Hydrogen-Rich Saline Attenuates Brain Injury Induced by Cardiopulmonary Bypass and Inhibits Microvascular Endothelial Cell Apoptosis Via the PI3K/Akt/GSK3β Signaling Pathway in Rats
Cellular Physiology and Biochemistry
Background/Aims: Cardiopulmonary bypass (CPB) is prone to inducing brain injury during open heart surgery. A hydrogen-rich solution (HRS) can prevent oxidation and apoptosis, and inhibit inflammation. This study investigated effects of HRS on brain injury induced by CPB and regulatory mechanisms of the PI3K/Akt/GSK3β signaling pathway. Methods: A rat CPB model and an in vitro cell hypoxia model were established. After HRS treatment, Rat behavior was measured using neurological deficit score;
... l deficit score; Evans blue (EB) was used to assess permeability of the blood-brain barrier (BBB); HE staining was used to observe pathological changes; Inflammatory factors and brain injury markers were detected by ELISA; the PI3K/Akt/GSK3β pathway-related proteins and apoptosis were assessed by western blot, immunohistochemistry and qRT -PCR analyses of brain tissue and neurons. Results: After CPB, brain tissue anatomy was disordered, and cell structure was abnormal. Brain tissue EB content increased. There was an increase in the number of apoptotic cells, an increase in expression of Bax and caspase-3, a decrease in expression of Bcl2, and increases in levels of Akt, GSK3β, P-Akt, and P-GSK3β in brain tissue. HRS treatment attenuated the inflammatory reaction ,brain tissue EB content was significantly reduced and significantly decreased expression levels of Bax, caspase-3, Akt, GSK3β, P-Akt, and P-GSK3β in the brain. After adding the PI3K signaling pathway inhibitor, LY294002, to rat cerebral microvascular endothelial cells (CMECs), HRS could reduce activated Chen et al.: HRS Protect Against Rat Brain Injury by PI3K/Akt/GSK3β Pathway During CPB Akt expression and downstream regulatory gene phosphorylation of GSK3β expression, and inhibit CMEC apoptosis. Conclusion: The PI3K/Akt/GSK3β signaling pathway plays an important role in the mechanism of CPB-induced brain injury. HRS can reduce CPB-induced brain injury and inhibit CMEC apoptosis through the PI3K/Akt/GSK3β signaling pathway. Fig. 2 . Different concentrations of HRS attenuated brain tissue injury after CPB. After establishment of the rat CPB model, animals were treated with 1 mL/kg, 4 mL/kg, or 6 mL/kg of HRS. At 24 h after CPB, the hippocampus was stained with HE to assess pathological changes. In addition, neurological function scores were determined, BBB permeability was measured using Evans Blue, and changes in levels of inflammatory factors and brain injury markers in serum were detected by ELISA. These data indicate that 6 mL/kg HRS significantly attenuated brain tissue injury after CPB. A: HE staining. B: Comparison of neurological function scores of rats in each group. C: Inflammatory factor (IL-1β, IL-6, TNF-α) expression levels measured by ELISA. D: Brain injury marker (S-100β, NSE) expression levels measured by ELISA.Compared with the sham group, *P<0.05. Compared with the CPB group,# P<0.05. Chen et al.: HRS Protect Against Rat Brain Injury by PI3K/Akt/GSK3β Pathway During CPB HRS inhibited neuronal apoptosis via the PI3K/Akt/GSK3β signaling pathway after CPB in rats To confirm whether the PI3K/Akt/GSK3β signaling pathway mediates neuronal apoptosis, we assessed expression levels of several key factors in the signaling pathway. In the CPB group, expression of Akt, GSK3β, P-Akt, and P-GSK3β increased. However, after Fig. 3 . HRS protected brain tissue from CPB-induced apoptosis. After establishment of the CPB model in rats, animals were treated with 6 mL/kg HRS. TUNEL staining was used to detect apoptotic cells in brain hippocampal tissue. Western Blot and real-time quantitative PCR were used to assess expression of Bax, Bcl2, and caspase-3 in the hippocampus. Data were collected from the sham, CPB, and HRS treatment groups. A: Apoptotic cells detected by TUNEL staining.B-C: Western blot analysis and quantitative real-time PCR of Bax, caspase-3, and Bcl2. Compared with the sham group, *P<0.05. Compared with the CPB group,# P<0.05.