| Part I Exenatide protects against hypoxia/reoxygenation-inducedinjury in H9c2cellsObjective: To investigate the protective effects of Glucagon-likepeptide-1(GLP-1) analogue, exenatide on H9c2cells in vitro model ofhypoxia/reoxygenation (H/R).Methods: H9c2cells were employed to establish an in vitro model ofH/R. The expression of GLP-1receptor (GLP-1R) was observed by laserscanning confocal microscope and western blot. After exenatidepretreatment, cell viability was assessed with the cell counting kit-8; therelease of lactate dehydrogenase (LDH) in the cultured supernatants andcaspase-3activity were determined by colorimetry; the release of creatinekinase-MB (CK-MB) in the cultured supernatants was determined by ELISA;the cell apoptosis ratio was measured by flow cytometry; the levels ofcleaved-caspase-3and cytochrome-c were determined by western blot.Results:①GLP-1R was expressed on H9c2cell membrane.② Comparing with Control group, H9c2cell viability in H/R group wassignificantly decreased (P<0.05); LDH and CK-MB levels, cell apoptosisratio, Capase-3activity, cleaved-caspase-3and cytochrome-c levels in H/Rgroup were significantly increased (P<0.05).③Compared with H/R group,H9c2cell viability in Exenatide+H/R group was significantly increased(P<0.05); LDH and CK-MB levels, cell apoptosis ratio, Capase-3activity,cleaved-caspase-3and cytochrome-c levels in Exenatide+H/R group weresignificantly decreased (P<0.05).Conclusion: Exenatide showed effectively protective effects on H9c2cells from H/R injury. Part II The protective effects of exenatide on mitochondrialfunction in H9c2cells subjected to H/RObjective: To investigate the protective effects of exenatide onmitochondrial function in H9c2cells subjected to H/R.Methods: H9c2cells were employed to establish an in vitro model ofH/R. After exenatide pretreatment, the intracellular reactive oxygen species(ROS) and reactive nitrogen species (RNS) levels, the opening ofmitochondrial permeability transition pore (mPTP), the mitochondrialcalcium (Ca2+m) and mitochondrial membrane potential (ΔΨm) weredetermined by flow cytometry in combination with laser confocalmicroscopy. The mitochondrial morphology was examined by transmissionelectron microscope. The malondialdehyde (MDA), the total superoxidedismutase (T-SOD) and ATPase activity were determined by colorimetry.Cellular ATP contents were measured using the ATP bioluminescent assaykit.Results:①Comparing with Control group, H/R injury resulted inswollen mitochondria which displayed spherical structures with disarrayedcristae, disorganized matrix, and more cytosolic vacuoles. The levels of ROS,RNS, Ca2+m and MDA in H/R group were significantly increased (P<0.05);T-SOD, ATP and ATPase activity in H/R group were significantly decreased (P<0.05).②Compared with H/R group, Exenatide treatment attenuatedmitochondrial swell, cristae disarray and membrane rupture in H9c2cellsafter H/R. The levels of ROS, RNS, Ca2+m and MDA in Exenatide+H/Rgroup were significantly decreased (P<0.05); T-SOD, ATP and ATPaseactivity in Exenatide+H/R group were significantly increased (P<0.05).Conclusion: Exenatide showed effectively protective effects onmitochondrial function in H9c2cells from H/R injury. Part III Exenatide protects mitochondrial function in H9c2cellsfrom H/R injury via the GLP-1R/cAMP/PKA PathwayObjective: To investigate the mechanism of exenatide onmitochondrial protection in H9c2cells subjected to H/R.Methods: H9c2cells were employed to establish an in vitro model ofH/R. After indicated pretreatment, the intracellular ROS and RNS levels, theopening of mPTP, the Ca2+m and ΔΨm were determined by flow cytometryin combination with laser confocal microscopy. The ATPase activity wasdetermined by colorimetry. Cellular ATP content was measured using theATP bioluminescent assay kit. Protein kinase A (PKA), uncouplingprotein-3(UCP-3) and nuclear respiratory factor-1(NRF-1) weredetermined by western blot.Results:①Comparing with Exenatide+H/R group, the levels of ROS,RNS and Ca2+m in Exendin-(9-39), Rp-cAMPS and H-89groups weresignificantly increased (P<0.05); ATP contents and ATPase activities inExendin-(9-39), Rp-cAMPS and H-89groups were significantly decreased(P<0.05).②Comparing with Control group, the levels of PKA, UCP-3andNRF-1in H/R group were significantly decreased (P<0.05); Comparingwith H/R group, the levels of PKA, UCP-3and NRF-1in Exenatide+H/Rgroup were significantly increased (P<0.05).③Comparing with Exenatide+H/R group, the levels of PKA, UCP-3and NRF-1inExendin-(9-39), Rp-cAMPS and H-89groups were significantly decreased(P<0.05).④the above results in Forskolin group were in line with those inExenatide+H/R group (P>0.05).Conclusion: Exenatide protects mitochondrial function in H9c2cellsfrom H/R injury via the GLP-1R/cAMP/PKA pathway. |
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