Upregulation of BCL-2 by acridone derivative through gene promoter i-motif for alleviating liver damage of NAFLD/NASH

Abstract

Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) are global epidemic public health problems with pathogenesis incompletely understood. Hepatocyte excessive apoptosis is a significant symbol for NAFLD/NASH patients, and therefore anti-apoptosis therapy could be used for NAFLD/NASH treatment. Up-regulation of BCL-2 has been found to be closely related with anti-apoptosis. BCL-2 gene promoter region has a C-rich sequence, which can form i-motif structure and play important role in regulating gene transcription. In this study, after extensive screening and evaluation, we found that acridone derivative A22 could up-regulate BCL-2 transcription and translation in vitro and in cells through selective binding to and stabilizing BCL-2 gene promoter i-motif. Our further experiments showed that A22 could reduce hepatocyte apoptosis in NAFLD/NASH model possibly through up-regulating BCL-2 expression. A22 could reduce inflammation, endoplasmic reticulum stress and cirrhosis in high-fat diet-fed mice liver model. Our findings provide a potentially new approach of anti-apoptosis for NAFLD/NASH treatment, and A22 could be further developed as a lead compound for NAFLD/NASH therapy. Our present study first demonstrated that gene promoter i-motif could be targeted for gene up-regulation for extended treatment of other important diseases besides cancer.

Figure 1.

A22 could specifically bind to and stabilize BCL-2 gene promoter i-motif. (A) Structure of compound A22. (B) Binding affinity of A22 to BCL-2 gene promoter i-motif was studied by using SPR with K_D value determined to be 3.56 μM. Bio-py39 was annealed in MES buffer at pH 5.5 for this experiment. (C) CD melting experiment was recorded at 288 nm for py39 annealed in 1× BPES buffer at pH 5.5 with or without A22, indicating that A22 could stabilize BCL-2 gene promoter i-motif. Py39 had its maximum absorption at 288 nm. (D) CD melting experiment was recorded at 266 nm for pu39 with or without A22, indicating that A22 had no significant effect on BCL-2 promoter G-quadruplex. Pu39 had its maximum absorption at 266 nm, which was annealed in buffer of 20 mM Tris–HCl, 100 mM KCl, pH 7.4. All the experiments were repeated for three times.

Figure 2.

ESI-MS spectra of oligomer py39 with or without addition of A22 at pH 6.0. (A) Oligomer py39 alone at pH 6.0. Py39 was annealed in 1× BPES buffer at pH 6.0. (B) Oligomer py39 with addition of A22 at pH 6.0. The mass of 12 221 Da corresponding to py39–A22 adduct was detected. All the experiments were repeated for three times.

Figure 3.

Effect of A22 on gene transcription and translation in HepG2 cells. The mRNA levels of BCL-2 and BAX (A), as well as C-KIT, KRAS, C-MYC and VEGF (B) in HepG2 cells were analyzed by using qRT-PCR after incubation with increasing concentration of A22 for 12 h. (C) Effects of A22 on protein expressions of C-MYC, VEGF, C-KIT and BCL-2 in the presence or absence of increasing concentration of A22 for 24 h, which were quantitatively analyzed (D). All the experiments were repeated for three times. The data are expressed as the mean ± SEM: (*) P < 0.05, (**) P < 0.01, significantly different from the control.

Figure 4.

Effect of A22 on anti-apoptosis in 0.5 mM palmitic acid oil (PA) induced cell model. (A) Effect of A22 on cell viability for anti-apoptotic protective effect. (B) Effect of A22 on transcription of BCL-2 and BAX with measurement of mRNA levels. (C) Effect of A22 on protein expressions related with apoptosis (left), which were quantitatively analyzed (right). All the experiments were repeated for three times. The data are expressed as the mean ± SEM: (*) P < 0.05, (**) P < 0.01, significantly different from the control.

Figure 5.

Effect of A22 on various body parameters in NAFLD/NASH mice model. Male adult C57/B6J mice were fed chow (CH) or high-fat (HF) diet for 16 weeks, and A22 was administrated in the last 6 weeks to two groups (10 and 40 mg/kg. i.p. every other day). Body/tissue weights and serum as well as liver parameters were analyzed in the end of the experiment after 8 h of fasting, and the data were obtained in following category: (A) body weight, (B) representative individuals of each group, (C) liver weight, (D) food-intake, (E) alanine aminotransferase (ALT), (F) aspartate aminotransferase (AST), (G) alkaline phosphatase (ALP), (H) total cholesterol, (I) triglyceride, (J) fat mass, (K) plasma glucose and (L) plasma insulin. Data are statistically analyzed as means ± SEM, and each circle or column indicated one group (N = 8 mice/group). * means P < 0.05, versus HF control mice; ** means P < 0.01, versus HF control mice.

Figure 6.

Effect of A22 on alleviating morphological changes of mice livers. (A) Representative images of livers (× 100 magnification) with oil red O staining, H&E staining, Sirius Red staining, and masson staining. (B–D) Ballooning (blue arrow indicated), hepatic steatosis (black arrow indicated), and fibrosis (blue arrow indicated) scores were obtained according to the NAFLD Activity Score (NAS) System as described in Methods. (E) Relative lipid droplet contents were determined. (F) Expressions of BCL-2 in livers of each group were determined by using immunohistochemistry. (G) Liver apoptosis was determined by using a TUNEL assay in situ. Representative images were captured with stained black dots indicated in the image. Data were statistically analyzed as means ± SEM, and each circle indicated one group (N = 8 mice/group). *P< 0.05, versus HF control mice; **P< 0.01, versus HF control group.

Figure 7.

Effect of A22 on ameliorating apoptosis, ER stress, inflammation, metabolic syndrome, and fibrogenesis in HF diet-fed mice. (A) Effect of A22 on BCL-2 gene transcription. (B) Effect of A22 on BAX gene transcription. (C) Effect of A22 on expressions of apoptosis-related proteins in liver. The extracted proteins from the liver were immunoblotted with specific antibodies, and quantified based on the loading control of ACTIN. (D) Effect of A22 on ER stress. The UPR proteins (IRE-1α, PERK, elF-2α and CHOP) were analyzed by using western Blot. (E) Effect of A22 on expressions of inflammatory factors. (F) Effect of A22 on expressions of fibrogenic proteins. Data were statistically analyzed as means ± SEM, and each column indicated one group (N = 8 mice/group). *P< 0.05, versus HF control mice; **P< 0.01, versus HF control group (N = 8 mice /group).