p < 0.01) increased serum TSH concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.01) decrease in serum TSH concentration compared to the PCOS rats (Table 3). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group. ANOVA was adopted. TSH: thyroid stimulating hormone; T3: triiodothyronine; T4: thyroxine.
Induction of PCOS significantly (p < 0.05) decreased serum T3 and T4 hormone concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.05) increase in serum T3 and T4 hormone concentration relative to the PCOS rats (Table 3).

Effect of M. Chamomilla and Metformin on PCOS-Associated Increased Estrogen Level
The serum estrogen level was significantly (p < 0.01) increased in PCOS rats compared to the control rats ( Figure 3). Administration of both M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.01) decrease in serum estrogen levels compared to the PCOS rats ( Figure 3). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group. ANOVA was adopted. TSH: thyroid stimulating hormone; T3: triiodothyronine; T4: thyroxine.

Effect of M. Chamomilla and Metformin on PCOS-Associated Increased Estrogen Level
The serum estrogen level was significantly (p < 0.01) increased in PCOS rats compared to the control rats ( Figure 3). Administration of both M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.01) decrease in serum estrogen levels compared to the PCOS rats ( Figure  3).

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Gland Histopathological Changes (H & E)
Control rats showed normal thyroid gland histology, where the follicles are lined by cuboidal epithelium enclosing normal luminal colloid ( Figure 4a). The connective tissue between follicles showed few capillaries and cells (Figure 5a

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Gland Ultrastructure Changes (Semithin Sections (Toluidine Blue) and Ultrathin Sections)
Follicular cells of control rat thyroid glands showed active euchromatic nuclei and cell organelles including rough endoplasmic reticulum (rER) and mitochondria with normal density and dense colloidal granules (Figure 6a,b). Induction of PCOS altered normal follicular cell's ultrastructure. Deformed nuclei with increased heterochromatin, decreased rER which looked dilated and increased dense cytoplasmic colloidal granules were observed. In addition, follicles filled with colloid contained cell debris and numerous desquamated swollen cells could be observed in semithin sections (Figure 6c,d). Semithin sections of M. chamomilla extract thyroid gland showed nearly normal follicles with homogenous colloid free of cell debris. Follicular cells were similar to those of the controls with an active euchromatic nucleus and normal non-dilated rER (Figure 6e,f). Furthermore, metformin administration showed a similar effect, where follicular cells appeared normal, having large nuclei and normal rER with few dense granules (Figure 6g,h).

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Gland Ultrastructure Changes (Semithin Sections (Toluidine Blue) and Ultrathin Sections)
Follicular cells of control rat thyroid glands showed active euchromatic nuclei and cell organelles including rough endoplasmic reticulum (rER) and mitochondria with normal density and dense colloidal granules (Figure 6a,b). Induction of PCOS altered normal follicular cell's ultrastructure. Deformed nuclei with increased heterochromatin, decreased rER which looked dilated and increased dense cytoplasmic colloidal granules were observed. In addition, follicles filled with colloid contained cell debris and numerous desquamated swollen cells could be observed in semithin sections (Figure 6c,d). Semithin sections of M. chamomilla extract thyroid gland showed nearly normal follicles with homogenous colloid free of cell debris. Follicular cells were similar to those of the controls with an active euchromatic nucleus and normal non-dilated rER (Figure 6e,f). Furthermore, metformin administration showed a similar effect, where follicular cells appeared normal, having large nuclei and normal rER with few dense granules (Figure 6g,h).

Effect of M. Chamomilla and Metformin on PCOS-Associated Thyroid Gland Caspase-3 and PCNA Immunoexpression
Induction of PCOS significantly (p < 0.01) increased thyroid gland caspase-3 expression as compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant decrease in thyroid gland caspase-3 expression compared to the PCOS rats (Figure 7 1,2).
Induction of PCOS significantly (p < 0.01) increased thyroid gland caspase-3 expression as compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant decrease in thyroid gland caspase-3 expression compared to the PCOS rats (Figure 7 1,2).
The induction of PCOS significantly (p < 0.01) decreased thyroid gland PCNA expression compared to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in a nonsignificant change in thyroid gland PCNA expression as compared to the PCOS rats. On the other hand, the administration of metformin extract to PCOS rats significantly (p < 0.01) increased thyroid gland PCNA expression compared to the PCOS rats (Figure 7 1,3). . Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. * significant from the control group. # significant from the PCOS group.

Effect of M. Chamomilla and Metformin on PCOS-Associated Oxidative Stress Status
The induction of PCOS significantly (p < 0.01) increased serum MDA concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.05) decrease in serum MDA concentration compared to the PCOS rats (Figure 8a).
The induction of PCOS significantly (p < 0.05) decreased serum GSH concentration compared to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in a significant (p < The induction of PCOS significantly (p < 0.01) decreased thyroid gland PCNA expression compared to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in a non-significant change in thyroid gland PCNA expression as compared to the PCOS rats. On the other hand, the administration of metformin extract to PCOS rats significantly (p < 0.01) increased thyroid gland PCNA expression compared to the PCOS rats (Figure 7 1,3).

Effect of M. Chamomilla and Metformin on PCOS-Associated Oxidative Stress Status
The induction of PCOS significantly (p < 0.01) increased serum MDA concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in a significant (p < 0.05) decrease in serum MDA concentration compared to the PCOS rats (Figure 8a).
The induction of PCOS significantly (p < 0.05) decreased serum GSH concentration compared to the control rats. Administration of M. chamomilla extract to PCOS rats resulted in a significant (p < 0.01) increase in serum GSH concentration compared to the PCOS rats. On the other hand, the administration of metformin to PCOS rats resulted in a non-significant change in serum GSH concentration compared to the PCOS rats (Figure 8b). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group. ANOVA was adopted. GPx: glutathione peroxidase; CAT: catalase; SOD: superoxide dismutase.

Discussion
This study clearly showed that the ethanolic extracts of M. chamomilla relieved the thyroid gland hypofunction demonstrated in estradiol valerate-induced PCOS in rats after 30 days of treatment. This therapeutic effect was characterized by restored serum TSH and estrogen levels, increased serum T3 and T4 levels, reduced oxidative stress, and improved antioxidant capacity. The thyroid gland histological study also showed significant improvements in the histology of thyroid follicles. Furthermore, M. chamomilla extract significantly reduced the apoptosis of the thyroid gland.
Several researchers have established an increased incidence of thyroid autoimmunity in females with PCOS. A previous study demonstrated higher concentrations of thyroid antibody in females with PCOS, increased thyroid size and increased incidence of hypoechogenic thyroid glands (favorable with thyroiditis). Moreover, in the same research, PCOS females showed an increased TSH concentration [20]. Likewise, this study showed increased thyroid gland weight and decreased serum TSH in PCOS rats suggesting that it may be autoimmune thyroiditis accompanying PCOS caused by estradiol valerate. So far, the pathophysiological mechanism that connects PCOS to hypothyroidism Induction of PCOS significantly (p < 0.01) decreased serum GPx and CAT concentration compared to the control rats. Administration of M. chamomilla extract and metformin to PCOS rats resulted in significant (p < 0.01) increases in serum GPx and CAT concentration compared to the PCOS rats. On the other hand, the serum concentration of SOD showed no significant changes across all the experimental groups (Table 4). Results are expressed as mean ± SE (n of 6 animals). The level of significance was fixed at p ≤ 0.05. a significant from the control group. b significant from the PCOS group. ANOVA was adopted. GPx: glutathione peroxidase; CAT: catalase; SOD: superoxide dismutase.

Discussion
This study clearly showed that the ethanolic extracts of M. chamomilla relieved the thyroid gland hypofunction demonstrated in estradiol valerate-induced PCOS in rats after 30 days of treatment. This therapeutic effect was characterized by restored serum TSH and estrogen levels, increased serum T3 and T4 levels, reduced oxidative stress, and improved antioxidant capacity. The thyroid gland histological study also showed significant improvements in the histology of thyroid follicles. Furthermore, M. chamomilla extract significantly reduced the apoptosis of the thyroid gland.
Several researchers have established an increased incidence of thyroid autoimmunity in females with PCOS. A previous study demonstrated higher concentrations of thyroid antibody in females with PCOS, increased thyroid size and increased incidence of hypoechogenic thyroid glands (favorable with thyroiditis). Moreover, in the same research, PCOS females showed an increased TSH concentration [20]. Likewise, this study showed increased thyroid gland weight and decreased serum TSH in PCOS rats suggesting that it may be autoimmune thyroiditis accompanying PCOS caused by estradiol valerate. So far, the pathophysiological mechanism that connects PCOS to hypothyroidism has not been strictly defined. Currently, the best explanation seems to be overlapping pathways with a complex interplay between PCOS, fattiness, thyroid hypofunction, and autoimmunity, operating to generate convergent clinical images [21]. A reasonable elucidation for higher thyroid autoimmunity in PCOS is probably due to the hyperestrogenic status of PCOS. Hyperestrogenism was suggested as a reason for enhanced autoimmune disorders in females contrasted with males [22,23]. Similarly, the results of this study showed increased serum concentration of estrogen in rats in the PCOS group together with the increased level of serum TSH, decreased T3 and T4 serum levels.
Treatment of PCOS rats with M. chamomilla extract significantly ameliorated the hypothyroid function as confirmed by the improved thyroid hormones and histological structure of thyroid follicles. Moreover, caspase-3 (apoptotic marker) expression was decreased in the thyroid gland of M. chamomilla rats compared to PCOS rats. This may be due to M. chamomilla caused a drop in the level of serum estrogen [15]. Phytoestrogens regulate the function of aromatase and the activity of major enzymes in estrogen biosynthesis. In the ovary, the enzyme converts androgens into estrogens. Thus, as phytoestrogens inhibit this enzyme, this process is blocked, thereby reducing the concentration of estrogen [24]. Chamomile extract constitutes the phytoestrogen active constituents; coumarins, which may decrease estrogen formation [25]. Furthermore, phytoestrogens inhibit the function of cytochrome P450 enzymes, thereby hampering cholesterol conversion to pregnenolone, and thus decreasing the production of estrogen [26,27]. In addition, the increase in free T3 and T4 is responsible for the decrease in TSH hormone.
Treatment of PCOS rats with metformin significantly ameliorated the hypothyroid function as confirmed by the decreased TSH and improved T3 and T4 hormones relative to PCOS rats. The results were in agreement with those of Xiaowen et al. [28], who reported that that metformin may directly affect the synthesis of thyroid hormone and then decrease the level of TSH. It has been demonstrated that metformin crosses the blood-brain barrier and a central mechanism of TSH inhibition may thus be an attractive explanation. Even though it activates AMPK in the periphery, metformin suppresses AMPK activity in the hypothalamus and possibly counteracts hypothalamic T3 action on TSH secretion [29].
Also, the caspase-3 expression was decreased in M. chamomilla rats compared to PCOS rats. These histological features were generally correlated to the thyroidal functional status because the serum T3 and T4 levels were enhanced in animals treated with estrogen or tamoxifen as compared to the vehicle-treated controls.
Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the activity of the antioxidant molecules and enzymes, resulting in injurious consequences for the body tissues and cells [30]. Studies have reported that PCOS was accompanied by oxidative stress that may result from obesity, hyperinsulinemia, and dyslipidemia. Administration of many antioxidants like omega-3 fatty acids, α-lipoic acid, and N-acetylcysteine ameliorated hyperlipidemia and insulin resistance in PCOS females [31]. Likewise, this study's results showed increased serum MDA and decreased the serum concentration of GSH, GPx, and CAT in PCOS rats. Treatment of PCOS rats with M. chamomilla extract significantly improved serum oxidant/antioxidant status, compared to PCOS rats, which may be correlated with the improved PCOS-associated thyroid gland hypofunction. Chamomile contains plentiful antioxidants' active constituents [32]. This study showed that the volatile chemical ingredients of M. chamomilla consist of sesquiterpenes compounds (α-bisabolol, bisabolol oxides, bisabolone oxide, trans-beta-farnesene), pentacosane, tetracosane, and tricosane [33][34][35]. The sesquiterpenes compounds may be essential for the antioxidant function of M. chamomilla extract [36,37].

Conclusions
The histological examination of the thyroid gland with both light and electron microscopy, as well as the hormone measurements, showed the efficacy of M. chamomilla ethanolic extract and metformin in improving thyroid hypofunction associated with PCOS in rats, through an antioxidant mechanism.