| Objective:Obstructive sleep apnea(OSA)is a common sleep breathing disease with high morbidity,disability and mortality.It is also an independent risk factor for many cardiovascular diseases.Its main pathophysiological feature is intermittent hypoxia.This hypoxia-reoxygenation is similar to ischemia-reperfusion and can lead to oxidative stress and vascular endothelial disorders.In recent years,a large number of studies have shown that quercetin has anti-inflammatory,anti-oxidant,anti-apoptosis,scavenging oxygen free radicals and immunomodulatory activities.At present,quercetin is mainly studied in the field of tumors,but it is rarely reported in obstructive sleep apnea.This study intends to establish an OSA model through SD rats and human umbilical vein endothelial cells(HUVECs)exposed in IH,and to explore the protective effect of quercetin on OSA to provide a new direction and experimental basis for the prevention and treatment of cardiovascular complications of OSA.Methods:Section 1:1.Male SD rats(n=24)were randomly divided into normal control group(NC group),quercetin group(Que group),intermittent hypoxia group(IH group),intermittent hypoxia + quercetin group(IH+Que group),a total of 6 weeks of exposure.The aorta of each group was stained by HE staining to observe the morphological.Peripheral blood was used to detect oxidative stress indicators(SOD,MDA),and western blot was used to detect the expression levels of apoptosis-related proteins cleaved caspase-3,Bax and Bcl-2.2.HUVECs cells were pretreated with different concentrations of quercetin(12.5,25,50,100 μM)and then intermittent hypoxia for 12 h.CCK-8 was used to determine the effect of quercetin on cell viability;the growth status was observed under light microscope;the oxidative stress levels of SOD and MDA were measured with the kit;DUFH-DA method to detect the level of ROS;apoptosis were detected with flow cytometry;Western blot were used to analyze the expression of apoptosis-related proteins.Section 2:1.SD rats were divided into NC group,Que group,IH group,intermittent IH+Que group,IH+Que+CQ group.After exposure,the number of autophagosomes/autophagolysosomes in the aorta were observed by TEM;the autophagy-related proteins LC3,Beclin-1,and p62 expression level were determined by western blot;immunohistochemical method was used to analyze the expression level of LC3.2.After HUVECs treatment,the expression levels of autophagy-related proteins LC3,Beclin-1,and p62 were determined by western blot;autophagy phenomena were observed by MDC staining;autophagy-related gene expression levels were detected by q RT-PCR;the LC3 level were analyzed by immunofluorescence.After pretreatment with 3-MA,the number of autophagosomes/autophagolysosomes in the cells were detected by TEM;western blot to analyzed the expression levels of autophagy and apoptosis-related proteins;SOD and MDA were detected with the kit;DUFH-DA method to detect the level of ROS.Section 3:1.After the animal exposure,the expression levels of SIRT1 and p-AMPK/AMPK in the aorta were determined by western blot.2.Divide HUVECs into NC,IH,IH+Que,IH+Que+EX527,IH+Que+Com C,and after adding SIRT1 small interfering(si RNA)to silence SIRT1 protein,Western blot was used to detect changes in the expression levels of p-AMPK,AMPK,SIRT1 and autophagy-related proteins in each group.Results:Section 1:1.HE staining of rat aorta: the IH group showed severe damage,with disordered vascular endothelial cells,swelling,degeneration,necrosis,and even shedding.and the quercetin can effectively improve the aortic endothelial injury.2.Compared with the NC group,the MDA level were decreased,and the SOD were increased in IH group;the MDA level were increased,and the SOD were decreased in the IH+Que group than the IH group(P<0.05).3.Compared with the NC group,the protein levels of cleaved caspase-3 were increased and the Bcl-2/Bax decreased in IH group.The expression of cleaved caspase-3 protein in rat aorta of IH+Que group were decreased and the expression of Bcl2-/Bax were increased(p<0.05).4.The CCK-8 assay showed that IH significantly reduced the viability of HUVECs cells,and quercetin showed a concentration-dependent inhibition of IHinduced reduction in HUVECs cell viability.5.After IH treatment,the number of HUVECs cells gradually decreased,the intercellular intervals increased,and some cells lost their original morphology and appeared floating.After pretreatment with different concentrations of quercetin,the cells showed improved morphology compared with the IH group.6.Compared with NC group,the MDA levels were decreased in IH group,while the SOD activity were increased.After the intervention of Que,the MDA levels increased significantly and SOD levels were decreased.It was observed that the number of ROS positive cells in IH group increased significantly than NC group,but higher than in IH+Que group.7.Compared with NC group,the protein of cleaved caspase-3 expression was increased and Bcl-2/Bax ratio was decreased in the IH group.After pretreatment of cells with quercetin,the expression of cleaved caspase-3 was decreased and the expression of Bcl-2/Bax was increased in a concentration-dependent manner compared with the IH group(p<0.05).8.The apoptosis rate of the IH group was higher than NC group,and the apoptosis rate was significantly decreased after adding quercetin pretreatment.Section 2:1.In the aorta,the number of autophagosomes/autophagolysosomes were increased of the IH group compared with NC group,and was further increased in IH+Que group than the IH group.2.Compared with NC group,the protein of LC3-II/LC3-I and Beclin-1 expression in aortic was increased,while p62 was decreased in the IH group.After pretreatment with quercetin,the expression of LC3-II/LC3-I and Beclin-1 was further increased and the expression of p62 was significantly decreased compared with the IH group.3.The results of LC3 immunohistochemistry showed that the expression of LC3 in the IH+Que+CQ group was higher than the IH+Que group.And western blot showed that the LC3-II/LC3-I ratio in the IH+Que+CQ group was higher than the IH+Que group(p<0.05).4.In HUVECs cells,compared with the NC group,the protein expression levels of LC3-II/LC3-I ratio and Beclin-1 were increased,while p62 was decreased in IH group.And the protein expression levels of LC3-II/LC3-I ratio and Beclin-1 were further increased after pretreatment with different concentrations of quercetin compared with the IH group.5.MDC staining showed that green fluorescence was observed in the IH group,and the green fluorescence was significantly increased in IH+Que group compared with IH group,and there was no significant difference between the 50 μM and 100 μM concentrations.6.Autophagy genes Beclin-1,Atg5,Atg7,and Atg12 were increased after IH treatment compared with the NC group,and the autophagy genes were further increased with the addition of different concentrations of Que pretreatment.7.Immunofluorescence results indicated that green fluorescence was increased in IH group,and after adding 50μM quercetin pretreatment,the green fluorescene was further increased than IH group.8.Compared with the IH+Que group,the LC3-II/LC3-I ratio and the protein expression level of Beclin-1 were decreased and the p62 protein expression was increased in the IH+Que+3-MA group.Transmission electron microscopy observed a large number of autophagosomes /autophagic lysosomes in the IH+Que group,while the number of autophagosomes/autophagic lysosomes was decreased after the addition of the autophagy inhibitor 3-MA.Section 3:1.In the aorta,compared with NC group,the p-AMPK/AMPK expression levels were upregulated in the IH group(p < 0.05),the SIRT1 expression levels were no significant differences between NC and IH group.The expression level of pAMPK/AMPK and SIRT1 were significantly increased in the IH+Que group.2.In HUVECs cells,compared with the IH group,the p-AMPK/AMPK ratio and the expression of SIRT1 were increased after pretreatment with different concentrations of Que.3.Compared with the IH+Que group,the protein ratio of LC3-II/LC3-I and Beclin-1 protein expression level were decreased,and p62 protein expression was increased after EX527 or Com C intervention.4.After SIRT1 si RNA intervention,SIRT1 expression was suppressed,the LC3-II/LC3-I ratio and Beclin-1 protein expression level were decreased,and p62 protein expression was increased(p<0.05).Conclusion:1.IH induces oxidative stress and apoptosis in rats and HUVECs.2.Quercetin ameliorates IH-induced oxidative stress and reduces apoptosis in a concentration-dependent manner.3.IH induces autophagy in rats and HUVECs..4.Quercetin activates endothelial cell autophagy,ameliorating the response to IHinduced endothelial cell injury.5.Quercetin plays a protective role by activating autophagy through activation of AMPK-SIRT1 signaling pathway. |
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