The Conditional Expression Of Pancreatic Lipase-related Proteins And Gene Therapy For Hepatic Fibrosis

Hepatic cirrhosis, one of the most dangerous diseases, has a high morbidity rate. The underlying mechanism of hepatic fibrosis remains unclear. Previous research has shown that hepatic stellate cell (HSC) play a critical role in the development of hepatic fibrosis. Inhibition the hydrolysis of hepatic retinyl ester which can inhibit the activation of HSC, has been considered as a therapy for hepatic fibrosis. However, the identity, function and regulation of hepatic enzymes potentially involved in catalyzing the hydrolysis of retinyl esters are not fully understood. Our previous study showed that the expression of mPlrp2 and mClps was activated in livers in response to the lack of light illuminating. The dissertation is mainly focused on the conditional expression of mPlrp2 and mClps and gene therapy for hepatic fibrosis.Firstly, the levels of serum retinoid of the mice under different illumination condition had been analysed by HPLC. Results showed that lack of light illuminating for 48 hours, the level of serum all-trans retinoic acid (ATRA) was decreased from 2.45 ng/mL to 1.75 ng/mL, and hepatic mPlrp2 and mClps expression were induced. Light exposure resumed the ATRA level to 2.43 ng/mL and inhibited hepatic mPlrp2 and mClps expression in mice under constant darkness. The data indicated that ATRA was a signal molecular involved in the regulation of hepatic mPlrp2 and mClps expression. Northern Blot and HPLC analysis showed ATRA regulated ecto-5'-nucleotidase (CD73) gene expression by retinoic acid receptor-alpha (RAR-alpha) and modulated serum 5'-AMP levels. The elevation of 5'-AMP was associated with mPlrp2 and mClps expression in the livers. Mice elevated expression of hepatic mPlrp2 and mClps, lowered hepatic and serum lipid levels and markedly elevated circulatory levels of all-trans retinol, indicated mPlrp2 and mClps was involved in hepatic lipid metabolism. These results suggest environmental light influence hepatic lipid homeostasis by modulating retinoic acid signaling was associate with mPlrp2 and mClps gene expression in livers.To determine the role of mPlrp2 and mClps in hepatic lipid metabolism, the animal model of vitamin A deficiency was established by feeding a vitamin A free diet. RT-PCR results showed that mice fed a VA-free diet for 7 days exhibited increased the expression hepatic mPlrp2 and mClps, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by methylthioadenosine (MTA), markedly decreased plasma level of retinol in these mice from 0.59μg/mL to 0.39μg/mL. HPLC analysis showed that conditional expression of mPlrp2 and mClps in VA-free mice were associated with the increased ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illuminating deficiency or administrating 5'-AMP elevated the ratio of AdoMet to AdoHcy, and induced mPlrp2 and mClps genes expression in livers, which could be blocked by ATRA. Activated HSC-T6 cell line specifically expressed mClps and mPlrp2. Hepatic stellate cells (HSCs) are primarily involved in the storage of retinol esters. HSC-T6 cells specifically expressed the mClps and mPlrp2 genes, whereas other cell lines, including several hepatic cell lines, failed to exhibit detectable expression of mClps and mPlrp2. Inhibition of mClps gene expressions by siRNA interference decreased hydrolysis of retinyl esters in HSC-T6 cell line. These data suggest that conditional expression of mPlrp2 and mClps is invoved in the hydrolysis of retinyl esters in mouse livers.The animal model of hepatic fibrosis was established by administrating CCl4 or bile duct ligation (BDL). RT-PCR results showed that mPlrp2 and mClps expreesion were activated in fibrotic liver, and there was a linear correlation between the expression of these two genes and Acta-2, a marker of activated HSCs. RNA interference of mPlrp2 ameliorated HSCs activation and hepatic fibrosis in BDL mice. AdoMet has anti-inflammatory properties and reduces HSCs activation in fibrotic liver. We found that administration of AdoMet could inhibit hepatic mPlrp2 and mClps expression. Taken together, the data suggest that mPlrp2 is involved in hepatic fibrosis and RNA interference of mPlrp2 ameliorates hepatic fibrosis, mPlrp2 and mClps are potential targets of hepatic fibrosis gene therapy.