Objective:To discuss whether safranal affects the proliferation,migration,and phenotypic transformation of the vascular smooth muscle cells(VSMCs)in a high-glucose environment and to clarify the function of safranal in the prevention and treatment of diabetic(DM)vascular complications.Methods:The SD rats were selected as experimental subjects;primary VSMCs were cultured from rat thoracic aortas and divided into control group,25 mmol·L-1 high glucose(HG)group,HG+20 μmol·L-1 safranal group,HG+40 μmol·L-1 safranal group,and HG+80 μmol·L-1 safranal group.The cells in control group received no treatment;the cells in 25 mmol·L-1 HG group were pretreated with 25 mmol·L-1 HG;the cells in HG+20,40,and 80 μmol·L-1 safranal groups were further treated with 20,40,and 80 μmol·L-1 safranal respectively for 48 h on the basis of 25 mmol·L-1 HG group.Cell counting kit-8(CCK-8)method was used to determine the appropriate concentration of safranal and detect the viabilities of the VSMCs in various groups;cell scratch healing assay was used to detect the scratch healing rates of the VSMCs in various groups;Transwell chamber assay was used to detect the numbers of the migration VSMCs in various groups;immunofluorescence method was used to detect the fluorescence intensities of alpha-smooth muscle actin(α-SMA)and rabbit anti-osteopontin(OPN)in the VSMCs in various groups;Western blotting method was used to detect the expression levels of OPN,α-SMA,and proliferating cell nuclear antigen(PCNA)in the VSMCs in various groups.Results:Under microscope,on the 4th day of in vitro culture,the spindle-shaped or triangular cells crawled out from the edge of the thoracic aorta tissue blocks,with long spindle being the most common morphology.On the 14th,the cells gradually covered the bottom of the dish;when cell density reached 80%-90%,the characteristic"hills and valleys"growth pattern appeared.Third-generation cells were taken for immunofluorescence identification;immunofluorescence staining with VSMC-specific marker α-SMA showed positive expression of α-SMA protein in the primarily cultured VSMCs.The CCK-8 assay results showed that compared with control group,the cell viability of the cells in 160 μmol·L-1 safranal group was significantly decreased(P<0.01),indicating toxic damage to the cells.Under the conditions of safranal concentrations at 20,40,and 80 μmol·L-1 respectively,after 48 h intervention on VSMCs,no significant adverse effect on cell viability was observed;considering both the effect and toxicity of safranal,these three concentrations were used in subsequent cell experiments.After 48 h intervention,compared with control group,the activity of the VSMCs in 25 mmol·L-1 HG group was increased(P<0.001);compared with 25 mmol·L-1 HG group,the activities of the VSMCs in HG+20,40,and 80 μmol·L-1 safranal groups were gradually decreased(P<0.05).The cell scratch healing assay and Transwell assay results showed that after 48 h intervention,the scratch healing rate of the VSMCs in 25 mmol·L-1 HG group was significantly higher than that in control group(P<0.01),and the number of transmembrane cells through the Transwell chamber was significantly increased(P<0.05);compared with 25 mmol·L-1 HG group,the scratch healing rates of the VSMCs in HG+20,40,and 80 μmol·L-1 safranal groups were gradually decreased(P<0.05),and the number of transmembrane cells was decreased(P<0.05).The immunofluorescence staining results showed that compared with control group,the fluorescence intensity of α-SMA protein in the VSMCs in 25 mmol·L-1 HG group was significantly weakened(P<0.001),while the fluorescence intensity of OPN protein was significantly enhanced(P<0.001);compared with 25 mmol·L-1 HG group,the fluorescence intensities of α-SMA protein in the VSMCs in HG+20,40,and 80 μmol·L-1 safranal groups were gradually increased(P<0.05),and the fluorescence intensities of OPN were gradually weakened(P<0.05).The Western blotting method results showed that compared with control group,the expression level of α-SMA protein in the VSMCs in 25 mmol·L-1 HG group was decreased(P<0.05),and the expression levels of PCNA and OPN proteins were increased(P<0.01);compared with 25 mmol·L-1 HG group,the expression level of α-SMA protein in the VSMCs in HG+20,40,and 80 μmol·L-1 safranal groups were increased(P<0.05),and the expression levels of PCNA and OPN proteins were decreased(P<0.05).Conclusion:Safranal can inhibit the proliferation,migration,and phenotypic transformation of the VSMCs induced by high glucose.

Objective:To investigate the effect of tumor necrosis factor α (TNF-α) on the permeability of blood labyrinth barrier in C57BL/6J male mice and its possible mechanism.Methods:As for the design of animal experiment, twenty male C57BL/6J mice aged 6-8 weeks were randomly divided into control group and cisplatin group with 10 mice in each group by software method. The control group was intraperitoneally injected with normal saline every day, and the cisplatin group was intraperitoneally injected with 4 mg/kg cisplatin for 4 consecutive days. After administration, auditory brainstem response (ABR) was used to detect hearing changes in mice. HE staining was used to observe the morphological changes of mouse cochlea vasculature. The expression of TNF-α was detected by immunohistochemistry and ELISA. The permeability of the blood labyrinth barrier was observed by Evans blue staining. With respect to the design of cell experiment, primarily cultured cochlea pericytes (PC) and endothelial cells (EC) were divided into EC group, EC+TNF-α group, EC+PC group, EC+PC+TNF-α group, EC+PC+TNF-α+SB-3CT (MMP-9/MMP-2 secretion inhibitor) group, PC group, PC+TNF-α group, PC+TNF-α+LY294002 (PI3K/AKT pathway inhibitor) group, PC+LY294002 group. The protein expressions of ZO-1, VE-cadherin, MMP-9, MMP-2, PI3K, p-PI3K, AKT, and p-AKT were detected by Western blot. TEER and FITC-dextran penetration experiment were used to detect EC resistance and monolayer EC permeability, respectively. The data were statistically analyzed using GraphPad Prism 8 software.Results:In animal experiment, compared with control group, the ABR response threshold of mice in cisplatin group was increased ( P<0.01). The vaccine ular structure of the cochlea was disordered red, wrinkled and vacuole increased. The extravasation of vascular red fluorescent dye increased ( P<0.05), and also, levels of serum TNF-α and cochlear veins increased ( P<0.01). In cell experiment, by treatment of EC with different concentrations of cisplatin, 20 μmol cisplatin led to the highest expression of TNF-α ( P<0.01). The expression of TNF-α was the highest after 3 h intervention in EC ( P<0.05). Compared with those in EC+PC group, the resistance value of EC was decreased, the permeability of monolayers EC was increased, the expression of ZO 1 and VE cadherin proteins was decreased, and however, the resistance value was increased and the permeability of EC was decreased after the intervention of SB-3CT in EC+PC+TNF-α group. The expressions of ZO-1 and VE-cadherin proteins were increased ( P<0.05). The expression of MMP-9 increased after TNF-α intervention ( P<0.05), the expression of MMP-2 had no significant change, and the expression of p-PI3K/PI3K and p-AKT/AKT were increased ( P<0.05). The expression of MMP-9 decreased in PC+TNF-α+LY294002 group ( P<0.05). Conclusion:The hearing loss of C57BL/6J male mice induced by cisplatin may be related to the increased release of TNF-α from the blood labyrinth barrier EC in the cochlear stria vascularis, and the activation of PI3K/AKT signaling pathway by TNF-α in PC, which increases the secretion of MMP-9 from PC, ultimately leads to the increased permeability of the blood labyrinth barrier.

Objective To investigate the mechanisms by which astaxanthin(AST)alleviates oxaliplatin(OXA)-induced neuropathic pain through antioxidant pathways so as to provide theoretical basis for clinical intervention.Methods Animal experiments:SD rats were divided into five groups(n=6):control group,OXA(4 mg/kg)group,OXA+Oil group,OXA+AST(5 mg/kg)group,and OXA+AST(10 mg/kg)group.Mechanical and cold pain thresholds were measured at day 0,7,14,and 21.Malondialdehyde(MDA)content and superoxide dismutase(SOD)activity in the dorsal root ganglia(DRG)were detected using the thiobarbituric acid(TBA)method and WST-1 assay,respectively.Western blotting was performed to analyze the expressions of Nrf2 and HO-1.Cell experiments:neuro-2a cells were divided into control group,OXA(50 μmol/L)group,AST(10 μmol/L)group,and OXA(50 μmol/L)+AST(10 μmol/L)group.Cells were treated with nerve growth factor(NGF,50 ng/mL)to induce growth,and morphological changes were observed under an inverted microscope.Intracellular reactive oxygen species(ROS)level and mitochondrial superoxide were measured using DCFH-DA fluorescent probe and MitoSOXTM red,respectively.Mitochondrial function was assessed by JC-1 assay.Western blotting was used to detect Nrf2 and HO-1 expressions.Results Animal experiments:① Mechanical and cold pain thresholds were reduced in OXA and OXA+Oil groups(P＜0.05),while AST significantly increased these thresholds in OXA-treated rats(P＜0.05).② SOD activity decreased while MDA content increased in the DRG of OXA-treated rats(P＜0.05).AST restored SOD activity and reduced MDA level(P＜0.05,P＜0.01).③ Western blotting showed elevated Nrf2 and HO-1 expressions in OXA group(P＞0.05),which were further upregulated by AST(P＜0.05,P＜0.01).Cell experiments:① OXA reduced the number of neurite-bearing cells and shortened the average neurite length(P＜0.05).Inverted microscopic observation revealed that AST intervention increased both parameters(P＜0.01,P＜0.001).② OXA increased intracellular and mitochondrial ROS fluorescence intensity(P＜0.05),which was attenuated by AST(P＜0.01).③ JC-1 assay revealed decreased mitochondrial membrane potential in OXA group(P＜0.01),which was partially reversed by AST(P＜0.05).④ Western blotting results showed that OXA upregulated Nrf2 and HO-1 expressions(P＜0.05,P＜0.01),and AST further enhanced their levels(P＜0.01).Conclusion AST alleviates OXA-induced neuropathic pain by promoting Nrf2/HO-1 expression,enhancing SOD activity,reducing lipid peroxidation and ROS production,and improving mitochondrial function.

Objective To investigate the mechanisms by which astaxanthin(AST)alleviates oxaliplatin(OXA)-induced neuropathic pain through antioxidant pathways so as to provide theoretical basis for clinical intervention.Methods Animal experiments:SD rats were divided into five groups(n=6):control group,OXA(4 mg/kg)group,OXA+Oil group,OXA+AST(5 mg/kg)group,and OXA+AST(10 mg/kg)group.Mechanical and cold pain thresholds were measured at day 0,7,14,and 21.Malondialdehyde(MDA)content and superoxide dismutase(SOD)activity in the dorsal root ganglia(DRG)were detected using the thiobarbituric acid(TBA)method and WST-1 assay,respectively.Western blotting was performed to analyze the expressions of Nrf2 and HO-1.Cell experiments:neuro-2a cells were divided into control group,OXA(50 μmol/L)group,AST(10 μmol/L)group,and OXA(50 μmol/L)+AST(10 μmol/L)group.Cells were treated with nerve growth factor(NGF,50 ng/mL)to induce growth,and morphological changes were observed under an inverted microscope.Intracellular reactive oxygen species(ROS)level and mitochondrial superoxide were measured using DCFH-DA fluorescent probe and MitoSOXTM red,respectively.Mitochondrial function was assessed by JC-1 assay.Western blotting was used to detect Nrf2 and HO-1 expressions.Results Animal experiments:① Mechanical and cold pain thresholds were reduced in OXA and OXA+Oil groups(P＜0.05),while AST significantly increased these thresholds in OXA-treated rats(P＜0.05).② SOD activity decreased while MDA content increased in the DRG of OXA-treated rats(P＜0.05).AST restored SOD activity and reduced MDA level(P＜0.05,P＜0.01).③ Western blotting showed elevated Nrf2 and HO-1 expressions in OXA group(P＞0.05),which were further upregulated by AST(P＜0.05,P＜0.01).Cell experiments:① OXA reduced the number of neurite-bearing cells and shortened the average neurite length(P＜0.05).Inverted microscopic observation revealed that AST intervention increased both parameters(P＜0.01,P＜0.001).② OXA increased intracellular and mitochondrial ROS fluorescence intensity(P＜0.05),which was attenuated by AST(P＜0.01).③ JC-1 assay revealed decreased mitochondrial membrane potential in OXA group(P＜0.01),which was partially reversed by AST(P＜0.05).④ Western blotting results showed that OXA upregulated Nrf2 and HO-1 expressions(P＜0.05,P＜0.01),and AST further enhanced their levels(P＜0.01).Conclusion AST alleviates OXA-induced neuropathic pain by promoting Nrf2/HO-1 expression,enhancing SOD activity,reducing lipid peroxidation and ROS production,and improving mitochondrial function.

Objective:To investigate the effect of tumor necrosis factor α (TNF-α) on the permeability of blood labyrinth barrier in C57BL/6J male mice and its possible mechanism.Methods:As for the design of animal experiment, twenty male C57BL/6J mice aged 6-8 weeks were randomly divided into control group and cisplatin group with 10 mice in each group by software method. The control group was intraperitoneally injected with normal saline every day, and the cisplatin group was intraperitoneally injected with 4 mg/kg cisplatin for 4 consecutive days. After administration, auditory brainstem response (ABR) was used to detect hearing changes in mice. HE staining was used to observe the morphological changes of mouse cochlea vasculature. The expression of TNF-α was detected by immunohistochemistry and ELISA. The permeability of the blood labyrinth barrier was observed by Evans blue staining. With respect to the design of cell experiment, primarily cultured cochlea pericytes (PC) and endothelial cells (EC) were divided into EC group, EC+TNF-α group, EC+PC group, EC+PC+TNF-α group, EC+PC+TNF-α+SB-3CT (MMP-9/MMP-2 secretion inhibitor) group, PC group, PC+TNF-α group, PC+TNF-α+LY294002 (PI3K/AKT pathway inhibitor) group, PC+LY294002 group. The protein expressions of ZO-1, VE-cadherin, MMP-9, MMP-2, PI3K, p-PI3K, AKT, and p-AKT were detected by Western blot. TEER and FITC-dextran penetration experiment were used to detect EC resistance and monolayer EC permeability, respectively. The data were statistically analyzed using GraphPad Prism 8 software.Results:In animal experiment, compared with control group, the ABR response threshold of mice in cisplatin group was increased ( P<0.01). The vaccine ular structure of the cochlea was disordered red, wrinkled and vacuole increased. The extravasation of vascular red fluorescent dye increased ( P<0.05), and also, levels of serum TNF-α and cochlear veins increased ( P<0.01). In cell experiment, by treatment of EC with different concentrations of cisplatin, 20 μmol cisplatin led to the highest expression of TNF-α ( P<0.01). The expression of TNF-α was the highest after 3 h intervention in EC ( P<0.05). Compared with those in EC+PC group, the resistance value of EC was decreased, the permeability of monolayers EC was increased, the expression of ZO 1 and VE cadherin proteins was decreased, and however, the resistance value was increased and the permeability of EC was decreased after the intervention of SB-3CT in EC+PC+TNF-α group. The expressions of ZO-1 and VE-cadherin proteins were increased ( P<0.05). The expression of MMP-9 increased after TNF-α intervention ( P<0.05), the expression of MMP-2 had no significant change, and the expression of p-PI3K/PI3K and p-AKT/AKT were increased ( P<0.05). The expression of MMP-9 decreased in PC+TNF-α+LY294002 group ( P<0.05). Conclusion:The hearing loss of C57BL/6J male mice induced by cisplatin may be related to the increased release of TNF-α from the blood labyrinth barrier EC in the cochlear stria vascularis, and the activation of PI3K/AKT signaling pathway by TNF-α in PC, which increases the secretion of MMP-9 from PC, ultimately leads to the increased permeability of the blood labyrinth barrier.

Objective:To study whether cisplatin may induce apoptosis in the pericytes of cochlear stria vascularis and underlying mechanisms.Methods:Twenty male C57BL/6J mice aged 6-8 weeks were divided into control group and a cisplatin group. Primary cultured mouse cochlear vascular peristriatal cells were identified and divided into control group, cisplatin group, and N-acetylcysteine+cisplatin group. Auditory brainstem response (ABR) was used to detect hearing in mice. Hematoxylin eosin (HE) staining was used to observe morphological changes in the stria vascularis of the cochlea. The total superoxide dismutase (SOD) activity test kit (WST-1 method) and thiobarbituric acid (TBA) method were used to detect SOD activity and Malondialdehyde (MDA) content, respectively. DCFH-DA fluorescence probe was used to detect the content of reactive oxygen species in peripheral cells. Hoechst 33 342 and flow cytometry were used to detect the apoptosis rate of pericytes. Immunofluorescence technology was used to detect the distribution and expression of apoptosis related proteins in pericytes of cochlear stria vascularis. Immunohistochemistry and Western blotting (WB) were used to detect the expression of apoptosis-and mitochondrial-related proteins. Mito SOX TM-red and JC-1 were used to detect the mitochondrial function of pericytes. Evans blue staining was used to observe the permeability of the blood labyrinth barrier (BLB). Statistical analysis was conducted using SPSS 18.0 software. Results:Compared with the control group, the cisplatin group significantly increased in the hearing threshold ( t=4.72, P<0.01), Ⅰ-wave latency ( t=12.25, P<0.05), and the levels of oxidative stress in the cochlea and pericytes ( t=38.34, P<0.01), and also cisplatin caused disorder and contraction of the cochlear stria vascularis structure, increased BLB permeability [Evans blue leakage (1.08±0.42) AU vs (0.55±0.23) AU, t=4.64, P<0.05], with a statistically significant difference, enhanced the expressions of apoptotic proteins c-Caspase-3 ( t=5.01, P<0.01) and Bax ( t=6.33, P<0.01) in the peristriatal cells of cochlear blood vessels in mice treated with cisplatin increased. And cisplatin can induce apoptosis of primary cultured pericytes and up-regulate the expression of c-Caspase-3 and Bax ( P<0.05). The NAC+cisplatin group partially reversed cisplatin-induced pericyte apoptosis ( P<0.05). Cisplatin caused damage to the mitochondrial function of peripheral cells, and induced the release of apoptosis-inducing factor (AIF) and cytochrome C (cyt-c) into the cytoplasm ( P<0.05). The NAC+cisplatin group partially reversed cisplatin induced pericyte apoptosis ( P<0.05) and mitochondrial damage ( P<0.05). Conclusion:Cisplatin can increase the level of oxidative stress in the cochlea and cause mitochondrial pathway apoptosis in C57BL/6J mouse cochlear vascular peristriatal cells.

Objective: To investigate the role of connexin 43 ( Cx43) in acute sympathetic stress induced by phe- nylephrine (PE) overactivation of α1-adrenergic receptor ( α1-AR) in cardiomyocytes． Methods: Cardiomyocytes of H9C2 rats were randomly divided into control group，PE alone treatment group，Gap26 ( Cx43 specific inhibitor) intervention group and Gap26 alone treatment group．PE alone treatment group was treated with 50 μmol / L PE for 15 min．The Gap26 intervention group was pretreated with 0. 5 μmol / L Gap26 for 30 min，and then treated with 50 μmol / L PE for 15 min．The protein and mRNA expression levels of Cx43，NLRP3 inflammasome，interleukin-1 β (IL-1 β) ，Caspase-1，interleukin-18 (IL-18 ) were detected by Western blot and qRT-PCR. The expression and co-location of Cx43 in cardiomyocytes were observed by immunofluorescence assay，and the expression of inflamma- tory cytokines IL-1 β and IL-18 in cardiomyocytes was detected by ELISA. Results: Compared with control group， the protein and mRNA levels of Cx43，NLRP3，Caspase-1 and IL-18 in PE group increased．Compared with PE a- lone treatment group，the protein and mRNA levels of Cx43，NLRP3，Caspase-1 and IL-18 decreased after Gap26 intervention，but they were still higher than those of control group． Similarly ，immunofluorescence showed that Cx43 protein expression increased in PE alone group，while Cx43 expression was down-regulated in Gap26 inter- vention group compared with PE alone group．ELISA results showed that the expression of IL-1 β and IL-18 was sig- nificantly up-regulated in PE alone group，but down-regulated in Gap26 intervention group． Conclusion Cx43 is involved in α1-AR activation induced cardiac acute sympathetic stress by regulating NLRP3 inflammasome.

Objective:To evaluate the relationship between transforming growth factor beta-3 (TGF-β 3)/mammalian homologs of the drosophila mad gene 3 (Smad3) signaling pathway and neuronal apoptosis during reduction of focal cerebral ischemia-reperfusion (I/R) injury by isoflurane postconditioning (ISO) in rats. Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 210-230 g, were randomly divided into 5 groups ( n=12 each): sham operation group (group S), cerebral I/R group (group I/R), cerebral I/R plus isoflurane postconditioning group (group I/R+ ISO), cerebral I/R plus pirfenidone group (group I/R+ P), and cerebral I/R plus isoflurane postconditioning plus pirfenidone group (group I/R+ ISO+ P). Local cerebral I/R was produced by middle cerebral artery occlusion for 1.5 h followed by 24-h reperfusion in anesthetized rats.Pirfenidone 5 μg/kg was injected into the lateral ventricle at 30 min before ischemia in I/R+ P and I/R+ ISO+ P groups.In I/R+ ISO and I/R+ ISO+ P groups, 1.5% isoflurane was inhaled for 1 h starting from the time point immediately after onset of reperfusion.Neuro-functional deficit was assessed using neurologic deficit scores (NDS) at the end of reperfusion.Then the animals were sacrificed, and brain tissues were removed for determination of the neuronal damage rate (by Nissl staining), neuronal apoptosis rate (by TUNEL), expression of TGF-β 3 (using immunofluorescence), and expression of TGF-β 3, phosphorylated Smad3 (p-Smad3), caspase-3, Bax and Bcl-2 (by Western blot). Results:Compared with group S, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3, caspase-3 and Bax was up-regulated, and the expression of p-Smad3 and Bcl-2 was down-regulated in group I/R ( P<0.05). Compared with group I/R, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly decreased, the expression of TGF-β 3, p-Smad3 and Bcl-2 was up-regulated, and the expression of caspase-3 and Bax was down-regulated in group I/R+ ISO, and the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3 and p-Smad3 was down-regulated, and the expression of caspase-3 was up-regulated in group I/R+ P ( P<0.05). Compared with group I/R+ ISO, the NDS, neuronal damage rate and apoptosis rate of neurons were significantly increased, the expression of TGF-β 3, p-Smad3 and Bcl-2 was down-regulated, and the expression of caspase-3 and Bax was up-regulated in group I/R+ ISO+ P ( P<0.05). Conclusion:Isoflurane postconditioning can inhibit neuronal apoptosis by activating the TGF-β 3/Smad3 signaling pathway, thus reducing focal I/R injury in rats.

Objective: The aging model of guinea pigs induced by D-galactose was set up to investigate the changes of BK_Ca expression and function on cochlear pericytes and their relationship with age-related hearing loss. Methods: Thirty healthy 8-week-old guinea pigs were randomly divided into three groups, with 10 in each group: D-galactose aging model group, subcutaneous injection of D-galactose (500 mg/kg) daily for 6 weeks; saline control group, the same amount of saline was injected into the neck of the aging model group for 6 weeks; the blank control group, no treatment was performed. The threshold of auditory brainstem response (ABR) was detected. The content of BK_Ca in the perivascular cells of the guinea pig cochlear cells was detected by immunofluorescence technique. The changes of peripheral current density and BK_Ca current were detected by patch clamp technique. The data were analyzed by GraphPad Prism software. Results: Compared with the saline group and the control group, the ABR threshold and the amplitude of the wave I were significantly decreased in the aging model group, and the difference was statistically significant (P<0.01). Compared with the control group, the expression of BK_Ca in the vascular pericytes of guinea pigs in the aging model group was significantly reduced (1.00±0.08 vs 0.27±0.03,the difference was statistically significant P<0.01), and the cell current density and BK_Ca net current value were also significantly reduced with statistically significant (P<0.01). Conclusions D-galactose can successfully induce guinea pig aging model, in which BK_Ca expression decreases and net current value decreases in pericytes of cochlear striavascularis, and changes in BK_Ca expression and function may be related to age-related hearing loss.

Objective To investigate whether Wnt/β-catenin signaling pathway mediating the neuroprotection of isoflurane post-conditioning in hippocampal neurons damage induced by ischemia/reperfusion injury in rats.Methods According to the randomized principle, 60 male Sprague-Dawley rats were randomly divided into five groups (12 rats in each group):sham group (group S), model group (group M), ISO+model group (group MI), ISO+model+DKK-1 group (group MDI) and model+DKK-1 group (group MD).A rat model of middle cerebral artery occlusion (MCAO) was established with 90 min ischemia followed by 24 hreperfusion.Group S was only exposed to one side of the internal carotid artery without fishing line.Isoflurane post-conditioning groups (group MI, MDI) were immediately treated with 1.5％isoflurane for 60 min at the onset of reperfusion.DKK-1 (5μg/kg) was injected intracerebroventricularly 30 min before the model established in group MDI and group MD.After reperfusion for 24 h, Longa score method was used for neurological deficit score.HE staining and Tunel fluorescence was employed to observe the morphological changes of neurons.Immunohistochemistry and Western Blot were applied to detect the expression of target protein in CA1 region.Results Compared with group S, the neurobehavioral score, the number of apoptosis and the expression of Bax and GSK-3βprotein in group M all increased (P<0.05), while the expression ofβ-catenin and Bcl-2/Bax ratio decreased (P<0.05) ;Compared with group M, the neurobehavioral score, the number of apoptosis and the expression of Bax protein were significantly decreased (P<0.05), while the expression of Bcl-2, β-catenin protein and the Bcl-2/Bax ratio were significantly increased (P<0.05) in group MI.Compared with group MI, the neurobehavioral score, the number of apoptosis, Bax and GSK-3βprotein in group MDI were significantly increased (P<0.05), while the Bcl-2, β-catenin protein expression, and Bcl-2/Bax ratio were significantly decreased (P<0.05).Conclusion Isoflurane post-conditioning may protect the hippocampus neurons against cerebral ischemic reperfusion-induced damage via the way that the Wnt/β-catenin signaling pathway regulates the expression levels of Bcl-2 and Bax proteins in rats.