Effects Of Myocardial Injury Induced By Cardiopulmonary Bypass In Rats And Mechanism Of Myocardial Protection Of HRS Via PI3K/Akt Signaling Pathway

Objective:Hydrogen-rich solution?HRS?,which is weakly alkaline,has a negative potential and contains the small molecule water.HRS possesses some characters including strong anti-inflammatory,anti-oxidative stress and anti-apoptotic.HRS exerts a protective effect on the brain,liver and intestines against ischemia-reperfusion injury,however,the underlying therapeutic mechanism remains unknown.The phosphatidylinositol 3'-kinase/protein kinase B?PI3K/Akt?signaling pathway plays an important role in the regulation of protective mechanisms of the myocardial ischemia-reperfusion injury.The mechanisms of myocardial injury induced by cardiopulmonary bypass?CPB?remain uncertain.There is no consensus about the physiological significance of expression of the aquaporin?AQP?in the myocardium and the myocardial protection of HRS by the PI3K/Akt signaling pathway after CPB.The present study will establish a rat model of CPB with cardioplegic arrest?CA?without blood priming,and investigate the changes of myocardial water content,myocardial injury index,inflammatory mediators,antioxidant capacity,and protein expression of AQP-1,4 after CPB.The further discussion will focus on the myocardial injury mechanism induced by CPB and the feasibility of myocardial protection of HRS by the PI3K/Akt signaling pathway after CPB at the different levels of cell,molecule,and gene.It may provide new ideas of myocardial protection strategies during CPB and new experimental basis on the clinical application of HRS.Methods:The first part of the present study includes establishment of a rat model of CPB with CA,hemodynamic changes observed during CPB,the myocardial water content that was detected in rats.The plasma cTnI,hFABP,TNF-?,IL-6,IL-10,8-iso-PGF2?levels were detected by radioimmunoassay?RIA?and enzyme linked immunosorbent assay?ELISA?,the malonaldehyde?MDA?,myeloperoxidase?MPO?and superoxide dimutase?SOD?concentration of myocardial tissue were detected by spectrophotometry,investigating the myocardial morphology by hematoxylin and eosin?HE?staining and electron microscopy,the myocardial protein expression of aquaporin-1?AQP-1?,aquaporin-4?AQP-4?were detected by immunohistochemistry and Western blot,the myocardial mRNA expression of AQP-1,AQP-4 were detected by real-time quantitative polymerase chain reaction?qRT-PCR?.The relationship between AQP-1,AQP-4 protein and myocardial water content,inflammatory mediators and peroxidation index were detected by immunohistochemistry,Western blot and qRT-PCR.The second part of the present study includes establishing the rat model of CPB with CA and give HRS treatment and monitor the hemodynamic changes of rats and determine the myocardial water content.Morphology of the myocardial tissues was observed by HE staining and Masson staining.The myocardial injury markers?cTnI,LDH,CK-MB and BNP?,inflammatory factors?IL-1?,IL-6 and TNF-??and oxidative stress factors?SOD,MDA and MPO?levels were detected by ELISA.Cell apoptosis was detected by TUNEL assay.Apoptosis-related proteins for myocardial tissue?Bcl-2,Bax and caspase-3?,aquaporins?AQP-1 and AQP-4?and PI3K signaling pathway related proteins?PI3K,Akt,p-Akt and HO-1?were detected by Western Blot.The third part of the present study based on the second part and gives the rats PI3K inhibitor treatment,then determine the myocardial water content.The myocardial injury markers?cTnI,LDH,CK-MB and BNP?,inflammatory factors?IL-1?,IL-6 and TNF-??and oxidative stress factors?SOD,MDA and MPO?levels were detected by ELISA.Morphology of the myocardial tissues was observed by Masson staining.Cell apoptosis was detected by TUNEL assay.Apoptosis-related proteins for myocardial tissue?Bcl-2,Bax and caspase-3?,aquaporins?AQP-1 and AQP-4?and PI3K signaling pathway related proteins?PI3K,Akt,p-Akt and HO-1?were detected by Western Blot.Furthermore,cell hypoxia/reoxygenation model were established.Cell viability were detected by 3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium bromide?MTT?and cell apoptosis were detected by flow cytometry.Apoptosis-related proteins for myocardial tissue?Bcl-2,Bax and caspase-3?,aquaporins?AQP-1 and AQP-4?and PI3K signaling pathway related proteins?PI3K,Akt,p-Akt and HO-1?were detected by Western Blot.Results:In the first part of the present study:1.Changes of myocardial injury in each group.Compared with the sham group,myocardial water content at each time point in CPB group significantly increased?P<0.05?.2.Changes of plasma cTnI,hFABP,TNF-?,IL-6,IL-10 and 8-iso-PGF₂?levels.Compared with the sham group,plasma cTnI,hFABP,TNF-?,IL-6 and 8-iso-PGF₂?levels of CPB group at each time point significantly increased and IL-10 level significantly decreased?P<0.05?.3.Changes of MDA,MPO and SOD levels in the cardiac tissue.Compared with the sham group MDA,MPO concentrations in myocardial tissue of CPB group at each time point significantly increased,and SOD activity significantly decreased?P<0.05?.4.HE staining observed by optical microscope and electron microscopy observation.The myocardial injury of CPB group was serious.5.Myocardial AQP-1 and AQP-4 protein expression detected by immunohistochemistry.Compared with the sham group,AQP-1 and AQP-4 protein expression significantly increased at each time point in CPB group cardiac tissue.6.Western blot of myocardial AQP-1 and AQP-4 protein.Compared with the sham group,AQP-1 and AQP-4 of CPB group significantly increased?P<0.05?.7.Real-Time PCR detection of myocardial AQP-1 and AQP-4 mRNA expression.AQP-1 and AQP-4mRNA expression levels of each group consistent with protein expression levels.8.The relationship between myocardial AQP-1,AQP-4 protein expression and myocardial injury.Myocardial AQP-1 and AQP-4 protein expression was positively correlated with myocardial water content,plasma cTnI,hFABP levels,plasma TNF-?and IL-6 levels,plasma 8-iso-PGF2?level and myocardial MDA concentration?P<0.05?.In the second part of the present study:The levels of rectal temperature,pH,PaCO₂and PaO₂ in rats were stable,and the HR,MAP,LVDP,+dP/dtmax and Hb levels of CPB rats were significantly increased after HRS treatment?P<0.05?.The myocardial water content of CPB rats significantly decreased after HRS treatment?P<0.05?.HE staining and Masson trichrome staining showed that HRS treatment improved myocardial injury in CPB rats.ELISA showed that HRS decreased myocardial injury correlation factor,oxidative stress correlation factor and inflammatory factor secretion in rats with CPB.TUNEL assay showed that HRS improved the apoptosis induced by CPB.Western blot experiment showed that after HRS treatment,the expression levels of Bax,caspase-3,AQP-1 and AQP-4 protein in CPB rats significantly decreased?P<0.05?,and the expression levels of Bcl-2,PI3K,p-Akt and HO-1 significantly increased?P<0.05?.In the third part of this study,PI3K inhibitors increased the HRS treatment of myocardial water content in rats;ELISA results showed that PI3K inhibitor reversed the inhibitory effect of HRS on myocardial injury related factors,oxidative stress correlation factors and inflammatory factors in CPB rats.Masson trichrome staining showed that PI3K inhibitor reversed the protective effect of HRS on myocardial injury in CPB rats.TUNEL assay test results showed that PI3K inhibitor reversed the inhibitory effect of HRS on apoptosis of CPB rats.Western blot the experimental results showed that after the injection of PI3K inhibitors,HRS processing of CPB rats Bax,caspase-3,AQP-1 and AQP-4 protein expression level significantly increased?P<0.05?,the Bcl-2,PI3K,and p-Akt and HO-1 protein expression level significantly decreased?P<0.05?.Conclusions:1.Cardiopulmonary bypass with cardioplegic arrest can cause myocardial injury.The possible mechanism is that CPB can increase inflammatory response,lipid peroxidation,and myocardial AQP-1 and AQP-4 protein expression.2.HRS via PI3K/Akt signaling pathway plays a protective role in myocardium after CPB.The main mechanism is inhibiting systemic inflammatory response,inhibiting ischemia-reperfusion injury and decreasing the expression of AQP-1 protein and AQP-4 protein.