Protective effects of physcion against cerebral injury induced by ischemia-reperfusion in rats

AIM: To explore the effect of physcion (P) on the level of IL-1β and expression of ICAM-1 and caspase-3 during cerebral ischemia-reperfusion injury. METHODS: The 91 healthy adult SD rats were selected, and were randomly divided into normal group, sham-operated group, cerebral ischemia-reperfusion group (model), low-dose physcion (PLD) and high-dose physcion (PHD) treatment group. The level of IL-1β was detected by radioimmunoassay. The expression of ICAM-1 and caspase-3 was detected by immunohistochemistry. The changes of tissue pathology were also investigated. RESULTS: The level of IL-1β reached the peak at 6 h after ischemia-reperfusion (IR). The protein expression of ICAM-1 and caspase-3 reached the peak at 24 h after IR. The level of IL-1β and the protein expression of ICAM-1 and caspase-3 in PHD group decreased obviously compared with those in model group (P<0.05 or P<0.01), infiltration and adhesiveness of neutrophils were less serious at the same time. CONCLUSION: Physcion decreases the level of IL-1β and the protein expression of ICAM-1 and caspase-3 to protect brain tissue from cerebral ischemia-reperfusion injury.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

The influence of anesthesia and means of postoperative pain control on T lymphocyte subtypes of blood in patients with rectal cancer

AIM: To evaluate the influence of anesthesia and different means of postoperative pain control on the T-lymphocyte during the perioperative period in patients with rectal cancer.METHODS: 40 adult patients, aged 65 or older, of American Society of Anesthesiologists (ASA) class 2-3 were divided into two groups according to the type and means of postoperative pain managements. Group Ⅰ (n=20) received intravenous anesthesia and patient controlled analgesia(PCA), fentanyl (13 μg/kg) for post pain; group Ⅱ (n=20) received intravenous anesthesia plus lumber epidural anesthesia and epidural PCA of morphine 5 mg plus ropivacaine 100 mg for post operative pain. Blood samples from internal jugular vein were obtained before surgery, at the completion of surgery and 24, 48, and 120 h post surgery for detecting CD3⁺, CD4⁺, CD4/CD8 counts of peripheral T-lymphocytes. In addition, blood cortisol level and pain intensity were assessed by visual analogue score (VAS)at each time point. RESULTS: Baseline(before anesthesia) values of CD3⁺,CD4⁺, CD4/CD8 in patients were messured and there was a significant decrease of all these values from completion of surgery to 48 h after surgery in both groups (P<0.01). However, group Ⅱ showed a higher CD4⁺ at 48 h, higher CD3⁺,CD4⁺, CD4/CD8 at 120 h post surgery than group Ⅰ (P<0.05). Patients in both groups obtained good pain relief post surgery,but VAS in group Ⅱ were significantly lower than those in group Ⅰ at 24 and 48 h post surgery (P<0.01). Compared with baseline, blood cortisol levels in both groups increased markedly at completion of surgery, and at 24, 48 h after surgery (P<0.01),while the increased cortisol level in group Ⅱ at completion of surgery and 24 h after surgery was less than that in group Ⅰ (P<0.05).CONCLUSION: Combined intravenous anesthesia with lumber epidural anesthesia appears to reduce the perioperative stress response and exerts less negative effects on the T-lymphocytes, suggesting that such a means of anesthesia might be more suitable to the elderly patients with rectal cancer.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

Effects of ambroxol combined with low-dose heparin on production of ICAM-1 and E,P-selectin in acute lung injury

AIM: To study the production of intercellular adhesion molecule-1(ICAM-1), E-selectin and P-selectin in serum, lung tissues and bronchoalveolar lavage fluid(BALF)of acute lung injury(ALI) model and to observe the effects of ambroxol combined with low-dose heparin on the changes of the 3 factors above.METHODS: Twenty-four healthy rabbits were randomly divided into 3 groups: normal saline control group (NC), oleic acid injury group (OA), ambroxol+ heparin treatment group (AH). The rabbit ALI model was induced by oleic acid injection through auricular vein. Partial pressure of O₂ in artery(PaO₂) was analyzed.The concentrations of ICAM-1 and E-selectin were detected by ELISA.The apoptosis index(AI) was measured by TUNEL method.The expression of P-selectin was determined by immunohistochemical method.The ultrastructural changes of the lung tissues were observed under electron microscope, and the lung wet/dry ratio(W/D) was calculated.RESULTS: PaO₂ in AH group and OA group was significantly lower (P<0.01) than that in NC group, and PaO₂ in AH group was significantly higher than that in OA group (P<0.01). The concentrations of ICAM-1 and E-selectin in serum, lung tissues and BALF, and AI and W/D in lung tissues in AH group were higher (P<0.05 or P<0.01) than those in NC group, and was lower than those in OA group (P<0.05 or P<0.01). In NC group, no significant change of the above parameters at all time points was observed (P>0.05). In OA group, PaO₂ was significantly decreased (P<0.01) with the pathological process developed, and the concentrations of ICAM-1 and E-selectin were significantly increased. In AH group, PaO₂ was decreased (P<0.05),and the concentrations of ICAM-1 and E-selectin were increased with the process of ALI developed. The P-selectin expression in lung tissues of OA group was distributed mainly in inflammatory cells, capillary endothelial cells and plasma. From low to high levels, the order was NC group < AH group < OA group in the expression of P-selectin. The most obvious apoptosis was observed in OA group. No apoptosis or occasional positive cells were found in NC group. The apoptotic rate in AH group was significantly reduced compared with that in OA group.CONCLUSION: In ALI induced by OA, ICAM-1, E-selectin and P-selectin are significantly increased and are involved in the occurrence and development of ALI. Ambroxol combined with low-dose heparin reduces the levels of ICAM-1, E-selectin and P-selectin, the pulmonary edema and the lung injury, improves pulmonary functions, and plays an important role in the prevention and treatment of acute lung injury.     

Effect of ligustrazine on expression of LFA-1 and ICAM-1 in bone marrow of radiation injured mice

AIM: To investigate the effect of ligustrazine on the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in bone marrow and on the mechanism of hematopoietic reconstitution in radiation injured mice.METHODS: The 24 mice (clean class) were randomly divided into 3 groups: normal group, radiation injured group and ligustrazine group. After irradiation by 6.0Gy ［⁶⁰Co］ γ-ray, the radiation injured animals were given normal saline (0.2 mL, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine through gastric tube (0.2 mL, twice a day). The mice in normal group received no treatment. At the 7th, 14th, 21st day after irradiation, the femur were taken and the bone marrow mononuclear cells (BMNCs) suspension were made to culture bone marrow stromal cells (BMSCs). The mRNA and protein expressions of ICAM in BMSCs were assayed by RT-PCR and Western blotting. The expression levels of LFA-1 in BMNCs were evaluated by flow cytometry analysis.RESULTS: In ligustrazine group the expression levels of LFA-1 at the 7th, 14th and 21st days after irradiation were higher than those in radiation injured group (P<0.01 or P<0.05). However, the expression level of ICAM-1 was lower than that in the compared group (P<0.01 or P<0.05).CONCLUSION: Ligustrazine can increase the LFA-1 expression level of BMNCs, decrease the ICAM-1 expression level in BMSCs, indicating that ligustrizine promotes the recovery of hematopoietic cells in bone marrow, then improves the bone marrow microenvironment and enhances hematopoietic reconstitution.     

Relationship between the E-selectin and oxidative stress in patients with obese type 2 diabetes mellitus

AIM: To investigate the relationship between the levels of soluble E-selectin and oxidative stress in patients with obese type 2 diabetes mellitus.METHODS: The level of E-selectin, the contents of ox-LDL and malondialdehyde (MDA) and activities of superoxide dismutase (SOD) were measured in patients with obese and non-obese type 2 diabetes mellitus.RESULTS: The levels of E-selectin, ox-LDL and MDA were higher in patients with obese type 2 diabetes mellitus than those in control group (P<0.05), and the contents of HDL-C, HDL2-C and HDL3-C were significantly lower than those in control group (P<0.01).The activity of SOD in patients with obese type 2 diabetes mellitus was significantly lower than that in control group.The contents of E-selectin and MDA were more markedly elevated in patients with obese type 2 diabetes mellitus than those in patients with non-obese type 2 diabetes mellitus (P<0.01,P<0.05) and the activity of SOD was also significantly lower than that in patients with non-obese type 2 diabetes mellitus (P<0.01).There was significantly positive correlation between E-selectin and HbA1c, waist circumference, TC, ox-LDL, MDA (r=0.352, P<0.05;r=0.634, P<0.05;r=0.517, P<0.05;r=0.480, P<0.05;r=0.572, P<0.05), and negatively correlation between E-selectin and HDL3-C (r=-0.374, P<0.05).CONCLUSION: The plasma level of E-selectin is markedly elevated in patients with obese type 2 diabetes mellitus.E-selectin is possibly associated with oxidative stress.     

Effects of dehydroepiandrosterone on expression of ICAM-1 in rabbit aorta and HUVECs induced by high lipid levels

AIM: To investigate the effects of dehydroepiandrosterone (DHEA) on the expression of intercellular adhesion molecule-1 (ICAM-1) induced by high lipid levels in rabbit aorta and human umbilical venous endothelial cells (HUVECs), and the effects of all-trans retinoic acid (ATRA) in this process.METHODS: For in vitro experiments, the cultured HUVEC were divided into control group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+DHEA group, ox-LDL+DHEA+ATRA group and DHEA group. The HUVECs in all groups were treated with the corresponding reagents for 24 h. The expression of ICAM-1 at mRNA and protein levels in all groups were determined by RT-PCR and ELISA, respectively. For in vivo experiments, the rabbits were divided into control group, high lipid group, high lipid+DHEA group, high lipid+DHEA+ATRA group and DHEA group. The rabbits in all groups were fed with the corresponding diets for 10 weeks. The expression of ICAM-1 in the rabbit aorta at mRNA and protein levels was determined by RT-PCR and immunohistochemistry. RESULTS: The expression of ICAM-1 in the HUVECs in ox-LDL group was significantly increased compared with control group (P<0.05). Compared with ox-LDL group, the expression of ICAM-1 in ox-LDL+DHEA group was obviously decreased (P<0.05). The expression of ICAM-1 was similar in both control group and DHEA group (P>0.05). The expression of ICAM-1 was similar in both ox-LDL+DHEA group and ox-LDL+DHEA+ATRA group (P>0.05). The expression of ICAM-1 in the rabbit aorta in high lipid group was significantly increased compared with control group (P<0.05). Compared with high lipid group, the expression of ICAM-1 in high lipid+DHEA group was obviously decreased (P<0.05). No remarkable difference in the expression of ICAM-1 between control group and DHEA group was observed (P>0.05), so did between high lipid+DHEA group and high lipid+DHEA+ATRA group (P>0.05). CONCLUSION: DHEA inhibits high lipid-induced ICAM-1 expression in rabbit aorta and HUVECs. That may be one of the mechanisms of antiatherosclerotic effect of DHEA. ATRA seems no positive effect on DHEA function.     

Renal protection of combination of irbesartan and sulodexide on diabetic rat

AIM: To assess renal protective effects of the combination of irbesartan and sulodexide on STZ-induced diabetic rats. METHODS: Diabetes was induced by injection of streptozotocin in rats. The animals were randomly divided into five groups: control (C), diabetes (D), diabetes treated with irbesartan (I), diabetes treated with sulodexide (S), and diabetes treated with combination of irbesartan and sulodexide (I+S). Urine albumin excretion rate (UAER), the level of malondialdehyde (MDA) and activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in renal tissues were determined, and renal tissue morphology was observed under light microscope after 12 weeks. Expression of ICAM-1 mRNA was examined by RT-PCR. NF-κB was evaluated using electrophoretic mobility shift assay (EMSA). RESULTS: Increased UAER and kidney pathologic injury were attenuated by treatment with either irbesartan or sulodexide alone and further reduced by using the combination of the two drugs. Elevated MDA level and decreased activities of SOD, CAT and GSH-PX in diabetic renal tissues were improved by irbesartan or sulodexide, and more effectively by combination of irbesartan and sulodexide. NF-κB activities were higher in renal tissue of diabetic rats than those in control group, and further abrogated by combination therapy in both cases (P<0.05). Over-expression of ICAM-1 mRNA observed in diabetic rats was attenuated by irbesartan or sulodexide to a similar level and further reduced by the combination of two drugs(P<0.05). CONCLUSION: The combination of irbesartan and sulodexide confers superiority over mono-therapies on the effect of renal protection. The mechanism may be at least partly correlated with synergestic suppression of increasing oxidative stress and NF-κB activities as well as over-expression of ICAM-1 mRNA in renal tissues.     

Poly (I∶C) promotes iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse

AIM: To investigate the effects of poly (I∶C) as virus mimics on iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse. METHODS: Female, 32 NOD mice were randomly divided into 4 groups: (1) control; (2) high iodine; (3) poly (I∶C); (4) high iodine+poly (I∶C). Nine weeks after administration, mice were sacrificed. The following parameters were determined: body weight, thyroid weight and anatomic form. Thyroid hormone (T4) in serum was measured by radioimmunoassay, the thyroid morphology was observed through HE staining, apoptosis was detected by TUNEL, the mRNA expression levels of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were determined by the method of real time RT-PCR. RESULTS: Compared to control group and poly (I∶C) group, the thyroid absolute weight and relative weight in high iodine group were increased (P<0.01), the level of total T4 in serum was decreased (P<0.05), inflammation and apoptosis were obviously observed, the mRNA expressions of TRAIL, TRAIL-sR1, CXCL10 and ICAM-1 were upregulated (P<0.05). Compared to high iodine group, thyroid absolute weight and relative weight in high iodine+poly (I∶C) group were further increased, the level of total T4 in serum was further decreased (P<0.05), the ratio of inflammatory degree Ⅳ increased to 50.0%, the numbers of apoptosis cells were further enhanced, the mRNA expressions of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were further upregulated (P<0.05). Otherwise, the tendency of all parameters in poly (I∶C) group was similar to that in control group (P>0.05). CONCLUSION: Poly (I∶C) aggravates chronic lymphocytic thyroiditis induced by excess of iodine associated with increase in infiltration of lymphocytes and induction of apoptosis.     

Cardioprotective effect of sevoflurane preconditioning on ROS production in rat heart with ischemia-reperfusion

AIM: To investigate the effects of sevoflurane preconditioning on the production of reactive oxygen species (ROS) and nitric oxide (NO), the activity of superoxide dismutase(SOD), glutathione peroxide(GPx) and catalase(CAT) in myocardial ischemia-reperfusion injury(IRI). METHODS: Sixty rats were randomly allocated to 8 groups. Following 2% sevoflurane preconditioning for 30 min, the left anterior descending artery was ligated for 30 min and then reperfused for 120 min in vivo. The infarction size of the hearts was measured with the staining of 2,3,5-triphenyltetrazoliumchloride. The myocardial apoptotic index was measured by the method of TUNEL. The ROS fluorescent probe dihydroethidium was used for the measurement of ROS. The myocardium was homogenized for the measurement of NO, SOD, GPx and CAT. To evaluate the effects of ROS and NO on the cardioprotection of sevoflurane preconditioning, ROS scavenger N-(2-mercaptopropionyl) glycine (2-MPG) or NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) were employed to block their actions. RESULTS: Compared with control group, the production of ROS was induced by sevoflurane preconditioning before ischemia-reperfusion injury (12.0±0.8 vs 2.6±0.5, P<0.05) and decreased after ischemia-reperfusion injury (16.2 ±0.9 vs 24.9±1.3, P<0.05). 2-MPG decreased the elevation of ROS caused by sevoflurane preconditioning before ischemia-reperfusion injury (5.1±0.7 vs 12.0±0.8, P<0.05). No difference of ROS production between treating with 2-MPG+Sevo+IRI and with IRI (24.9±1.4 vs 24.9±1.3, P>0.05) was observed. Compared with control group, sevoflurane preconditioning also induced the generation of NO (34.5±3.2 vs 15.9±1.4, P<0.05) and the activity of SOD(1.5±0.5 vs 0.6±0.2, P<0.05), GPx(22.8±2.5 vs 12.7±2.2, P<0.05) and CAT(15.5±1.8 vs 11.2±1.4, P<0.05). 2-MPG blocked the increase in NO production and inhibited the activity of SOD,GPx,CAT. L-NAME also attenuated the activity of SOD,GPx,CAT. CONCLUSION: Sevoflurane preconditioning protects the rat heart against ischemia-reperfusion injury by reducing the infarction size and apoptosis. Production of ROS at sub-injury dose induced by sevoflurane preconditioning stimulates the myocardium to create SOD,GPx,CAT and NO, thus inhibiting the further formation of ROS and protecting the heart under the condition of ischemia-reperfusion.     

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.     

Effect of hydrogen molecule on apoptosis-related proteins and Nrf2 signaling pathway in glomerular mesangial cells cultured with high glucose

AIM: To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular mesangial cells cultured with high glucose and to explore its possible mechanism. METHODS: Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group (C group, 5.5 mmol/L glucose), mannitol group (G group, 5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (H group, 25 mmol/L glucose), high glucose+hydrogen-rich water group (HH group, 25 mmol/L glucose+hydrogen-rich water), and cultured for 48 h. The protein levels of Bax, Bcl-2, cleaved caspase-3, nuclear factor E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1) were determined by Western blot, and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR. The level of intracellular reactive oxygen species (ROS) was detected by dihydroethidium method, and the activity of superoxide dismutase (SOD) was measured by WST-8 assay. RESULTS: Compared with C group, the protein levels of Bax and cleaved caspase-3 were up-regulated, and Bcl-2 was down-regulated in H group (P <0.05). No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group, the protein levels of Bax and cleaved caspase-3 were down-regulated, and Bcl-2 was up-regulated in HH group (P <0.05). The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group (P<0.05). However, there was no difference of the SOD activity between C group and HH group. The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group (P<0.05). Compared with C group, clearly reduced protein expression of Nrf2, HO-1 and NQO-1, and decreased mRNA expression of HO-1 and NQO-1 in H group were observed (P<0.05). Compared with H group, the protein levels of Nrf2, HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group (P<0.05).CONCLUSION: Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic proteins in glomerular mesangial cells cultured with high glucose. The mechanism may be related to activation of Nrf2 signaling pathway.     

JNK pathway promotes apoptosis of rat hippocampal neurons after cerebral ischemia and reperfusion

AIM:To investigate the effect of c-Jun N-terminal kinase(JNK) pathway on the apoptosis of hippocampal neurons after cerebral ischemia-reperfusion(IR) in SD rats. METHODS:Ninety rats were randomly divided into 5 groups:sham group, cerebral IR group,cerebral IR+JNK inhibitor(SP600125) group,cerebral IR+JNK agonist(anisomycin) group and cerebral IR+vehicle group. The brain samples were collected 24 h after reperfusion. The protein level of caspase-3 in hippocampal neurons was measured by immunohistochemical and Western blotting techniques. The mRNA expression of caspase-3 in the hippocampus was determined by real-time fluorescence quantitative PCR. The apoptosis of hippocampal neurons was detected by TUNEL staining. RESULTS:Compared with sham group, the expression of caspase-3 at mRNA and protein levels in cerebral IR group increased obviously(P<0.05). Compared with cerebral IR group, the expression of caspase-3 at mRNA and protein levels in cerebral IR+JNK inhibitor group decreased obviously(P<0.05), and those in cerebral group increased obviously(P<0.05). However, the expression of caspase-3 at mRNA and protein levels in cerebral IR+vehicle group had no obvious change(P>0.05).The apoptosis of hippocampal neurons in each group was consistent with the changes of caspase-3 at mRNA and protein levels. CONCLUSION:Activation of JNK pathway enhances caspase-3 expression in rat hippocampal neurons after cerebral IR,thus promoting the apoptosis of the neurons.     

Intracellular Ca2+ mobilization in mouse CD8+ T- lymphocytes immunized with gp96-peptide complexes

AIM: To further investigate the effect and mechanism of gp96-peptide complexes by observing ［Ca2+］i mobilization in CD8+ T-lymphocytes. METHODS: Spleen T-lymphocytes from the mice immunized with gp96-peptide complexes were used. The cell was stained with quantum-red-labeled anti-CD8 mAb and loaded with Fluo-3/AM. The double-color flow cytometry analysis method was used to detect ［Ca2+］i changes in CD8+ T-lymphocytes from the mice immunized with gp96-peptide complexes. A23187, MnCl2 and Con A were used as stimulators. RESULTS: The ［Ca2+］i in immunized CD8+T-lymphocytes was higher than that in non-immunized cells. Compared with the control group, A23187 and Con A induced higher ［Ca2+］i increases in spleen CD8+ T-lymphocytes of gp96-peptide complexes-stimulated mice than that in non-immunized cells. CONCLUSION: CD8+T-lymphocytes activation is likely due to an elevation of ［Ca2+］i. It suggests that gp96-peptide complexes play a significant role in the modulation of CD8+ T-lymphocytes responses.     

Injury effect of recombinant soluble human CD40 ligand on human umbilical vein endothelial cells

AIM: To investigate the damage in human umbilical vein endothelial cells (HUVECs) induced by recombinant soluble human CD40 ligand (rshCD40L). METHODS: The cultured HUVECs were treated with rshCD40L for 12 h. The survival activity of the HUVECs was observed by MTS assay. The expression of E-selectin, intercellular adhesion molecule (ICAM)-1, tissue factor (TF) and tissue factor pathway inhibitor (TFPI) was measured by ELISA. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were detected by the methods of thibabituric acid (TBA). RESULTS: Compared with normal group, different concentrations of rshCD40L (0.5, 1, 2, 3 mg/L) had no obvious effect on the survival activity of the HUVECs (P>0.05). rshCD40L at concentration of 0.5 mg/L promoted the secretion of E-selectin, sICAM-1, TF and TFPI in the HUVECs (P<0.01). rshCD40L at concentration of 0.5 mg/L also increased MDA content and reduced the activity of SOD in the HUVECs (P<0.05). CONCLUSION: 0.5~3mg/L rshCD40L has no obvious effect on endothelial cell survival, but already causes endothelial dysfunction by increasing endothelial inflammation and exogenous coagulation reaction, inducing lipid peroxides injury and reducing antioxidant capacity.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.