糖化终末产物对INS-1细胞基质金属蛋白酶2表达的影响

目的 探讨糖化终末产物(AGEs)对大鼠胰岛细胞株INS-1基质金属蛋白酶2(MMP-2)表达量的影响.方法 培养INS-1细胞,检测MMP-2表达;制备糖化胎牛血清,通过流式细胞仪检测细胞内活性氧簇(ROS)水平,通过逆转录PCR、实时定量PCR和Western印迹方法观察细胞中MMP-2表达量的变化.结果 INS-1细胞中存在MMP-2的表达;AGEs刺激后ROS明显升高(P<0.05),刺激组细胞内MMP-2mRNA表达量和蛋白表达水平均显著高于对照组(P<0.05).结论 提示胰岛β细胞内存在MMP-2表达,AGEs可诱导β细胞MMP-2表达量增高,MMP-2在介导胰岛氧化应激损伤中可能起着重要作用.

Abstract：

Objective To explore the impact of advanced glycation end-products ( AGEs) on matrix metalloproteinase 2 ( MMP-2) expression in cultivated INS-1 cells. Method INS-1 cells were cultivated and MMP-2 expression was analyzed. Glycated serum was prepared for incubating with INS-1 cell. Reactive oxygen species (ROS) was detected by flow cytometry. The intracelluar MMP-2 expression was analyzed by RT-PCR, realtime PCR and Western blot. The MMP-2 cDNA was expressed in cultivated INS-1 cells. Result The level of ROS treated with AGEs was significantly higher than that in the control( P<0.05 ) , and the levels of MMP-2 and its protein expressions turned out as well( P<0. 05). Conclusion The results suggest that MMP-2 was expressed in INS-1 cells. Increased MMP-2 expression in ?cells may be induced by AGEs, suggesting that MMP-2 might play an important role in oxidative stress-mediated islet injury.     

In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells

AIM: To isolate, culture and identify the human fetal pancreatic ductal stem cells in vitro, and to observe the potency of these multipotential cells differentiation into insulin-producing cells.METHODS: The human fetal pancreas was digested by i g/L collagease type IV and then 2.5 g/L trypsin was used to isolate the pancreatic ductal stem cells, followed by culture in serum-free, glucose-free DMEM media with some additional chemical substrates in vitro (according to the different stage). The cells were induced by glucose-free (control),5 mmol/L, 17.8 mmol/L and 25 mmol/L glucose, respectively.The cell types of differentiated cells were identified using immunocytochemical staining.RESULTS: The shape of human fetal pancreatic ductal stem cells cultured/n vitro was firstly fusiform in the first 2 wk,and became monolayer and cobblestone pattern after another 3 to 4 wk. After induced and differentiated by the glucose of different concentrations for another 1 to 2 wk,the cells formed the pancreatic islet-like structures. The identification and potency of these cells were then identified by using the pancreatic ductal stem cell marker, cytokeratin-19 (CK-19), pancreatic β cell marker, insulin and pancreatic cell marker, glucagons with immunocytochemical staining.At the end of the second week, 95.2% of the cells were positive for CK-19 immunoreactivity. Up to 22.7% of the cells induced by glucose were positive for insulin immunoreactivity, and less than 3.8% of the cells were positive for glucagon immunoreactivity in pancreatic isletlike structures. The positive ratio of immunoreactive staining was dependent on the concentration of glucose, and it was observed that the 17.8 mmol/L glucose stimulated effectively to produce insulin- and glucagons-producing cells.CONCLUSION: The human fetal pancreatic ductal stem cells are capable of proliferation in vitro. These cells have multidifferentiation potential and can be induced by glucose and differentiated into insulin-producing cells in vitro.     

Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells

Objective: To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2) induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1α mRNA expressions were detected by real time PCR.Results: Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells; while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions: Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.     

波动性高糖对胰岛β细胞增殖及Skp2-p27表达的影响

目的 探讨波动性高糖对INS-1细胞增殖、凋亡及对细胞周期进程的影响,并研究其可能的分子机制.方法 采用细胞计数试剂盒(cell counting kit-8)检测细胞增殖活性,流式细胞仪测定细胞周期及细胞ROS水平,Annexin-V/PI双标流式细胞术检测细胞凋亡.应用Western印迹检测细胞周期调控蛋白p27及Skp2的表达水平.结果 (1)波动性高糖及持续性高糖均明显抑制INS-1细胞的生长,且波动性高糖对细胞增殖的抑制作用更为显著.(2)波动性高糖及持续性高糖均明显增加INS-1细胞的凋亡,且波动性高糖作用更为显著.(3)波动性高糖及持续性高糖能明显抑制细胞周期进程,使INS-1细胞周期更多滞留在G0/G1期,G2/M期与S期细胞比例下降,波动性高糖作用更显著.(4)波动性高糖及持续性高糖均能显著增强细胞周期调控蛋白p27的表达,同时减弱Skp2蛋白的表达水平.结论 波动性高糖较持续性高糖更能够抑制INS-1细胞的增殖和诱导凋亡,可能是通过减弱Skp2蛋白的表达水平,增加p27蛋白的活性,使细胞阻滞在G0/G1期,抑制细胞周期进程,从而减弱细胞的增殖活性.

Abstract：

Objective To investigate the effect of intermittent high glucose on proliferation, apoptosis, and cell cycle progression of INS-1 cells, and the possible intracellular pathways activated by intermittent high glucose. Methods Cell viability was evaluated by cell counting kit, the cell cycle was determined by flow cytometry,Annexin-V/PI double-labeled cell apoptosis detection kit was used to monitor cell apoptosis. Cell cycle related protein Skp2 and p27 expressions were detected by Western blot. Results ( 1 ) Both intermittent and constant high glucose significantly inhibited the growth of INS-1 cells, and the former effect was more significant. ( 2 ) Intermittent and constant high glucose levels significantly increased apoptosis in INS-1 cells, and the former effect was more significant. (3) Intermittent and constant high glucose levels significantly inhibited the cell process, the G0/G1 cell cycle arrest also was induced by intermittent high glucose, resulting in lowered proportion of the G2/M phase and S phase of INS-1 cells. (4) Intermittent and constant high glucose significantly decreased the level of protein Skp2 and increased the level of cell cycle related protein p27. Conclusion Intermittent high glucose levels affect INS-1 cell growth and proliferation, as well as induce cell apoptosis, probably by decreasing the level of protein Skp2 and increasing the level of p27 in the cells, resulting in arrest of progression through the G1 phase to the S phase of INS1 cells, and thus impairment of cell proliferation.     

Inhibition of KL-6/MUC1 glycosylation limits aggressive progression of pancreatic cancer

AIM:To evaluate the significance of KL-6/MUC1(a type of MUC1)glycosylation in pancreatic cancer progression.METHODS:KL-6/MUC1 expression was detected by immunohistochemistry in 48 patients with pancreatic duct cell carcinoma.The N-/O-glycosylation inhibitors(tunicamycin and benzyl-N-acetyl-α-galactosaminide)were then used to interfere with KL-6/MUC1 glycosylation in two pancreatic carcinoma cell lines,and the effects on KL-6/MUC1 expression,and cell adhesion and invasion were determined.In addition,protein expression of epithelial-mesenchymal transition markers,E-cadherin and vimentin,were evaluated in cells after treatment with glycosylation inhibitors.RESULTS:Overexpression of KL-6/MUC1 was found in all pancreatic cancer tissues,but not in the surrounding normal pancreatic tissues.The expression profile of KL-6/MUC1 was significantly decreased after treatment with the inhibitors.The adhesion and invasive ability of cancer cells were significantly decreased after drug treatment,and increased E-cadherin and decreased vimentin expression were found.CONCLUSION:KL-6/MUC1 glycosylation is involved in pancreatic cancer metastasis and invasion.Therapeutic strategies which target this may help control the aggressive behavior of pancreatic cancer cells.     

Identification of effective siRNA against K-ras in human pancreatic cancer cell line MiaPaCa-2 by siRNA expression cassette

AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.     

In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells

AIM: To isolate,culture and identify the human fetal pancreaticductal stem cells in vitro,and to observe the potency of thesemultipotential cells differentiation into insulin-producingcells.METHODS: The human fetal pancreas was digested by 1 g/Lcollagease type Ⅳ and then 2.5 g/L trypsin was used toisolate the pancreatic ductal stem cells,followed by culturein serum-free,glucose-free DMEM media with some additionalchemical substrates in vitro (according to the differentstage).The cells were induced by glucose-free (control),5 mmol/L,17.8 mmol/L and 25 mmol/l_ glucose,respectively.The cell types of differentiated cells were identified usingimmunocytochemical staining.RESULTS: The shape of human fetal pancreatic ductal stemcells cultured in vitro was firstly fusiform in the first 2 wk,and became monolayer and cobblestone pattern afteranother 3 to 4 wk.After induced and differentiated by theglucose of different concentrations for another 1 to 2 wk,the cells formed the pancreatic islet-like structures.Theidentification and potency of these cells were then identifiedby using the pancreatic ductal stem cell marker,cytokeratin-19 (CK-19),pancreatic β cell marker,insulin and pancreaticα cell marker,glucagons with immunocytochemical staining.At the end of the second week,95.2% of the cells werepositive for CK-19 immunoreactivity.Up to 22.7% of thecells induced by glucose were positive for insulinirnrnunoreactivity,and less than 3.8% of the cells werepositive for glucagon immunoreactivity in pancreatic islet-like structures.The positive ratio of immunoreactive stainingwas dependent on the concentration of glucose,and itwas observed that the 17.8 mmol/L glucose stimulatedeffectively to produce insulin- and glucagons-producing cells.CONCLUSION: The human fetal pancreatic ductal stemcells are capable of proliferation in vitro.These cells havemultidifferentiation potential and can be induced by glucoseand differentiated into insulin-producing cells in vitro.     

甲状旁腺素受体1表达对高糖状态下INS-1细胞功能影响研究

目的 观察甲状旁腺受体1(PTH1R)的表达对胰岛β细胞胰岛素合成与分泌功能影响.方法 构建出PTH1R基因沉默的细胞模型后,分别应用放射免疫法观察25 mmol/L D-葡萄糖处理后对照组、阴性基因克隆组(siPTH1R-NC)、PTHrP组及阳性基因序列组(siPTH1R)细胞内胰岛素含量、葡萄糖刺激胰岛素分泌能力、应用Fluo-3/AM 检测INS-1细胞内钙离子浓度及应用INS-1细胞2-脱氧-[3H]-葡萄糖摄入率检测INS-1细胞葡萄糖转运能力.结果 PTHrP组胰岛素分泌能力高于其他3组,siPTH1R组则低于对照组及siPTH1R-NC组(均P＜0.01);PTHrP组中细胞内胰岛素含量显著高于对照组、siPTH1R-NC及siPTH1R组(均P＜0.01),其他3组间差异无统计学意义;PTHrP组中钙离子浓度水平高于其他3组,siPTH1R组低于对照组及siPTH1R-NC组.PTHrP组中细胞2-脱氧-[3H]-葡萄糖摄入率高于其他3组.结论 高糖状态下PTH1R表达水平与INS-1细胞胰岛素合成与分泌功能有关,可能为INS-1细胞自我保护的一种作用.

Abstract：

Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.     

Uncoupling protein 2 deficiency reduces proliferative capacity of murine pancreatic stellate cells

BACKGROUND: Uncoupling protein 2(UCP2) has been suggested to inhibit mitochondrial production of reactive oxygen species(ROS) by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells(PSCs), which are involved in pancreatic wound repair and fibrogenesis.METHODS: PSCs were isolated from 12 months old(aged) UCP2~(-/-) mice and animals of the wild-type(WT) strain C57BL/6. Proliferation and cell death were assessed by employing trypan blue staining and a 5-bromo-2'-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of m RNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2', 7'-dichlorofluorescin diacetate. Expression of senescence-associated β-galactosidase(SA β-Gal) was used as a surrogate marker of cellular senescence.RESULTS: PSCs derived from UCP2~(-/-) mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dosedependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2~(-/-) mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2~(-/-) mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker α-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-β1.CONCLUSIONS: A reduced proliferative capacity of PSC from aged UCP2~(-/-) mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2~(-/-) mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.     

Overexpression of Caspase-1 in adenocarcinoma of pancreas and chronic pancreatitis

AIM: To identify the expression of Caspase-l(interleukin-1β converting enzyme) and its role in adenoma of the pancreas and chronic pancreatitis. METHODS: The expression of Caspase-1 was assessed in 42 pancreatic cancer tissue samples, 38 chronic pancreatitis specimens, and 9 normal pancreatic tissues by immunohistochemistry and Western blot analysis. RESULTS: Overexpression of Caspase-1 was observed in both disorders, but there were differences in the expression patterns in distinct morphologic compartments. Pancreatic cancer tissues showed a clear cytoplasmatic overexpression of Caspase-1 in tumor cells of 71% of the tumors, whereas normal pancreatic tissues showed only occasional immunoreactivity. In chronic pancreatitis, overexpression of Caspase-1 was found in atrophic acinar cells (89%), hyperplastic ducts (87%), and dedifferentiating acinar cells (84%). Although in atrophic cells a clear nuclear expression was found, hyperplastic ducts and dedifferentiating acinar cells showed dear cytoplasmic expression. Western blot analysis revealed a marked expression of the 45 kDa precursor of Caspase-1 in pancreatic cancer and chronic pancrealJtis (80% and 86%, respectively). Clear bands at 30 kDa, which suggested the p10-p20 heterodimer of active Caspase-1, were found in 60% of the cancer tissue and 14% ofbhe pancreatifis tissue specimens, but not in normal pancreatic tissues. CONCLUSION: Overexpression of Caspase-1 is a frequent event in pancreatic disorders and its differential expression patterns may reflect two functions of the protease. One is its participation in the apoptotic pathway in atrophic acinar cells and tumor-surrounding pancreatitis tissue, the other is its possible role in proliferative processes in pancreatic cancer cells and hyperplastic duct cells and dedifferentiating acinar cells in chronic pancreatitis.     

Evaluation of first phase insulin secretion by a nateglinide-intravenous glucose insulin release test in newly diagnosed type 2 diabetics

Objective To evaluate the function of the first phase of insulin secretion of pancreatic B cells in newly diagnosed type 2 diabetics using nateglinide-intravenous glucose insulin release test (NG-IVGIRT). Methods NG-IVGIRT and intravenous glucose insulin release test (IVGIRT) were done in 8 patients with newly diagnosed type 2 diabetes mellitus and NG-IVGIRT was done in 8 normal people. Insulin and glucose of blood were deter-     

Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells

AIM: To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment. METHODS: Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-XL and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots. RESULTS: Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or antiFas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-XL expression. CONCLUSION: Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.     

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

AIM： To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats. METHODS： In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS： Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides （100 mg/L） had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance. CONCLUSION： Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.     

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes

AIM: To evaluate/isolate cancer stem cells(CSCs) from tissue or cell lines according to various definitions and cell surface markers. METHODS: Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. RESULTS: Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabineresistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. CONCLUSION: Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.     

Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation.METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclin D1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy.RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediatedthrough p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase(acetyl K68) and nuclear factor-κB p65(acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models.CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis.     

Uncoupling protein 2 deifciency reduces proliferative capacity of murine pancreatic stellate cells

BACKGROUND: Uncoupling protein 2 (UCP2) has been sug-gested to inhibit mitochondrial production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deifciency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells (PSCs), which are involved in pancre-atic wound repair and ifbrogenesis.
METHODS: PSCs were isolated from 12 months old (aged) UCP2-/- mice and animals of the wild-type (WT) strain C57BL/6. Proliferation and cell death were assessed by em-ploying trypan blue staining and a 5-bromo-2’-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immuno-blotting and immunolfuorescence analysis. Intracellular ROS levels were measured with 2’, 7’-dichlorolfuorescin diacetate. Expression of senescence-associatedβ-galactosidase (SAβ-Gal) was used as a surrogate marker of cellular senescence.
RESULTS: PSCs derived from UCP2-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose-dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, ex-pression of the activation markerα-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleu-kin-6 and transforming growth factor-β1.
CONCLUSIONS: A reduced proliferative capacity of PSC from aged UCP2-/- mice may contribute to the retarded regenera-tion after acute pancreatitis. Apart from their slower growth, PSC of UCP2-/- mice displayed no functional abnormalities. The antiifbrotic potential of UCP2 inhibitors deserves further attention.     

Sheng-jiang powder ameliorates obesity-induced pancreatic inflammatory injury via stimulating activation of the AMPK signalling pathway in rats

AIM To investigate the mechanisms by which Sheng-jiang powder(SJP) ameliorates obesity-induced pancreatic inflammatory injury.METHODS Sprague-Dawley rats were randomized into three groups: normal group(NG), obese group(HLG), or SJP treatment group(HSG). Obesity was induced by feeding a high-fat diet in the HLG and HSG, while the NG received standard chow. Rats were euthanized after 12 wk, and blood and pancreatic tissues were collected for histopathological analyses. Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB) and transforming growth factor beta(TGF-β) expression, serum triglyceride and adiponectin levels, and apoptosis in pancreatic acinar cells were assessed. A high-fat AR42 J acinar cell injury model was established using very low-density lipoprotein(VLDL). AR42 J acinar cell culture supernatant, treated with different interventions, was applied to seven groups of pancreatic stellate cells(PSCs). The proliferation of PSCs and the expression of fibronectin and type I collagenase were assessed.RESULTS Compared with the NG, we found higher pathological scores for pancreatic tissues, lower serum adiponectin levels, higher expression levels of NF-κB in pancreatic tissues and TGF-β in pancreatic inflammatory cells, and increased apoptosis among pancreatic acinar cells for the HLG(P 0.05). Compared with the HLG, we found reduced body weight, Lee's index scores, serum triglyceride levels, and pathological scores for pancreatic tissues; higher serum adiponectin levels; and lower expression levels of NF-κB, in pancreatic tissue and TGF-β in pancreatic inflammatory cells for the HSG(P 0.05). The in vitro studies showed enhanced PSC activation and increased expression levels of fibronectin and type I collagenase after SJP treatment. An adenosine 5‘-monophosphate-activated protein kinase(AMPK) inhibitor inhibited PSC activation.CONCLUSION SJP may ameliorate obesity-induced pancreatic inflammatory injury in rats by regulating key molecules of the adiponectin-AMPK signalling pathway.