The Role And Mechanism Of Primary Cilia In The Anti-hypoxia Injury Of Pulmonary Microvascular Endothelial Cells

Hypoxia is an important cause of many acute and chronic diseases.In the state of hypoxia,the body is in an oxidative stress state.Furthermore,lipid peroxidation,protein oxidation,saccharification and oxidation,and DNA oxidative damage caused by reactive oxygen species(ROS)and other free radicals may cause apoptosis and necrosis,and then cause degeneration of organizational structure and function.Plateau hypoxia can cause temporary or permanent changes in multiple systems in the body,and even induce altitude sickness such as high altitude cerebral edema and high altitude pulmonary edema.The body has a natural antioxidant defense system,including superoxide dismutase(SOD),glutathione peroxidase(GPH-PX),vitamin E and ascorbic acid,which can eliminate or neutralize free radicals,used to against oxidative damage.Previous research by our group found that primary cilia play an important role in cellular anti-oxidative stress.After the primary cilia is interfered,the anti-anocix ability of cells is lost,and the relevant antioxidant signaling pathway is also interrupted,but the specific mechanism within this change is still unclear.This paper will conduct further research on this topic.In this experiment,we treated the pulmonary microvascular endothelial cells(PMVEC)that had grown to the confluence state as follows:(1)The PMVEC cells were cultured in a closed hypoxic device with an O2 concentration of 2% for 0,6,12,18,and 24 h;(2)The primary cilia of PMVEC cells were first inhibited by siRNA,and then placed in a closed hypoxic device for the same period of time.The morphology of the cells treated with hypoxia was observed under an inverted microscope.The occurrence rate and length of primary cilia were observed by immunofluorescence staining and laser confocal microscopy.The nuclear translocation of anti-oxidation signaling pathway-related factors and the location of primary cilia were studied.To acknowledge the oxidant and antioxidant index,we detect the enzyme activity of antioxidation glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)and the content of oxidation index including malondialdehyde(MDA)and reactive oxygen species(ROS).Besides,The protein and gene expression of anti-oxidation signaling pathwayrelated factors were analyzed by Western blot and RT-PCR.The results showed that after hypoxia treatment,the occurrence rate of primary cilia of PMVEC increased,the length increased,and the activity of antioxidant enzymes increased firstly and then decreased gradually.The content of oxidation products increased significantly and then decreased in the same trend.After interfering IFT88 expression,immunofluorescence staining showed that the primary cilia,antioxidant enzyme activity and oxidation product content of the transfected control group(Negative control,NC group)were consistent with the normal control group(without transfection),but in siRNA group,the activity of antioxidant enzymes was not significantly increased,and the content of oxidation products was always higher than that in the NC group.The protein expression of Nrf2,HIF-1? and HIF-2? in the anti-hypoxia signal pathway of NC group increased firstly and then decreased,and significant nuclear translocation occurred;the protein expression level of the above proteins in the siRNA group showed almost no change,also no nuclear translocation occurred.The gene expression levels of Nrf2,HIF-1? and HIF-2? in the NC group showed a significant increase and then decreased,but RNAi showed almost no change.The above experimental results showed that PMVEC cells which lost primary cilia had lower antioxidant enzyme activity than normal cells,the protein and gene expression levels of anti-oxidative stress related signaling factors decreased,and nuclear translocation disappeared,indicating that the cells cannot initiate their intrinsic anti-hypoxia injury and antioxidant stress defense system after the cells lose primary cilia.This result preliminarily proves that the primary cilia is the oxygen receptor of cells,and also provides new ideas for the development of anti-hypoxia drugs.