Research On The Protective Effects And Mechanism Of Lipoxin A4 For Skin Flap And Skeletal Muscle Ischemia Reperfusion Injury In Rats

Part Ⅰ: Research on the protective effects and mechanism of Lipoxin A4 in skin flap ischemia reperfusion injury in ratsObjective: To investigate the protective effects of Lipoxin A4(LA4)in rats with ischemia reperfusion(I/R)-injured skin flap.Methods: It is to establish rat abdominal artery skin flap ischemia reperfusion injury model by intravenous injection of LA4.After undergoing 72 h of I/R management of an abdominal artery skin flap,we calculated flap survival area by perusal surgery.A laser Doppler flowmeter was used to measure flap blood supply,and hematoxylin and eosin(H&E)staining was used to observe morphological changes.TdT-mediated dUTP-X nick end labeling(TUNEL)staining was also used to observe early apoptosis and is presented as the percentage of TUNEL-positive cells.The change of Nrf2,HO-1,Bcl2 and Bax protein content were detected by western blotting.Moreover,The MDA content and SOD activity were also measured.And the content of LC3 were determined by immunohistochemical staining and western blotting.Results: Compared to the I/R group,LA4 treated group presented a larger survival area and better blood perfusion with less inflammatory infiltration such as IL-1β,IL-6 and TNF-α and cell apoptosis,a higher expression of Bcl2 and a lower expression of Bax,and a higher SOD activity and a lower MDA content.LA4 treated group exhibited a prominent enhanced nuclear accumulation of nuclear factor erythroid 2-related factor 2(Nrf 2)and expression of heme oxygenase 1(HO-1).LA4 also promotes LC3Ⅰ converting to LC3Ⅱ,improving autophagy in I/R injured skin flap.Conclusion: LA4 can inhibit inflammatory reaction,oxidative stress,cell apoptosis,improve autophagy and protect ischemia/reperfusion(I/R)-injured skin flap.Our study shows that LA4 may have the potential as a therapeutic agent for I/R-injured skin flap.Part Ⅱ: Research on the protective effects and mechanism of Lipoxin A4 for ischemia reperfusion injured limb in ratsObjective: To investigate the protective effects and underlying mechanism of Lipoxin A4(LA4)in rats with ischemia/reperfusion(I/R)-injured lower limb.Methods: A rat model of I/R-injured skeletal muscle was obtained by subjecting rats to a 3-h ligation of the right femoral artery followed by 3 h of reperfusion.We observed the edema of lower limb muscle and neutrophil infiltration.HE staining was used to observe the injury of muscle tissue,and ELISA was used to detect the levels of inflammatory factors TNF-α,IL-Iβ and IL-6 in muscle tissue.At meanwhile,the activities of SOD,CAT and GSH-Px were detected,and the percentage of muscle cells’ apoptosis were observed using TUNEL staining.The changes of expression Nrf2,HO-1,LC3,Beclin1,Bax and Bcl2 were detected by Western blotting.The number of autophagosome was observed by transmission electron microscope.Results: Treatment with LA4 significantly ameliorated histological damage scores and neutrophil infiltration in I/R-injured skeletal muscle.LA4 treatment resulted in remarkable decreases in the wet weight/dry weight ratio(W/D ratio),inflammatory response,oxidative stress,and cell apoptosis.In addition,treatment with LA4 was accompanied by a prominently enhanced nuclear accumulation and expression of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in the I/R-injured skeletal muscle.However,these protective effects were reversed by zinc protoporphyrin-IX(ZnPP),a specific HO-1 inhibitor.LA4 can promote the formation of autophagy in muscle tissue of ischemia reperfusion injury,promote the transformation of LC3 II to LC3 I,and increase the expression of Beclin1.Conclusion: LA4 can inhibit inflammatory reaction,oxidative stress,cell apoptosis and protect ischemia/reperfusion(I/R)-injured skeletal muscle by improving autophagy.Our study shows that LA4 may have the potential as a therapeutic agent for I/R-injured muscle tissue via activation of the Nrf2/HO-1 signaling pathway.Part Ⅲ: Research on the protective effects of LA4 against oxidative stress-injured skeletal muscle cells.Objective: To observe the protective effects of LA4 with oxidative stress-injured skeletal muscle cells.Methods: We use different concentrations of H2O2(0,25,50,100,200,400 umol/L)to stimulate rat myoblasts(L6 cells)to simulate oxidative stress-injured skeletal muscle cells.MTT assay was used to observe the changes of cell activity,the level of ROS was evaluate by detecting the level of oxidative stress.Morphological changes of cells under oxidative stress injury were observed by light microscopy,and CK and LDH concentrations in culture medium were measured to ascertain the severity of cell injury.The content of GSH in cells were measured to evaluate the cell’s ability of anti-oxidative stress.TUNEL staining was used to observe the apoptosis ratio of skeletal muscle cells,and the change of apoptosis related protein Bcl2 and Bax content were detected by Western Blotting.Results: The optimal concentration of H2O2 oxidative stress injury of skeletal muscle cells was 200 μmol/L,and the optimal effect time was 4 hours,and the cell activity was decreased by about 55% to negative controls.LA4(10 nmol/L)significantly increased the activity of H2O2-injured skeletal muscle cells.LA4 can also inhibit the generation of ROS induced by H2O2,inhibit the release of CK and LDH into the culture medium,and promote the consumption of GSH in the cell.LA4 can promote the expression of Bcl2,inhibit the expression of Bax and decrease the percentage of apoptosis of L6 cells induced by H2O2.Conclusion: LA4 has a potential protective effect on oxidative stress-injured skeletal muscle cells in vitro,which were protectived by increasing the cell activity,inhibiting the production of ROS and increasing the antioxidant stress ability of cells.Part Ⅳ: Research on protective mechanism of Lipoxin A4 in oxidative stress-injured skeletal muscle cellsObjective: To study the specific protective mechanism of LA4 in the oxidative stress-injured skeletal muscle cells in vitro.Methods: We detected Nrf2/HO-1,ERK,P38,JNK and PI3K/AKT signaling pathway and studied the level of autophagy.Results: In the oxidative stress injury model of skeletal muscle cells in vitro,LA4 could activate the Nrf2/HO-1 signaling pathway,promote the transfer of Nrf2 into the nucleus,increase the expression of HO-1 protein.LA4 can increase the level of autophagy in skeletal muscle cells under oxidative stress.LA4 activates the Nrf2/HO-1 signaling pathway by activating the ERK pathway,and LA4 has no significant effect on P38,JNK and PI3K/AKT signaling pathways.LA4 increased the level of autophagy in skeletal muscle by oxidative stress injury by activating ERK/Nrf2 signaling pathway,and decreases the level of autophagy after the ERK/Nrf2 signaling pathway was inhibited.Conclusion: Our study showed that LA4 may have the potential as a therapeutic agent for oxidative stress-injured muscle tissue via activation of the ERK/Nrf2 signaling pathway and improved level of autophagy.