Quercetin activates mitochondrial autophagy via PINK1/parkin pathway to alleviate cerebral ischemia-reperfusion injury in rats

AIM To analyze the regulatory effect of quercetin （QUE） on PTEN-induced putative kinase 1 （PINK1）/parkin mitochondrial autophagy pathway, and to explore the mechanism of quercetin in relieving cerebral ischemia/reperfusion （I/R） injury. METHODS Sixty SD male rats were randomly divided into sham operation group, model group （I/R group）, QUE group,3-methyladenine （3-MA） group and QUE+3-MA group. Administration started in each group 3 days before modeling, once a day, at 30 min after the last administration,except sham group, the other groups used 4-vessel blockage method to establish the whole brain I/R model. On the day after modeling, the neural function was evaluated by neuropathy disability score （NDS）. The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride （TTC） staining. The morphological changes of mitochondria in hippocampus were observed by transmission electron microscopy. The contents of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in hippocampus were measured by ELISA. The activity of superoxide dismutase （SOD） and contents of malondialdehyde （MDA） in hippocampus were detected by xanthine oxidase method, thiobarbituric acid condensation method. Western blot was used to detect the proteinex pression of PINK1, parkin and LC3-II in brain tissue. RESULTS Compared with sham group, the hippocampus of the rats in I/R group and QUE+3-MA group showed swelling of mitochondria, destruction or disappearance of internal crista and other pathological damage,also the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA, the protein expression levels of PINK1, parkin and LC3-II were increased （P<0.05）, while NDS score and activity of SOD were decreased （P<0.05）. Compared with I/R group and QUE+3-MA group, the pathological damage degree of hippocampus in QUE group was reduced, the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA were decreased （P<0.05）, the proteinexpression levels of PINK1, parkin and LC3-II, and NDS score and activity of SOD were increased （P<0.05）.The above indexes in 3-MA group were opposite to QUE group. No significant difference in the above indexes between I/R group and QUE+3-MA group was observed （P>0.05）. CONCLUSION Quercetin activates mitochondrial autophagy and reduces cerebral I/R by regulating the expression of PINK1/parkin pathway proteins.     

Protective effect of atorvastatin on blood brain barrier in rats with focal cerebral ischemia-reperfusion injury

AIM: To explore the protective effects of atorvastatin on blood brain barrier(BBB) in cerebral ischemia-reperfusion(IR) injury and the potential mechanisms involved. METHODS: SD rats were divided into sham group, IR group and atorvastain group. Intraluminal suture method was used to establish cerebral IR model, and the ischemic brain was reperfused for 72 h after the occlusion. The rats in atorvastatin group were administered with atorvastatin(20 mg·kg^-1·d^-1) by gavage once a day for 3 consecutive days after operation. At 72 h after reperfusion, neurological function scores, the water content of the brain tissue, Evans blue(EB) content of ischemic hemisphere, the expression of tight junction(TJ)-associated protein occludin and inflammation factor phosphatidylinositiol 3-kinase-p110 gamma(PI3K-p110γ) were tested and analyzed. RESULTS: In IR group, the rats showed elevated neurological function scores(P<0.01), brain tissue water content(P<0.01) and EB content(P<0.01), accompanied with the down-regulation of occludin expression(P<0.01) and up-regulation of PI3K-p110γ(P<0.01) at 72 h after reperfusion. Compared with IR group, decreased brain edema(P<0.01) and EB leakage(P<0.01) were observed in atorvastatin group, accompanied with increased occludin expression(P<0.01) and decreased PI3K-p110γ expression(P<0.01). However, no statistical difference of the neurological function scores between the 2 groups was observed. CONCLUSION: Atorvastain attenuates cerebral IR injury, which may be associated with the inhibition of inflammatory reactions and the up-regulation of TJ-asso-ciated proteins to maintain the stability of BBB.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Protective effect of gabexate mesilate on blood-brain barrier in rats with cerebral ischemia-reperfusion

AIM To investigate the protective effects of gabexate mesilate （GM） on blood-brain barrier （BBB） permeability in rat model with cerebral ischemia-reperfusion （I/R）. METHODS Adult male SD rats （n=180） were randomly divided into sham group, I/R group, nimodipine （NMP; 2 mg·kg^-1·d^-1） group and GM （5, 10 and 20 mg·kg^-1·d^-1） groups （n=30 in each group）. The rat model of cerebral I/R was established by blocking the middle cerebral artery with thread plug for 2 h. Ten min before modeling, the drugs were given intraperitoneally. The nerve function was detected by Longa scoring method. The permeability of BBB was measured by Evans blue permeation method, and the brain water content was measured by dry-wet weight method. The activity of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, and the content of malondialdehyde （MDA） in brain tissue were determined by biochemical analysis. The content of tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β） and IL-6 was measured by ELISA. The mRNA expression of matrix metalloproteinase-2 （MMP-2）, MMP-9 and nuclear factor-κB （NF-κB） was detected by RT-PCR. The protein levels of MMP-2, MMP-9 and NF-κB were determined by Western blot. RESULTS Compared with I/R group, the Longa score, permeability of Evans blue and brain water content of the rats in GM （10 and 20 mg·kg^-1·d^-1） and NMP （2 mg·kg^-1·d^-1） groups were decreased. The activity of SOD and GSH-Px was increased, while the content of MDA was decreased. The content of TNF-α, IL-1β and IL-6 was decreased, and the mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were significantly down-regulated. Compared with NMP （2 mg·kg^-1·d^-1） group, the Longa score and permeability of Evans blue were decreased in GM （20 mg·kg^-1·d^-1） group, the activity of SOD was increased, and the content of MDA and TNF-α was decreased. The mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were down-regulated. All of the differences were significant （P<0.05 or P<0.01）. CONCLUSION GM has protective effect on BBB in the rats with cerebral I/R. Its mechanism may be related to inhibition of oxidative stress and inflammation, and down-regulation of MMP-2, MMP-9 and NF-κB expression.     

Astrocyte activity and autophagy after radiation-induced brain injury in rats

AIM: To investigate the activity of astrocytes and autophagy-related changes after radiation-induced brain injury (RBI) in rats.METHODS: A total of 36 Sprague-Dawley rats, weighing 180~200 g, were trained for 4 d in the Morris water maze. They were randomly divided into sham group, model group and 3-methyladenine (3-MA) group. The rats in model group and 3-MA group were given single whole-brain X-ray irradiation at a dose of 20 Gy after intraperitoneal anesthesia. After the irradiation was completed, the rats in model group was given 5 μL of NaCl into the lateral ventricle, and the rats in 3-MA group was injected with 3-MA at 600 nmol into the lateral ventricle. After 8 weeks of feeding, Morris water maze was used for measuring the learning and memory abilities. The brain tissues were taken and HE staining was used to observe the pathological changes of the hippocampus. The protein level of GFAP was determined by immunohistochemistry and Western blot for evaluating astrocyte activity. Dual fluorescence staining of GFAP and LC3 was performed for evaluating the changes of autophagy in the astrocytes. The protein level of cleaved caspase-3 detected by Western blot and TUNEL staining in the ipsilateral hippocampus were used to evaluate the apoptosis. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were examined by ELISA to assess the inflammatory response in the hippocampus.RESULTS: Radiation inhibited astrocyte activity, activated autophagy in astrocytes, and aggravated brain damage. 3-MA promoted the activation of astrocytes and promoted the repair of brain tissue damage.CONCLUSION: The injury of rat hippocampus after radiation is obvious, and the number of astrocytes is significantly reduced. 3-MA significantly attenuates the damage. This finding may provide a new approach for the treatment of radiation-induced brain injury.     

Role of autophagy in ursolic acid-induced injury of human umbilical vein endothelial cells

AIM: To investigate the role of autophagy in the injury of human umbilical vein endothelial cells (HUVECs) induced by ursolic acid (UA). METHODS: HUVECs were cultured in vitro with UA at various concentrations for 36 h and the proliferation inhibitory rate of HUVECs was determined by MTT method. The change of ultrastructure was observed under transmission electronic microscope (TEM). The autophagy was observed using fluorescent microscope by monodansylcadaverin (MDC) staining. The protein level and mRNA expression of microtubule-associated protein light chain 3(LC3) and Beclin-1 were detected by Western blotting and RT-PCR, respectively. Cell apoptotic rate was measured by flow cytometry analysis. RESULTS: UA at various concentrations showed significantly dose-dependent inhibitory effect on the proliferation of HUVECs. Autophagy was induced in HUVECs treated with UA as detected by MDC staining and TEM. The protein level and mRNA expression of LC3 and Beclin-1 in HUVECs were significantly increased following the treatment with UA, which was also in a time-dependent manner. Compared with UA group, addition of 3-methyladenine(3-MA) inhibited the increase in autophagic vacuoles and exacerbated the apoptosis. CONCLUSION: Autophagy shows protective effect on the proliferation inhibition of HUVECs induced by UA and the proliferation inhibition can be enhanced by the autophagy inhibitor 3-MA. 3-MA may enhance the apoptotic rate of HUVECs induced by UA.     

Tetramethylpyrazine combined with bone marrow mesenchymal stem cell transplantation inhibits neuronal apoptosis in rats with cerebral ischemia

ATM: To investigate the effects of tetramethylpyrazine (TMP) combined with bone marrow mesenchymal stem cells (BMSCs) on neuronal apoptosis, and Bcl-2 and Bax expression in rats with cerebral ischemia. METHODS: The BMSCs were isolated by the whole bone marrow adherent method and cultured, and those in the 3rd passage were used for tail-vein transplantation. The rats were subjected to right middle cerebral artery occlusion (MCAO) using suture method, and the rats except sham group were randomly divided into model group, BMSCs (1×10⁹ cells/L) group, TMP (40 mg/kg) group and combination (TMP+BMSCs) group with 12 rats in each group. Neurological function was evaluated by modified neurological severity scoring (mNSS) on 1 d, 7 d and 14 d after cerebral ischemia. Toluidine blue staining was performed to detect cerebral infarct volume, HE staining was used to observe brain histopathological change, neuronal apoptosis was observed by TUNEL staining, and the mRNA and protein expression of Bcl-2 and Bax was detected by real-time fluorescence quantitative PCR and Western blot at 14 d after cerebral ischemia. RESULTS: Compared with BMSCs group and TMP group, TMP combined with BMSCs significantly reduced the score of mNSS (P<0.01) and the infarct volume (P<0.01), alleviated the pathological damage in the peripheral area of cerebral ischemia, decreased the number of TUNEL positive cells (P<0.01), increased the expression of Bcl-2 and decreased the expression of Bax at mRNA and protein levels (P<0.01).CONCLUSION: Tetramethylpyrazine combined with transplantation of BMSCs improves the functional recovery, reduces the infarct volume, relieves the ischemic injury of the brain tissue, and attenuates neuronal apoptosis in the rats with cerebral ischemia. The mechanism may be related to regulating the expression of Bcl-2 and Bax.     

Effect of DADLE on lung injury in rats with acute global cerebral ischemia-reperfusion

AIM：To observe the effects of δ opioid receptor agonist DADLE on acute lung injury (ALI) induced by acute global cerebral ischemia-reperfusion in rats. METHODS：SD rats (n=30) were randomly divided into sham group, model (I/R) group and DADLE treatment group. Global cerebral ischemia-reperfusion model was established by a modified 2-vessel occlusion plus hypotension. DADLE (5 mg/kg) treatment was performed via the left jugular injection before reperfusion. After 120-min reperfusion, the pathological changes of the lung tissues were observed under light microscope and electronic microscope. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) level were detected. The partial pressure of arterial oxygen (PaO2) was also measured. RESULTS：In I/R group, widened alveolar septum, capillary dilatation and congestion, endovascular and perivascular cells in the lung with neutrophil infiltration, and significantly reduced type II epithelial cell surface microvilli, alveolar lumen cavity and trachea with serous exudate were observed. SOD activity decreased, but the MDA level increased. Compared with I/R group, the SOD activity increased and MDA level decreased in DADLE treatment group, with significantly reduced lung congestion, the degree of lung injury, and the infiltration of neutrophils. Compared with I/R group, the PaO2 and oxygenation index in DADLE treatment group were increased. CONCLUSION：Various degrees of pulmonary injury were observed in acute global cerebral ischemia reperfusion model. DADLE might have a protective effect on lung tissues of ALI in rats.     

Effects of bFGF on brain edema,nerve function injury and autophagy related protein in rats with traumatic brain injury

AIM:To study the effects of basic fibroblast growth factor (bFGF) on brain edema, nerve function damage and autophagy related proteins in rats with head injury. METHODS:The rat model of craniocerebral injury (CI) was constructed. The rats were divided into control group, CI group, and low-, middle-and high-dose bFGF groups (n=10). The CI model was established in CI group, while the rats in control group were not given epidural impact. The rats in low-dose, middle-dose and high-dose bFGF groups were given bFGF at 2, 4 and 6 μg, respectively, by intraperitoneal injection after 30 min. The neurological function in the rats was evaluated by improved neurological function scoring. The rat brain tissues were taken, and the water content was detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the brain tissue were measured by ELISA. The malondialdehyde (MDA) content was analyzed by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was examined by WST-8 assay. The glutathine peroxidase (GSH-Px) activity was detected by colorimetric method. The protein levels of autophagy related proteins LC3-Ⅱ and beclin-1 in the brain tissues were determined by Western blot. RESULTS:The neurological function score was increased significantly of the rats in CI group. The rat model of craniocerebral injury was successfully constructed. Neurological function scores in the rats in low-dose, middle-dose and high-dose bFGF groups were reduced, the water content of the brain tissue was also reduced (P<0.05). The levels of TNF-α, IL-6 and IL-1 β were decreased in the brain tissues (P<0.05), the content of MDA was declined (P<0.05), the activities of SOD and GSH-Px were increased (P<0.05), the protein levels of LC3-Ⅱ and beclin-1 were decreased, compared with the untreated rats in CI group (P<0.05). CONCLUSION:bFGF improves the nerve function of the rats with craniocerebral injury, reduces the water content of the brain tissue, reduces the expression of autophagic protein LC3-Ⅱ and beclin-1.The mechanism is related to the inhibition of inflammatory reaction and oxidative damage.     

Celecoxib inhibits viability,induces apoptosis and inhibits autophagy in acute myeloid leukemia cell lines HL-60 and HL-60A

AIM: To investigate the effects of celecoxib on viability, apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A. METHODS: The HL-60 cells and HL-60A cells were cultured with various concentrations (0, 20, 40, 60, 80 and 100 μmol/L) of celecoxib. The inhibitory effect of celecoxib on the cell viability was evaluated by MTT assay. Apoptosis was analyzed by Annexin-V/PI staining. Apoptosis-related and autophagy-related proteins were determined by Western blot. RESULTS: IC₅₀ of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1 μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively. For HL-60A cells, the corresponding IC₅₀ were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively. The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ⁺ PI^-, Annexin-Ⅴ⁺ PI⁺ and Annexin-Ⅴ^-PI⁺ cells were increased in the HL-60 cells, and those of Annexin-Ⅴ⁺PI^- and Annexin-Ⅴ⁺ PI⁺ cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib, the induction of apoptosis was observed, the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated, the autophagy-related proteins LC3 II and P62 were both increased, and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed, indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement. CONCLUSION: Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis. Celecoxib inhibits mTOR-independent autophagy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells.     

All-trans retinoic acid attenuates blood-brain barrier injury induced by cerebral ischemia-reperfusion in rats through JNK/P38 MAPK signaling pathway

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on blood-brain barrier after cerebral ischemia-reperfusion (CIR) injury in rats and its possible role mechanism.METHODS: Male SD rats were randomly divided into sham group, model (CIR) group and CIR+ATRA (10, 30 and 90 mg/kg) groups. The rat model of CIR injury was established by MCAO thread occlusion method. After ischemia for 1.5 h and reperfusion for 24 h, the neurological functional behavioral score, cerebral infarction volume, brain water content and Evans blue content were determined. The activity of matrix metalloprotein-9 (MMP-9) was measured by gelatin zymography. The protein levels of claudin-5, occludin, ZO-1, JNK, p-JNK, P38, p-P38 and MMP-9 in the brain tissues were determined by Western blot.RESULTS: Compared with CIR model group, ATRA at 30 mg/kg significantly improved neurological function, and decreased cerebral infarction volume, brain water content, Evans blue content and the degradation of tight junction proteins in ischemic area (P<0.01). The activity and protein expression of MMP-9 in ischemic brain tissue were decreased (P<0.01). The phosphorylation of JNK and P38 was inhibited and the protein levels of p-JNK and p-P38 were decreased (P<0.01).CONCLUSION: ATRA reduces the damage of brain tissue and the destruction of blood-brain barrier induced by CIR in rats. The protective effect may be related to inhibiting the activation of JNK/P38 MAPK signaling pathway and MMP-9.     

Effect of 3-n-butylphthalide pretreatment on structure and function chan-ges of blood brain barrier in rat model of focal cerebral ischemia-reperfusion injury

AIM: To investigate whether pretreatment with 3-n-butylphthalide (NBP) ameliorates blood brain barrier (BBB) dysfunction in a rat model of focal cerebral ischemia-reperfusion injury (CIRI). METHODS: Male SD rats (n=120, 24 rats in each group) were randomly divided into sham operation group (sham group), model group (IR group), low dose group of NBP pretreatment (NBP I group), medium dose group of NBP pretreatment (NBP II group) and high dose group of NBP pretreatment (NBP III group). The model of CIRI was established by a suture method. After ischemia for 2 h and reperfusion for 24 h, the contents of water and Evans blue (EB) were detected. The pathological changes of the BBB ultrastructure were observed under transmission electron microscope. The protein level of matrix metalloproteinases 9 (MMP-9) was measured by immunohistochemical technique. The mRNA expression of MMP-9 was determined by real-time PCR. RESULTS: After CIRI, the content of water and EB was progressively increased, the BBB was damaged seriously, and the expression of MMP-9 was significantly up-regulated compared with sham group (all P<0.01). Pretreatment with NBP significantly decreased the contents of water and EB, relieved morphological damage of the BBB, and reduced the expression of MMP-9 obviously (all P<0.01). Compared with NBP I group, the changes in NBP II and III group were remarkable (P<0.05), but the difference between NBP II group and NBP III group was not obvious (P＞0.05). CONCLUSION: Pretreatment of 3-n-butylphthalide has preventive effect against cerebral ischemia reperfusion injury in the rats, which may be related to decrease the expression of MMP-9 and reduce the permeability of blood brain barrier.     

Autophagy induces mitochondrial dysfunction in neurons from neonatal rats with hypoxic ischemic encephalopathy

AIM: To explore the influence of autophagy on the induction of mitochondrial dysfunction in the neurons in a neonatal rat hypoxic ischemic encephalopathy (HIE) model. METHODS: Ten-day-old rat pups (n=30) were randomly divided into sham group and model group. The rats in the latter group were subject to hypoxia-ischemia treatment via unilateral common carotid artery ligation. The rats were sacrificed for brain pathological examination, and the protein levels of cleaved caspase-3 and LC3B-II were detected by immunohistochemical analysis. For the in vitro experiments, the autophagy of primarily cultured rat neurons was observed after hypoxia, and Western blot and mitochondrial function testing were also performed. RESULTS: Compare with sham group, the hypoxia-ischemia treatment caused atrophy and apoptosis of neurons, and ventricular area enlargement of rat brains. Immunohistochemical results demonstrated significantly higher levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase-3 and LC3B-II (P<0.01). In vitro experiments demonstrated that hypoxia induced autophagy and apoptosis in the neurons. Compared with sham group, there were higher levels of reactive oxygen species and mitochondrial superoxide, and lower mitochondrial membrane potential in the model group (P<0.01). CONCLUSION: In neonatal HIE rat model, the hypoxia-induced mitochondrial dysfunction is related to apoptosis and autophagy. It will provide a new idea for administration of autopahgy inducer agents in treatment of HIE.     

Protective effects of glutamine pretreatment on occludin protein in rats with intestinal ischemia-reperfusion injury

AIM: To determine the effects of glutamine(Gln) pretreatment on occludin protein in the rats with intestinal ischemia-reperfusion(I/R) injury. METHODS: Male Wistar rats(n=30) were randomly divided into 3 groups(n=10):sham group, I/R group and Gln pretreatment group. The rats in Gln pretreatment group were pretreated with Gln at dose of 1 g·kg^-1·d^-1 by orogastric route for 7 d, and those in the other 2 groups were pretreated with the same volume of normal saline. Intestinal I/R was induced by 30-min occlusion of the superior mesenteric artery followed by 24 h of reperfusion. After the operation, the levels of IL-10, IL-2, TNF-α, SOD and MDA were measured. The occludin protein was determined by the methods of immunohistochemistry and Western blotting. RESULTS: The occludin protein level in I/R group was significantly lower than that in sham group and Gln group(P<0.05). The levels of MDA and TNF-α in I/R group were significantly higher than those in sham group and Gln group(P<0.05). The levels of SOD, IL-10 and IL-2 in I/R group were significantly lower than those in sham group and Gln group(P<0.05). CONCLUSION: Glutamine has a protective effect on occludin protein in intestinal ischemia-reperfusion injury. The mechanism may be rela-ted to oxidative stress response and inflammatory inhibition.     

Effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion-induced early liver injury in rats

AIM：To explore the effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion (IR)-induced early liver injury. METHODS：Adult SD rats (n=35) were randomized into 5 groups with 7 rats each: sham operation group (S group), IR group, cromolyn sodium treatment group (IR+C group, 25 mg/kg), ketotifen treatment group (IR+K group, 1 mg/kg), compound 48/80 treatment group (IR+CP group, 0.75 mg/kg). IR was induced by superior mesenteric artery occlusion for 75 min followed by 4 h of reperfusion. The agents were intravenously administered 5 min before reperfusion. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histamine, and the liver levels of lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed. The liver histopathologic changes were also evaluated. RESULTS：IR resulted in severe liver injury as demonstrated by great increases in injury scores, concomitant significant increases in serum levels of AST, ALT and histamine, and liver levels of LDH, TNF-α, IL-8, and MDA, accompanied by reduced SOD activity (all P<0.05 vs S group). Treatment with cromolyn sodium or ketotifen markedly alleviated IR-mediated liver injury as confirmed by significant reduction of the above biomedical changes, whereas compound 48/80 further aggravated liver injury by dramatically enhancing the biomedical changes (all P<0.05 vs IR group). CONCLUSION：Inhibition of mast cell function before reperfusion may reduce early liver injury induced by intestinal ischemia reperfusion. Histamine, oxidative stress and inflammatory response may provide promising effects on it.     

Effects of simvastatin on expression of Bcl-2 and Bax in myocardium of rats with renal ischemia-reperfusion injury

AIM: To observe the effect of simvastatin on myocardial tissue after renal ischemia-reperfusion injury and its mechanism. METHODS: A rat model of renal ischemia-reperfusion injury was prepared by clamping the bilateral renal arteries for 45 min. The rats (n=36) were randomly divided into sham operation group, renal ischemia-reperfusion (I/R) group and simvastatin group with 12 rats in each group. The content of serum creatinine (SCr), blood urea nitrogen (BUN) and myocardial tissue malondialdehyde (MDA), the myocardial activity of lactate dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD), and the myocardial protein expression of Bcl-2 and Bax were detected. RESULTS: Compared with sham operation group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in I/R group were significantly increased (P<0.05), and the activity of SOD was significantly decreased (P<0.05). Compared with I/R group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in simvastatin group were significantly decreased (P<0.05), while SOD activity was enhanced (P<0.05). The protein expression of Bcl-2 and Bax in sham operation group was less than that in I/R group (P<0.05), and the protein level of Bax in simvastatin group was significantly lower than that in I/R group (P<0.05), while the protein level of Bcl-2 was increased (P<0.05). CONCLUSION: Simvastatin has a protective effect on the myocardium of the rats with renal ischemia-reperfusion injury, and the protective mechanism may be related to the elimination of free radicals by simvastatin, increase in the protein expression of Bcl-2 and decrease in the protein expression of Bax.     

PI3K/Akt signaling pathway regulates autophagy induced by acute kidney injury in septic rats

AIM：To investigate the autophagy induced by sepsis and acute kidney injury, and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process. METHODS：The rats were subjected to cecal ligation and puncture (CLP) or sham operation. Histopathologic changes of the renal tissues were examined by HE staining. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by chemical colorimetry. The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting. In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy. The protein expression of LC3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting. These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected. RESULTS：Compared with sham group, the severe changes of renal histopathological injuries in CLP groups were observed, the levels of BUN and SCr in CLP groups were significantly increased. LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP. After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose- and time-dependent fashion. The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment. Treatment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells. CONCLUSION：Autophagy in the kidney is induced by sepsis and acute kidney injury. PI3/Akt signaling pathway may be involved in this process.     

Effect of microRNA-155 on regulation of angiogenesis in diabetic rats with cerebral ischemic injury

AIM: To evaluate the effect of microRNA-155(miRNA-155) on the regulation of angiogenesis in diabetic rats with cerebral ischemic injury. METHODS: Adult male Sprague-Dawley rats were randomly divided into 5 groups:sham group, cerebral ischemia group, diabetic cerebral ischemia group, diabetic cerebral ischemia+miRNA-155 inhibitors group and diabetic cerebral ischemia+scramble group. Diabetes model was made by injection of streptozocin and permanent cerebral ischemic model was developed by suture-occluded method. The scores of neurological deficit and infarct volume were estimated at 24 h after cerebral ischemia. miRNA-155 level was detected by real-time polymerase chain reaction. The expression of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31) and vascular endothelial growth factor(VEGF) was detected by Western blotting. RESULTS: miRNA-155 inhibitor significantly reduced miRNA-155 levels in the ischemic cortex(P<0.05), improved the scores of neurological deficit, reduced infarction size and upregulated the levels of CD31 and VEGF(P<0.05). CONCLUSION: miRNA-155 has a critical role in the regulation of angiogenesis in diabetic rats with cerebral ischemia. Down-regulation of miRNA-155 using miRNA-155 inhibitor attenuates brain infarct injury in diabetic rats.