H3 Lab Report Wu Yanlong

NTU H3 MOLECULAR BIOLOGY

Investigating the effects of Aspirin on Apolipoprotein AI synthesis in Human Intestinal Cells Wu Yanlong

1. Introduction 1.1 Literature Review Overview of lipoprotein metabolism (Robert, 2009): The main lipids in lipoproteins are free and esterified cholesterol and triglyceride. In triglyceride metabolism, fats are broken down during digestion, enter intestinal cells, and are re-packaged in chylomicrons. Chylomicrons, secreted via the lymphatic system into bloodstream, enter the vena cava and circulate until they interact with lipoprotein lipase (LPL), releasing free fatty acids, which are taken up by cells that store fats. CM remnants (CMRs) are taken up by liver cells. Triglycerides are packaged with cholesterol and are eventually transported in bloodstream in LDL (low-density lipoprotein). LDL transports cholesterol from the liver to all other parts of the body. LDL can be taken in by cells via endocytosis. In HDL cholesterol metabolism, HDL, via APOA-I (A1), mediates reverse cholesterol transport by interacting with receptors on non-hepatic cells, which are cells that are not from the liver. Atherosclerosis (Lusis, 2000): LDL may start depositing along the walls of coronary artery. As they start to become oxidized, it triggers an inflammatory response which recruits macrophages. The macrophages attempt to engulf the deposited fats but are overwhelmed by the amount of fats, thereby forming foam cells. As LDL and foam cells continue to build up, the smooth muscle cells migrate and form a fibrous cap over the deposition. The smooth muscle cells also deposit calcium. The fibrous cap may rupture and expose the contents within, leading to thrombosis (blood clot). This leads to complete blockage of coronary artery which leads to heart attack. HDL is highly protective against atherosclerosis because it helps to remove cholesterol from artery wall and peripheral tissues, and it also inhibits oxidation of LDL, which is the trigger for inflammation. Aspirin: Aspirin is an anti-inflammatory drug. Previous research has shown that it helps to stabilize atherosclerotic plaques by down regulation of MMP-2 and MMP-9 expression (Yiqin, 2009). Matrix metalloproteinase (MMP)-2 and MMP-9 can degrade the extracellular matrix and lead to disruption or rupture of plaque. Aspirin was also shown to increase expression of Scavenger receptor class B type I (SR-BI) in macrophages, which helps to promote cholesterol efflux, thereby giving it atheroprotective properties (Tancevski, 2006).

1

1.2 Hypothesis Aspirin increases Apo-AI synthesis in human intestinal cells. 1.3 Rationale and aim of study Given that cardiovascular disease is the leading cause of death worldwide, the use of aspirin as a potential pharmaceutical drug to increase HDL levels among patients has important implications. In this study, gene expression and protein expression of Apo-AI is being investigated. 2. Methods Intestinal cells (Caco-2) were incubated without or with aspirin (5 mM) for a total period of 10 hours, after which total RNA was extracted. The extracted mRNA was reverse transcribed to cDNA and subsequently subjected to PCR using specific primers for apoA-I. The DNA was run in agarose gel electrophoresis to visualize the DNA as bands. Intensity (optical density) of the bands was quantified by densitometric scanning using the Bio-Rad GS-800 densitometer and Quantity One software; 5 replicates were obtained. This quantifies gene expression. Protein samples from intestinal cells were incubated with primary antibody followed by Protein A-agarose beads for protein immunoprecipitation. The precipitated proteins were visualized as bands via polyacrylamide gel electrophoresis (SDS-PAGE). Similarly, the Intensity (optical density) of the bands was quantified by densitometric scanning using the Bio-Rad GS-800 densitometer and Quantity One software; 8 replicates were obtained. This quantifies protein expression. Mann-Whitney U test was used to check for significant difference, instead of t-test, because sample size is small; hence we are unable to assume that data is parametric or follows normal distribution. A one-tailed test was conducted since the hypothesis is directional.

2

3. Results

Intensity (Optical Density)

70 60 50 40 30 20 10 0

Figure 1. Gene expression of ApoAI. Column 1: DNA ladder. Column 2: Aspirin absent. Column 3: Aspirin present

A

B

Figure 2. Gene expression of Apo-AI. Mean optical density of gel bands from 5 replicates. A: Aspirin absent. B: Aspirin present. Bars represent standard error of mean (SEM).

As seen in Fig 1, gene expression of Apo-AI is higher in intestinal cells incubated with aspirin compared to those that are incubated without aspirin. Fig 2 shows that the optical density of the bands has a significant difference (Mann-Whitney p value of 0.00604, p value