The Study Of Inflammatory Mechanism In Endothelial Cells Injury Induced By The Interaction Of Endothelial Cells And Polymorphonuclear Leukocytes From Rats Exposed To Chronic Intermittent Hypoxia

Background and objectiveObstructive sleep apnea syndrome(OSAS) is a Common diseases, characterized by repeatedly apnea and hypopnea during sleep. It’s important pathophysiology change is chronic intermittent hypoxia(CIH) by repeatedly apnea. CIH injure the vascular endothelial causes by a series of oxidative stress and inflammation which significant increase coronary artery atherosclerosis, hypertension and other cardiovascular disease risk. Oxidative stress and inflammatory reaction bioactive substances mainly come from activated polymorphonuclear(PMN). PMN is activated by various stimulated factors including CIH. It takes up nearly 60% of the total number of circulating white blood cells and direct contacts with the vascular endothelial cells. The interaction of PMN and endothelial cells is the basic pathophysiology of many diseases. Therefore, we respectively isolate and purify the PMN from normoxia exposed rats and CIH exposed rats, then respectively co-cultured with normoxia exposed and CIH exposed rat aortic endothelial cells by direct co-cultivation and transwell co-cultivation. The aim of this experiment is to discuss the interaction and mechanism of PMN with endothelial cells during CIH by observing the TNF-α and NF-κB and provide the theory basis for preventing and treatmenting of OSAS and its complications for clinical. Content1.The comparative study of TNF-α level in supernatant when endothelial cells and polymorphonuclears exposed to different intermittent hypoxia and co-cultured.2. The comparative study of NF-κB level in endothelial cells when endothelial cells and polymorphonuclears exposed to different intermittent hypoxia and co-cultured. Method18 male Wistar rats were randomly divided into normal oxygenic control group and chronic intermittent hypoxia(CIH) group. CIH group was exposed in intermittent hypoxia environment. The oxygen concentration fluctuated within the range of 5.4%-20.7%. The frequency of hypoxia was 30 times/h and 8h/d to simulate the process of OSAS patients with intermittent hypoxia. Normal oxygenic control group’s cabin were filled compressed air with corresponding frequency and flow change. Rats were executed after 6 weeks, and blooded from abdominal aorta. Isolate and purify the PMNs by double Ficoll- Histopaque density gradient centrifugation. Purifed PMNs were suspened in RPMI- 1640 medium. Adjust the the cell concentration to 1 x 106/ml, for co-culturing with rat aortic endothelial cells.Rat aortic endothelial cells were cultured with RPMI- 1640 culture medium containing 10% fetal bovine serum. Endothelial cell intermittent hypoxia group was exposed to intermittent hypoxia environment. 300 seconds for a cycle, in 45 seconds before each cycle mixed gas was filled into hypoxia cabin wich containing 5% CO2, 1.5%O2 and 93.5% N2.The next 255 seconds was mixed gas containing 5% CO2, 21% O2 and 74% N2 to simulate the endothelial cells’ s intermittent hypoxic process in body. 48 cycles and 4 hours total. The number of intermittent hypoxia times is 12 per hour.Co-culture modes are divided into direct co-culture and transwell co-culture. Direct co-culture was directly adding the PMN suspension into cell culture holes with rat aortic endothelial cell. Transwell co-culture was adding the PMN suspension into transwell closet which place in cell culture holes befour.The ratio of the PMN and endothelial cell is about 10:1. The cell culture plate was putted into standard cell culture box after sampling for 4 hours. Extract culture supernatant after co-culture.Enzyme-linked immunosorbent assay(ELISA) was applied to test the changes of TNF-α level in co-culture supernatant. Western blotting was applied to test the NF-κB p65 concentration in endothelial cells. Grouping as follows: 1.normal oxygenic endothelial cells+isopyknic culture medium group(transwell co-culture) 2.normal oxygenic endothelial cells+isopyknic culture medium group( direct co-culture) 3.CIH endothelial cells+ isopyknic culture medium group(transwell co-culture) 4.CIH endothelial cells+ isopyknic culture medium group(direct co-culture) 5.normal oxygenic endothelial cells+ normal oxygenic PMN group(transwell co-culture) 6.normal oxygenic endothelial cells+ normal oxygenic PMN group(direct co-culture) 7.CIH endothelial cells + normal oxygenic PMN group(transwell co-culture,endothelial cells actived only) 8、CIH endothelial cells + normal oxygenic PMN group(direct co-culture,endothelial cells actived only) 9、normal oxygenic endothelial cells+CIHPMN group(transwell co-culture,PMN actived only) 10. normal oxygenic endothelial cells+CIHPMN group(direct co-culture,PMN actived only) 11.CIH endothelial cells +CIHPMN group(transwell co-culture,endothelial cells and PMN actived both) 12.CIH endothelial cells +CIHPMN group(direct co-culture,endothelial cells and PMN actived both) Result: TNF-α1. The TNF-α level in supernatant has no significant difference by means of two different co-culture modes in addition to CIH endothelial cells + CIHPMN group when compared intra-class.CIH endothelial cells + CIHPMN direct co-culture group’s TNF-α level in supernatant was higher than traswell group(P = 0.010).2.Different CIH exposed groups’ s TNF-α level in supernatant was higher than normal oxygenic endothelial cells+ normal oxygenic PMN group(all P < 0.05) when compared within direct co-culture mode.TNF-α level in CIH endothelial cells + CIHPMN group got highest.And it was higher than CIH endothelial cells + normal oxygenic PMN group and normal oxygenic endothelial cells+CIHPMN group(all P < 0.05). CIH endothelial cells + normal oxygenic PMN group express more TNF-α than normal oxygenic endothelial cells+CIHPMN group(P < 0.05).3.Different CIH exposed groups’ s TNF-α level in supernatant was higher than normal oxygenic endothelial cells+ normal oxygenic PMN group(all P < 0.05) when compared within transwell co-culture mode.TNF-α level in CIH endothelial cells + CIHPMN group got highest.And it was higher than CIH endothelial cells + normal oxygenic PMN group and normal oxygenic endothelial cells+CIHPMN group(all P < 0.05). CIH endothelial cells + normal oxygenic PMN group express more TNF-α than normal oxygenic endothelial cells+CIHPMN group(P < 0.05).4.The TNF-α level has no statistical differences when comparing normal oxygenic endothelial cells+isopyknic culture medium group and normal oxygenic endothelial cells+ normal oxygenic PMN group by both Co-culture modes, as well as comparing CIH endothelial cells+ isopyknic culture medium group and CIH endothelial cells + normal oxygenic PMN group.5.The TNF-α level has no statistical differences between transwell Co-culture mode and direct co-culture Co-culture mode in normal oxygenic endothelial cells+ isopyknic culture medium group as well as in CIH endothelial cells + isopyknic culture medium group.NF-κBp651. The NF-κBp65 level in endothelial cell has no significant difference by means of two different co-culture modes when endothelial cells co-coltued with normal oxygenic PMN(P>0.05).But when endothelial cells co-coltued with CIH PMN direct co-culture group’s NF-κBp65 level in endothelial cell is higher than traswell group(P <0.05).2.Comparison in direct co-culture mode:The levels of NF-κBp65 was no statistical significance between CIH endothelial cells + normal oxygenic PMN group and normal oxygenic endothelial cells + CIH PMN group(P>0.05). But significantly increased compared to the normal oxygenic endothelial cells + normal oxygenic PMN group. The levels of NF-κBp65 in the CIH endothelial cells + CIHPMN group was significant increased than the other three groups( P<0.05).3. Comparison in transwell co-culture mode:The levels of NF-κBp65 in turned up as normal oxygenic endothelial cells + normal oxygenic PMN group, normal oxygenic endothelial cells + CIH PMN group, CIH endothelial cells + normal oxygenic PMN group and CIH endothelial cells + CIHPMN group, and hadstatistically significant between each other(P < 0.05).Conclusion1. PMN and endothelial cells directly co-colture mode expressed more inflammatory cytokines than transwell co-colture mode. It prompted that intercellular adhesion effect plays an important role in endothelial injury by CIH.2. When both cells were experienced the CIH exposure and co-coltured directly levels of NF-κBp65、TNF-αget highest. Moreover, endothelial cells experienced the hypoxia exposure could induce more Inflammatory cytokines than PMNs experienced the hypoxia exposure.3. Not activated PMN does not aggravate the inflammatory reaction. The experiment used transwell closet will not affect the expression of inflammatory cytokines.