Effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation after isoflurane-induced postconditioning.
BACKGROUND
Isoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic-ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat's brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown.


METHODS
A total of 80 healthy Sprague-Dawley rats weighing 50-70g were randomly divided into 10 groups of 8: normal control, oxygen and glucose deprivation (OGD), 1.5% ISPOC, 3.0% ISPOC, 4.5% ISPOC, blocker of activin A (SB431542), blocker of ERK1/2 (U0126), 3.0% ISPOC+SB431542, 3.0% ISPOC+U0126, and vehicle (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin-eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-PCR) were performed to validate the protein expression levels of activin A, Smad2/3, P-Smad2/3, ERK1/2, and phosphorylation ERK1/2 (P-ERK1/2).


RESULTS
The number of damaged neurons and mean fluorescence intensity(MFI) of PI staining increased, but formazan generation, expression levels of activin A and P-ERK1/2 protein, and mRNA synthesis level of activin A decreased in the OGD group compared with the normal control group (p<0.05). The number of damaged neurons and MFI of PI staining decreased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A increased significantly in the 1.5% ISPOC and 3.0% ISPOC groups (p<0.05) compared with the OGD group. The result in the 4.5% ISPOC group, was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups. The number of damage neuron and MFI of PI staining increased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A decreased in the 4.5% ISPOC group. However, the expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group (p<0.05). The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group. The MFI of PI staining increased, but the expression levels of activin A, P-Smad2/3, and P-ERK1/2 decreased (p<0.05). The expression level of ERK1/2 protein in all groups exhibited no change (p>0.05).


CONCLUSIONS
Results of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane. Activin A/Smad 2/3 and activin A/ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection.
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China. Electronic address: 313111941@qq.com.
Qin
Q
Wang
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China. Electronic address: 313111941@qq.com.
Jiangwen
J
Yin
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China. Electronic address: iamsheng2006@163.com.
Sheng
S
Wang
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China.
Di
D
Cui
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China.
Hong
H
Lin
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China.
Mingyue
M
Ge
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China.
Zhigang
Z
Dai
Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832002, China.
Liping
L
Xie
Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi 832002, China.
Junqiang
J
Si
Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi 832002, China.
Ketao
K
Ma
Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi 832002, China.
Li
L
Li
Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi 832002, China.
Lei
L
Zhao
D000818:Animals; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000172:deficiency; D006624:Hippocampus / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D020925:Hypoxia-Ischemia, Brain / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D066298:In Vitro Techniques; D028322:Inhibin-beta Subunits / Q000235:genetics / Q000378:metabolism*; D007530:Isoflurane / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009410:Nerve Degeneration; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010766:Phosphorylation; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D013997:Time Factors
D018696:Neuroprotective Agents; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; C103008:inhibin beta A subunit; D028322:Inhibin-beta Subunits; D007530:Isoflurane; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D005947:Glucose
Activin A; Extracellular signal-regulated kinase 1/2; Hypoxic-ischemic brain damage; Isoflurane postconditioning; Oxygen and glucose deprivation