巯基丙酮酸硫基转移酶介导PI3K/AKT信号对急性胰腺炎细胞模型凋亡和自噬的影响

目的:研究巯基丙酮酸硫基转移酶(MPST)介导PI3K/AKT信号对急性胰腺炎(AP)细胞模型凋亡和自噬的影响。方法:构建AP体外细胞模型,分组为CON组、AP组、AP+siMPST组和AP+oeMPST组。采用CCK-8法检测细胞活力;采用ELISA法检测细胞上清炎症因子TNF-α、IL-1和IL-6水平;采用流式细胞仪检测细胞凋亡水平;采用Western blotting检测腺泡细胞LC3Ⅱ/Ⅰ、beclin1、ATG5、MPST、PI3K、p-PI3K、AKT、p-AKT的表达水平。结果:AP组、AP+siMPST组和AP+oeMPST组的细胞存活率均低于CON组(P<0.01),而细胞凋亡率则均高于CON组(P<0.01);AP+siMPST组细胞存活率、AP+oeMPST组细胞凋亡率均高于AP组(P<0.01),而AP+siMPST组细胞凋亡率、AP+oeMPST组细胞存活率均低于AP组(P<0.01)。AP组、AP+oeMPST组的腺泡细胞TNF-α、IL-1和IL-6、LC3Ⅱ/Ⅰ、beclin1、ATG5、MPST、p-PI3K/PI3K、p-AKT/AKT水平明显高于CON组(P<0.01);上述指标水平AP+siMPST组均低于AP组(P<0.01),而AP+oeMPST组则均高于AP组(P<0.01)。结论:MPST可通过PI3K/AKT信号诱导胰腺腺泡细胞凋亡、自噬和炎症反应;抑制MPST可能对AP具有治疗意义。

Coexpression of cholecystokinin-B/gastrin receptor and gastrin gene in human gastric tissues and gastric cancer cell line

AIM:To compare the expression patterns of cholecystokinin-B (CCK-B)/gastrin receptor genes in matched human gastric carcinoma and adjacent non-neoplastic mucosa of patients with gastric cancer,inflammatory gastric mucosa from patients with gastritis,normal stomachs from 2 autopsied patients and a gastric carcinoma cell line (SGC-7901), and to explore their relationship with progression to malignancy of human gastric carcinomas.METHODS:RT-PCR and sequencing were employed to detect the mRNA expression levels of CCK-B receptor and gastrin gene in specimens from 30 patients with gastric carcinoma and healthy bordering non-cancerous mucosa,10 gastritis patients and normal stomachs from 2 autopsied patients as well as SGC-7901.The results were semi-quantified by normalizing it to the mRNA level of β-actin gene using Lab Image software.The sequences were analyzed by BLAST program.RESULTS:CCK-B receptor transcripts were detected in all of human gastric tissues in this study, including normal,inflammatory and malignant tissues and SGC-7901. However,the expression levels of CCK-B receptor in normal gastric tissues were higher than those in other groups (P<0.05),and its expressions did not correlate with the differentiation and metastasis of gastric cancer (P>0.05).On the other hand,gastrin mRNA was detected in SGC-7901 and in specimens obtained from gastric cancer patients (22/30)but not in other gastric tissues,and its expression was highly correlated with the metastases of gastric cancer (P<0.05).CONCLUSION:Human gastric carcinomas and gastric cancer cell line SGC-7901 cells coexpress CCK-B receptor and gastrin mRNA. Gastrin/CCK-B receptor autocrine or paracrine pathway may possibly play an important role in the progression of gastric cancer.

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

AIM: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-κB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-κB p50 subunit.METHODS: Pancreatitis was induced in male Wistar rats by administering 2×100 μg/kg body weight of cholecystokininoctapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK.RESULTS: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity,pancreatic levels of TNF-α and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-κB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide.According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis.CONCLUSION: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-κB might be clinically useful for the suppression of the severity of acute pancreatitis.

Interplay between nitric oxide and VIP in CCK-8-induced phasic contractile activity in the rabbit sphincter of Oddi

AIM: The sphincter of Oddi (SO) plays an important role in delivery of bile into the duodenum. To establish whether vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) were involved in phasic contractile activity of the rabbit SO stimulated by cholecystokinin-octapeptide (CCK-8). METHODS: Isolated SO muscle rings were cleaned of fat and mounted horizontally on two small L-shaped hooks one of which was connected to a force transducer for the measurement of isometric tension.The experiments were carried out in a thermostatically controlled (37±0.2℃) organ bath (5 mL) containing Krebs solution.The organ fluid was gassed with 95% O2 and 50 mL/L CO2 to keep the pH at 7.40±0.05. Contractile responses to CCK-8 (1 μmol/L) were evaluated in the presence and absence of NG-nitro-L-arginine (LNNA), an inhibitor of NO synthase (100 μmol/L), and (p-chloro-D-Phe6-Leu17)-VIP (VlPa, 30 μmol/L), a VIP receptor antagonist. RESULTS: CCK-8 stimulated the phasic activity of the SO. NO synthase inhibition increased the frequency and amplitude of contractions with a slight increase in developed tension. Pre-incubation with VlPa also attenuated this CCK-8 effect. The combined application of LNNA and VlPa abolished the phasic activity of the muscle rings with a marked increase in tension in response to CCK-8. CONCLUSION: VIP and NO together contribute to an increase in phasic activity of SO.

Effects of gastrin 17 on β-catenin/Tcf-4 pathway in Colo320WT colon cancer cells

AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor （CCK-2R）, was transfected into a colonic cancer cell line Colo320 by Lipofectamine ^TM 2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a time-dependent manner （0, 1, 6, 12, 24 and 48 h）, then with L365,260 （Gastrin17 receptor blocker） for 30 rain, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS： Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION： Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.

Presence of CCK-A, B receptors and effect of gastrin and cholecystokinin on growth of pancreatobiliary cancer cell lines

AIM: To investigate the effects of gastrin and cholecystokinin (CCK) and their specific antagonists on the growth of pancreatic and biliary tract cancer cell lines. METHODS: Five pancreatic and 6 biliary cancer cell lines with 2 conrtol cells were used in this study. Cell proliferation study was done using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test and direct cell count method. Reverse transcription-polymerase chain reaction (RT-PCR) and slot blot hybridization were performed to examine and quantify the expression of hormonal receptors in these cell lines. RESULTS: SNU-308 showed a growth stimulating effect by gastrin-17, as did SNU-478 by both gastrin-17 and CCK-8. The trophic effect of these two hormones was completely blocked by specific antagonists (L-365, 260 for gastrin and L-364, 718 for CCK). Other cell lines did not respond to gastrin or CCK. In RT-PCR, the presence of CCK-A receptor and CCK-B/gastrin receptor mRNA was detected in all biliary and pancreatic cancer cell lines. In slot blot hybridization, compared to the cell lines which did not respond to hormones, those that responded to hormones showed high expression of receptor mRNA. CONCLUSION: Gastrin and CCK exert a trophic action on some of the biliary tract cancers.

Effect of cholecystokinin on experimental neuronal aging

AIM: To observe the effect of cholecystokinin (CCK) on lipofusin value, neuronal dendrite and spine ultrastructure, and total cellular protein during the process of experimental neuronal aging. METHODS: Experimental neuronal aging study model was established by NBA2 cellular serum-free culture method. By using single intracellular lipofusin value from microspectrophotometry, morphology of neuronal dendrites and spines from the scanner electron microscopy, and total cellular protein as the indexes of experimental neuronal aging, we observed the effect of CCKs on the process of experimental neuronal aging. RESULTS: Under the condition of serum-free culture, intracellular fluorescence value (%) increased with the extension of culture time (1 d 8.51±3.43; 5 d 10.12±3.03; 10 d 20.54±10.3; 15 d 36.88±10.49; bP<0.01). When CCK was added to serum-free culture medium, intracellular lipofusin value (%) decreased remarkably after consecutive CCK reaction for 10 and 15 d (control 36.88±10.49; 5 d 32.03±10.01; 10 d 14.37±5.55; 15 d 17.31±4.80; bP<0.01). As the time of serum-free culturing was prolonged, the number of neuronal dendrite and spine cells decreased. The later increased in number when CCK8 was added. CCK8 could improve the total cellular protein in the process of experimental neuronal aging. CONCLUSION: CCKs may prolong the process of experimental neuronal aging by maintaining the structure and the number of neuronal dendrite and spine cells and changing the total cellular protein.

INTRAPANCREATIC CHOLECYSTOKININ MEDIATES VAGALLY STIMULATED EXOCRINE SECRETION FROM THE RAT PANCREAS

Although cholecystokinin is localized within neuronal fibres of the pancreas, a physiological role for intrapancreatic cholecystokinin has not been identified. The strategy of this study was to elicit pure vagal stimulation electrically, and to use specific receptor antagonists to identify the mediators of exocrine pancreatic secretion. We conclude that vagal stimulation of the rat pancreas involves ganglionic neurotransmission and release of acetylcholine and cholecystokinin from intrapancreatic, postganglionic fibres. To our knowledge, this is the first study to demonstrate a physiological role for intrapancreatic cholecystokinin.

生喂大豆会降低其营养价值

给鸡饲喂生大豆是有害的，特别是在头几周。这是因为生大豆中含有一定量的抗胰蛋白酶的物质。摄入生大豆会使胰蛋白酶消化蛋白质的能力丧失。这种能力的丧失会引起粪便中胰蛋白酶排泄量的增加以及必需氨基酸的流失。而这些反应是由小肠分泌的肠促胰酶肽激素的大量释放引起...