The Expression And Significance Of GSK3β,CLIP-170 In The Process Of Injury And Reparation Of Airway Epithelia In Mice

The acute and chronic bronchitis causes the injury of airway mucous epithelia. In afterward repair process, the peripheral epithelial cells move, splits, proliferate and differentiate to overlay the damaged district again. The cell appearance changes when cell move, pustute of cell membrane direct and the central bodies polarize. When splitting chromosome succeeds in catching and the spindle is formated and directed. These cellses activity prcess is depended on the microtubule, microfilament, Rho GTP enzyme and the complicated interaction between the microtubule associated proteins. Rho GTP enzyme is one of the little-molecular-weight G protein members of Ras super family. They are a group of guanosine monophosphate binding protein whose molecular weight is about 20-25kd that serve as message transmitting organs or molecular switch in the message transmitting path of cell and produce all kinds of biological effect through effecting cystoskeleton or its target protein. These proteins include Cdc42, RhoA, Rac etc. Among these Cdc42 is studied more. Microtubule associated protein includes APC(adenomatous polyposis coli), EB1(end- binding protein), CLIP-170(cytoplasmic linker protein-170) and CLASP(CLIP associated protein) etc. They bind with the positive end of microtubule, participating in regulating the function of microtubule dynamics. GSK3 (glycogen synthase kinase 3) shows constituting activation in resting cell, whose substrates are APC and CLASP. The GSK3 activity changes and its phosphorylation on some MAPs can affect the function of microtubule dynamics, then affect the life activity such as the migration and splitting of the cell further.ObjectiveIn order to study the expression and significance of GSK3β,Cdc42 and CLIP-170 in the process of injury and reparation of airway epithelia in mice to further clarify the molecule mechanism of injury and reparation of epithelia.MethodsThirty-six KM mice in good conditions were treated with bleomycin intratracheally (5mg/kg, body weight) after abdominal anesthesia, inducing its acute injury of airway epithelia. After killed in batch, the lung organizes and trachea of mice were obtained with six normal mice as control. S-P immunohistochemical method were respectively used to examine the expression and distribution of GSK3βand CLIP-170 in the airway epithelial cell. Western blot technique were used to examine the expression change of GSK3β,P-GSK3β,Cdc42 and CLIP-170 in the injury and reparation process of airway epithelial cell. By double-label immunofluorescence technique and Confocal Laser Scanning Microscopy (CLSM) we explored the colocational relationship between GSK3β,CLIP-170 andβ-tubulin respectively.Results1. Observation on the wholeThe two lungs of normal mice are bloom, the surface is smooth and glossy and the elasticity is well. The lungs of experimental mice appear different degree pathological change from dotted hemorrhage,swelling to atrophy and hard etc.2. Morphologic changesThe normal respiratory epithelia cells arranged orderly under light microscope. It was visible as pseudostratified ciliated columnar epithelium or ciliated columnar epithelium. The lungs of experimental mice was infiltrated with many inflammated cells. The epithelium cells of airway and pulmonary alveoli necrosis and shed off with cilia lodging. Subsequently the cells of inflammation obviously decrease, then epithelia is more complete, then fibroblast proliferate, and eventually lung tissue fibrosis overall.3. The expression of GSK3βand CLIP-170 by immunohistochemistry stainingThe expression of GSK3βin cytoplasm is higher in the normal control group, then decrease significantly(P<0.01) after stimulated by Bleomycin four and seven days later. However, at subsequent fourteen and tweenty-eight days the expression have a little bit trend to back up again(P<0.01), which was still lower than normal group. The expression of CLIP-170 in cytoplasm is lower in the normal control group, then increase gradually with the development of injury(P<0.01).4. The results of western blotGSK3βdecreases after injury, but return to increase in last phase of reparation, but the P-GSK3βshows the contrary change. Cdc42 goes up after injury, then it appears a descending trend gradually. Inversely CLIP-170 display a continously advancing trend.5.Results of double-label immunofluorescence technique and CLSMβ-tubulin distributes more dispersedly in the epithelia of normal mice than stimulated by BLM. In BLM 4d and 7d groupβ-tubulin which also expressed in the epithelium cells of pulmonary alveoli distributes like a line obviously along under the cell membrane and lateral margin. CLIP-170 and GSK3βdistribute in accordance with theβ-tubulin. The trend of moving to cytoplasm is existed in BLM 14d and 28d in these three protein.Conclusions1,The phosphorylation of GSK3βis Cdc42-depended.2,The phosphorylation and dephosphorylation of GSK3βcan regulate MAPs concentrating and binding with the end of microtubule, so play a vital role in the injury and reparation process of airway epithelia.3,CLIP-170, who expresses more after injury, binds withβ-tubulin and reinforces the stabilization of microtubule. It can make microtubule to grow,lengthen,fasciculate and concentrate on cellular periphery. CLIP-170 also play important role in the injury and reparation process of mice airway epithelia.