The Changing Regulation of Autophagy in Atherosclerosis in ApoE Gene Knockout Mice

Aim To investage the changes of autophagy in different stages of atherosclerosis (AS) in Apolipoprotein E gene knockout (ApoE -/) mice. Methods 6-weeks-old male ApoE -/mice (No.=40) were randomly divided into two groups and fed with common adaptability diet for 2 weeks. The mice of the control group (No.=10) received a sham operation and the common diet for another 8 weeks. While the model mice (No.=30) received a right common carotid artery cannulation and randomly subdivided into three groups (the 2 weeks, the 4 weeks and the 8 weeks) and fed with the high fat diet separately for 2 weeks, 4 weeks and 8 weeks. The blood samples obtained from femoral arteries were studied via the biochemical analysis. The right common carotid arteries were split out for histopathological study. Real-time quantitative polymerase chain reaction (qRT-PCR) and the western blot were used to detect the relative expression levels of mRNA and protein about mTOR. Results As the operation time prolonged the lipid levels especially TG and LDL_c were time relative increased. The histopathological analysis results showed that there was a small amount of cells infiltrated in the common carotid artery in the 2 weeks, although the wall was still unspoiled. The vascular wall in the 4 weeks was messy and there was thrombus in the vascular lumen. The thickness of the right common carotid artery in the 8 weeks was higher than others and its elastic membranes significantly decreased. The qRT-PCR and western blot detection suggested the mRNA and protein expression of mTOR in the 4 weeks was higher than the 2 weeks and the 8 weeks, and the expression in the 8 weeks was also higher than those in the 2 weeks. Conclusions Autophagy was continuously stimulated during AS formation,however, the levels of autophagy will decrease after reaching the peak at a time.


Introduction
AS has been one of the most dangerous diseases among the world and it also is the reason for some fatal diseases, such as stroke and coronary heart disease [1,2] .
The cause of AS is unclearly now, however, there are some theories about it, like the oxidative stress, the immune and the inflammation [3][4][5][6] . Autophagy is a cellular housekeeping process which can swallow and degrade the damaged proteins and other cellular macromolecular particles [7,8] . Autophagy can be regulated by a series of signaling molecules and one of them is the mammalian target of rapamycin (mTOR). If The Changing Regulation of Autophagy in Atherosclerosis in ApoE Gene Knockout Mice http://www.ijSciences.com Volume 5 -November 2016 (11) 50 there are inadequate nutrients or in the presence of mTOR inhibitors (eg.rapamycin), mTOR is not activated and autophagy is induction [9][10][11] .
According to the published research, autophagy is stimulated after the AS happened. Perrotta I [12] found autophagosome can be found in all kinds of cells which involved in AS. Ouimet M et al [13]  Materials and methods

Animals
The SPF levels ApoE -/mice (male) and its high-fat diet (0.25% cholesterol +15% fat) was purchased from Beijing HFK Bioscience. All mice were feed separately (22~25℃)with free drinking water and feeding.

Reagents
Reverse

Tissue pathological section
The common carotid artery isolated from the ApoE -/mice was washed with physiological saline and then immediately immersed in 30% of formaldehyde Solution. The artery was dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin before making paraffin section and the next thing was xylene dewaxing, gradient ethanol hydration, hematoxylin-eosin stainning, then gradient alcohol and xylene dehydration and mounting.

The qRT-PCR
The total RNA was isolated from the common carotid artery using Trizol reagent and the RNA concentration was tested by the spectrophotometer. Next, the quantitative RNA (about 1ug) was used to detect the relative mRNA expression levels of mTOR. Finally, the relative mRNA expression levels were calculated using 2^-△△ Ct [14] method and the mRNA levels of GAPDH were used as an internal control. The producer was pre-denaturation at 95℃ for 30s, denaturation at 95℃ for 5s and annealing at 60℃ for 20s, totally 40 cycles.
The mTOR primers was that: forward

The western blot
Tissue was washed with pre-cooled PBS for 2 times and then lysed in RIPA buffer with protease inhibitors.
After centrifugation at 12000g for 10 minutes at 4°C,

Data analysis
SPSS 19.0 was used for the statistical analysis, and the single factor variance analysis was used to analyze statistical differences. P < 0.05 was accepted as significant. The data were expressed as the mean  sd.

The detection of blood about ApoE -/mice
The detection indicated the level of TG, TC, LDL_c in the model was higher than the control (P<0.05) and the level was gradually increased with the extension of the high fat feeding time after the common carotid artery cannula operation. It means the level of TG, TC, LDL_c in the 4 weeks was higher than the 2 weeks (P<0.05)but lower than the 8 weeks (P<0.05).

The expression mTOR
The analysis of western blot bands suggested the p-mTOR/mTOR in the control was higher than the 4 weeks and the 8 weeks (P<0.05) and the ratio in the 8 weeks was higher than the 4 weeks but lower than the 2 weeks (P<0.05). and it usually regarded as a survival mechanism [15,16] .
The occurrence of autophagy is guided by a series of signaling pathways and the most classical signaling pathway is a signal system that takes the mammalian target of rapamycin (mTOR) as the center. The mTOR can supress autophagy by phosphorylating autophagy related gene 13 (ATG13) and decreasing the activity of ATG1 kinase in normal circumstances. The mTOR protein kinase is the target of the antifungal drug rapamycin so it can induce autophagy by inhibiting the activity of mTOR, dephosphorylating ATG13 and activating ATG1 [5,7,17] .
Recent studies suggest that autophagy plays an important role in the formation and development of AS.
Such as, Verheye et al [18] found autophagy presents in Martinet et al [19] thought that oxidized low density lipoprotein (ox-LDL), inflammation and metabolic stress can promote the autophagy in the plaque cells and plaque cell autophagy is a cellular device that prevents the external oxidative stress by degrading the damaged cells. However, the levels of autophagy will not always rise but reduce at a time. In this research we found the relative expression level of mRNA and protein of autophagy related protein mTOR in the 8 weeks was higher than the 4 weeks. De Meyer et al [20] found that oxidative damage of the lysosomal membrane will lead to the release of lysosomal hydrolases, which causes damage of cytosolic proteins The Changing Regulation of Autophagy in Atherosclerosis in ApoE Gene Knockout Mice http://www.ijSciences.com Volume 5 -November 2016 (11) 53 and organelles and finally induces apoptosis. Li W et al [21] found the expression of ATG5 in advanced AS was lower than in the early stage and the expression of p62 in the advanced AS was higher than in the early by immunohistochemistry. In conclusion, we suggested autophagy will not always rise in the whole stage of AS and it will reduce at a peak.