Construction of COX-2 short hairpin RNA expression vector and its inhibitory effect on hepatic fibrosis

<p>The aim of this study was to construct recombinant adenovirus vector carrying a short hairpin RNA (shRNA) that can exclusively target cyclooxygenase-2 (COX-2) gene expression and observe its inhibitory effect on hepatic fibrosis. We designed and synthesized three oligonucleotide sequences, and cloned those into a shuttle vector, pYr-1.1-hU6-EGFP, after annealing. The restriction-enzyme digestion and sequencing analyses confirmed that the constructed recombinant eukaryotic expression vector was correct. A recombination reaction using LR Clonase was performed for the pYr-1.1-hU6-EGFP COX-2shRNA and the adenovirus vector pAd/BL-DEST to form Ad-COX-2shRNA. The adenoviruses containing the recombinant plasmids were transfected into hepatic stellate cells (HSCs). The transfection efficiency of the three COX-2 shRNAs exceeded 70%. Reverse transcription PCR (RT-PCR) and western blot confirmed that the target gene expression was decreased at the level of mRNA and protein, and the interference effect of COX-2 shRNA-1 was better than that of the other two COX-2 shRNAs. COX-2 shRNA-1 recombinant adenovirus vectors (1 × 10⁹ PFU/mL) were injected via the tail vein into rats fed a high-fat diet with a 40% carbon tetrachloride peanut oil lavage, which induced liver fibrosis. Rats were euthanized at the end of the 12th week, and their liver was removed. Liver expression of COX-2 mRNA and protein was detected by RT-PCR and immunohistochemistry, respectively. RT-PCR and immunohistochemistry showed that COX-2 shRNA-1 inhibited COX-2 expression in liver tissue. Hematoxylin/eosin and Masson staining showed that COX-2 shRNA-1 ameliorated the severity of liver fibrosis. The COX-2 shRNA eukaryotic expression vectors were constructed successfully and COX-2 shRNA-1 ameliorated liver fibrosis.</p

NC enhances intracellular ox-LDL degradation through facilitation of lipophagy.

<p>(A) Representative photomicrographs of colocalization of lipid droplets (LDs) with LC3-II in THP-1 cells. After THP-1 cells were transfected with GFP-LC3II for 24 h, cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing with PBS, cells were fixed with 4% paraformaldehyde, and then stained with Nile Red (10 ng/ml) for 30 min to evaluate the accumulation of LDs. The colocalization of LDs with LC3II was examined by immunocytochemistry as described in Methods section. (B) The percentage of colocalization of LDs with LC3-II was quantified with Image J software (n = 16 cells/group). (C) Representative photomicrographs of LD accumulation in THP-1cells. Cells were incubated with ox-LDL (100 μg/ml) conjugate without or with NC of 36 h, or pre-incubated with ox-LDL for 4 h, and then treated without or with NC for additional 36 h. The intracellular LD accumulation was evaluated by Nile Red (10 ng/ml) staining. (D) Average number of LDs in THP-1 cells was quantified (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Autophagy prevents ox-LDL-induced foam cell formation in THP-1 cells.

<p>THP-1 cells were treated with the vehicle solution (control), control + CQ (10 μM), ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + CQ (10 μM), ox-LDL (100 μg/ml) + Rap (20 μM) for 36 h, respectively. (A) LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels were detected by western blot analysis. Each lane contained 20 μg proteins for all experiments. (B) and (C) The LC3-II/LC3-I ratio and p62 level were quantified with Sigma Scan Pro5 software. Each lane was normalized to that of GAPDH (kDa). (D) Oil red O staining was used to evaluate THP-1 foam cell formation (magnification × 200). (D) Intracellular total cholesterol content was determined by enzymatic assay. All the data were shown as mean ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC promotes cholesterol efflux via restoring autophagy flux.

<p>(A)and (B) THP-1 cells were incubated in medium containing 100 μg/ml ox-LDL that was labeled with 0.5 μ Ci/mL ³H-cholesterol (PerkinElmer) for an additional 30 h and then treated with vehicle, NC (10 μM), NC (10 μM) +3-MA (10 mM), and NC (10 μM) + CQ (20 μM) for additional 6 h. Subsequently, ApoA1- or HDL-mediated cholesterol efflux was analyzed by liquid scintillation counting assay. The efflux is expressed as the percentage of effluxed ³H-cholesterol/total cell cholesterol ³H-cholesterol content (effluxed ³H-cholesterol + intracellular ³H-cholesterol) × 100%. All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC reduces ox-LDL accumulation in THP-1 cells via activation of autophagy.

<p>(A) Representative photomicrographs of THP-1 cells loaded with Dil-ox-LDL. Cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing 3 times, cell lysates were collected for the measurement of fluorescence. Nuclei were counterstained with DAPI. (B) Quantification of fluorescence intensity from experiments as described in (A). (C) TEM was used to evaluate foam cell formation and autophagy alteration. THP-1 cells were treated with vehicle, ox-LDL (100 μg/ml), ox-LDL + NC (10 μM)), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA, ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h, respectively. Mitochondria (M), the nucleus (N), lysosomes (L), autophagosomes (APs), autophagolysosomes (ALs), and lipid droplets (LDs) were indicated. (D, E, and F) Average number of APs, ALs, and LDs was quantified as described in Methods section (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC rescues autophagy flux via inhibiting the PI3K /m-TOR pathway.

<p>(A) THP-1 cells were treated with vehicle, ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) +NC (10 μM), ox-LDL (100 μg/ml) +NC (10 μM) + 740Y-P (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. Cell lysates were collected and analyzed by western blotting assay for PI3K (85 kDa), p-mTOR (289 kDa), p-p70S6K (70 kDa), LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels. Each lane was loaded with 20 μg proteins for all experiments. (B, C, D, and E) The relative optical density values of PI3K, p-mTOR, p-p70S6K, LC3-II, LC3-I, and p62 to GAPDH, respectively, were quantified with Sigma Scan Pro5 software. All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC rescues the impaired autophagy flux in ox-LDL-treated THP-1 cells.

<p>(A) THP-1 cells were treated with different concentration of NC (0, 1, 5, 10, 20 μM) in the presence of ox-LDL (100 μg/ml) for 36 h. Cell lysates were analyzed by western blotting assay for LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels. Each lane was loaded with 20 μg proteins in all experiments. (B) and (C) The LC3-II and p62 levels were quantified with Sigma Scan Pro5 software. (D) Confocal images of representative images of GFP and RFP fluorescent puncta in THP-1 cells transfected with GFP-RFP-LC3II for 24 h, and then treated with indicated reagents for 36 h. (E) Quantification of GFP/RFP double-positive and RFP single-positive puncta in each cell treated with indicated reagents for 36 h (n = 23 cells/group). All the data were shown as mean ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Highly efficient and green esterification of carboxylic acids in deep eutectic solvents without any other additives

<p>A protocol that carboxylic acids esterifies with the quaternary ammonium salt of deep eutectic solvent (DES) is presented, which opens a new access to ester using DES as alkylating agent, solvent, and catalyst. The reaction runs smoothly in DES without any other additives. Substituted cinnamic acids, aromatic acids, and aliphatic acids can be esterified in moderate to good yields. The advantages of this reaction include excellent functional group compatibility and simple reaction procedure.</p

EGCG inhibits CUMS–induced activation of the mTOR pathway in CA1.

<p>(A) The effects of EGCG on p-mTOR (289 kDa) and p-p70S6K (70 kDa) protein levels in CA1 were investigated by western blot analysis. Each lane contained 30 µg proteins for all experiments. (C, D) Densitometry analysis of p-mTOR and p-p70S6K protein levels was performed using three independent experiments, respectively. GAPDH was used as control for protein loading. Control, control group; CUMS, chronic unpredictable mild stress group; CUMS + CQ, CQ administration followed by CUMS group; CUMS + EGCG, EGCG treatment followed by CUMS group; CUMS + EGCG + CQ, co-administration with EGCG and CQ followed by CUMS group; Vehicle, vehicle treatment followed by CUMS group. The drugs (EGCG and CQ) were given to rats 30 min before the stress exposure. Data are shown as mean ± SEM (n = 3 for each group). *<i>P</i><0.05 versus CUMS group.</p

EGCG effects on CUMS–induced changes in ultra-structure and autophagic flux in CA1.

<p>TEM and western blotting were conducted to evaluate autophagic flux. (A) TEM was used to examine autophagosome formation. Control group shows ultra-structure of neuronal cells in the CA1 hippocampus under control conditions, characterized by normal structure of the mitochondria (M) and the nucleus (N) with evenly distributed chromatin. CUMS group, CUMS + CQ, CUMS + EGCG + CQ and CUMS + vehicle group show ultra-structurally changed neuronal cells in the CA1 sector of the hippocampus, including abnormal mitochondria (M), heterogeneous lysosomes, and increased autophagic vacuoles (AVs)-containing impaired mitochondria (M) and unrecognized aggregate of electron-dense material compared with the control. CUMS + EGCG group indicates that EGCG-treatment markedly attenuates CUMS-induced ultra-structure impairment in CA1, characterized by decreased swollen mitochondria, heterogeneous lysosomes, and dysfunctional lysosomes-containing dense granules compared with those in the CUMS group rats. The presented photos are representative of the three animals used in each experimental group. (B) Western blotting results showing the effects of EGCG on LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels in CA1. Each lane contained 30 µg proteins for all experiments. (C), and (D), Densitometry analysis of LC3-II and p62 protein levels was performed using three independent experiments. GAPDH was used as control for protein loading. Control, control group; CUMS, chronic unpredictable mild stress group; CUMS + CQ, CQ administration followed by CUMS group; CUMS + EGCG, EGCG treatment followed by CUMS group; CUMS + EGCG + CQ, co-administration with EGCG and CQ followed by CUMS group; Vehicle, vehicle treatment followed by CUMS group. The drugs (EGCG and CQ) were given to rats 30 min before the stress exposure. Data are shown as mean ± SEM (n = 3 for each group), and one-way ANOVA with Newman–Keuls post hoc analysis was used. NS means no significant difference. *<i>P</i><0.05 versus CUMS group, ^#<i>P</i><0.05 versus CUMS + EGCG group, ^Δ<i>P</i><0.05 versus CUMS + CQ group. Scale bar, 500 m.</p