Alteration of Structure and Function of Tight Junction and Gap Junction in Alveolar Epithelial Cells during Acute Lung Injury
|School||First Military Medical University|
|Course||Respiration within the science|
|Keywords||Acute lung injury Alveolar epithelial cell Tight junction Gap junction Junction protien|
BACKGROUND:Acute lung injury (ALI) refers to the syndrome of acute respiration failure characterized by edema of interstitial lung tissue and injure of cells in lung parenchyma. At last the ALI develops into acute respiratory distress syndrome (ARDS). The clinical characters are urgent starting, dangerous conditions and high death rate (almost 50%). But the pathogenesis is not completely interpreted yet and there is no special treating method in clinical. So it is a tough urgent to be solved problem in clinical to study the pathogenesis and treatment methods. The junctions between alveolar epithelial cells include tight junctions (TJ) and gap junctions (GJ). The two methods of junction guarantee the integrality of the blood-gas barrier and regulate the distribution and transference of the fluid, ions and metabolism product. TJs have two functions, the barrier (or gate) function (BF) and the fence function (FF). The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. The cell passage formatted by GJs is called gap junction channels. The gap junction channels allow the molecules which are about 1000Da to pass through and make the hydrophilic ions, molecules, metabolism product or signal transduction molecules to scatter directly. So the gap junction channels act as the gate to regulate the transportation and distribution of the ions, electric current and low molecule metabolism product among cells.Many studies have proved that the TJs are composed by some proteins including occludin, claudin, zonula-1 (zo-1), cingulin, symplekin, and et al. Occlludin was the first identified protein in 1993, from then on, there are some sub-models of occluding and 24 claudins were identified. The claudin protein is considered to be the key protein in the structure of TJ. GJ is special membrane channel in the two adjacent cells. Their two symmetry components are called as connexons and hemichanels which biochemistry components are 28KDa membrane protein. Each connexon composes of six dumbbell sub-unit—connexin. These six sub-units make up a hexagonal hydrophilia hemichannel, two hemichannel connect to form one hydrophilia channel (gap junction) which diameter is 15A or so.In the patients of ALI or ARDS, the barrier composed by alveolar epithelial cells and endotheliocytes was gravely damaged, which result in liquid containing protein abundantly getting into the alveolus. Because of different influencing factor, the clinical process of ALI or ARDS is different. But one important mechanism is that the intensity of the injury on the alveolar epithelial cell barrier determines the level of lung injury. In the previous studies on junction protein, too many were focus on the beginning of tumor, angiocardionpathy, development of organ and et al. But studies on the relation between the junction and lung injury were too few. This study investigated the relationship between the ALI and TJs and GJ of alveolar epithelial cells by observing the change of the component proteins of the TJ and GJ during the ALI of Wistar rat.OBJECTIVE:1. Replication the Wistar rat model of ALl by oil acid, and observe the change of alveolus structure in the normal and injured lung by light microscope; Probe the change of ultramicro structure in ALI by observing the ultramicro structure of nomal and injured alveolus by TEM and SEM.2. Find out the relationship between the ALl and GJ by studying the change of Cx32mRNA and Cx26 expression in rat lung in normal and injured lung.3. Find out the relationship between the ALI and TJ by studying the change of claudin-3 expression in rat lung in normal and injured lung.METHODS:1. Replication of the animal model: reproduce the ALI model by the slow injection of oil acid via the rat tail vein. Take out the lung on the time 1, 5, 12, 24 and 48 hours after the injection. Observe structure change of the lung tissue by HE staining, observe ultramicro structure change of the lung tissue by scanning electron microscope and transmission electron microscope.2. Detect the expression of Cx26 and Claucin-3 by SP-immunohistochemistry in the normal and ALI lung tissue, and observe the expression by light microscope.3. Detect the expression of Cx32mRNA by in situ hybridization in the normal and ALI lung tissue, and observe the expression by light microscope.4. Statistics process: The SPSS10.0 statistical software was used in this study and one-way ANOVA was used to compare the means of the groups. If the difference was significant, the multiple comparison process was used further. Homogeneity of variance assumed, the LSD method was used; homogeneity of variance not assumed, the Tamhane’sT2 method was used. And the significant level was 0.05. RESULTS:1. In the injured groups, the rats appeared, to some sentence, accelerated and embarrassed breath, cyanopathy, foam-blood phlegm and et al. But the rats of the normal group didn’t show the symptom. The injured lung tissue became larger in volume, duller in the lung verge. Hyperemia, edema, hemorrhage were appeared in the injured lung. There was some pink foam phlegm in the trachea. These changes were greater in the middle and inferior injured lung. By light microscope, in the HE staining sections increasing of the alveolus wall width, hyperemia and hemorrhage of lung mesenchymal, soakage of inflammation cells and edema could be seen in the injured lungs. In the alveolus, there were light red edema liquid and neutrophil cells. And there was no obviously change in the structure of normal lungs.2. Ultramicro structure change of the lung tissue by electron microscope: By transmission electron microscope (TEM) in the ALI rat lung tissues, quantity of lung mesenchymal cells were destroyed, which shape and structure could not be recognized. There was no integrate junctional structure could be seen. In alveolar epithelial typeⅠcells (AECⅠ), cell membrane was broken and nucleus were dissolved. While in the normal rat lung tissue, the morphosis of the epithelium and the junctional structure was integrated. By scanning electron microscope (SEM) in the ALI rat lung tissue, the alveoli were filled by floccule and drop materials. These materials connected with epithelium or mesenchymal and formed globulate drops or reticulate structure. While in the normal rat lung tissue, the wall of the alveoli was smooth and glossy, and there was no extravasate in alveoli.3. Expression and change of Cx26: Immunohistochemistry displayed that the Cx26 positive staining was localized in the cytoplasm and membrane of the alveolar epithelial cells. The ratios of Cx26 positive expression in normal group and 1, 5, 12, 24, 48 hour groups were (12.25±1.12)%,(9.11±0.62)%, (5.71±0.52)%, (6.22±0.77)%, (6.64±0.53)%, (8.25±0.53)% respectively. The variance of Cx26 positive ratios of the groups were homogeneous (P=0.393), and the analysis of variance displayed the difference was significant (F=115.638, P＜0.001), so the LSD method was used to compare the means of the groups. The difference of Cx26 positive ratios between normal group and other groups was significant (P＜0.001); the difference between 5th hour group and 12th hour group, 12th hour group and 24th hour group was not significant (the P value was 0.115 and 0.193 respectively); the difference between other groups was significant (P＜0.05). And from the 5th group the ratio of Cx26 positive expression had a rising tendency.4. Expression and change of Cx32mRNA: In situ hybridization displayed that the Cx32mRNA positive staining was localized in the cytoplasm of the alveolar epithelial cells. The ratios of Cx32mRNA positive expression in normal group and 1, 5, 12, 24, 48 hour groups were (13.95±1.57)%, (5.53±0.98)%, (4.83±1.00)%, (5.27±0.93)%, (6.80±0.87)%, (8.92±0.73)% respectively. The variance of Cx32mRNA positive ratios of the groups were homogeneous (P=0.201), and the analysis of variance displayed the difference was significant (F=109.804, P＜0.001), so the LSD method was used to compare the means of the groups. The difference of Cx32mRNA positive ratios between normal group and other groups was significant (P＜0.001); the difference of 48th group between other groups was significant (P＜0.001); there was no significant difference between the 1st hour group and the 5th hour group, the 1st hour group and the 12th hour group, the 5th group and the 12th groups (the P value was 0.140, 0.585 and 0.193 respectively); the difference between others was significant (P＜0.05). From the 5th group the ratio of Cx32mRNA positive expression had a rising tendency.5. Expression and change of claudin-3: Immunohistochemistry displayed that the claudin-3 positive staining was localized in the cytoplasm and membrane of the alveolar epithelial cells. The ratios of claudin-3 positive expression in normal group and 1, 5, 12, 24, 48 hour groups were 08.45±2.37)%, (12.08±1.10)%, (9.19±1.08)%, (7.75±1.70)%, (11.81±2.07)%, (13.54±1.21)% respectively. The variance of claudin-3 positive ratios of the groups were homogeneous (P=0.127), and the analysis of variance displayed the difference was significant (F=50.523, P＜0.001), so the LSD method was used to compare the means of the groups. The difference of claudin-3 positive ratios between normal group and other groups was significant (P＜0.001); the difference between 1st hour group and 24th hour group, 1st hour group and 48th hour group, 5th hour group and 12th hour group was not significant (the P value was 0.714, 0.055 and 0.058 respectively); the difference between others was significant (P＜0.05). From the 12th group the ratio of claudin-3 positive expression had a rising tendency.Conclusion:1. By observing the ultramicro structure of the ALI lung tissue by electron microscope, the structure of TJ and GJ had been destroyed gravely. The destroy of TJ and GJ maybe one of the reasons that lead to edema in lung mesenchymal and alveoli.2. The expression of Cx26 and Cx32mRNA declined sharply at the beginning stage of ALI, but worked up at the recovery stage, which illustrated that damage and recovery of GJ has intimate touch with the beginning and recovery of ALI.3. The change of claudin-3 in groups suggested that damage and recovery of TJ has intimate touch with the beginning and recovery of ALI.