Study Of Role And Mechanism Of Hedgehog Signaling Pathway In Rat Acute Lung Injury Induced By Sea Water Drowning

Backgroud and Objective:sea water drowning is not only an important cause of death in the accident of civil sailing, wrecking on the sea, but also the key factor of army non-military depletion of numbers in training or fighting on the sea. As the consequence or result of sea water drowning, lung injury, secondary pulmonary edema or sea water respiratory distress syndrome(SW-RDS) occurs in most of patients, and leads to the challenge of treatment. Alveolus capillary barrier injure plays a key role in the process of severe pulmonary edema. The proliferation and repair of Pulmonary microvascular endothelial cells can improve the permeability of the barrier and alleviate the degree of pulmonary edema. Therefore, the understanding of signaling mechanism involved in PMVEC reparation after sea water drowning injure will provide new treatment strategies.The Hedgehog signaling pathway plays a fundamental role in the development of various embryonic tissues development. It is composed of Hh protein,the transmembrane receptor protein patched (ptc) and smoothend(smo) and the transcription factor Gli. Hh is a protein secreted by cells. There are three kinds of it: Sonic Hedgehog(Shh),Indian Hedgehog (Ihh) and Desert Hedgehog(Dhh).Recent investigations found that main component molecules of Hh pathway are present in many mature tissue and regulat cell proliferation and renewal in the process of repairation after injure. The activation of Hh signaling pathway can increase PMVEC proliferation and induce capillary remodeling. Thus we hypothesize that activation of Hh pathway can improve the PMVEC proliferation (survival) and reparation in the process of sea water drowning induced lung injure. Therefore we investigate the effect of Hh signaling pathway on proliferation and apoptosis of PMVEC, and the improvements of pulmonary microvascular permeability and pulmonary edema in rat sea water drowning model. Methods:1. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripheral lung tissue pieces used for cell culture were examined. Used rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as control, CD34, lectin from Bandeiraea simplicifolia and factorⅧrelated antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morphology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively.2. PMVEC were divided randomly five groups:normal control, sea water treatment 2h,sea water treatment 4h,sea water treatment 8h and sea water treatment 16h. The expression of Shh and Ptc-1 mRNA was detected by RT-PCR and the protein expression by Western blot . The changes of cell morpha and ultrastructure were investigated with microscope or electron microscope.3. PMVEC were divided randomly sis groups: normal control, Shh 50 ng/ml treatement group, Shh 100 ng/ml treatement group, Shh 400 ng/ml treatement group,and cyclopamine treatment group.MTT was used to detect the proliferational activity of PMVECs.4. PMVEC were divided randomly seven groups: normal control, sea water treatment group,Shh 50 ng/ml+sea water treatment group,Shh 100 ng/ml+sea water treatment group,Shh 200 ng/ml+sea water treatment group,Shh 400 ng/ml+sea water treatment group,and cyclopamine +sea water treatment group. MTT was used to detect the proliferational activity of PMVEC.TUNNL was used to examinate the apoptosis of PMVECs.5. A rat model of SW-ALI was established by inhalating 4ml/Kg sea water. Sixty SD rats were randomly divided to two groups: normal control and sea water treatment group. At 1h,2h,4h and 8h behind inhalating sea water, PaO2,the wet/dry ratio of lung tissue,the pulmonary microvascular permeability,the MPO activity and level of MDA in lung tissue,the level of TNF-αand IL-1βin serum and BALF were examined.The expression of Shh,Ptc-1 and Gli1 was detected. The histological changes of lung tissue were observed under optical microscope.6. Twenty SD rats were divided randomly four groups: control group,Shh treatment group,sea water treatment group,Shh+sea water treatment group. Rats in Shh and Shh+sea water treatment groups was jugular injection of recombinant human Shh at the dose of 6mg/kg. The rat model of SW-ALI was established ditto.PaO2,the wet/dry ratio of lung tissue,the pulmonary microvascular permeability,the expression of Ptc-1 and Gli1 were compared between the control group,Shh treatment group,sea water treatment group,Shh+sea water treatment group.Results:1. Histological sections showed that tissue pieces were scissored from periphery lung lobes accurately.Ⅷfactor related antigen and CD34 expression detected by immunofluorescence was positive,and BSI binding test was positive,too.Transmission electron microscope showed lots of protuberance on cell membrane and pinosomes in cytoplasm.2. Compared with normal control group,the expression of Shh,Ptc-1and Gli1 in mRNA and proterin level in PMVEC decreased significantly in sea water treatment groups. Microscope showed PMVEC with sea water treatment crenulated, the distance between cells shortened. Transmission electron microscope showed protuberance on cell membrane increased, mitochondrion collapsed and crista missed, pinosomes in cytoplasm and heterochromatin in nucleus increased.3. Shh increased significantly the proliferation of normal rat PMVEC.the effect of Shh shows a dose-dependent model.Cyclopamine can remarkably inhibit the proliferation of Shh100 group.4. Compared with normal control group,the all group of sea water treatment has a lower proliferation of PMVEC,but the groups of Shh treatment has a higher proliferation than sea water treatment group.After sea water treatment, apoptosis of PMVEC dereased significantly compared with normal control group,but apoptosis in Shh treatment groups were lower than sea water treatment groups, apoptosis in Cyclopamine treatment group were higher than Shh treatment groups5. Compared with normal control group, PaO2 decreased and W/D ratio, pulmonary microvascular permeability, MPO activity and the level of MDA, the level of TNF-αand IL-1βin sea water groups increased;the expression of Shh,Ptc-1and Gli1 in mRNA and proterin level in lung tissue of decreased significantly in sea water groups. 6. Compared with sea water group, PaO2 increased significantly and W/D ratio and pulmonary microvascular permeability decreased significantly in Shh+sea water treatment group. The expression of Ptc-1and Gli1 in mRNA and proterin level in the lung tissue of Shh+sea water treatment group was higher than sea water treatment group.Conclusion:1. The proliferation of rat PMVEC was inhibited and the apoptosis of rat PMVEC was promoted by the stimulation of sea water.2. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of normal rat PMVEC and inhibit the apoptosis of normal rat PMVEC.3. The expression of Hedgehog signalling decreased in PMVEC treated by sea water. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of PMVEC and inhibit the apoptosis of t PMVEC treated by sea water.4. Rat model of SW-ALI was successfully established. The pulmonary microvascular permeability gradually increased and the pulmonary edema aggravated with the increase of time. The expression of Hedgehog signalling decreased in the lung tissue of SW-ALI rat model. The pulmonary microvascular permeability and pulmonary edema can be improved by recombinant human Shh in rat model of SW-ALI. This effect of Hedgehog signaling pathway may be mediated by the protective role on PMVEC.