Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway.

BACKGROUND: The incidence rate of acute pancreatitis (AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit anti-tumor activity. However, exosome-derived miR-125b-5p in AP has not been reported. AIM: To elucidate the molecular mechanism of exosome-derived miR-125b-5p promoting AP exacerbation from the perspective of the interaction between immune cells and acinar cells. METHODS: Exosomes derived from AR42J cells were isolated and extracted in active and inactive states by an exosome extraction kit, and were verified via transmission electron microscopy, nanoparticle tracking analysis, and western blotting. RNA sequencing assay technology was used to screen differentially expressed miRNAs in active and inactive AR42J cell lines, and bioinformatics analysis was used to predict downstream target genes of miR-125b-5p. The expression level of miR-125b-5p and insulin-like growth factor 2 (IGF2) in the activated AR42J cell line and AP pancreatic tissue were detected by quantitative real-time polymerase chain reaction and western blots. The changes in the pancreatic inflammatory response in a rat AP model were detected by histopathological methods. Western Blot was used to detect the expression of IGF2, PI3K/AKT signaling pathway proteins, and apoptosis and necrosis related proteins. RESULTS: miR-125b-5p expression was upregulated in the activated AR42J cell line and AP pancreatic tissue, while that of IGF2 was downregulated. In vitro experiments confirmed that miR-125b-5p could promote the death of activated AR42J cells by inducing cell cycle arrest and apoptosis. In addition, miR-125b-5p was found to act on macrophages to promote M1 type polarization and inhibit M2 type polarization, resulting in a massive release of inflammatory factors and reactive oxygen species accumulation. Further research found that miR-125b-5p could inhibit the expression of IGF2 in the PI3K/AKT signaling pathway. Additionally, in vivo experiments revealed that miR-125b-5p can promote the progression of AP in a rat model. CONCLUSION: miR-125b-5p acts on IGF2 in the PI3K/AKT signaling pathway and promotes M1 type polarization and inhibits M2 type polarization of macrophage by inhibiting IGF2 expression, resulting in a large release of pro-inflammatory factors and an inflammatory cascade amplification effect, thus aggravating AP.

Thromboxane A2 receptor contributes to the activation of rat pancreatic stellate cells induced by 8-epi-prostaglandin F2α.

BACKGROUND: Pancreatic stellate cells (PSCs) activation plays a critical role in the development of chronic pancreatitis. Previous studies confirmed that thromboxane A2 receptor (TxA2r) was overexpressed in activated PSCs in rats. The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α (8-epi-PGF2α). METHODS: TxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay. Isolated PSCs were treated with 8-epi-PGF2α (10, 10, 10 mol/L) for 48 h, and SQ29548 (10, 10, and 10 mol/L), a TxA2r-specific antagonist for 48 h, respectively, to identify the drug concentration with the best biological effect and the least cytotoxicity. Then isolated PSCs were treated with SQ29548 (10 mol/L) for 2 h, followed by 10 mol/L 8-epi-PGF2α for 48 h. Real-time polymerase chain reaction was performed to detect the messenger RNA (mRNA) levels of α-smooth muscle actin (α-SMA) and collagen I. Comparisons between the groups were performed using Student's t test. RESULTS: TxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs (all P < 0.001). Compared with the control group, different concentrations of 8-epi-PGF2α significantly increased mRNA levels of α-SMA (10 mol/L: 2.23 ±â€Š0.18 vs. 1.00 ±â€Š0.07, t = 10.70, P < 0.001; 10 mol/L: 2.91 ±â€Š0.29 vs. 1.01 ±â€Š0.08, t = 10.83, P < 0.001; 10 mol/L, 1.67 ±â€Š0.07 vs. 1.00 ±â€Š0.08, t = 11.40, P < 0.001) and collagen I (10 mol/L: 2.68 ±â€Š0.09 vs. 1.00 ±â€Š0.07, t = 24.94, P < 0.001; 10 mol/L: 2.12 ±â€Š0.29 vs. 1.01 ±â€Š0.12, t = 6.08, P < 0.001; 10 mol/L: 1.46 ±â€Š0.15 vs. 1.00 ±â€Š0.05, t = 4.93, P = 0.008). However, different concentrations of SQ29548 all significantly reduced the expression of collagen I (10 mol/L: 0.55 ±â€Š0.07 vs. 1.00 ±â€Š0.07, t = 10.47, P < 0.001; 10 mol/L: 0.56 ±â€Š0.10 vs. 1.00 ±â€Š0.07, t = 6.185, P < 0.001; 10 mol/L: 0.27 ±â€Š0.04 vs. 1.00 ±â€Š0.07, t = 15.41, P < 0.001) and α-SMA (10 mol/L: 0.06 ±â€Š0.01 vs. 1.00 ±â€Š0.11, t = 15.17, P < 0.001; 10 mol/L: 0.28 ±â€Š0.03 vs. 1.00 ±â€Š0.11, t = 11.29, P < 0.001; 10 mol/L: 0.14 ±â€Š0.04 vs. 1.00 ±â€Š0.11, t = 12.86, P < 0.001). After being treated with SQ29548 (10 mol/L) and then 8-epi-PGF2α (10 mol/L), the mRNA levels of α-SMA (0.20 ±â€Š0.08 vs. 1.00 ±â€Š0.00, t = 17.46, P < 0.001) and collagen I (0.69 ±â€Š0.13 vs. 1.00 ±â€Š0.00, t = 4.20, P = 0.014) in PSCs were significantly lower than those of the control group. CONCLUSIONS: The results show that 8-epi-PGF2α promoted PSCs activation, while SQ29548 inhibited PSCs activation induced by 8-epi-PGF2α. The result indicated that TxA2r plays an important role during PSC activation and collagen synthesis induced by 8-epi-PGF2αin vitro. This receptor may provide a potential target for more effective antioxidant therapy for pancreatic fibrosis.

Upregulation of the checkpoint protein CHFR is associated with tumor suppression in pancreatic cancers.

The checkpoint with forkhead-associated (FHA) domain and RING-finger (CHFR) protein was identified as a cell cycle checkpoint protein and E3 ubiquitin ligase. In the present study, the potential functions of CHFR in pancreatic cancer were investigated. CHFR expression was measured in five pancreatic cancer cell lines by reverse transcription- quantitative polymerase chain reaction and western blotting. Capan-1 cells stably expressing CHFR were established by lentiviral vector transfection. Cell proliferation was assessed using Cell Counting Kit-8, and cell migration/invasion assay was determined using Transwell assays. Cell cycle and apoptosis induced by gemcitabine or docetaxel were evaluated using flow cytometry. CHFR expression levels were also evaluated in pancreatic ductal adenocarcinoma (PDAC) tumor samples as well as adjacent non-tumor tissues by immunohistochemistry. The significance of CHFR expression was determined, with respect to clinicopathological features and overall survival. Overexpression of CHFR in Capan-1 cells led to a decreased proliferative rate and reduced cell migration and invasion abilities. Results also indicated an increase in G1 phase cells in Capan-1 cells overexpressing CHFR. Docetaxel-induced apoptosis was inhibited in Capan-1 cells with CHFR-overexpression. A reduction in CHFR expression was detected in 51.9% of patients with PDAC, which significantly correlated with later T-stage. The results show CHFR functions as a tumor suppressor in pancreatic cancer, suggests its potential role in controlling the cell cycle of pancreatic cancer cells; however, CHFR overexpression is not a favorable factor in apoptosis induced by docetaxel.

Post-translational modifications of α-synuclein contribute to neurodegeneration in the colon of elderly individuals.

Synucleinopathies and abnormalities in the nerves of the enteric nervous system are hypothesized to be involved in age-associated motility disorders. The aim of the present study was to investigate the expression of various antigens, including α­synuclein (Syn) and its post­translational modified forms, in the human colon at various ages. In addition, the study aimed to correlate the expression of Syn with neurodegeneration. Immunohistochemistry was used to detect the expression of neurofilament (NF), Syn, as well as its nitrated (N) form in the healthy colonic tissue of 12 young (34.08±5.12 years), 10 middle­aged (51.80±3.52 years), and 11 elderly (75.82±7.70 years) individuals. To the best of our knowledge, the current study is the first to demonstrate the presence of N­Syn in the colonic tissue. N­Syn was identified in the upper layer of the mucosa and submucosa layer. Furthermore, Syn (wild­type) was present in the mucosa and submucosa. The number of NF­positive neurons in the submucosal layer declined significantly with age (P<0.01). In addition, Syn and N­Syn significantly increased during aging (P<0.01). Furthermore, a negative correlation was identified between neuron number and synucleinopathies, indicating the abnormal accumulation of both wild-type Syn and N­Syn in the mucosa, submucosa, muscle layer and myenteric plexus. The present study demonstrates that the Syn pathology may be linked to colic neuronal degeneration during normal aging, and this link may cause functional deficits.

Silencing pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic adenocarcinoma upregulated factor (PAUF) is a new oncogene that activates signaling pathways that play a critical role in resistance to gemcitabine. We thus speculated that PAUF also plays a role in resistance to gemcitabine of pancreatic cancer cells. We established BxPC-3 cell lines with stable PAUF knockdown (BxPC-3_shPAUF) and controls (BxPC-3_shCtrl) and evaluated sensitivity to gemcitabine in vitro by MTT and flow cytometry. We established a xenograft model of human pancreatic cancer to examine PAUF function in gemcitabine resistance in vivo. Gene chip microarrays were performed to identify differentially expressed genes in BxPC-3_shPAUF and BxPC-3_shCtrl cells. Silencing PAUF increased the sensitivity of BxPC-3 cells to gemcitabine in vitro and in vivo. PAUF-knockdown BxPC-3 cell lines treated with gemcitabine showed increased proliferation inhibition and apoptosis compared with controls. Gemcitabine exhibited a more pronounced effect on reduction of BxPC-3_shPAUF tumors than BxPC-3_shCtrl tumors. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays confirmed a significantly higher apoptotic rate of BXPC-3_shPAUF tumors compared with BXPC-3_shCtrl tumors. Gene array showed that PAUF function in gemcitabine sensitivity might involve MRP2, MRP3, MDR1, PIK3R1, and NFkB2 genes. PAUF could be considered as a key molecular target for sensitizing pancreatic cancer cells to gemcitabine.

Histological and mechanical properties of autologous living tissue biotubes.

The aim of this study was to explore and evaluate biotubes consisting of autologous tissues. The biotubes were prepared by intra-abdominally embedding silicon rods as moulds. The specimens were analyzed by mechanical tests, histological observation and superficial study. The intra-abdominal implantation of the silicone tubes readily stimulated the development of the biotubes. The biotubes consisted of collagen-rich extracellular matrices. Myofibroblasts appeared as elongated cells with circumferential or longitudinal orientations. Subsequent to one month of embedding, the thickness of the tube wall was 70-250 µm. The burst strength was 1100±187 mmHg and the suturability was excellent. Biotubes that have the ability to be widely variable in their shapes are composed of autologous cells and glomerular extracellular matrices. Biotubes are ideal grafts for tissue engineering as they are able to avoid immunological rejection and are of sufficient mechanical strength.

[The role of oxide stress during the pathogenesis of chronic pancreatic injuries induced by chronic high-fat diets in rat].

OBJECTIVE: To provide more detailed information on the roles of lipid peroxidation in the pathogenesis of chronic pancreatic injuries in a pre-clinical rat model. METHODS: Totally 72 rats were divided into 6 groups (12 in each group) Rats in 5 experimental groups (n = 12) were fed with a high-fat diet (1% cholesterol, 10% lard, 0.3% sodium tauroglycocholate, 87.3% standard rodent chow as the control group) for 2, 4, 6, 10 and 16 weeks, respectively. Morphological studies in the pancreas tissue samples from rats were investigated by using various histological methods. Pancreatic stellate cells (PSCs) were identified by immunohistochemical staining for Desmin and α-smooth muscle actin (α-SMA). The expression of the lipid peroxidation was detected by immunostaining for 4-hydroxy-2-nonenal (4-HNE) and thromboxane A2 receptor (TxA2r). The co-localization of α-SMA and 4-HNE or α-SMA and TxA2r in PSCs was also analyzed in this study. RESULTS: Pancreatic cells with positive staining for Desmin and α-SMA in HFD rats were distributed in a more extensive way when compared to that in the control group. The levels of pancreatic 4-HNE and TxA2r were increased in rats from HFD groups significantly. The co-localization of 4-HNE and TxA2r were also found within activated PSCs in both of groups. CONCLUSION: The results showed that a chronic HFD feeding may increase the lipid peroxidation process and collagen synthesis through a critical signaling pathway of activated PSCs following pancreatic injuries in rats.

Increased expression of α-synuclein in aged human brain associated with neuromelanin accumulation.

Although the increased prevalence of Parkinson's disease (PD) with aging suggests that aging processes predispose dopamine neurons to degeneration, the mechanism involved remains unknown. Dopamine neurons contain significant amounts of neuromelanin, and the amount of neuromelanin increases with aging. In the present study, age-related changes in the number of nigral neurons expressing neuromelanin (NM), α-synuclein, and tyrosine hydroxylase (TH) were stereologically analyzed in the postmortem brains of 28 healthy humans with an age range of 17-84 years. Stereological counting of NM content, α-synuclein content, and TH immunoreactivity revealed significant accumulation of NM and α-synuclein in neurons during the aging process. In cells containing a large amount of NM, α-synuclein-immunoreactive cells in aged individuals outnumbered those of younger individuals. In non-NM cells, the α-synuclein expression profile was similar across age groups. Furthermore, TH-immunoreactive neurons decreased significantly with aging, which was associated with accumulation of NM and α-synuclein. Our results suggest that age related accumulation of NM might induce α-synuclein over-expression and thereby make dopamine neurons more vulnerable to injuries.

Preliminary research on the pathological role of cathepsin-B in subcutaneous heteroplastic pancreatic carcinoma in nude mice.

BACKGROUND: Cathespin-B (cath-B) is an important proteolytic enzyme involved in the disease course of invasion in many types of cancer. Cath-B expression in subcutaneous heteroplastic pancreatic carcinoma in nude mice has not been studied. We investigated the role of cath-B in a model of heteroplastic pancreatic carcinoma in BALB/c nude mice. METHODS: Thirty-two six-week-old female BALB/c nude mice were equally divided into four groups. PANC-1 cells were inoculated subcutaneously in the left axillary region. Besides volume, weight of subcutaneous tumor, and change in body weight, cath-B expression in each group was measured by immunohistochemical staining, PCR and Western blotting. Its relationship to microvessel density (MVD), CD44v6, and placenta growth factor (PLGF) was also examined. CA-074Me, a specific inhibitor of cath-B, was injected intraperitoneally (i.p.) at different stages of tumor growth in group B and C. Gemcitabine (GEM), was also injected (i.p.) in group D to compare anti-tumor efficacy with CA-074Me. RESULTS: Expression of cath-B at different levels was related to tumor growth, MVD, and PLGF expression. In group A (control group), cath-B expression was enhanced more than that seen in other groups. CA-074Me clearly inhibited cath-B expression and tumor growth in group B. There was no difference between group C and D with respect to anti-tumor effect. CONCLUSIONS: Cath-B correlates with the growth and angiogenesis of tumors, but not with the adhesion induced by CD44v6. CA-074Me clearly inhibited cath-B expression and demonstrated an anti-neoplastic and anti-angiogenesis effect.