5.3 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
5.4.2 Effect of ethanol and lauric acid on LXRA mRNA expression and the correlation of CYP3A4
As shown in Figure 4.6, lauric acid treatment suppressed the gene expression level of LXRA in alcohol-induced HepG2 cells. Significant suppression of lauric acid on LXRA gene expression in alcohol-induced HepG2 cells was observed after 10 µM lauric acid treatment. Five µM of lauric acid did not
54 give any effect in reducing the LXRA gene expression level. Besides that, treatment with 20 µM lauric acid and resveratrol alone, did not give significant changes to LXRA gene expression as compared to untreated samples. This indicates that the lauric acid and resveratrol alone would not activate LXRA gene expression in normal physiology condition.
As mentioned by Watanabe et al. (2013), LXRA is said to regulate the CYP3A4 by either inhibiting function of Pregnane X Receptor (PXR) or by directly increasing the CYP3A4 gene expression during cholesterol homeostasis. PXR is the main transcription factor in regulating the CYP3A4 gene expression. Tompkins and Wallace (2007) showed that PXR activates CYP3A4 gene expression by binding on PXR response element present in CYP3A4 promoter region. In Figure 4.7, LXRA was increased 1.59-fold in gene expression when the cells were treated with 2% (v/v) ethanol. It showed that LXRA is not a negative regulator of CYP3A4 because if LXRA is a repressor of PXR, it is a repressor of CYP3A4. However, prior senior’s study showed increasing of CYP3A4 gene expression level in 2% (v/v) ethanol treated HepG2 cells.
On the other hand, if LXRA is acting as a negative regulator, the gene expression level of LXRA should be increased while the lauric acid concentration increased. This is due to LXRA has a similar binding motif of PXR in the responsive element of CYP3A4 gene and also same binding partner (RXR) with PXR (Watanabe et al., 2013). This leads to the
55 competition between LXRA and PXR for their binding promoter region (Miao et al, 2006; Watanabe et al., 2013). In short, the increase in LXRA should reversibly inhibit PXR function in activating CYP3A4 gene expression and thus decrease the CYP3A4 formation.
In contrast, the LXRA gene expression level decreased when the concentration of the lauric acid was increased. This showed similar trend to that of CYP3A4 gene expression (Lim, 2017). Thus, the similarity in patterns could confirm that LXRA acts as a positive regulator to induce the activation of CYP3A4. In detail, the lauric acid will decrease the gene expression of LXRA through suppress the CYP3A4 in 4β-hydroxylation of cholesterol thus decrease the formation of 4β-hydroxycholesterol in the liver (Bodin et al, 2001; Honda et al, 2011). The metabolite, 4β-hydroxycholesterol, is the ligand of LXRA and it will help in regulating the cholesterol homeostasis. Cholesterol formation in the alcohol-induced HepG2 is due to the high formation of acetyl-CoA during oxidation of ethanol which is initiated by alcohol dehydrogenase (ADH).
Acetyl-CoA is the basic component that used in synthesis the cholesterol compound (King, 2017).
Furthermore, since lauric acid is an antioxidant or a dietary saturated fat, it may play an important role in alteration of fatty acid synthesis which results from chronic alcoholic consumption (Ronis et al., 2004). As suggested by previous study, alcohol steatosis might be induced by the activation of SREBP-1c that lead to synthesis of lipogenic enzyme such as fatty acid
56 synthases (You et al., 2012). According to Ronis et al. (2004), the increase in lauric acid intake will not only improve the liver resistance towards oxidative stress but it should also decrease the fatty acid synthesis in the liver by increasing the fatty acid oxidation and lipid export.
Compared to 20 µM lauric acid treatment in alcohol-induced HepG2 cells, 20 µM resveratrol had no effect on inducing significant changes of LXRA gene expression level in alcohol-induced HepG2 cells. Resveratrol, a well-known antioxidant in treating the alcoholic fatty liver disease, could protect the hepatocytes from oxidative stress due to chronic alcohol consumption (Ajmo et al., 2008). This can be supported by previous senior study that showed that down regulation of CYP3A4 gene expression in HepG2 cells treated with 20 µM resveratrol and 2% (v/v) ethanol (Lim, 2017). However, in my study showed no significance different in LXRA gene expression level in 2% (v/v) ethanol induced HepG2 cells co-treated with 20 µM resveratrol. Therefore, it is assumed that resveratrol do not act on LXRA in regulating the CYP3A4 gene expression as lauric acid did. This can be supported by Deng et al. (2014), showed that resveratrol down regulate CYP3A4 through inhibiting the function of PXR, the main inducer that activate CYP3A4 gene expression.
57 5.5 Future studies
In this study, the exact mechanism of changes of LXRA gene expression was not studied clearly in every treatment. The LXRA function is still not identified in regulating the CYP3A4 gene expression especially with the treatment of 20 µM resveratrol and 2% (v/v) ethanol in HepG2 cells. Thus, the mechanism of LXRA in alcohol-induced cells can be further studied in details.
Besides that, the relationship of CYP3A4, cholesterol metabolism, alcohol metabolism and LXRA function can be studied to identify the exact pathway of alcohol-induced cholesterol biosynthesis that involved CYP3A4 and LXRA.
These studies can be done by knockdown LXRA gene in alcohol-induced HepG2 cells and identify the gene expression of CYP3A4 and also SREBP-1c.
By observing physiological and molecular changes in both CYP3A4 and SREBP-1c, the mechanism of changes of LXRA gene expression in alcohol induced HepG2 cells can be identified.
In addition, a time dependent experiment can be done to identify the LXRA gene expression level in alcohol-induced HepG2 cells with the presence or absence of lauric acid. The main purpose of this experiment is to determine the gene expression level of LXRA before the ethanol was fully metabolised in HepG2 cells. When the ethanol was fully eliminated in HepG2 cells, there may have some changes in the gene expression of LXRA. Thus, a time dependent experiment on alcohol-induced HepG2 cells can be done to investigate the changes of LXRA gene expression level.
58 CHAPTER 6
CONCLUSION
A study regarding the effect of lauric acid on Liver X Receptor α (LXRA) mRNA expression in alcohol-induced HepG2 cells was done. This study showed that alcohol induced LXRA mRNA expression in HepG2 cells at the concentration of 1%, 2% and 5% (v/v). The highest induction of LXRA mRNA expression was shown in 2% (v/v) ethanol-induced HepG2. This result tallied with previous senior’s study which showed CYP3A4 mRNA expression was increased in alcohol-induced HepG2 cells. In the presence of lauric acid, LXRA mRNA expression in alcohol-induced HepG2 cells was reduced. The most significant reduction was shown at 20 µM lauric acidco- treatment. The reduction in LXRA correlated with reduction in CYP3A4 in previous study. This hypothesises that LXRA mRNA expression level might affect the CYP3A4 mRNA expression in alcohol-induced HepG2 cells and LXRA is the direct regulator of CYP3A4 in alcohol-induced HepG2 cells. In a nut shell, lauric acid can act as potential antioxidant in reducing liver injury that is caused by ethanol consumption.
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