# Effects of _Aloe vera_ on the Regulation of Thyroxine Release in FRTL-5 Thyroid Cells
## 4 Discussion
Conversely, anti-TPO autoantibodies attack the enzyme that synthesizes thyroid hormones, leading to too few thyroid hormones in hypothyroidism [28]. Symptoms of hypothyroidism are treated with hormone replacement therapy using levothyroxine, which is a synthetic thyroxine. It has been reported that _Aloe barbadensis_ Linne juice inhibits TPO auto antibodies, and improves thyroid function in subclinical hypothyroidism patients [24]. We suggest that AV can be used to treat hypothyroidism due to its ability to increase TPO protein expression. The ability of AV to increase TPO could be induced by the inhibition of TPO autoantibodies. Thyroid peroxidase could be a key factor in AV-regulated thyroxine release and the presence of tyrosine, the main constituent of the thyroid hormone, in _Aloe barbadensis_ leaves could be one of the reasons for its curative effects on hypothyroidism [24]. In thyroid cells, cAMP-mediated signaling pathways that activate PKA and ERK mediate thyroid cell proliferation and function induced by TSH [29]. CREB, a transcription factor, plays important role in controlling the growth and function of FRTL-5 cells [30]. Our observations showed that AV affected ERK phosphorylation in both 5H and 6H media and CREB phosphorylation in 6H media, but not Akt phosphorylation in 5H and 6H media (Figure 4). Akt is also known to play an important role in FRTL-5 cells growth and cell cycle progression [30]. Thus, ERK and CREB, but not Akt, may be related to AV-regulated thyroid hormone. Akt might be related to the proliferation of FRTL-5 cells induced by TSH. We hypothesized that AV did not affect cell proliferation (Figure 2), because there was no change in Akt phosphorylation levels. Results from a few previous studies also suggest that quercetin inhibits cell proliferation by down regulating Akt phosphorylation in FRTL-5 cells [23]. To determine whether AV-induced ERK activation in 5H media is dependent on PKA or PKC, we inhibited PKA and PKC by H89 and GF109203X inhibitors, respectively. Phosphorylation of ERK was inhibited by the H89 inhibitor, but not by the GF109203X inhibitor. These results indicate that AV-induced ERK phosphorylation occurs mainly through cAMP-mediated signaling pathways. AV may induce thyroxine release via the PKA/ERK pathway and TPO (Figure 6A). Moreover, it may reduce thyroxine release by inhibiting the PKA/CREB, PKA/ERK pathways and TPO (Figure 6B). Figure 6: Proposed model showing the pathway by which Aloe (AV) regulate thyroid hormone production in FRTL-5 cells. (**A**) pathways induced by AV in 5H media. AV-induced ERK phosphorylation  
occurs mainly through cAMP-mediated signaling pathways and AV may induce thyroxine release via the PKA/ERK pathway. (**B**) pathways induced by AV in 6H media. AV may reduce thyroxine release by inhibiting the PKA/CREB, PKA/ERK pathways and TPO. Abbreviations: cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; ERK, extracellular signal-related kinase; PKA, protein kinase A; PKC, protein kinase C; TPO, thyroid peroxidase; TSHR; thyroid-stimulating hormone receptor. This study shows that AV could potentially act as an alternative therapy for both hypo- and hyperthyroidism. Nevertheless, there is a limitation in vitro that could be addressed in future research. A study focused on the stimulation of the production and release of thyroid hormone following the administration of AV extract should be performed in vivo. Conceptualization and design of experiments: [PERSON]-S.K. ; Conducting of experiments: [PERSON] and [PERSON]-A.R. ; Analysis of data: [PERSON] and [PERSON]-A.R. ; and Manuscript writing: [PERSON] and [PERSON]-A.R. All authors have read and agreed to the published version of the manuscript. This study was supported by the Korea Institute of Oriental Medicine under Grant KSN1515290 and the Korea Research Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT under Grant 2015H1D3A1066241. Not applicable.