Effects Of AC2-26 At Different Concentrations On The Injury Of Rat Alveolar Type Ⅱ Epithelial Cells Induced By CPB Serum

Objective:This experiment intends to use adult CPB serum to culture rat alveolar type II epithelial cells(AEC II)to simulate clinical CPB damage to AEC II;after the cell injury model is successfully established,add different concentrations of annexin A1 peptidomimetic AC2-26 to observe whether it can Reduce the damage of AECⅡ in rats,and find the best drug concentration to alleviate the alveolar type II cells in rats,and provide a theoretical basis for exploring CPB lung protection measures and experimental research.Methods:Select 200g～300g healthy SD rats after anesthesia to take lung tissues from both lungs for isolation,purification and culture of alveolar type II epithelial cells.16 patients with rheumatic heart disease undergoing surgery were selected.After the CPB bypass was stopped,10 ml of blood was taken,the serum was separated after centrifugation,and 10% CPB serum was prepared and added to the culture plate to co-culture with alveolar type Ⅱ epithelial cells to establish alveolar type Ⅱ epithelial cell injury model.Alveolar type Ⅱ epithelial cells were cultured for 48 hours and the culture medium was aspirated,and they were divided into control group(group C),AEC Ⅱ injury group(group B),AC2-26 group(group A),and control group(group C)only as primary primary Alveolar type Ⅱ epithelial cells were cultured,AEC Ⅱ injury group(group B)was the postoperative serum and alveolar type Ⅱ epithelial cell co-culture group,group A was the CPB serum,drug and cell co-culture group,group A was randomly divided according to different drug concentrations 0.125μmol/L for group A1,0.25μmol/L for group A2,0.5μmol/L for group A3,1.0μmol/L for group A4,2.0μmol/L for group A5,4.0μmol/L for group A6,8.0μmol/L for group A7,Each group was cultured for 12 h.Select alveolar typeⅡ epithelial cells cultured for 48 hours to observe AEC Ⅱ ultrastructure lamellar bodies under electron microscope;select 24 h,48h,72 h,96h alveolar type Ⅱ epithelial cells to observe cell morphology and growth under an inverted microscope;select co-culture The12-hour alveolar type Ⅱ epithelial cells were tested for CCK-8 cell viability;flow cytometry was used to test the cell apoptosis rate;ELISA was used to test the TNF-α and IL-8 in the culture medium.Results:1.Culture and identification of alveolar type Ⅱ epithelial cells(1)Under the inverted microscope,the cells showed a cobblestone-like morphology,scattered and distributed.As time passed,the alveolar type Ⅱ epithelial cells gradually proliferated into polygonal,irregular shapes,distributed in islands,and even connected into pieces to cover the entire field of vision.Obviously,there are abundant small particles around the nucleus.(2)Microvilli on the cell surface can be seen under the electron microscope,the cell membrane is intact,the cells can be tightly connected,the nucleus is large,irregular,the chromatin is uniform,the nuclear membrane is clear,the mitochondrial structure is fusiform,the number is abundant,and the cristae The lines are clear,and the specific lamellar bodies in the cytoplasm can also be seen,varying in number,distributed in an "onion skin" shape.(3)SPC immunofluorescence staining picture shows that the alveolar type Ⅱ epithelial cell SPC shows red fluorescence,and the nucleus shows blue fluorescence staining.SPC is mainly distributed around the cytoplasm,and the fluorescence intensity varies.2.Effects of AC2-26 at different concentrations on the injury of rat alveolar type Ⅱ epithelial cells induced by CPB serum(1)The growth of each group of cells under an inverted microscope,The cells in group C are tightly arranged and adhere well to the wall.The cells are interconnected and the number of cells is abundant.The number of cells in group B was significantly reduced,and the arrangement was disorderly.Cells in groups A5,A6,and A7 are severely damaged,the normal structure of cells disappears,the arrangement is disorderly,the cells appear poorly adherent,even anoikis,the intercellular space is significantly enlarged,the connections between cells are broken,and the cells in groups A1 and A2 are sparse,The cell gap widens,the cell membrane shrinks,the cell morphology is irregular,and grows in a long strip and spindle shape.The damage of the cells in the A3 and A4 groups is significantly reduced,the cells are tightly arranged,and the cell structure is good.(2)Comparison of cell viability of each group: Compared with group C,the cell viability of group B and group A was significantly reduced(P<0.05);the cell viability of groups A1-A7 was significantly higher than that of group B(P<0.05);Compared with A1,the cell activity of A2 group,A3 group and A4 group was increased,and the difference was statistically significant(P<0.05).There was no significant difference in cell activity of A5-A7 group(P>0.05).,A2 group,A3 group,A4 group three groups of cell viability comparison,no significant difference(P>0.05).(3)Comparison of cell apoptosis rate of each group: Compared with group C,the cell apoptosis rate of group B and A1-7 increased significantly(P<0.05);compared with group B,group A1,group A5,group A6 There was no significant change in the apoptotic rate of the A7 and A7 groups(P>0.05);while the A2,A3 and A4 groups had a significant decrease in the apoptotic rate,and the difference was statistically significant(P<0.05);while A1-7Compared with each group,there was no significant difference in the apoptosis rate between the A1 group and the A6 group.The apoptosis rate of the A1 group was lower than that of the A7 group,and the apoptosis of the other groups was significantly reduced compared with the A1 group.(P<0.05),the apoptosis rate of A2 group and A4 group was not significantly different(P > 0.05);but compared with A5 group,cell damage was significantly reduced(P<0.05),A3 group apoptosis rate(17.8± 0.68)Significantly lower than the other groups(P<0.05).(4)Comparison of TNF-α content changes in each group: Compared with group C,the TNF-α content of group B and group A increased(P<0.05);each group of group A compared with group B,A1-A5 The content of TNF-α in group B was lower than that in group B(P<0.05).There was no significant change in TNF-α content in group A6-A7 compared with group B;the comparison between groups in group A showed that the TNF-α content of group A2,group A3 and group A4-α content was significantly lower than A1 and A5 groups(P<0.05),A6 and A7 groups were significantly higher than A1 group TNF-α content(P<0.05),A3 group TNF-α content was significantly lower than A2,A4 group,The difference was statistically significant(P<0.05).(5)Comparison of IL-8 content changes in each group: Compared with group C,the expression of IL-8 in group B and group A increased(P<0.05);compared with group B,group A2,group A3,group A4 The IL-8 content of group was significantly decreased(P<0.05),but there was no significant difference in IL-8 content of group A1,A5,A6,A7 and group B(P>0.05);comparison between groups in group A,Compared with the A1 group,the IL-8 content of the A2-A4 group was significantly decreased,the IL-8 content of the A5-A7 group was significantly increased,and the IL-8 content of the A2 group was increased compared with the A3 and A4 groups,the difference was statistically significant(P<0.05),and there was no significant difference in IL-8 expression between the A3 group and the A4 group(P>0.05).Conclusion:1.The rat AECⅡ was successfully cultured.2.AC2-26 at concentrations of 0.25 μmol/L,0.5 μmol/L,and 1.0 μmol/L can alleviate the damage of rat alveolar type II epithelial cells induced by CPB serum,and the concentration of 0.5 μmol/L has the best effect.3.AC2-26 at concentrations of 2.0,4.0 and 8.0 μmol/L can not reduce the damage of AECⅡ in rats...