| Endotoxic shock is a clinically systemic disease with high mortality.Endotoxin shock patients are often associated with myocardial inflammatory injury and cardiac insufficiency,which is the main reason for the increase in mortalitycompared with those without cardiovascular impairment.Therefore,it is of great significance to improve the prognosis of patients with endotoxemia by improving endotoxin-induced cardiac dysfunction.In this study,we used Ad.Prx II as the target gene carrier,lipopolysaccharide(LPS)as the induction factor of inflammation and the rat cardiomyocytes as the research subject,which were obtained by enzymatic digestion.The aim is to investigate the characteristics of cardiomyocytes with inflammatory after LPS stimulation,to explore whether overexpression of Prx II prevents endotoxin-induced myocardial dysfunction via inhibition of the activation of NF-?B signal pathway and further to elucidate its mechanism.Methods:Part 1 The amplification and purification of adenovirus and the detection of its titer In this part,Ad.GFP and Ad.Prx II in the lab were amplified by HEK293 cells,and then purified by adenovirus purification kit.Finally,their titers were measured by green fluorescent protein labeling method and calculated afterwards.Part 2 Isolation and culture of adult rat cardiomyocytes The adult rat cardiomyocytes were isolated and cultured by enzymatic digestion,and the contractibility of the newly isolated cardiomyocytes was measured with the video-based motion adge-detection system so that we can determine whether they could be used for subsequent experiments.Part 3 Overexpression of Prx II protects cardiomyocytes against LPS-induced myocardial dysfunction First,the cardiomyocytes were treatment and cultured with different concentrations ofLPS(0,1,5,10 ?g/ml)for 24 h,and the protein of Prx II was extracted and measured by Western Blot.Secondly,the cardiomyocytes were infected with Ad.Prx II or control Ad.GFP at a multiplicity of infection(MOI)of 200 for 24 h.The myocytes were then harvested for quantitative immunoblotting,which is used to evaluate whether the Prx II is overexpressed.Thirdly,the cardiomyocytes were first infected with Ad.prx II or control Ad.GFP at a MOIof 200,and then added with LPS(10?g/ml)for establishing the inflammatory model,meanwhile,the cardiomyocytes were managed with a similar treatment without LPS as control.After 24 h,we can evaluate the cell survival by MTT,harvest the myocytes for quantitative immunoblotting,which is used to evaluate the changes of the expression of Prx II,Bcl-2,I?B?,p-I?B? and NF-?B and the activation of NF-?B signaling pathway.At the same time,LDH levels in the culture medium could be detected.Results: 1.The titers of Ad.GFP and Ad.Prx II were 1.8×1011/ml and 1.08×1010/ml,respectively,both of which were above 1×1010/ml,and could be used in the following experiments.2.The video-based motion adge-detection system showed that FS%,+d L/dtand-d L/dtof the isolated cardiomyocytes were11.30%±0.94%,122.36±11.19 ?m/s,133.00±12.16 ?m/s,respectively.3.(1)The optimal concentration of LPS was 10 ?g/ml in the inflammatory model.The expression of Prx II was down-regulated in a concentration dependent manner after LPS stimulation,which suggested thatdown-regulation of Prx II is involved in LPS-induced cardiomyocyte injury under stress.(2)MTT showed that Prx II can improve the viability of cardiomyocytes and prevent LPS-induced myocardial injury.(3)Quantitative immunoblotting that Prx II was overexpressed in cardiomyocytes successfully,which contributed to inhibiting the phosphorylation of I?B? and the activation of NF-?B.(4)The test of LDH showed that overexpression of Prx II significantly reduced the concentration of LDH in the culture medium,compared with the model group.Conclusions: Prx II prevents endotoxin-induced myocardial dysfunction and reduces theinflammatory factors caused by LPS stimulation,which improves the myocardial injury effectively.The mechanism may be related to Prx II inhibition of the activation of NF-?B signal pathway. |
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