Establishment of pancreatic acinar cell line AR42J with stable knockdown of Beclin1 / 中华胰腺病杂志

Objective To silence the beclin1 gene expression by using RNA interference technology in AR42J rat pancreatic acinar cells,and build a stable AR42J line silencing beclin1.Methods Three kinds of shRNA targeting rat beclin1 mRNA and negative control shRNA were designed and synthesized,and were inserted into the plasmids GV112,respectively.The recombinant plasmids were named as p-sh-Beclin1-1,psh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC.Lipo3000 was used to transfect the recombinant plasmid into AR42J cells,the expression of beclin1 mRNA were detected by RT-PCR to screen for the most efficient silencing plasmid,and then it was packaged into lentiviral (LV).AR42J cells were infected with LV and screened by puromycin.Beclin1 mRNA and protein expression was determined by RT-PCR and Western blot.Results The recombinant plasmid was confirmed by agarose gel electrophoresis and sequencing showed that shRNA sequences were in line with expectations.The beclin1 mRNA inhibition rates of AR42J cells after p-sh-Beclin1-1,p-sh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC transfection were (17.8 ± 4.0) ％,(30.6 ± 2.8) ％,(45.8 ± 7.7) ％,(7.0 ± 11.8) ％,respectively.The inhibition rates of three p-sh-Beclin1 transfection cells were significantly higher than that in non-transfection cells,and the difference was statistically significant (P ＜ 0.05).While the inhibition rate of p-shRNA-NC transfection cells was not significantly different from that of non-transfection cells,p-sh-Beclin 1-3 with highest rate of inhibition was packaged by LV,and infected AR42J cells,then puromycin was applied to screen,inhibition rate of beclin mRNA expression in LV infection cells was (86.1 ± 1.2) ％,and the protein expression inhibition rate was (87.9 ± 2.8) ％,and the difference between infection and non-infection groups was statistically significant (P ＜ 0.05).Conclusions The stable AR42J line silencing beclin1 is successfully established,which can provide a new cell model for future research of the role of beclin1 in the pathogenesis of acute pancreatitis.

Effects of pioglitazone pre-treating on acinar cells apoptosis induced by caerulein in acute pancreatitis / 中华消化杂志

Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein.Methods AR42J cells were divided into blank control group (with normal culture),pioglitazone group (40 μmol/L),caerulein control group (1 × 10-8 mol/L),pioglitazone+ caerulein group (40 μmol/L pioglitazone + 1 × 10-8 mol/L caerulein) and pioglitazone + GW9662+caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662 + 1 × 10-8 mol/L caerulein).Pioglitazone and GW9662 were added 30 minutes earlier than caerulein.Cell proliferation rate of each group was determined by MTT assay at three,six,12 and 24 hour.The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining.The activity of Caspase 3,8 and 9 of each group was measured.Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining.Single factor analysis of variance and LSD test were performed for data analysis.Results At six,12 and 24 hour,the cell proliferation rate of pioglitazone group and pioglitazone + caerulein group was 0.19±0.02,0.22±0.02,0.36±0.02 and 0.20±0.04,0.23±0.02,0.38±0.02,respectively,which were significantly lower than those of blank control group (0.25 ±0.04,0.28 ± 0.03 and 0.46±0.02) and caerulein group (0.23±0.02,0.29±0.01 and 0.46±0.05,t lgroup=-3.16,-4.61 and-6.25,tcaerulein group =-1.58,-4.61 and-6.15,all P＜0.05).And the cell proliferation rates of pioglitazone+GW9662+caerulein group at six,12 and 24 hour (0.23±0.02,0.27±0.02 and 0.45±0.01) were significantly higher than those of pioglitazone+caerulein group (t=2.25、3.87、4.56,all P＜0.05).There was no significant difference in cell apoptosis rate detected by flow cytometry with Annexin Ⅴ/PI staining between pioglitazone group ((11.80 ± 0.47) ％,(9.62 ± 2.63) ％ and (14.92 ± 2.52) ％) and pioglitazone+caerulein group ((8.78±0.47)％,(11.89±2.80)％ and (14.25±2.67)％,all P＞0.05),but cell apoptosis of these two groups were higher than those of control group ((5.52± 0.64)％,(5.30±0.97)％ and (5.47±0.88)％) and caerulein group ((5.98±1.21)％,(7.47± 0.58) ％ and (8.11 ± 1.32) ％) respectively,and the differences were statistically significant (t l group =9.81,4.45 and 10.74,tcaerulein group =4.38,7.62 and 6.98,all P ＜0.05).There was no significant difference in apoptosis rate between pioglitazone+GW9662+caerulein group ((5.82±0.26) ％,(6.05± 0.83) ％ and (9.23±0.90)％) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63,-10.07 and-5.70,all P＜0.05).At 12 hour,the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93 ± 0.40)％) was significantly higher than that of control group ((2.73 ±0.68) ％),the apoptosis rate of pioglitazone+ caerulein group ((8.43 ± 1.65)％) was significantly higher than that of caerulein group ((2.80 ± 0.56)％),the apoptosis rate of pioglitazone+GW9662+caerulein group ((3.87±0.35)％) was lower than that of pioglitazone+ caerulein group (t=7.93,8.92,-5.35,all P＜0.05).At 24 hour,the activity of Caspase 3,8 and 9 of pioglitazone+ caerulein group (1.28 ± 0.05,1.38 ± 0.04 and 1.53 ± 0.09) significantly increased compared with those of caerulein group (1.12±0.88,1.22±0.02 and 0.53±0.07,t=3.20,8.62 and 1.29,all P＜0.05).After treated with GW9662,part of activity of Caspase enzymes recovered.The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone + caerulein group was more than that of caerulein group (28.50±0.91)％ and (28.20±2.56)％ vs (15.00±3.67)％) and part of membrane potential recovered after GW9662 added ((20.67 ± 2.20) ％).Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential.And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone.

Establishment of a stable AR42J cell line expressing EGFP LC3 / 中华胰腺病杂志

Objective To establish a stable AR42J cell line expressing EGFP LC3.Methods The EGFP LC3 overexpressed Lentivirus was constructed and transfected into pancreatic acinar cells (AR42J) of rats.The rats with Lentiviral EGFP transfection were treated as negative control.The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The EGFP LC3 protein expression in the stable cell lines were analyzed by Western blot.The cells were treated with thapsigargin to establish endoplasmic reticulum stress model,and the LC3,PERK protein expressions were detected by Western blot.Results The transfection efficiency of Lentiviral EGFP LC3 of AR42J cell was ＞ 85％,which could achieve stable passage.The expression of LC3 mRNA of AR42J cells transfected with Lentiviral EGFP LC3 was 9.14 ±0.32 folds higher than that of negative control,which had no expression of LC3 protein,only EGFP expression.However,compared with non-transfection group,the LC3 mRNA expression in EGFP group was not significantly different.Conclusions A pancreatic acinar cell line (AR42J) of rat stably expressing EGFP LC3 protein is successfully constructed.And it may provide a new model for further research of the relationship between acute pancreatitis and autophagy.

Effect of lipopolysaccharide on toll like receptor 7 and toll like receptor 9 in AR42J cell lines / 中华胰腺病杂志

Objective To investigate the roles of toll like receptor7 (TLR7) and toll like receptor 9 (TLR9) in the pathogenesis of acute pancreatitis.Methods AR42J cells were treated by lipopolysaccharide at different dosages (0,1,10,100 mg/L),and cell model of acute pancreatitis in vitro was established.AR42J cells without lipopolysaccharide treatment were as control.Cells and culture supernatant were collected after 24 hours cultivation.TLR7,TLR9 mRNA and protein expressions were detected by RT-PCR and Western Blot,and levels of TNF-α,IL-10 in culture supernatant were measured by ELISA.Results The TLR 7 mRNA expression levels in control group,1,10,100 mg/L lipopolysaccharide group were 0.12 ± 0.09,0.28 ± 0.06,0.49 ± 0.04,0.78 ± 0.04,and the TLR9 mRNA expression levels were 0.06 ± 0.02,0.32 ± 0.03,0.56 ± 0.14,0.84 ± 0.12; the TLR7 protein expression levels were 0.04 ± 0.01,0.26 ± 0.05,0.49 ±0.04,0.77 ±0.16,and the TLR9 protein expression levels were 0.10 ±0.14,0.62 ±0.23,1.21 ± 0.26,1.75 ± 0.13 ; the TNF-α levels in culture supernatant were (8.01 ± 5.32),(25.64 ± 8.71),(49.06 ± 10.23),(75.83 ± 6.65) ng/L,and the IL-10 levels were (155.54 ± 25.47),(105.16 ± 10.49),(69.36 ± 8.19),(14.07 ± 9.06)ng/L.The expression levels of TLR7 and TLR9's mRNA,protein in cell,as well as the levels of TNF-α in culture supernatant increased with the lipopolysaccharide concentration,while the levels of IL-10 in culture supernatant decreased with the lipopolysaccharide concentration,and the difference among these groups was statistically significant (P ＜ 0.01).Conclusions The expressions of TLR7 and TLR9 in AR42J cells treated by using lipolysaccharide are obviously up-regulated,and it suggests that TLR7 and TLR9 may be vital in the pathogenesis of acute pancreatitis.

Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial beta-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

Effect of EGCG on Expression of Neurogenin 3 via the MAP Kinase Signaling Pathway in AR42J Cells, a Rat Pancreatic Tumor Cell Line

Epigallocatechin gallate (EGCG), or epigallocatechin 3-gallate, is the ester of epigallocatechin and gallic acid, and is a type of catechin. EGCG may be therapeutic for many disorders including diabetics and some types of cancer. However it is unknown whether EGCG can induce transdifferentiation of pancreatic cells in pancreatitis. The aim of this study was to investigate the effects of EGCG on the expression of pancreatic regenerating related markers in pancreatic AR42J cells, a model of pancreatic progenitor cells. AR42J cells, differentiated with betacellulin and activin A, were cultured with/without EGCG in a time-dependent manner. Cell growth rate, levels of mRNA, and protein expression were examined with the MTT assay, quantitative PCR, and Western blots, respectively. The results showed that AR42J cell growth rates were inhibited by EGCG in a dose-dependent manner. mRNA and protein expression of amylase, insulin and neurogenin 3 (ngn 3) increased in AR42J cells treated with EGCG. Additionally, we demonstrated that the signal transduction pathway of mitogen-activated protein (MAP) kinase is active in EGCG-treated AR42J cells. ERK and JNK phosphorylation decreased in cells treated with EGCG but not p38 phosphorylation. Activation of the p38 MAP kinase pathway was confirmed by specific MAP kinase pathways inhibitors: U0126 for ERK, SP600126 for JNK, and SB203580 for p38. Activated p38 phosphorylation was inhibited by the specific p38 inhibitor SB203580 but p38 phosphorylation was inhibited with increased EGCG treatment. The ERK and JNK MAP kinase pathways were not affected by EGCG treatment. Although further studies are needed, these results suggest that EGCG affects the induction of pancreatic cell regeneration by increasing the ngn 3 protein and mRNA expression and activating the p38 MAP kinase pathway.

Lipopolysaccharide stimulates the expressions of nuclear factor- κB mRNA and tumor necrosis factor-α mRNA in pancreatic acinus AR42J cell line of rats / 中华急诊医学杂志

Objective To investigate lipopolysaccharide (LPS) stimulates the expression of nuclear factor-KB (NF-κB) mRNA and tumor necrosis factor-a( TNF-α) mRNA in rat's pancreatic acinus AR42J cell line, and further address the pathogenesis of acute pancreatitis. Method The AR42J cell line was stimulated with different concentrations of LPS (0.001, 0.01, 0.1, 1.0, 10 and 100 mg/L) for 18 hours, or stimulated with 10 mg/L of LPS for different lengths of time (2, 6, 12,18 and 24 hrs) .Then, the expressions of NF-κB-P65 mRNA and TNF-α mRNA were determined by using RT-PCR, the levels of TNF-α protein in the culture supernatant were measured with radio-immuno assay (RIA), and the correlation between the expressions of TNF-α mRNA and NF-κB mRNA was analyzed. Results Both the expressions of NF-κB mRNA and TNF-α mRNA were up-regulated when AR42J cell line was stimulated with 10 mg/L of LPS for 2 hours or with 0.001 mg/L of LPS for 18 hours in both dose-dependent and time-dependent manners. Similarly, the levels of TNF-α protein were up-regulated when AR42J cell line was stimulated with 0.01 mg/L of LPS for 18 hours or with 10 mg/L of LPS for 6 hours in both dose-dependent and time-dependent manners. Statistical analysis revealed the positive correlation between the expressions of TNF-α mRNA and NF-κB-P65 mRNA(r = 0.962, P < 0.01). Conclusions LPS stimulates the expressions of TNF-α mRNA and NF-κB mRNA in both dose-dependent and time-dependent manners, and their expressions are closely correlated, suggesting the inhibition of their expressions as a potential therapeutic target for acute panceatitis.

Tat-NBD peptide inhibits inflammation effects related to NF-kB on AR42J cell / 中华急诊医学杂志

Objective To investigate the effects of Tat-NBD(NEMO-binding domain,NBD)peptide on LPS-stimulated AR42J acinus cells inflammatory response.Method Lipopolysaccharide(LPS)was added to culture media at doses of 10 mg/kg for 24 hours to stimulate the AR42J cells.For pretreatment.cells were incubated with different peptides for 2 hours before LPS stimulation.The expression of TNF-α mRNA WaS conducted using a semi-quantitative RT-PCR method.TNF-α protein in culture medium were detected by enzyme linked immunosorbent assay(ELISA).The expression and translocation of the NF-kB-p65 protein of AR42J was determined by Strept Actividin-Biotin Complex(SABC)method.Results LPS(10 mg/L)resulted in an increase of TNF-αmRNA and TNF-αprotein,whereas significant inhibitory effects were observed when cells were incubated with Tat-NBD(WT)just on dose of 0.1 me/L(P＜0.05).The Tat-NBD(WT)peptide decreased inflammatory cytokine expression by a dose-dependent manner and its peak role was on dose of 100 mg/L.Consisting with TNF-α expression decrease,NF-kB-p65 expression signitieantly decreased in Tat-NBD(WT)pretreatment group,especially on the largest dose.NO significant changes in the control peptide group.Conclusions Tat-NBD(WT)peptide can inhibit the inflammation of acinus simulated by LPS.