Study Of Mechanism Of Luteolin Applied To Acute Lung Injury Induced By Extracorporeal Circulation In Mice

Background:Approximately 25% patients underwent extracorporeal circulation(ECC)reported have significant respiratory damage within one week after surgery.If these patients cannot be treated in time,Acute lung injury(ALI)will proceed which seriously affecting prognosis.Finding suitable treatments for ALI induced by ECC is particularly important for improving the prognosis of patients after cardiac surgery.During ECC,ischemia-reperfusion,contacts of blood and tube can promote the production of pro-inflammatory factors and endotoxins which can activate the complement system.Under the circumstance,the macrophages are proved to be activated,and the activated macrophages will produce more pro-inflammatory factors,such as TNF-α and IL-6,which can aggravate the inflammatory waterfall effects and promote the activation,migration,adhesion,and accumulation of neutrophils(NE).They will also stimulate macrophages to continue secreting pro-inflammatory factors,repeatedly aggravating the process above.Activation of macrophages is definitely important in inflammations.Some reports have shown that the Activation of macrophages is related to the reorganization of Actin Filaments(F-actin).The previous research of our team has found that,Factin reorganization induced the activation of macrophages,thereby promoting the release of inflammatory cytokines,suggesting that F-actin reorganization of macrophages may have played an important role during ECC-induced ALI.Therefore,inhibition of F-actin reorganization in macrophages may be used as a new therapeutic target to alleviate ECC-induced ALI.The F-actin reorganization is regulated by Rho small G protein which has a prominent effect in regulating cytoskeleton changes.After Rho protein phosphorylates downstream Rho-associated kinases(ROCK),the activated ROCK will further phosphorylate and activate downstream LIM kinases(LIMK).The main function of phosphorylated LIMK is to phosphorylate cofilin to inactivate cofilin.Inactivated cofilin cannot cut down F-actin chains,so that F-actin reorganization occurs and cytoskeleton changes.In recent years,finding high-efficiency,low-toxicity and cheap antiinflammatory drugs has always been one of the concerns of researchers.The flavonoid compound Luteolin(Lut)is highly effective in antioxidant,anti-inflammatory,antiviral,and anti-tumor which is favored by researchers.A large number of studies have proved that Lut has a good anti-inflammatory effect in different inflammation models,but in the ALI model induced by ECC,it is unclear whether Lut has the same therapeutic effect,and its therapeutic mechanism and target are still inconclusive.If Lut has a therapeutic effect,does it inhibit macrophage activation by inhibiting Factin recombination of macrophages? And is the anti-inflammatory mechanism related to the Rho/ROCK pathway? Our research will explore these problems.Materials and Methods:Chapter 1: We randomly divided 24 Kunming mice into 4 groups including Base line(BL)group,Luteolin(Lut)group,ECC group and ECC+Lut group.Each group contains 6 mice.When experiments ended,mice were sacrificed to collect samples of blood,bronchoalveolar lavage fluid(BALF)and lung tissues.The experimental pathology(plasma,BALF,lung tissue inflammatory factor levels,flow cytometry of neutrophils and macrophages)and anatomic pathological indicators(pulmonary tissue pathological injury score,immunohistochemical analysis)were measured to explore SIRS and ALI degree and mechanism after ECC in mice.Chapter 2: We selected RAW264.7 cells as experiment objects for this part and we also used lipopolysaccharide(LPS)to seduce inflammations in cells to build cell inflammation model.We set up 4 groups of BL group,Lut group,LPS group and LPS+Lut group.When experiments ended,TNF-α、IL-6 released in cell supernatant were detected by ELISA;RNA levels of TNF-α、IL-6 in cells were detected by q PCR;western blot analysis were used to examine TAK1,IKKβ,p65 contents of NF-κB pathway in cells.Immunofluorescence technique was used to observe F-actin morphologic changes in different groups.Chapter 2:(1)Step 1: We planted RAW264.7 cells into 6 cell plates and divided into 6groups,BL group,Lut group,LPS group,LPS+Lut group,LPS+Y-27632 group,LPS+Lut+Y-27632 group.Y-27632 is a kind of ROCK inhibitors.When experiments ended,TNF-α、IL-6 released in cell supernatant were detected by ELISA;RNA levels of TNF-α、IL-6 in cells were detected by q PCR;western blot analysis were used to examine Rho,ROCK1,p-LIMK,p-cofilin and cofilin contents of Rho/ROCK pathway in cells.Immunofluorescence technique was used to observe F-actin morphologic changes in different groups.(2)Step 2: We successfully made up ROCK1 overexpression plasmid and transfected ROCK plasmid into RAW264.7 cells to explore influences of overexpression of ROCK.To make sure the successful rate and transfection efficiency,we set up 3 groups including Control group,Empty-load group and Plasmid group.We used q PCR technique to detect m RNA levels of Rho,ROCK,Cofilin in Rho/ROCK pathway to make sure whether ROCK plasmid was successfully transfected.(3)Step 3: We set up 3 groups to identify the accurate target of Lut including Lut+LPS+pc DNA3.1 group,Lut+LPS+pc ROCK1 group,Lut+LPS+pc ROCK1+CB group.CB,abbreviation of cytochalasin B,is regarded as a specific inhibitor of Factin.When experiments ended,TNF-α、IL-6 released in cell supernatant were detected by ELISA;RNA levels of TNF-α、IL-6 in cells were detected by q PCR;western blot analysis were used to examine Rho,ROCK1,p-LIMK,p-cofilin and cofilin contents of Rho/ROCK pathway in cells.Immunofluorescence technique was used to observe F-actin morphologic changes in different groups.Results:Chapter 1(1)Continuous arterial blood pressure and heart rate monitor: It has a limited influence on the MAP and HR of mice during the procedure of ECC,but the influence gradually disappears over time.(2)Inflammation factors level in blood and lung: Administration of Lut alone did not affect the levels of inflammatory factors either in the blood or in lung tissues;The levels of TNF-α and IL-6 in peripheral blood and lung tissues in mice undergoing ECC are significantly higher than those did not undergo ECC,however,upon administrating Lut,levels of TNF-α and IL-6 both in blood and lung are significantly reduced.(3)Pulmonary tissue pathological injury score: Pulmonary tissue pathological injury score of ECC group is much higher than BL and Lut group.When Lut was applied to mice before ECC,the score has dropped clearly.It revealed that ECC could induce ALI in mice,and undergoing treatment of Lut,ALI of mice could be significantly attenuated.(4)Flow cytometry: Administration of Lut alone could not activate and accelerate neutrophils and macrophages in both blood and BALF.When mice underwent ECC,the proportions of activated neutrophils and macrophages in both blood and BALF were much larger than BL and Lut group,while once upon Lut was administrated in mice,the situation referred above was reversed.(5)Immunohistochemical cell staining: The amounts of activated neutrophils and macrophages in BL and Lut groups were much less than ECC group,when Lut was administrated in mice before ECC,the amounts of activated neutrophils and macrophages were apparently reduced.Chapter 2(1)MTT examination: It has no toxic effects on RAW264.7 cells when Lut drug concentration was set to 10μmol/L.To guarantee enough RAW264.7 cell viability and make sure LPS can induce inflammation response,the best drug concentration of LPS was set to 10ng/ml.(2)Inflammation factors in cell supernatant: Administration of Lut alone did not affect the levels of inflammatory factors in cell supernatant.When LPS was added up to cell plates,the levels of IL-6 and TNF-α were apparently raised,while Lut was administrated in cell before LPS adding up,the levels of IL-6 and TNF-α reduced as a result.(3)q PCR detection: Administration of Lut alone did not affect the IL-6 and TNF-α contents of m RNA in cells.When LPS was added up to cell plates,contents of m RNA of IL-6 and TNF-α in cell were significantly raised,while Lut was administrated in cell before LPS adding up,the contents of IL-6 and TNF-α reduced again.(4)Western blot: There were no differences of every protein contents between BL and Lut group.After administrating LPS,the contents of TAK1,IKKβ and p65 were significantly lower than BL and Lut group.When Lut was applied firstly to cells,the contents of TAK1,IKKβ and p65 were reversed comparing to LPS group.(5)Immunofluorescence staining of F-actin: Most cells in BL group are round and the F-actin were distributed around the nucleus evenly.The cell morphology of the Lut group is similar to BL group.In LPS group,the cell morphologies changed and the distributions of F-actin became chaotic,and most cells generated lamellipodia,leading-edges,and so on.After pretreatment of Lut,most cells in Lut+LPS group did not be affected by LPS and progressed to irregular shapes.Compared with the LPS group,the production of lamellipodia and leading-edges were significantly reduced and the distribution of F-actin in cells was also more even.Using Image J software to calculate the Mean Fluorescence Intensity(MFI)of each cell slices.There were no differences of MFI between BL and Lut group.In LPS group,MFI aroused than BL and Lut group,while Lut was applied firstly to cells,MFI of F-actin was lower than LPS group.Chapter 3Step 1:(1)Analysis of m RNA of proteins in Rho/ROCK pathway: There were no differences in m RNA contents of Rho and cofilin proteins between every groups.In LPS group,m RNA contents of ROCK in cells were increased,and upon Lut or Y-27632 was applied,expression of ROCK m RNA was significantly suppressed.(2)Western blot of proteins in Rho/ROCK pathway: There were no differences in contents of Rho and cofilin proteins between every groups.In LPS group,contents of ROCK,p-LIMK,p-cofilin in cells were increased,and upon Lut or Y-27632 was applied,expression of ROCK,p-LIMK,p-cofilin were significantly suppressed.(3)Immunofluorescence staining of F-actin: Pretreatment of Y-27632,cell morphologies remained to be stable,even within stimulation of LPS,the production of lamellipodia and leading-edges were significantly reduced.Co-treatment of Lut and Y-27632 showed no different capacities in attenuating F-actin reorganization.MFI detection revealed that pretreatment of Y-27632 or co-treatment of Lut and Y-27632 both had lower MFI than LPS group.Step 2:Analysis of m RNA contents in 3 groups showed that in Plasmid group,m RNA contents of ROCK in cells significantly more than cells in the other two groups.Step 3:(1)Analysis of m RNA of proteins in Rho/ROCK pathway: There were no differences in m RNA contents of Rho and cofilin proteins between every groups.In Lut+LPS+pc ROCK1 group and Lut+LPS+pc ROCK1+CB group,m RNA contents of ROCK in cells were increased apparently.(2)Western blot of proteins in Rho/ROCK pathway: There were no differences in contents of Rho and cofilin proteins between every groups.Both in the Lut+LPS+pc ROCK1 and Lut+LPS+pc ROCK1+CB group,contents of ROCK,pLIMK,p-cofilin in cells were clearly increased.(3)Immunofluorescence staining of F-actin: In Lut+LPS+pc DNA3.1 group,cells are round and the F-actin were distributed around the nucleus evenly.While in Lut+LPS+pc ROCK1 group,most cell morphologies changed and the distributions of F-actin became chaotic,and most cells generated lamellipodia,leading-edges.In Lut+LPS+pc ROCK1+CB group,when CB was applied in cells,F-actin reorganization was suppressed and cell cytoskeleton remained stable,F-actin distributed evenly around the nucleus.MFI detection revealed that in Lut+LPS+pc DNA3.1 group,MFI of F-actin showed no differences compared to Lut+LPS group.When pc ROCK1 plasmid was transfected into cells,MFI of F-actin roused up again.At last,when CB was administrated into cell plates,MFI of cells in Lut+LPS+pc ROCK1+CB reduced as a result.Conclusions:1.The ECC model of mice can successfully induce SIRS and ALI.During the process,the activation of macrophages plays an important role.The amount of activated macrophages both in blood and lung tissues after ECC obviously increased.After applying Lut,the symptoms of SIRS and ALI were alleviated after ECC.It is likely to be achieved by inhibiting the activation of macrophages in both circulation and organs.The number of activated macrophages in the blood and lung is significantly reduced.2.When RAW264.7 macrophages were stimulated by LPS,the macrophages were activated and F-actin was reorganized.As a result,the cytoskeleton changed and the canonical NF-κB signaling pathway was activated.After pretreating Lut to the cells,the F-actin reorganization of macrophages was inhibited.At the same time,the activity of the NF-κB pathway was down-regulated,finally,the inflammatory response of macrophages was relieved.3.The mechanism by which Lut inhibits F-actin reorganization is related to the Rho/ROCK pathway,and the specific target may be the ROCK protein.Luteolin inhibits the activation of ROCK protein and inhibits phosphorylation of LIMK and cofilin,finally inhibiting F-actin reorganization.