The Study Of Effects Of Bovine Lactoferricin In IEC-6

BackgroundNecrotizing enterocolitis (NEC) is a medical condition primarily seen in premature infants, where portions of the bowel undergo necrosis. The condition is typically seen in premature infants, and the timing of its onset is generally inversely proportional to the gestational age of the baby at birth (i.e. the earlier a baby is born, the later signs of NEC are typically seen). Initial symptoms include feeding intolerance, increased gastric residuals, abdominal distension and bloody stools. Symptoms may progress rapidly to abdominal discoloration with intestinal perforation and peritonitis and systemic hypotension requiring intensive medical support.More recently ultrasonography has proven to be useful as it may detect signs and complications of NEC before they are evident on radiographs, specifically in cases that involve a paucity of bowel gas, a gasless abdomen, or a sentinel loop. Diagnosis is ultimately made in 5-10% of very low-birth-weight infants (<1,500g).Once a child is born prematurely, thought must be given to decreasing the risk for developing NEC. Toward that aim, the methods of providing hyperalimentation and oral feeds are both important. Despite decades of research on necrotizing enterocolitis, we still do not fully understand the pathogenesis of the disease, how to prevent or how to treat the disease. The main factors thought to be involved in the pathogenesis of NEC are:intestinal immaturity, enteral feeds, the intestinal microbiome, inflammation and local ischaemia and/or reperfusion injury. Histologically, there is a massive intestinal inflammatory response in NEC. The fetal gut develops in an environment where exposure to microbes is limited. Therefore, premature infants are exposed to a much greater diversity and quantity of bacteria, viruses and fungi. The premature infant gut displays an excessive inflammatory response, and toll-like receptor 4 seems to play a key role in this inflammatory response. Neonatologists at the University of Iowa reported on the importance of providing small amounts of trophic oral feeds of human milk starting as soon as possible, while the infant is being primarily fed intravenously, in order to prime the immature gut to mature and become ready to receive greater oral intake. Human milk from a milk bank or donor can be used if mother’s milk is unavailable. The gut mucosal cells do not get enough nourishment from arterial blood supply to stay healthy, especially in very premature infants, where the blood supply is limited due to immature development of the capillaries, so nutrients from the lumen of the gut are needed.In summary, our study tried to investigate the beneficial effects of bovine lactoferrin in IEC-6 based on the inflammation model in vitro induced by LPS. The study will be the basic research to develop a new drug target to cure the necrotizing enterocolitis.Objective1. To investigate the influence of bovine lactoferrin on the cell viability in IEC-6.2. To evaluate the effects and its underlying mechanisms of the bovine lactoferrin about the capacity of IEC-6 proliferation.3. To assess the protective effects of bovine lactoferrin in IEC-6 stimulated by LPS and its further influencent pathways.Methods1. Cell viability assayWe used MTT, a colorimetric assay to assess cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor,1-methoxy phenazine methosulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport.[1] Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. In this experiment, we compared the different OD value in the group with or without bovine lactoferrin.2. Cell proliferation assayThe BrdU Cell Proliferation Assay detects 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody. When cells are cultured with labeling medium that contains BrdU, this pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells. After removing labeling medium, cells are fixed and the DNA is denatured with our fixing/denaturing solution. Then a BrdU mouse mAb is added to detect the incorporated BrdU (The denaturing of DNA is necessary to improve the accessibility of the incorporated BrdU to the detection antibody). Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate 1MB is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation.3. Cell cycle assayThe cell cycle or cell-division cycle is the series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two daughter cells. Cell cycle can be subdivided into interphase (G0/G1, S and G2) and mitotic (M) phase (prophase, metaphase, anaphase and telophase). Cell Cycle Analysis Kit provides a quick and easy method to detect the number of cells in a cell population, which are at a specific stage of the cell cycle. We used a nuclear dye PI, the binding of which to nucleic acids in the cell results in fluorescence signal, which is proportional to cellular DNA content. The percentages of cells in different phases of the cell cycle (G0/G1, S, and G2/M) can be quantified by flow cytometry.4. PCRQuantitative PCR was used in our study the specific gene mRNA level in IEC-6 in different group.5. ELISAThe enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies and color change to identify a substance. We used such kind of assay to assess the cytokines level in the supernatants of different groups.6. Western BlotWestern blot technique was utilized to find the difference of proteins in IEC-6 with different treatments.7. Statistical analysisFor database management and statistical analysis, we used GraphPad prism 5.01. Data were expressed as means±SD. Difference between means were examined using one-way ANOVA followed by Newman Keuls. A value of P<0.05 was considered statistically significant.Results1. Bovine lactoferrin significantly increased the cell viability of IEC-6.2. Bovine lactoferrine promoted IEC-6 proliferation via regulating the cell S stage in the whole cell cycle.3. Bovine lactoferrine could downregulate the phospholation of MAPK and decrease the nuclear translocation of NF-κB to show the protective effects in EEC-6 treated by LPS.ConclusionBovine lactoferrin could show the beneficial effects in IEC-6 by promote the cell viability, cell proliferation, and anti-inflammationm, which would be of importance potentially to cure necrotizing enterocolitis.