Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.     

抗坏血酸对苹果组培苗耐热性的生理效应

用0.5 mmol/L抗坏血酸(ASA)处理苹果组培苗,在42℃的条件下进行不同时间高温胁迫处理,研究了ASA 处理对高温胁迫下苹果细胞膜相对透性、丙二醛(MDA)含量、抗氧化酶活性以及抗氧化剂含量的影响。结果表明, ASA处理可以有效地抑制抗氧化酶活性的下降,提高酶活性;同时还抑制ASA含量的下降,延缓了膜透性和丙二醛 含量的增加;但抑制了GSH含量的增加。这些结果说明,ASA处理可以增强苹果的耐热性。     

Effect of salvianolic acid B on vasodilatory function,NF-κB activation and inflammatory cytokine expression in diabetic rats

AIM: To investigate the ameliorative effect of salvianolic acid B on vasodilatory function in diabetic rats and the possible mechanisms. METHODS: SD rats (n=40) were fed on high-sugar and high-fat diet for 4 weeks, followed by a single intraperitoneal injection of streptozotocin (40 mg/kg). The rats with random blood glucose level over 16.7 mmol/L were considered diabetic and randomly allocated to 3 groups, namely model group, low dose (80 mg·kg^-1·d^-1) of salvianolic acid B group and high dose (160 mg·kg^-1·d^-1) of salvianolic acid B group. The rats in salvianolic acid B groups were intragastrically administered with corresponding doses of salvianolic acid B for 6 weeks. Vasodilatory function was measured as endothelium-dependent and-independent vasodilation of the aortic rings. The primary histopathological changes of aorta were observed by HE staining. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were measured by ELISA. The levels of total antioxidant capacity, malondialdehyde (MDA) and nitric oxide (NO) in aortic tissues were evaluated by colorimetric assays. The protein levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1), and the activation of nuclear factor-κB (NF-κB) were determined by Western blot. RESULTS: Treatment with salvianolic acid B evidently ameliorated endothelium-dependent diastolic function and pathological changes of aorta in diabetic rats (P<0.05 or P<0.01). Supplementation with salvianolic acid B resulted in significant increases in NO content and total antioxidant capacity in aortic tissues, accompanied by marked decreases in the level of MDA in aorta tissues and the serum levels of IL-6, TNF-α and CRP (P<0.05 or P<0.01). Salvianolic acid B markedly down-regulated NF-κB p65 nuclear translocation and protein expression of ICAM-1 and MCP-1 in aorta tissues (P<0.05 or P<0.01). CONCLUSION: Salvianolic acid B effectively ameliorates endothelium-dependent diastolic function of aorta in diabetic rats, which might be attributed to suppression of NF-κB activation and subsequent expression of inflammatory cytokines. The beneficial effect of salvianolic acid B on vascular endothelium might be derived from its antioxidant capacity.     

Synthetic kainate acid induces seizures in rats

AIM: To observe the effect of synthetical kainic acid (SKA) on inducing epileptic seizure in rats. METHODS: Forty Wistar rats were divided into normal control group, 5 mg/kg, 10 mg/kg and 12 mg/kg SKA-treated groups and kainic acid (KA) positive control group. The SKA was injected intraperitoneally. The changes of the epileptic behaviors were observed for 8 h successively and the electroencephalography was recorded for 3.5 h. RESULTS: SKA induced epileptic seizures at the dose of 5-12 mg/kg by intraperitoneal injection. No apparently difference between SKA and KA on the changes of epileptic behaviors or electroencephalography was observed. However, the epileptic seizure induced by SKA showed obvious stages, regular pattern and action steady. In addition, SKA caused lower fatality than KA did. CONCLUSION: SKA induces epileptic seizure in rats and the dose of 10 mg/kg by intraperitoneal injection is the optimal.     

Effects of riboflavin and ascorbic acid on apoptosis and proliferative inhibition of mouse thymocytes induced by deoxynivalenol in vivo

AIM: To explore the effects of riboflavin and ascorbic acid on the apoptosis induced by deoxynivalenol(DON) in mouse thymocytes. METHODS: The effects of riboflavin and ascorbic acid on the apoptosis and proliferation inhibition of thymocytes induced by DON in KM mice were studied with animal experiment, DNA agarose gel electrophoresis and flow cytometric DNA content analysis. RESULTS: Apoptosis rate of thymocytes in DON (4 mg/kg) treated group was13.73%±15.3% The percentages of apoptosis in riboflavin (1.25 mg/kg-10mg/kg) and ascorbic acid (25 mg/kg-100mg/kg) pretreated thymocytes groups were significantly lower than that in DON group (P <0.05). The result of DNA agarose gel electrophoresis showed that the characteristic ladder pattern of apoptosis was found in DON-treated thymocytes, but not in control and riboflavin pretreatment and ascorbic acid pretreatment groups The significant differences in proliferation index were not found among DON-treated thymocytes and riboflavin and ascorbic acid-pretreated thymocytes CONCLUSION: Pretreatment with riboflavin and ascorbic acid inhibit apoptosis of mouse thymocytes induced by DON in certain extent and have no effect on proliferation inhibition by DON.     

Effect of oxidative stress-induced autophagy on proliferation and apoptosis of MSCs

AIM: To investigate whether oxidative stress is able to induce autophagy in mesenchymal stem cells (MSCs), and to explore the effects of autophagy on MSC proliferation and apoptosis under oxidative stress circumstance as well as the underlying mechanism for promoting the therapeutic effects of transplanted MSCs on treating diabetes mellitus erectile dysfunction (DMED). METHODS: Hydrogen peroxide (H₂O₂) was applied to simulate the oxidative stress circumstance. The effects of H₂O₂ at concentration of 0, 50, 100, 200, 400 μmol/L on the viability of MSCs were tested by the method of Trypan blue exclusion and MTT assay respectively . The methods of MTT assay, Western blot and transmission electron microscope (TEM) were used to explore the effects of H₂O₂ on MSC apoptosis and autophagy. RESULTS: The proliferation of MSCs was obviously inhibited by H₂O₂ in a dose-dependent manner (P<0.01) and the 50% inhibiting concentration (IC₅₀) was (384.58±16.89) μmol/L. H₂O₂ induced apoptosis and autophay of MSCs. The proliferation rate of MSCs was suppressed by H₂O₂ significantly (P<0.05), with a further decline by blockade of autophagy (P<0.05) whereas increased by blockade of apoptosis (P<0.05). H₂O₂ induced MSCs apoptosis obviously (P<0.05), with an augment of apoptosis (P<0.05) by blockade of autophagy. Furthermore, the H₂O₂ increased expression of cleaved caspase-3 and cleavage of poly ADP-ribose polymerase 1 (PARP1), Which were decreased by apoptosis blockade whereas were enhanced by blockade of autopahgy. CONCLUSION: Oxidative stress plays a dual role in MSC survival, which induces MSC apoptosis and autophagy. Moreover, blockade of autophagy intensifies MSC apoptosis. Therefore, it is a promising method to ameliorate the effects of stem-cell based therapy on DMED by enhancing protective autophagy to increase the survival rate of transplanted MSCs against oxidative stress circumstance caused by diabetes mellitus.     

Effect of insulin on early myocardial oxidative stress in severely burnt rats

AIM：To investigate the effect of insulin on early myocardial oxidative stress in severely burnt rats. METHODS：Twenty-four Sprague-Dawley rats were randomly divided into three groups (8 rats in each group): control group (sham scald group), scald injury group and scald injury + insulin group. The rats in the latter two groups were subject to third-degree burn with 30% total burn surface area (TBSA) on the back, and then received intraperitoneal injection of normal saline (40 mL/kg) immediately. The rats in scald injury + insulin group were subcutaneously injected with insulin (1 U/kg), while those in scald injury group received subcutaneous injection of the same volume of normal saline. All rats were sacrificed 24 h after scald, and blood samples from abdominal aorta and myocardial tissues were taken. Blood glucose (BG) content, blood lactate dehydrogenase (LDH) and creatine kinase (CK) activity, and myocardial oxidative and antioxidative indexes, including malondialdehyde (MDA), xanthine oxidase (XO), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GPx), were detected by spectrophotometry. RESULTS：(1) Compared with control group, BG levels in scald injury group and scald injury + insulin group were significantly elevated (P<0.05). But BG in scald injury + insulin group was significantly lower than that in scald injury group (P<0.05). (2) Compared with control group, the activity of LDH and CK in scald injury group was significantly increased (P<0.05), while that in scald injury + insulin group was significantly lower than that in scald injury group (P<0.05). (3) Compared with control group, the MDA content and the XOD and MPO activity in scald injury group were significantly increased (P<0.05), while the activity of SO, CAT and GPx was significantly decreased (P<0.05). Compared with scald injury group, the MDA content and the XO and MPO activity in scald injury + insulin group were significantly reduced (P<0.05), while the activity of SOD, CAT and GPx was significantly elevated (P<0.05). CONCLUSION：Insulin intervention attenuates early myocardial oxidative stress in burnt rats and decreases the rise in myocardial enzyme activity, thus exerting a cardioprotective effect.     

Characterization of blueberry monodehydroascorbate reductase gene and changes in levels of ascorbic acid and the antioxidative capacity of water soluble antioxidants upon storage of fruits under various conditions

Blueberry (Vaccinium corymbosum) is among the richest fruits in ascorbic acid (AA), which is the most important antioxidant involved in the ascorbate–glutathione cycle. In this cycle monodehydroascorbate reductase (MDAR) is the enzymatic component involved in the regeneration of reduced ascorbate. Here we report on the isolation of a full-length cDNA from blueberry encoding a protein of 433 amino acids homologous to the MDARs of Pisum sativum and Vitis vinifera. To assess changes in the expression of blueberry MDAR after harvest, a storage trial was initiated, and the major results were: (1) A dramatic loss in AA occurred under all storage conditions. However, storing fruit under low O₂, combined with high CO₂ level (up to 18%) resulted in better preservation of AA. (2) The antioxidative capacity of water soluble antioxidants (ACW) decreased under all storage conditions, even after 3 weeks storage time, and decreasing O₂ levels did not result in preservation of the ACW. The northern blot hybridization showed a clear differential expression between freshly harvested and stored fruits as well as between fruits stored under various storage condition, in accordance with the above-mentioned changes.     

Effect of taurine on kidney injury induced by paraquat poisoning in rats

AIM:To study the effects of taurine at different doses on renal oxidative stress and inflammation induced by paraquat in rats.METHODS:Male SD rats (n=48) were randomly divided into 4 groups:negative control group,paraquat group,paraquat+low-dose taurine group,and paraquat+high-dose taurine group.The serum levels of creatinine and urea nitrogen were detected by a biochemical analyzer.The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by colorimetry.The plasma concentrations of IL-6 and intercellular adhesion molecule (ICAM)-1 were detected by ELISA.Renal reactive oxygen species (ROS) was checked by fluorescence probe dihydroethidium (DHE).The protein levels of renal p-P38 MAPK,p-ERK1/2 and p-JNK were determined by Western blot.The mRNA expression of TNF-α,IL-6 and TGF-β1 was detected by real-time PCR.RESULTS:Serum creatinine and urea nitrogen increased after paraquat poisoning,and decreased after feeding with taurine in poisoned rats,with better result in high-dose taurine group.Taurine reduced the oxidative stress and inflammation in the renal tissue,and also reduced the protein levels of p-JNK,p-ERK1/2 and p-P38 in the kidney of paraquat-poisoned rats.CONCLUSION:Taurine attenuates renal injury induced by paraquat poisoning in rats.The mechanism may be related to reducing renal MAPK activity,oxidative stress and inflammatory response.     

Role of chlorogenic acid in endothelial dysfunction in db/db mice

AIM: To explore the effects of chlorogenic acid (CGA) on endothelial dysfunction in db/db mice and the possible mechanism. METHODS: Male db/db mice (n=12) were divided into control group and CGA group, with 6 mice in each group. The mice in CGA group were treated with diet containing 0.02% CGA, while the mice in control group were given normal diet only. The observation period was 12 weeks. Fasting blood glucose level, tail blood pressure and the body weight were analyzed each week. At the end of the 12th week, the mice were anesthetized and blood was taken from carotid artery. The plasma levels of heme oxygenase-1 (HO-1), catalase (CAT), NAD(P)H dehydrogenase quinone 1 (NQO1) and glutathione peroxidase-1 (GPx-1) were measured by ELISA. The mouse aortas were isolated, and the superoxide anion and nitric oxide (NO) levels were measured by DHE and DAF-2 DA staining, respectively. Wire Myograph System was used to detect the vasorelaxation of db/db mouse aorta. The protein levels of peroxisome proliferator-activated receptor α (PPARα), nuclear factor E2-related factor 2 (Nrf2), phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated endothelial NO synthase (p-eNOS), P22^phox and P47^phox were determined by Western blot. RESULTS: Dietary CGA decreased fasting blood glucose and body weight in db/db mice as compared with control group (P<0.01 or P<0.05). The plasma levels of HO-1, CAT, NQO1 and GPx-1 in CGA group were higher than those in control group (P<0.01 or P<0.05). Administration of CGA for 12 weeks attenuated superoxide anion level, increased NO level in the mouse endothelium and improved endothelium-dependent relaxation of the db/db mouse aorta. CGA also increased the protein levels of PPARα, Nrf2, p-AMPK and p-eNOS, and decreased P22^phox and P47^phox levels (P<0.01). CONCLUSION: Dietary CGA improves db/db mouse endothelium-dependent relaxation. This effect may be related to the increases in the levels of antioxidant molecules PPARα, Nrf2 and p-AMPK, and the up-regulation of antioxidant capacity, thus decreasing the oxidative stress, promoting eNOS phosphorylation, and increasing NO level.     

Inhibitory effect of salidroside on liver oxidative stress in rats with non-alcoholic steatohepatitis

AIM：To explore the effects of salidroside (SDS) on the oxidative stress in liver tissues from rats with non-alcoholic steatohepatitis (NASH). METHODS：The experimental animal model of NASH was established in SD rats fed on high-fat and high-cholesterol diet (HFHCD) for 14 weeks. SDS (300 mg·kg-1·d-1) was administered via gavage daily from the 8th week after HFHCD feeding. At the end of the 14th week, serum samples were taken for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC). Liver tissues were taken for TG, TC, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) detection. The content of 8-isoprostaglandin F2α (8-iso-PGF2α) in liver tissues was determined by ELISA. The liver histopathological changes were observed under microscope with HE staining. The expression of 8-hydroxydeoxyguanosine (8-OHdG) in liver tissues was determined by immunohistochemical staining. RESULTS：At the end of the 14th week, ALT, AST, TG and TC in serum, and TG, TC, MDA and 8-iso-PGF2α in liver homogenate in NASH model group were significantly increased compared with control group, while SOD and GSH in liver tissues were significantly decreased. The liver expression of 8-OHdG in NASH model group was higher than that in control group. Compared with NASH model group, SDS significantly inhibited the elevation of serum ALT, AST, TG and TC, and liver TG, TC, MDA and 8-iso-PGF2α, but increased the levels of SOD and GSH in liver tissues. Meanwhile, the liver histopathological score and 8-OHdG expression were decreased in SDS treatment group. CONCLUSION：Salidroside can effectively inhibit steatohepatitis induced by HFHCD, and its antioxidant effect may be one of the mechanisms.     

Effects of apelin-13 on oxidative stress induced by high uric acid in adipocytes

AIM: To study the effects of apelin-13 on oxidative stress induced by high uric acid in 3T3-L1 adipocytes and its underlying mechanisms. METHODS: 3T3-L1 adipocytes were stimulated with uric acid at 10 mg/dL for 48 h. Some of the adipocytes were administered with 1 μmol/L apelin-13 in the presence of uric acid at 10 mg/dL. The adipocytes stimulated with 100 μmol/L H₂O₂ were served as positive controls. The intracellular reactive oxygen species (ROS) concentrations were detected by flow cytometry. The biochemical kits were used to measure the activities of superotide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and NADPH oxidase (NOX) activity, and the content of malondialdehyde (MDA) in the cell lysate and the supernatant. The mRNA levels of renin-angiotensin system (RAS) components, including angiotensinogen (AGT), angiotensin-converting enzyrne1 (ACE1), angiotensin II type 1 receptor (AT1R) and AT2R, as well as angiotensin II receptor -like 1 (APJ) were measured by real-time PCR. The concentrations of angiotensin II (AngⅡ) in the cell lysate and the supernatant were measured by ELISA. RESULTS: Adipocytes stimulated with uric acid at 10 mg/dL had lower activities of antioxidant enzymes (SOD, GSH-PX and CAT) and higher levels of NOX activity and MDA content (P < 0.05). Accordingly, the intracellular ROS levels were found to be dramatically increased. However, apelin-13 administration attenuated uric acid-induced oxidative stress in the 3T3-L1 adipocytes. Uric acid at 10 mg/dL upregulated the mRNA expression of local RAS, enhanced AngⅡ concentrations both in the cell lysate and the supernatant, and down-regulated the mRNA level of APJ in the adipocytes (P < 0.05). Conversely, apelin-13 partially reversed these parameters. CONCLUSION: Apelin-13 attenuates oxidative stress induced by uric acid, may be via down-regulation of local RAS expression in the 3T3-L1 adipocytes.     

Effect of endoplasmic reticulum stress on brain injury following chronic intermittent hypoxia in growing rats

AIM: To explore the role of endoplasmic reticulum stress (ERS) in brain injury following chronic intermittent hypoxia in growing rats and the protective effect of treatment with salubrinal. METHODS: Healthy male SD rats (3~4-week-old, 100~120 g, n=64) were randomly assigned to 8 groups (8 rats in each group):the groups of intermittent hypoxia for 2 and 4 weeks (2IH and 4IH), the groups of control (C) for 2 and 4 weeks (2C and 4C), the groups of dimethylsulfoxide (DMSO) for 2 and 4 weeks (2DMSO and 4DMSO) and the groups of salubrinal for 2 and 4 weeks (2SAL and 4SAL). The 8-arm radial maze was used to assess the working memory error (WME), reference memory error (RME) and total error (TE) of the rats. The changes of neuronal apoptosis in the hippocampus were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The activity of superoxide dismutase (SOD), and the protein levels of endoplasmic reticulum stress marker compounds, C/EBP homologous protein (CHOP), phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2α) and phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), were analyzed. RESULTS: Chronic intermittent hypoxia (CIH) significantly increased RME, WME, TE and neuronal apoptotic index (AI) (P<0.01), and decreased the activity of SOD in the hippocampus and serum (P<0.01). The protein levels of p-PERK and CHOP progressively increased in hippocampus in IH groups (P<0.01), and p-eIF2α was downregulated (P<0.05). Treatment with salubrinal significantly decreased RME (P<0.05), WME (P<0.05), TE (P<0.01) and AI (P<0.01), and increased the activity of SOD (P<0.01). Salubrinal induced the phosphorylation of eIF2α significantly after CIH in hippocampus and downregulated the level of CHOP (P<0.01). CONCLUSION: Chronic intermittent hypoxia upregulates the protein levels of p-PERK and CHOP in the hippocampus, and decreases p-eIF2α protein and the activity of SOD. Salubrinal, a selective inhibitor of eIF-2α dephosphorylation, increases the activity of SOD and prevents CHOP protein activation throughout CIH exposure. Our findings suggest ERS-mediated cell apoptosis is one of the underlying mechanisms of cognitive dysfunction in OSAHS children. Further, a specific ERS inhibitor salubrinal should be tested for neuroprotection against CIH-induced brain injury.     

Effect of cimicifugoside H-1 on amino acid neurotransmitters in striatum of rats with cerebral ischemia

AIM: To study the neuroprotective effect of cimicifugoside H-1 and to explore the mechanism involved by determining the variation of amino acid neurotransmitters in extracellular fluid in the striatum of rats with cerebral ischemia. METHODS: The rats were randomly divided into sham-operated, cerebral ischemia, high-, middle- and low-dose cimicifugoside H-1, and ginkgo groups. Focal cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) with sutures. Normal saline was intraperitoneally injected into the rats in sham-operated group and cerebral ischemia group, while ginkgo and different doses of cimicifugoside H-1 were injected into the rats in ginkgo group and high-, middle- and low-dose cimicifugoside H-1 groups, respectively, once a day for 7 d. The striatal fluids were gained in vivo by brain microdialysis. The contents of aspartic acid, glutamic acid, glycine and γ-aminobutyric acid were tested by high-performance liquid chromatography electrochemical detector system. RESULTS: Compared with sham-operated group, the contents of excitatory amino acids (aspartic acid and glutamic acid) were significantly increased 2 h after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid were significantly decreased 2 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid did not show significant decrease 2 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. Compared with sham-operated group, the contents of inhibitory amino acid (γ-aminobutyric acid and glycine) were significantly decreased 3 h after cerebral ischemia in cerebral ischemia group (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine were significantly increased 3 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine did not show significant decrease 3 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. CONCLUSION: Cimicifugoside H-1 restrains the excessive releases of excitatory amino acids and increases the contents of inhibitory amino acids during cerebral ischemia. It doesn't only penetrate through the blood brain barrier, but also regulates the disorder of excitatory amino acid during cerebral ischemia, thus showing the protective function to cerebral neuron during cerebral ischemia.     

Protective effect of salidroside on rats with hypoxic pulmonary hypertension by suppressing oxidative stress

AIM: To study whether salidroside plays a protective role in hypoxia-induced pulmonary hypertension by suppressing oxidative stress. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia (N) group, hypoxia for 4 weeks (H₄) group, low-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 16 mg/kg, H₄S16) group and high-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 32 mg/kg, H₄S32) group. The mean pulmonary arterial pressure (mPAP), the weight ratio of right ventricle/(left ventricle+septum)[RV/(LV+S)] and vessel wall area/vessel total area (WA/TA) were evaluated. The levels of malondialdehyde (MDA) in the serum and lung tissues were detected by colorimetric method. The levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in the serum and lung tissues were measured by ELISA. The activity of superoxide dismutase (SOD) in the serum was analyzed by hydroxylamine method. The expression of NAPDH oxidase 4 (NOX4) and SOD1 in the lung tissues was determined by Western blot. RESULTS: Compared with N group, the levels of mPAP, RV/(LV+S) and WA/TA in H₄ group were significantly increased, which were apparently attenuated by salidroside injection in a dose-dependent manner. Meanwhile, salidroside administration apparently decreased the levels of MDA and 8-iso-PGF_2α in the serum and lung tissues, as well as the expression of NOX4 in the lung tissues. Besides, compared with N group, the activity of SOD in the serum and the expression of SOD1 in the lung tissues in H₄ group were significantly decreased, while administration of salidroside increased the activity of SOD in the serum and the expression of SOD1 in the lung tissues in a dose-dependent manner. CONCLUSION: Salidroside protects the pulmonary vessels from remodeling and attenuates hypoxia-induced pulmonary hypertension by inhibiting oxidative stress.     

Effects of glutamine pretreatment on intestinal ischemia-reperfusion injury in rats

AIM：To determine the effects of glutamine (Gln) pretreatment on intestinal ischemia-reperfusion (I/R) injury in the rats. METHODS：Thirty male Wistar rats 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 1 g·kg-1·d-1 Gln by orogastric route for 7 d, the rats in the other 2 groups were pretreated with 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 plasma endotoxin, serum D-lactic acid, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured. The intestinal mucosal injury was observed with HE staining and evaluated using Chius scoring. RESULTS：Serum D-lactic acid, endotoxin level, MDA level and Chiu's score in I/R group were significantly higher than those in sham group and Gln group (all P<0.05). Serum SOD activity was significantly lower than that in sham group and Gln group (P<0.05). CONCLUSION：Glutamine has a protective effect on the intestines during ischemia-reperfusion injury. The mechanism may be related to oxidative stress response.     

Effects of cyclosporin on oxidative stress and mitochondrial energy metabolism in rat hippocampus after status epilepticus

AIM：To investigate the effects of cyclosporin on oxidative stress and mitochondrial energy metabolism in the rat hippocampus after status epilepticus. METHODS：Status epilepticus was induced by pilocarpine. The changes of malondialdehyde and superoxide dismutase in the rat hippocampus with or without cyclosporin injection were evaluated. Additionally, the mitochondrial permeability transition, the activity of mitochondrial respiratory chain complex I/III and ATP content in the rat hippocampus were detected. RESULTS：Cyclosporin significantly inhibited mitochondrial permeability transition in the rat hippocampus after status epilepticus. Decreased malondialdehyde and increased superoxide dismutase levels were detected in cyclosporin treatment group compared with status epilepticus group (P<0.05). More-over, the activity of mitochondria respiratory chain complex I, not III, in the mitochondrial fraction increased after cyclo-sporin treatment (P<0.05). In addition, cyclosporin significantly prevented the decrease of ATP content in rat hippocampus after status epilepticus (P<0.05). CONCLUSION：Cyclosporin suppresses oxidative stress in the rat hippocampus after status epilepticus. Cyclosporin alleviates the impairment of mitochondrial energy metabolism in rat hippocampus after status epilepticus.     

Effect of TLR4 on regulation of autophagy in renal tubular epithelial cells by soluble uric acid

AIM To investigate the role of Toll-like receptor 4 (TLR4) in autophagy induced by soluble uric acid in renal tubular epithelial HK-2 cells. METHODS After HK-2 cells were co-stimulated with soluble uric acid or/and chloroquine (CQ), the protein expression of LC3-Ⅱ and P62 was determined by Western blot. Autophagosomes and autophagolysosomes were observed by transmission electron microscopy. Next, HK-2 cells were co-stimulated with soluble uric acid and TLR4 inhibitor TAK242. The mRNA expression of TLR4 was detected by RT-qPCR, the protein expression of TLR4, LC3-Ⅱ and P62 was determined by Western blot, and the autophagic flux was observed by the method of mRFP-GFP-LC3. RESULTS The expression of LC3-Ⅱand P62 in the HK-2 cells was up-regulated by soluble uric acid. The expression of LC3-Ⅱ and P62 was further increased after co-stimulated with uric acid and CQ,and the number of autophagic body was increased while the number of autophagolyososome was decreased as observed by TEM, indicating that the autophagic flux was blocked. The expression levels of TLR4, LC3-Ⅱ and P62 in soluble uric acid group were higher than those in control group. Meanwhile, TAK242 inhibited the up-regulation of TLR4, LC3-Ⅱ and P62 by soluble uric acid. The results of mRFP-GFP-LC3 experiment showed that the levels of autophagosomes were significantly higher in soluble uric acid group than that in control group, and the levels of autophagolysosomes were lower. Compared with soluble uric acid group, the level of autophagosomes was decreased, and autophagolysosomes was increased in TAK242+soluble uric acid group, indicating that the fusion of autophagosomes and lysosomes was increased, and the process of autophagolysosome formation was smoother. CONCLUSION Soluble uric acid leads blocked autophagic flux in renal tubular epithelial cells. Soluble uric acid mediates abnormal tubular autophagic flux through TLR4.