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APS endotoxin induced intestinal epithelial cell injury

Author: YuanZuo
Tutor: SunMei
School: China Medical University
Course: Pediatrics
Keywords: APS Intestinal epithelial cells Cells Tumor Necrosis Factor Interleukin-8 Nuclear factor -κB Mitogen-activated protein kinase Extracellular signal- regulated kinase cJun N-terminal kinase P38 protease
CLC: R2855
Type: PhD thesis
Year: 2008
Downloads: 457
Quote: 0
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Abstract


Introduction intestinal epithelial cells are an important component of intestinal mucosal barrier and, while that of the host mucosal surface is a natural and acquired immune system play a central regulatory role of host and pathogen defense first bidirectional link. Some intestinal diseases and non-enteric diseases can lead to bowel dysfunction, various pathological factors such as trauma, shock, severe infections and other severe stress, the body immune function, a large number of intestinal bacteria and endotoxin LPS intrusion circulation and intestinal tissue, resulting in bacterial translocation and intestinal endotoxemia, thus further increasing the damage of intestinal epithelial cells, activating a series of intracellular immune response, resulting in some inflammatory mediators such as TNF-α, IL-6, IL- 8, platelet-activating factor, a large number of production and release, causing systemic inflammatory response syndrome (SIRS), start and accelerate multi-system organ failure (MSOF). Research LPS-mediated intestinal epithelial cell injury has become a widely noted research. Much progress has been made in recent years. NF-κB is associated intestinal immune function of many key regulator of gene expression, which for many cytokines (IL-1, IL-2, IL-6, IL-8, IL-12, etc.) in the lymphocytes, epithelial cells, monocytes plays an indispensable role. Is now clear, LPS under the action of intestinal epithelial cells can be activated NF-κB, efficient induction of various cytokines (IL-1, IL-6, IL-8, TNF, granulocyte - macrophage colony stimulating factor ), adhesion molecules such as increased gene expression, while involved in inflammation cascade waterfall effect amplification and gene expression in a variety of enzymes also play an important role in regulating. Because NF-κB activation is our present understanding of the important causes of intestinal inflammatory damage pathways, and therefore this pathway starting to study blocking NF-κB activation pathway in regulating inflammation blocking protein synthesis, to inhibit inflammatory response is the treatment of intestinal damage a promising approach. Mitogen-activated protein kinase (MAPK) are a class of widely distributed intracellular serine / threonine residues of the protein kinase, is a family of cell surface receptors and decisive connection between the expression of genes important signal regulating enzymes. In mammalian cells have been cloned at least four MAPK subfamilies. Respectively, extracellular signal-regulated kinase (ERK1/ERK2), cJun N-terminal kinase (JNK1/JNK 2), P38 MAPK (α, β) and ERK5. MAPK is activated can stay in the cytoplasm, activating a range of other protein kinases, phosphorylation of the cytoskeletal components, but also nuclear translocation into the nucleus via activation of nuclear transcription factor respective to phosphorylation, which initiates a These gene expression, protein synthesis and promoting the channel is changed, the completion of extracellular stimuli. Grishin and other scholars in vitro using LPS-stimulated IEC-6 cells found, P38MAPK phosphorylation and COX-2 expression increased in a dose-dependent manner. From the perspective of this study confirmed the P38MAPK vitro in LPS-induced intestinal epithelial cell injury. MAPK through a multi-level, multi-faceted signal conditioning, affecting gene expression, media releases and other intracellular signaling pathways involved in the intestinal cells of the release of proinflammatory cytokines such as injury, and therefore blocking signaling pathways and regulation of MAPK signaling levels molecule expression and activity of intestinal injury treatment will provide new ideas and approaches. Astragalus is China's traditional Chinese medicine, because of its low toxicity and side effects is small and has enhanced and restore immune function, and is currently used for control of the country has its oncology, immune dysfunction and immune deficiencies and other diseases, to play its enhanced immune and immune regulation agent. Astragalus Astragalus polysaccharide is an important role of the monomer component. Wang Lixin, etc. to deal with mice injected with endotoxin APS and found that it endotoxin-induced liver homogenates malondialdehyde (MDA) and elevated glutathione (GSH) decreased endotoxin deal with small rat liver mitochondria injury has a protective effect. Lu Jing Tao study confirmed APS can inhibit bacterial lipopolysaccharide-induced rat peritoneal macrophages to release tumor necrosis factor, interleukin and nitric oxide secretion in vivo studies have demonstrated so APS can inhibit the secretion of inflammatory cytokines thereby reducing tissue damage to exert its effects on immune protection. At home and abroad about the effects and mechanism of APS more, but yet on whether APS on LPS induced intestinal injury in rats, and studies whether this protective effect signal transduction pathway. This study is based on IEC-6 intestinal epithelial cells for the study, research LPS lead IEC-6 cells after injury, APS is able to effectively promote epithelial cell proliferation and thus play a role in mucosal barrier repair; whether through inhibition of MAPK / NF -κB signaling pathway to inhibit the release of inflammatory cytokines play its intestinal immune protection. APS through the study was to investigate the role played intestinal protective mechanism of the regulation, which provides a theoretical basis for its clinical application. An experimental method, materials, IEC-6 cells were purchased from the Chinese Academy of Medical Sciences Cancer Hospital biological testing center. The recovery of IEC-6 cells were cultured with 10% FBS DMEM medium with 0.01mg/ml insulin, in a CO2 incubator (37 ℃, 5% CO2) in the culture; medium was changed the next day, to about 80% confluence with 0.25% trypsin digestion and passage, five days passaged once and passaged five times the cells used in the experiment group. The cultured cells were randomly divided into five groups: Group 1: control group: DMEM medium was the control; 2 groups: pure LPS group: LPS concentration of 10 ug / ml; 3 groups: simple APS group: Divided into four subgroups: ① APS50 ug / ml; ② APS100 ug / ml; ③ APS200ug/ml; ④ APS500 ug / ml; 4 groups: LPS with different concentrations of APS group: Divided into four subgroups; ① LPS10 ug / ml APS50ug/ml group; ② LPS10 ug / ml APS 100 ug / ml group; ③ LPS10 ug / ml APS 200 ug / ml group; ④ LPS10ug/ml APS 500 ug / ml group; 5 Group: APS with different concentrations of LPS group: Sanya divided into groups: ① APS500 ug / ml LPS 5 ug / ml; ② APS500 ug / ml LPS 10 ug / ml; ③ APS500 ug / ml LPS 20 ug / ml. Second, the method (A) MTT method to detect cell proliferation rate is 1, the normal IEC-6 cells were CO2 incubator (37 ℃, 5% CO2) in different concentrations APS (50 ug / ml, 100 ug / ml, 200 ug / ml, 500 ug / ml) were cultivating 24 hours, 48 ​​hours, 72 hours, MTT was detected pure APS IEC-6 cells of normal proliferation rate; 2, the IEC-6 cells were incubated with LPS (10ug / ml) for 1 hour after the culture system by adding various concentrations of APS (50 ug / ml, 100 ug / ml, 200 ug / ml, 500 ug / ml) were cultivating 24 hours, 48 ​​hours, 72 hours, MTT method APS detected after injury to LPS IEC-6 cell proliferation; 3, the IEC-6 cells cultured with APS500ug/ml pretreated for 24 hours, then to different concentrations of LPS (respectively 5ug/ml, 10ug/ml, 20ug / ml), was observed 1 hours, MTT was measured after pretreatment of the LPS injury APS IEC-6 cell proliferation rate. (B) RT-PCR method for detection of TNF-α and IL-8mRNAIEC-6 cells in DMEM medium with different concentrations of APS (50 ug / ml; 100 ug / ml; 200 ug / ml; 500 ug / ml) Cultivation 24 hours. 1, given LPS (10ug/ml) stimulated for 1 hour, the cells were collected, extracted and determination of total RNA, synthesized cDNA, PCR amplification of TNF-α, IL-8 and GAPDH, agarose gel electrophoresis analysis of the APS IEC injury LPS -6 secreted TNF-α and IL-8mRNA of expression; 2, were given LPS (10ug/ml) stimulated for 1 hour, after 4 hours the cells were collected, extracted and determination of total RNA, synthesis of cDNA, PCR amplification of TNF- α, IL-8 and GAPDH, agarose gel electrophoresis analysis at different time points APS on LPS injury IEC-6 secreted TNF-α and IL-8mRNA changes. (C) Western-Blotting detected P-ERK1 / 2, P-JNK, P-P38 and NF-κB, IκB-α protein expression IEC-6 cells in DMEM medium with different concentrations of APS (50 ug / ml; 100 ug / ml; 200 ug / ml; 500 ug / ml) cultivation for 24 hours. 1, given LPS (10ug/ml) stimulated for 15 minutes, cells were collected, total protein was extracted and quantified, SDS gel electrophoresis, transferred to a membrane and closed, specific antibodies were incubated, after the color of the APS gel imager LPS injury IEC-6 secretion IκB-α protein expression influence; 2, given LPS (10ug/ml) stimulation for 30 minutes, with the former method, nuclear protein extraction and quantitative detection of APS on LPS injury of IEC-6 secretion NF- κB protein expression effect; 3, given LPS (10ug/ml) stimulation for 1 hour, with the former method, the detection APS on LPS injury IEC-6 secretion in P-ERK1 / 2, P-JNK, P-P38 Protein Expression . Third, the statistical analysis of all data as mean ± standard deviation ((?) ± s) said that, with the SPSS11.5 statistical package using single-factor analysis of variance by LSD test. P <0.05 was significant difference. The experimental results a, LPS IEC-6 under the action of morphological changes in normal intestinal epithelial cells by a homogeneous population of cells composed of epithelioid, cobblestone mosaic arrangement of non-overlapping, typical monolayer cells irregular polygon, clear boundary , large nuclei, showing oval cells connected to each other, showing strong proliferative activity. LPS injury in intestinal epithelial cells presenting cells fuzzy boundaries, cells from polygonal into round, a large number of particles within the cytoplasm like substance, some membrane rupture, cell morphology incomplete. Two, APS on LPS injury IEC-6 cells Effect 1, APS normal IEC-6 cells promote proliferation. MTT assay showed that: APS at different times on different cell proliferation, and after treatment with different concentrations of APS on cell proliferation was significant difference (P <0.05). APS of IEC-6 cells proliferation in a dose-dependent effect, the treatment time is prolonged gradually weakened, the performance at 24 hours is shown APS50ug/ml proliferation effect, and in the first 72 hours, APS concentration of 200ug/ml only showed significant effect in promoting proliferation. 2, APS on LPS IEC-6 cells after injury has no significant role in promoting cell proliferation. MTT results showed that: the role of cells by LPS only in the first 24 hours the concentration of APS manifestations when 200ug/ml and 500ug/ml proliferation effect, whereas in 48 hours and 72 hours time points and different concentrations of test APS group were not seen significant proliferation effect (P> 0.05). Therefore, when IEC-6 cells by LPS injury, APS failed to show a significant role in promoting proliferation. 3, APS pretreatment increased the LPS injury IEC-6 cell proliferation activity. MTT results showed that: in effect 5ug/ml by LPS when compared with control group, APS is not promoting proliferation of pretreatment group (P> 0.05). In effect by LPS10ug/ml and 20ug/ml when compared with control group, APS pretreatment significantly promoting proliferation (P <0.01). Three, APS on LPS-stimulated IEC-6 cells produced TNF-α, IL-8 mRNA of 1, RT-PCR analysis showed that when LPS-stimulated IEC-6 cells, TNF-α, IL-8 mRNA was secretion induced significant increase (P <0.01), and astragalus polysaccharide (APS) can inhibit the secretion of this increase, and a dose-dependent manner: 50ug/ml APS partially IEC-6 inhibited LPS-stimulated TNF-generated α and IL-8 mRNA levels, while 200ug/ml and 500ug/ml APS with increasing concentration, the inhibition of TNF-α and IL-8 mRNA levels gradually increased (P <0.01). 2, RT-PCR test results also showed that: Astragalus polysaccharide (APS) inhibition of TNF-α, IL-8 mRNA increased secretion in a time-dependent: LPS-induced TNF-αmRNA APS expression is effectively suppressed, 1 hour and 4 hours inhibition rates were 10.3% and 25.5%, LPS-induced IL-8 mRNA expression was equally effective inhibition of APS: 1 hour and 4 hours inhibition rates were 15.3% and 18.8%. Four, APS on LPS-induced IEC-6 cells produced by NF-κB protein expression analysis of Western-Blotting method affect test results show that when the LPS-stimulated IEC-6 cells, NF-κB inhibitory protein IκB-a decreased expression, NF -κB protein with significant increase (P <0.01), astragalus polysaccharide (APS) significantly inhibited this effect in a dose-dependent manner and, as the concentration increases, 500ug/mlAPS showed a stronger inhibition of NF-κB role of protein expression (P <0.01). Five, APS on LPS stimulated cells of IEC-6 P-ERK1 / 2, P-JNK, P-P38 protein expression analysis of the role of LPS induced by detecting the APS of IEC-6 cells P-P38 at the protein level impact study found that when LPS stimulation IEC-6 cells, P38 protein phosphorylation enhanced secretion is induced significant increase (P <0.01), APS inhibiting phosphorylation of P38 protein, with the increase of the concentration of APS, the inhibition of P38 protein phosphorylation gradually increased, which is the concentration of APS in 500ug/ml strongest inhibition. Similarly, LPS induced IEC-6 cells secrete ERK1 / 2 and JNK phosphorylation also significantly expressed, and the difference was statistically significant (P <0.05), but caused by APS on LPS-induced ERK1 / 2 and JNK phosphorylation expression but no significant effect (P> 0.05). Conclusion 1, astragalus polysaccharides on normal IEC-6 cells promote cell proliferation; when cells by LPS injury, APS did not show a significant role in promoting proliferation repair, suggesting APS promoting cell proliferation is dependent on cell normal physiological state; APS LPS pretreatment increased after injury of IEC-6 cell proliferation, indicating that early intervention can reduce APS LPS on IEC-6 cells showing injury protective effect on cells, APS therapeutic effect also depends on the severity of damage of LPS. 2, LPS effect on intestinal epithelial cells, cytokines TNF-α and IL-8 mRNA expression in a large number, and the APS can inhibit LPS-stimulated cells secrete TNF-α and IL-8 mRNA excessive production, and inhibition of this APS role in a concentration and time dependent. Description of astragalus polysaccharides on intestinal epithelial cells of the immune protective effect may be through inhibition of cytokine TNF-α, IL-8, etc. overexpression, thereby reducing its inflammatory tissue damage. 3, LPS stimulated IEC-6 时, IκB-a decreased expression, NF-κB nuclear protein expression, the APS can effectively inhibit the expression of NF-κB protein growth and decreased expression of IκB-a, and with the APS concentration, the inhibition of NF-κB protein effect gradually. Description APS inhibition of cytokine overexpression may be through inhibition of nuclear factor NF-κB signaling pathway to achieve. 4, LPS acts on the intestinal epithelial cells, which secrete protein phosphorylation P38MAPK enhanced, APS can significantly inhibit this secretion increased in a dose-dependent manner, indicating that APS on LPS-induced IEC-6 cells of the immune protective effect may also be achieved through the signal path P38MAPK. LPS acts on the intestinal epithelial cells, the secretion of ERK1 / 2 and JNK phosphorylation also increased, but the APS on ERK1 / 2 and JNK phosphorylation without significant impact. Description APS on LPS-induced IEC-6 cells of the immune protective effect not through ERK1 / 2 and JNK signaling pathway.

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