| Objective The oxidative stress model of rat exhaustive exercise and the rat thoracic aortic endothelial cell?RTAEC?oxidative stress model were established to explore the induction of excessive oxidative stress by Lycium Barbarum Polysaccharide?LBP?protective effect of cardiovascular endothelial cell damage and its molecular mechanism.Methods In vivo experimental studies 60 male Sprague Dawley?SD?rats were selected and randomly divided into:Normal control group,Aerobic exercise group,Exhaustive exercise group,Exhaustive exercise+LBP(200mg·?kg·d?-1)group.Except for the control group,the other three groups were trained for 35 days?1h per day?.The exhaustive exercise group and the exhaustive exercise+LBP group were trained to exhaustion at the last swim.Rat weight was measured before each swim.Angiotensin II?Ang II?and oxidative stress indicators Glutathione?GSH?,Malondialdehyde?MDA?,Total Antioxidation Capacity?T-AOC?,Peroxidation Catalase?CAT?content changes.The morphology and structure of thoracic aorta vessels and myocardial tissues were observed by HE.TUNEL staining was used to detect apoptosis of rat thoracic aorta vascular cells.IF detected Bcl-2-associated x protein?Bax?,B-cell lymphoma-2?Bcl-2?,Cysteineas partate 3?Caspase3?expression level.Western Blot detection of thoracic aorta vessels and myocardial Tumor Necrosis Factor??TNF-??,Kelch like ECH associated protein1?Keap1?,and Nuclear factor erythroid-2 related factor 2?Nrf2?,Phosphorylated nuclear factor erythroid-2 related factor 2?P-Nrf2?,Glutamate-Cysteine Ligase,Catalytic subunit?GCLC?,Recombinant NADH Dehydrogenase,Quinone 1?NQO1?,Glutamate-Cysteine Ligase,Modifier Subunit?GCLM?Protein expression level.IHC was used to detect the expression levels of TNF-?,Keap1,Nrf2,GCLC,NQO1,and GCLM in the thoracic aorta and myocardium of rats.In vitro experimental research uses Ang?to establish RTAEC oxidative stress model,which is divided into:Blank control group,LBP?3200?g/ml?group,Ang?(10-4mol/l)group,Ang?+LBP group.The content of GSH,MDA,T-AOC and CAT was detected by ELISA.Western Blot was used to detect the expression levels of TNF-?,Keap1,Nrf2,P-Nrf2,GCLC,NQO1,and GCLM.IF detected the expression levels of Nrf2 and GCLM.RT-qPCR to detect mRNA expression of TNF-?,Interleukin-6?IL-6?,Interleukin-1??IL-1??,Keap1,Nrf2,GCLC,NQO1,and GCLM Level.In addition,in vitro experiments were performed using small interfering RNA?SiRNA?50nmol??to transfect RTAEC and gene silencing of Nrf2.The experimental protocol was:1.Detection of transfection efficiency group:Blank control group,LiPofectamine 2000?LP2000??-?group,LP2000?+?group.Observe the efficiency of transfection of endothelial cells with the help of LP2000 under the help of LP2000.2.Screen the best SiRNA group:Blank control group,SiNC group,SiRNA3 group,SiRNA4 group,SiRNA5 group.Western Blot was used to detect the protein expression levels of Nrf2 and P-Nrf2.RT-qPCR was used to detect the protein expression level of Nrf2.3.Silent Nrf2 gene grouping:Blank control group,Ang?group,Ang?+LBP group,SiNC group,SiNC+Ang?+LBP group,SiRNA group,SiRNA+Ang?+LBP group.Western Blot was used to detect the protein expression levels of Nrf2 and P-Nrf2 in Ang?+LBP group and SiRNA+Ang?+LBP group.The content of GSH,MDA,T-AOC and CAT was detected by ELISA.RT-qPCR was used to detect the mRNA expression levels of TNF-?,IL-6 and IL-1?.Flow cytometry was used to detect the number of TNF-?positive cells.Results1.There was no significant change in the weight of the rats in each group before the training.From the third week,the weight of the rats in the Exhausted exercise group was significantly behind that of the Exhausted exercise+LBP group,but there was no significant difference between the two groups?P>0.05?.2.ELISA results showed that:Compared with the Exhausted exercise group,the levels of serum Ang?and MDA in the Exhausted exercise+LBP group decreased?P<0.05?,the expression of CAT increased?P<0.01?,and the expression of T-AOC increased.?P<0.001?,GSH expression level increased.Compared with the Ang?group,the CAT expression level in the supernatant of Ang?+LBP group increased?P<0.05?,the MDA expression level decreased?P<0.05?,the T-AOC expression level increased?P<0.05?,and the GSH expression level increased High?P<0.05?.Compared with Ang?+LBP group,the expression levels of CAT,T-AOC and GSH in the supernatant of SiRNA+Ang?+LBP group decreased?P<0.01?,and the expression of MDA increased?P<0.05?.3.The HE results showed that the wall of the thoracic aorta in the Normal control group was intact.There was no change in vascular structure in the Aerobic exercise group.Endothelial cells in the vascular wall of the Exhausted exercise group were obviously lacking,and the elastic fibers were disordered and broken.Vascular endothelial cells were more complete and elastic fibers were more regular in the Exhaustive exercise+LBP group.The myocardium of Normal control rats was arranged in a reticular pattern.There was a small amount of inflammatory cell infiltration between the myocardium in the Aerobic exercise group.Myocardial fibers in the Exhausted exercise group were disordered,and a large number of inflammatory cells infiltrated.Myocardial fibers swelled and a small amount of inflammatory cells infiltrated in the Exhausted exercise+LBP group.4.IF results showed that compared with the Exhausted exercise group,the positive expression of Bax and Caspase3 in myocardium of the Exhausted exercise+LBP group decreased,while the positive expression of Bcl-2 increased.Compared with the Ang?group,the positive expression of Nrf2 in the cytoplasm and nucleus of the Ang?+LBP group increased,and the positive expression of GCLM in the cytoplasm increased.It was observed under fluorescence microscope that the efficiency of SiRNA transfection of endothelial cells was more than 80%.5.TUNEL staining results showed that compared with the Exhaustive exercise group,the positive expression of thoracic aorta in the Exhaustive exercise+LBP group was significantly reduced.6.Western Blot results showed that compared with the Exhausted exercise group,the expressions of TNF-?and Keap1 protein in the thoracic aorta of the Exhausted exercise+LBP group decreased?P<0.05,P<0.01?,Nrf2,P-Nrf2 The expressions of GCLC,GCLC,NQO1and GCLM protein were increased?P<0.05?.Compared with the Exhausted exercise group,the expressions of TNF-?and Keap1 in the myocardial tissue of the Exhausted exercise+LBP group decreased?P<0.05?,the expression of Nrf2 increased?P<0.01?,and the expression of P-Nrf2 and GCLM increased.GCLC,NQO1 protein expression increased?P<0.05?.Compared with the Ang?group,the expression of TNF-?and Keap1 protein in the Ang?+LBP group decreased?P<0.05?,the expression of Nrf2,NQO1 protein increased,and the expression of P-Nrf2,GCLC,GCLM protein increased?P<0.05?.Compared with the Blank control group,the expressions of Nrf2 and P-Nrf2 in the SiRNA5 group were more significantly reduced?P<0.05,P<0.01?.Compared with the Ang?+LBP group,the expression of Nrf2 protein in the SiRNA+Ang?+LBP group decreased?P<0.05?,and the expression of P-Nrf2 protein decreased.7.IHC results showed that compared with the Exhaustive exercise group,the positive expression of TNF-?in the thoracic aorta of the Exhaustive exercise+LBP group decreased,and the positive expression of Nrf2,GCLC,NQO1,and GCLM increased.Compared with the Exhausted exercise group,the positive expression of TNF-?nucleus decreased,the positive expression of Nrf2 increased,and the positive expression of NQO1 and GCLM increased in the Exhausted exercise+LBP group.8.RT-qPCR results showed that compared with the Ang?group,the mRNA expression of TNF-?and IL-6 in the Ang?+LBP group decreased?P<0.05?,and the mRNA expression of IL-1?and Keap1 decreased?P<0.01?.Nrf2 The mRNA expression of NQO1 and NQO1increased?P<0.01?,and the mRNA expression of GCLC and GCLM increased?P<0.05?.Compared with the blank Control group,the mRNA expression of Nrf2 in the SiRNA5 group was more significantly reduced?P<0.05?.Compared with the Ang?+LBP group,the mRNA expression of TNF-?,IL-6,and IL-1?in the SiRNA+Ang?+LBP group increased?P<0.05?.9.The results of flow cytometry showed that compared with the Ang?+LBP group,the expression of TNF-?positive cells in the SiRNA+Ang?+LBP group increased?P<0.001?.Conclusion LBP enhances the expression of the Keap1/Nrf2 anti-oxidative stress signal pathway in endothelial cells by improving the oxidative stress state and inflammatory response of endothelial cells,thereby improving the anti-oxidative stress capacity of endothelial cells and alleviating the oxidative stress state and the damage and apoptosis of myocardial vascular tissue cells ultimately play a role in protecting the cardiovascular system. |
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