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.

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.

Efficacy and safety of endovascular treatment versus intravenous thrombolysis for acute ischemic stroke: a meta-analysis of randomized controlled trials.

BACKGROUND AND PURPOSE: Although endovascular therapy (ET) is increasingly used in patients with moderate to severe acute ischemic stroke, its efficacy and safety remains controversial. We performed a meta-analysis aiming to compare the benefits and safety of endovascular treatment and intravenous thrombolysis in the treatment of acute ischemic stroke. METHODS: We systematically searched PubMed, Embase, Science direct and Springer unitil July, 2013. The primary outcomes included good outcome (mRS ≤ 2) and excellent outcome (mRS ≤ 1) at 90 days or at trial end point. Secondary outcomes were occurrence of symptomatic hemorrhage and all-cause mortality. RESULTS: Using a prespecified search strategy, 5 RCTs with 1106 patients comparing ET and intravenous thrombolysis (IVT) were included in the meta-analysis. ET and IVT were associated with similar good (43.06% vs 41.78%; OR=1.14; 95% CI, 0.77 to 1.69; P=0.52;) and excellent (30.43% vs 30.42%; OR=1.05; 95% CI, 0.80 to 1.38; P=0.72;) outcome. For additional end points, ET was not associated with increased occurrence of symptomatic hemorrhage (6.25% vs. 6.22%; OR=1.03; 95% CI, 0.62 to 1.69; P=0.91;), or all-cause mortality (18.45% vs. 17.35%; OR=1.00; 95% CI, 0.73 to 1.39; P=0.99;). CONCLUSIONS: Formal meta-analysis indicates that there are similar safety outcomes and functional independence with endovascular therapy and intravenous thrombolysis for acute ischemic stroke.

[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.

Protective effect of ambroxol against paraquat-induced pulmonary fibrosis in rats.

OBJECTIVE: To evaluate the possible therapeutic effect of ambroxol on pulmonary fibrosis induced by paraquat. METHODS: Adult male Sprague-Dawley rats (n=144, 200-250 g) were divided into four groups (Control, Ambroxol, Paraquat, and Paraquat+Ambroxol group) and sacrificed on day 1, 3, 5, 7, 14 and 28. Several significant oxidant stress markers (MDA, SOD and GSH-PX), MPO activity, cytokines (TNF-α, MCP-1, TGF-ß1, MMP-2 and TIMP-1), total inflammatory cell count, hydroxyproline content, collagen I and III mRNA were analyzed. RESULTS: In Paraquat group, the MDA, MPO activity, hydroxyproline contents, the mRNA expression of TNF-α, MCP-1, TGF-ß1, MMP-2, TIMP-1, collagen I, collagen III and the number of total inflammatory cells were up-regulated in lung tissue, but SOD and GSH-PX activity were down-regulated in lung tissue compared with Control group (p<0.05). In paraquat+ambroxol group, the MDA, MPO activity, hydroxyproline content, the mRNA expression of TNF-α, MCP-1, TGF-ß1, MMP-2, TIMP-1 collagen I, collagen III and the number of total inflammatory cells were significantly decreased, while the SOD and GSH-PX activities in lung tissue were increased compared with Paraquat group (p<0.05). Histological examination of paraquat-treated rats showed lung injury with interstitial edema and widespread inflammatory cell infiltration in the alveolar space and septum, as well as pulmonary fibrosis. Ambroxol could markedly reduce such damage in lung tissue and prevent pulmonary fibrosis. CONCLUSION: The results of this study indicated that ambroxol could reduce lung damage and prevent pulmonary fibrosis induced by paraquat.

Effect of penehyclidine hydrochloride on patients with acute lung injury and its mechanisms.

OBJECTIVE: To assess the effects of penehyclidine hydrochloride on patients with acute lung injury (ALI), to observe the expression of Toll-like receptor 4 (TLR4) on the peripheral monocytes of ALI patients and changes of inflammatory and anti-inflammatory cytokines and to investigate the mechanism of TLR4 in ALI. METHODS: Forty-five patients with ALI were randomly divided into penehyclidine hydrochloride treatment group (P group, n equal to 21) and conventional treatment group (control group, C group, n equal to 24). Patients in both groups received conventional treatment, including active treatment of the primary disease, respiratory support, nutritional support and fluid management therapy, while those in P group were given penehyclidine hydrochloride (1 mg, im, q. 12 h) in addition. The TLR4 expression of 20 healthy volunteers were detected. The clinical effect, average length of stay in ICU and hospital, values of PaO2 and PaO2/FiO2, expression of TLR4 on the surface of peripheral blood mononuclear cells and some serum cytokines were evaluated for 48 h. RESULTS: The general conditions of the two groups were improved gradually and PaO2 increased progressively. Compared with 0 h, PaO2 and PaO2/FiO2 at 6, 12, 24 and 48 h after treatment were significantly increased (P less than 0.05). The improvement in P group was obviously greater than that in C group (P less than 0.05). The average length of hospitalization showed no difference between the two groups, but penehyclidine hydrochloride significantly decreased the average length of stay in ICU (t equal to 3.485, P less than 0.01). The expression of TLR4 in two groups were both obviously higher than that of healthy volunteers (P less than 0.01). It decreased significantly at 24 h (t equal to 2.032, P less than 0.05) and 48 h (t equal to 3.620, P less than 0.01) and was lower in P group than in C group. The patients who showed a higher level of TLR4 expression in early stage had a worse prognosis and most of them developed acute respiratory distress syndrome (ARDS). The incidence of ARDS was 23.8% in P group and 29.17% in C group at 24 h. Untill 48 h, there were other two patients developing ARDS in control group. Serum IL-1, IL-8 and TNF-alpha expressions reduced after 24 h in both groups. The reduction in P group was more obvious than that in C group (P less than 0.05). IL-13 increased gradually from 0 h to 24 h, and decreased slightly at 48 h, which showed no difference between two groups (t equal to 1.028, P larger than 0.05). CONCLUSIONS: Penehyclidine hydrochloride improves the arterial oxygen pressure, down-regulates the expression of TLR4 and restrains the inflammatory cytokines in the downstream of TLR4 signaling pathway. It prevents the development of ALI and can be considered as an important drug in ALI treatment.

Triptolide prolonged allogeneic islet graft survival in chemically induced and spontaneously diabetic mice without impairment of islet function.

BACKGROUND: Triptolide (TPT) is a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook. F. It exhibits potent immunosuppressive and anti-inflammatory properties. This study was undertaken to investigate its effects on prolongation of islet allograft survival in rodents. Additionally, we investigated whether TPT would be toxic to islet function in vivo. METHODS: We transplanted BALB/c islets to either chemically induced diabetic C57BL/6 mice or spontaneously diabetic nonobese diabetic (NOD) mice. TPT was injected within 2 weeks or continuously, until rejection, in the two combinations. Then, we evaluated the toxicity of TPT on islet function by daily injection to naive BALB/c or diabetic BALB/c that was cured by syngeneic islet transplantation under the kidney capsule. Mice injected with cyclosporine A (CsA) or vehicle served as controls. Intraperitoneal glucose tolerance tests (IPGTTs) performed at 4 and 8 weeks in the naive BALB/c group, and at 2, 4, 6, and 8 weeks in the syngeneic transplanted group. RESULTS: The medium survival time of islets allograft from TPT treated C57BL/6 and NOD recipients were 28.5 days (range 24-30 days, n=10) and 33.0 days (range 15-47 days, n=6), respectively, and they were significantly different from those of the vehicle treated controls, which were 14.0 days (range 13-16 days, n=6) and 5.0 days (range 4-10 days, n=6), respectively (all P<0.0001). The IPGTT demonstrated that there was no difference between the TPT treated and vehicle treated groups, either in the normal or syngeneic transplanted islet BALB/c mice. However, CsA injection impaired islet function in both normal and syngeneic transplanted mice as early as 4 weeks. CONCLUSION: TPT prolonged islets allograft survival in a chemically induced diabetic or an autoimmune diabetic murine model without impairment of islet function.

[Effect of Caveolin-1 on growth and apoptosis of human breast carcinoma cell line Hs578T/Dox].

BACKGROUND AND OBJECTIVE: Caveolin-1 is a marker protein of caveolae which is related with oncogenesis as a signal transduction hinge. This study was to investigate the effect of Caveolin-1 on the growth and apoptosis of doxorubicin-resistant human breast carcinoma cell line Hs578T/Dox. METHODS: Plasmids pCI-neo-caveolin-1 and pCI-neo-vector (control) were transfected into Hs578T/Dox cells, respectively. The expression of Caveolin-1 was detected by Western blot. Cell proliferation was detected by MTT assay. Cell cycle was detected by flow cytometry (FCM). Colony formation potential on soft agar was evaluated. Cell apoptosis was detected by FCM when cells were cultured for 48 h or cultured with staurosporine for 8 h. RESULTS: Caveolin-1 was overexpressed in Hs578T/Dox-cav-1 cells. The proliferation of Hs578T/Dox-cav-1 cells was obviously promoted when compared with that of Hs578T/Dox-vector cells (P<0.01). Colony size was larger in Hs578T/Dox-cav-1 group than in Hs578T/Dox-vector group. More colonies were formed in Hs578T/Dox-cav-1 group as compare with those in Hs578T/Dox-vector group (983.6+/-75.0 vs. 700.8+/-78.9, P<0.01). The proportions of cells at S and G2/M phases were higher in Hs578T/Dox-cav-1 group than in Hs578T/Dox-vector group. The proliferation rate of Hs578T/Dox-cav-1 cells was also higher than that of Hs578T/Dox-vector cells [(76.6+/-4.0)% vs. (58.0+/-4.1)%]. Over-expressed Caveolin-1 significantly reduced apoptosis index when the cells were cultured for 48 h [(5.7+/-0.5)% vs. (11.3+/-0.8)%] or cultured with staurosporine for 8 h [(13.8+/-1.2)% vs (21.4+/-1.9)%]. CONCLUSION: Overexpression of Caveolin-1 protein may promote the growth and anti-apoptosis ability of Hs578T/Dox cells.

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.

[Surface modification of vascular tissue engineering biomaterial by low temperature plasma with NH3, CO2 and O2].

OBJECTIVE: To study the biocompatibility of poly (hydroxybutyrate-hydroxyvalerate) (PHBV) modified by low temperature plasma with NH3, CO2, and O2 with canine Endothelial cells (ECs) and provide the better materials for blood vessel tissue engineering via surface modification. METHODS: PHBV was modified by low temperature plasma with NH3, CO2, and O2, for 0, 5, 10, 20, and 30 min respectively, PHBV not modified was used as control group. The contact angle was measured. X-ray photoelectron spectroscopy was used to detect the surface elements. Canine endothelial cells (ECs) were cultured on the surface of PHBV and were stained with fluorescence isothiocyanate (FITC). Their morphological characteristics were observed with fluorescence microscopy, and the cell proliferation was detected with MTT assay. RESULTS: The surface contents of carbon of the PHBV modified by low temperature plasma with O2 and CO2 were 63.75% and 69.72%, both lower than that before modification (77.97%). The surface contents of oxygen of the PHBV modified by low temperature plasma with O2 and CO2 were 30.72% and 28.48%, both higher than that before modification (21.74%). The surface content of nitrogen of the PHBV modified by low temperature plasma with NH3 was 3.25%, remarkably higher than that before modification (0). The contact angles of different modification groups, especially those of the 5 min groups, were all significantly smaller than that of the unmodified group (P < 0.05 or P < 0.01). Compared with the control group, the cytocompatibility was better in the low temperature plasma modified groups. CONCLUSION: The surface of PHBV modified by low temperature plasma with NH3, CO2, and O2 has active groups and good biocompatibility, so the surface modification of low temperature plasma with NH3, CO2, and O2 can be a kind of effective method for the tissue engineering blood vessel.

[Role of trefoil peptides in modulation of gastric adaptation to stress].

OBJECTIVE: To investigate the role of trefoil peptides in modulation of gastric adaptation to water restraint stress (WRS) in rats. METHODS: Wistar rats were exposed to single or repeated WRS for 4 hours every other day for up to 6 days, gastric mucosal blood flow (GMBF) was measured by LDF-3 flowmeter, the extent of gastric mucosal lesions was evaluated grossly and histologically, and expression of PS2 intestinal trefoil peptide (ITF), cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) and transferase growth factor-alpha (TGF-alpha) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: One application of WRS produced extensive gastric mucosal erosion. With repeated WRS, the gastric mucosa became adapted to the stress of WRS, and the ulcerative index (UI) was reduced by 22.0% that of one WRS challenge after four consecutive WRS. Expression of PS2 was markedly decreased and expression of ITF, COX-2, iNOS and TGF-alpha were markedly increased after single stress. But this adaptation to WRS was accompanied by increased GMBF and active cell proliferation in the neck region of gastric glands, and by increased expression of PS2, ITF, TGF-alpha, but reduced expression of COX-2 and iNOS. CONCLUSION: Gastric adaptation to WRS injury involves enhanced cell proliferation, increased expression of PS2, ITF, TGF-alpha and reduced expression of COX-2 and iNOS, suggesting trefoil peptides might play an important modulating role in this phenomenon.

Serial analysis of gene expression in mice with lipopolysaccharide-induced acute lung injury.

OBJECTIVE: To monitor the systemic gene expression profile in a murine model of lipopolysaccharide-induced acute lung injury. METHODS: Acute lung injury was induced by intratracheal injection of lipopolysaccharide in 3 mice. Another 3 normal mice receiving same volume of normal saline were taken as the controls. The comprehensive gene expression profile was monitored by the recently modified long serial analysis of gene expression. RESULTS: A total of 24,670 tags representing 12,168 transcripts in the control mice and 26,378 tags representing 13,397 transcripts in the mice with lung injury were identified respectively. There were 11 transcripts increasing and 7 transcripts decreasing more than 10 folds in the lipopolysaccharide-treated mice. The most overexpressed genes in the mice with lung injury included serum amyloid A3, metallothionein 2, lipocalin 2, cyclin-dependent kinase inhibitor 1A, lactate dehydrogenase 1, melatonin receptor, S100 calcium-binding protein A9, natriuretic peptide precursor, etc. Mitogen activated protein kinase 3, serum albumin, complement component 1 inhibitor, and ATP synthase were underexpressed in the lung injury mice. CONCLUSIONS: Serial analysis of gene expression provides a molecular characteristic of acute lung injury.

Cyclooxygenase 2, pS2, inducible nitric oxide synthase and transforming growth factor alpha in gastric adaptation to stress.

AIM: To determine the role of mucosal gene expression of cyclooxygenase 2 (COX-2), pS2 (belongs to trefoil peptides), inducible nitric oxide synthase (iNOS) and transforming growth factor alpha (TGFalpha) in gastric adaptation to water immersion and restraint stress (WRS) in rats. METHODS: Wistar rats were exposed to single or repeated WRS for 4 h every other day for up to 6 d. Gastric mucosal blood flow (GMBF) was measured by laser Doppler flowmeter-3. The extent of gastric mucosal lesions were evaluated grossly and histologically and expressions of COX-2, pS2,iNOS and TGFalpha were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The damage to the surface of gastric epithelium with focal areas of deep haemorrhagic necrosis was induced by repeated WRS. The adaptative cytoprotection against stress was developed with activation of cell proliferation in the neck regions of gastric glands. The ulcer index (UI) in groups II, III and IV was markedly reduced as compared with group I (I: 47.23+/-1.20; IV: 10.39+/-1.18,P<0.01). GMBF significantly decreased after first exposure to WRS with an adaptive increasement of GMBF in experimental groups after repetitive challenges with WRS. After the 4th WRS, the value of GMBF almost restored to normal level (I: 321.87+/-8.85; IV: 455.95+/-11.81, P<0.01). First WRS significantly decreased the expression of pS2 and significantly increased the expressions of COX-2, iNOS and TGFalpha. After repeated WRS, pS2 and TGFalpha expressions gradually increased (pS2: I: 0.37+/-0.02; IV: 0.77+/-0.01; TGFalpha: I: 0.86+/-0.01; IV: 0.93+/-0.03, P<0.05) with a decrease in the expressions of COX-2 and iNOS (COX-2: I: 0.45+/-0.02; IV: 0.22+/-0.01; iNOS: I: 0.93+/-0.01; IV: 0.56+/-0.01, P<0.01). Expressions of pS2, COX-2, iNOS and TGFalpha showed regular changes with a good relationship among them. CONCLUSION: Gastric adaptation to WRS injury involves enhanced cell proliferation, increased expression of pS2 and TGFalpha, and reduced expression of COX-2 and iNOS. These changes play an important role in adaptation of gastric mucosa after repeated WRS.

Role of TFF in healing of stress-induced gastric lesions.

AIM: To determine the changes of pS2 and ITF of TFF expression in gastric mucosa and the effect on ulcer healing of pS2, ITF to Water-immersion and restraint stress (WRS) in rats. METHODS: Wistar rats were exposed to single or repeated WRS for 4 h every other day for up to 6 days.Gastric mucosal blood flow (GMBF) was measured by LDF-3 flowmeter and the extent of gastric mucosal lesions were evaluated grossly and histologically. Expression of pS2 and ITF mRNA was determined by RT-PCR. Immunohistochemistry was used to further detect the expression of pS2 and ITF. RESULTS: WRS applied once produced numerous gastric mucosal erosions, but the number of these lesions gradually declined and GMBF restored at 2, 4, 8 h after stress. The area of gastric mucosal lesion was reduced by 64.9 % and GMBF was increased by 89.8 % at 8 h. The healing of stress-induced ulcerations was accompanied by increased expression of pS2 (0.51+/-0.14 vs 0.77+/-0.11, P<0.01) and ITF (0.022+/-0.001 vs 0.177+/-0.010, P<0.01). The results were demonstrated further by immunohistochemistry of pS2 (0.95+/-0.11 vs 1.41+/-0.04, P<0.01) and ITF (0.134+/-0.001 vs 0.253+/-0.01,P<0.01). With repeated WRS, adaptation to this WRS developed, the area of gastric mucosal lesions was reduced by 22.0 % after four consecutive WRS. This adaptation to WRS was accompanied by increased GMBF (being increased by 94.2 %), active cell proliferation in the neck region of gastric glands, and increased expression of pS2 (0.37+/-0.02 vs 0.77+/-0.01, P<0.01) and ITF (0.040+/- 0.001 vs 0.372+/-0.010, P<0.01). The result was demonstrated further by immunohistochemistry of pS2 (0.55+/-0.04 vs 2.46+/-0.08, P<0.01) and ITF (0.134+/-0.001 vs 0.354+/-0.070, P<0.01). CONCLUSION: TFF may not only participate in the early phase of epithelial repair known as restitution (maked by increased cell migration), but also play an important role in the subsequent, protracted phase of glandular renewal(made by cell proliferation).