Asiaticoside Alleviates Cerebral Ischemia-Reperfusion Injury via NOD2/Mitogen-Activated Protein Kinase (MAPK)/Nuclear Factor kappa B (NF-κB) Signaling Pathway.

BACKGROUND Cerebral ischemia-reperfusion injury (CIRI) remains a serious health problem. Centella asiatica formulations are used to treat central nervous system disorders. In the present study, asiaticoside, an extract of the plant Centella asiatica, was investigated in CIRI in vivo and vitro. MATERIAL AND METHODS We made a CIRI model in vivo in SD rats treated by middle cerebral artery occlusion, and a cell model of ischemia-reperfusion injury was made in PC12 cells treated by deprivation of oxygen and glucose/restoration. CIRI in vivo was assessed by scores of neurological functions, encephaledema, and cerebral infarction area. Inflammation level and oxidative stress level were detected by the appropriate kits. TUNEL assay was performed for assessment of cell apoptosis and Western blot analysis was performed to assess protein expression levels. CCK8 assay was performed for evaluation of cell survival and flow cytometer was used to detect cell apoptosis in vitro. RESULTS Nervous function injury, brain edema, cell apoptosis, infarct size, apoptosis-related protein expressions, and protein expressions of the NOD2/MAPK/NF-kappaB signaling pathway in the CIRI model were all reversed by asiaticoside in rats. The cell apoptosis, inflammation level, and oxidative stress level in the model of cerebral ischemia-reperfusion injury were reduced by asiaticoside. The effects of asiaticoside on CIRI were reversed by NOD 2 agonists. CONCLUSIONS Asiaticoside showed a protective effect against cerebral ischemia-reperfusion injury via the NOD2/MAPK/NF-kappaB signaling pathway. These findings are vital for future research on use of asiaticoside in CIRI, providing a new avenue for alleviating CIRI.

Curcumin Prevents Brain Damage and Cognitive Dysfunction During Ischemic-reperfusion Through the Regulation of miR-7-5p.

OBJECTIVE: This study was to investigate the potential protective effects of curcumin in cerebral ischemia-reperfusion (CIR) and its regulation of miR-7. METHODS: Rats were occluded by middle cerebral artery occlusion (MCAO) for 1.5 h and reperfused for 2 h to establish a local CIR model. After 24 hours of model establishment, MCAO rats were given curcumin for 3 days by intragastric administration. PC12 cells were cultured for 6 h in oxygen-glucose deprivation medium and then reoxygenated for 24 h to establish an oxygenglucose deprivation/reoxygenation (OGD/R) model. The OGD/R model cells were treated with curcumin for 48 h. RESULTS: Curcumin inhibited the decrease of miR-7-5p expression and an increase of RelA p65 expression induced by CIR and ODG/R. RelA p65 was a target of miR-7-5p. MiR-7-5p antagonists were able to counteract the effect of curcumin on the expression of RelA p65 in ischemic brain tissue of MCAO rats and OGD/R model cells. Curcumin improved OGD/R-induced inhibition of cell activity, necrosis and apoptosis. Curcumin significantly reduced the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß, reactive oxygen species (ROS) and malondialdehyde (MDA) and increased the activity of superoxide dismutases (SOD) and catalase (CAT) in OGD/R-induced cells. Curcumin may inhibit OGD/R-induced cell damage by regulating miR-7-5p. Curcumin improved cerebral infarction, nerve damage and cognitive dysfunction in rats with CIR, which may be related to the regulation of miR-7-5p/RelA p65 axis. CONCLUSION: Curcumin exerts cerebral protection by attenuating cell necrosis and apoptosis, inflammatory response and oxidative stress following CIR, which may be related to its regulation of the miR-7/RELA p65 axis.

Effects of anisodamine on the expressions of vascular endothelial growth factor and intercellular adhesion molecule 1 in experimental infusion phlebitis.

BACKGROUND: Infusion phlebitis is the most common side effect of clinical intravenous drug therapy and several clinical studies have demonstrated that anisodamine can effectively prevent the occurrence of infusion phlebitis. This study was designed to investigate effects of anisodamine on the expressions of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) in a rabbit model of infusion phlebitis and to analyze the mechanisms of anisodamine effect on the prevention and treatment of experimental infusion phlebitis. METHODS: Twenty-four specific pathogen-free male Japanese white rabbits were randomly assigned to the control group, the model group, the magnesium sulfate group and the anisodamine group. The rabbit model of infusion phlebitis, induced by intravenous administration, was established and expressions of VEGF and ICAM-1 were determined and contrasted with the control group treated with normal saline. We evaluated expression by histopathology, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting assay. RESULTS: Pathohistological changes of the model group were observed, such as loss of venous endothelial cells, inflammatory cell infiltration, edema and thrombus. The magnesium sulfate group and the anisodamine group showed significant protective effects on vascular congestion, inflammatory cell infiltration, proliferation, swelling of endothelium and perivascular hemorrhage. The model group showed the highest expressions of VEGF and ICAM-1 of the four groups (P < 0.01). On the contrary, anisodamine alleviated the inflammatory damage by significantly reducing the expressions of VEGF and ICAM-1 compared with the model group (P < 0.01). There was no significant difference in the expressions of VEGF and ICAM-1 between the magnesium sulfate group and the anisodamine group (P > 0.05). CONCLUSION: Anisodamine alleviates inflammatory damage by significantly reducing the expressions of VEGF and ICAM-1, and shows significant protective effects in an animal model of infusion phlebitis.

Cigarette smoke extract promotes proliferation of airway smooth muscle cells in asthmatic rats via regulating cyclin D1 expression.

BACKGROUND: Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle. METHODS: ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE + pcDNA3.1 group and CSE + pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting. RESULTS: (1) The percentage of S + G2M phase, absorbance value at 490 nm wavelength (A(490)) and the expression rate of PCNA protein in CSE group were (31.22 +/- 1.17)%, 0.782 +/- 0.221, (90.2 +/- 7.0)% respectively, which were significantly increased compared with those of control group ((18.36 +/- 1.02)%, 0.521 +/- 0.109, and (54.1 +/- 3.5)%, respectively) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S + G2M phase, A(490) and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P < 0.01). (2) The ratios of A(490) of cyclin D1 mRNA in CSE group was 0.288 +/- 0.034, which was significantly increased compared with that of control group (0.158 +/- 0.006) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P < 0.01). (3) The ratios of A(490) of cyclin D1 protein expression in CSE group was 0.375 +/- 0.008, which was significantly increased compared with that of control group (0.268 +/- 0.004) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P < 0.01). CONCLUSION: CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

[Effects of Ginkgo biloba extract on proliferation and apoptosis of T lymphocytes in vitro in rats with asthma].

OBJECTIVE: To explore the partial therapeutic mechanism of Ginkgo Biloba extract (GBE) in treating asthma. METHODS: Fourteen SD rats were randomly divided into two groups, 7 rats were sensitized as the asthmatic model group and the others taken as the healthy control group. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) of the rats, and were cultured in vitro with Ginkgolide B (BN-52021 group) or Ginkgo Biloba extract 761 (EGb761 group) in different concentrations or without any of them (control group). T lymphocytes proliferation in groups were measured by using MTT assay and the effect of BN-52021 on T lymphocytes apoptosis was analyzed by flow cytometry at various times. RESULTS: Compared with the control group, BN-52021 could significantly inhibit the proliferation of T lymphocytes in both healthy and asthmatic rats in vitro (P <0. 05). The effects were enhanced as the concentration increasing and the time prolonging, the effects to the latter were higher than those to the former, showing significant difference between them ( P <0.05 ). However, the effect of EGb761 was varied with the concentrations. EGb761 could promote T lymphocytes proliferation at low concentration but inhibit it at high concentration, there was a significant difference as compared with that in the control group ( all P < 0. 05). The apoptotic rate of T lymphocytes rose as the concentration of BN-52021 increasing (P < 0. 01). CONCLUSION: GBE has different effects on T lymphocytes proliferation since the different ingredients and the concentrations in vitro, and it also has different effects between healthy and asthmatic rats. Ginkgolide B is the main active ingredient among them, it can not only inhibit T lymphocytes proliferation but also increase the apoptotic rate.

[Effects of Shen-Mai injection on sternohyoid contractile properties in chronic intermittent hypoxia rat].

OBJECTIVE: To investigate the effect of Shen-Mai injection (SMI) on sternohyoid contractile properties in rats with chronic intermittent hypoxia. METHODS: Thirty healthy male SD rats were randomly assigned to three equal groups, the control group (A group), the chronic intermittent hypoxia group (B group) and the SMI group(C group). Rats in B group and C group were exposed to alternating periods of hypoxia and normoxia once per minute for 8 h/d for 5 weeks in order to mimic the intermittent hypoxia of obstructive sleep apnea-hypopnea syndrome (OSAHS) in humans. Isometric contractile properties were determined by electrostimulating the strips of isolated sternohyoid muscles at different frequencies (from 10 Hz to 100 Hz) to observe the changes of the sternohyoid contractile properties. RESULTS: (1) The tension of sternohyoid muscle in A group at different frequencies was (23.2 +/- 5.6), (26.2 +/- 5.0), (35.1 +/- 5.4), (46.0 +/- 8.5), (57.0 +/- 9.9), (69.9 +/- 9.7), (79.2 +/- 9.5), (85.7 +/- 7.6), (87.9 +/- 7.9), and (86.6 +/- 12.4) g/cm(2). The tension of sternohyoid muscle in B group [(19.5 +/- 4.7), (23.8 +/- 4.7), (33.0 +/- 5.1), (45.1 +/- 5.9), (54.2 +/- 7.0), (66.1 +/- 9.1), (74.2 +/- 9.1), (79.7 +/- 9.0), (82.0 +/- 8.4), and (80.7 +/- 11.8) g/cm(2)] was not significantly different from those in A group respectively (all P > 0.05); while the tension of sternohyoid muscle in C group [(30.5 +/- 2.3), (40.0 +/- 5.4), (56.2 +/- 7.6), (72.2 +/- 6.4), (82.0 +/- 5.5), (92.4 +/- 4.6), (98.1 +/- 4.0), (99.2 +/- 7.4), (101.8 +/- 3.9), and (102.2 +/- 4.0) g/cm(2)] was significantly different from those in B group respectively (all P < 0.05). (2) In fatigue test, the tension percentages of sternohyoid muscle in A group at 1, 2, 3, 4, and 5 min were (87.9 +/- 5.7)%, (72.1 +/- 11.5)%, (55.6 +/- 9.6)%, (39.7 +/- 10.7)%, (33.2 +/- 10.2)%. Compared with A group, the tension percentages of sternohyoid muscle in B group at 1, 2, 3, 4, and 5 min [(75.6 +/- 8.5)%, (41.6 +/- 7.3)%, (29.0 +/- 2.7)%, (20.4 +/- 2.9)%, (18.5 +/- 2.5)%, respectively] decreased significantly (all P < 0.05). Compared with B group, the tension percentages of sternohyoid muscle in C group [(87.9 +/- 4.4)%, (67.9 +/- 14.1)%, (48.4 +/- 9.9)%, (38.2 +/- 7.0)%, (33.8 +/- 9.3)%, respectively] increased significantly (all P < 0.05). CONCLUSIONS: Chronic intermittent hypoxia can increase upper airway muscle fatigue. SMI can significantly increase the contractile properties of upper airway muscle and resist the fatigue of upper airway muscle.

[Regulatory function of nuclear factor kappa B on lymphocyte proliferation and apoptosis in bronchial asthmatic rats and effect of triptolide on the regulation].

OBJECTIVE: To investigate whether nuclear factor kappa B (NF-kappa B) participates in the regulatory function of lymphocyte proliferation and apoptosis in bronchial asthma and whether the regulatory effect of triptolide on lymphocyte proliferation and apoptosis is conducted through NF-kappa B. METHODS: Intervention with dexamethasone, triptolide and PDTC, a NF-kappa B inhibitor, were used to treat astmatic rats respectively. Pathological examination, airway response were determined, the NF-kappa B P65 expression in lung tissue and splenic lymphocytes by immunofluorescent assay were adopted, proliferative cell nuclear antigen (PCNA) in splenic lymphocytes was measured by immunohistochemistry, apoptosis of splenic lymphocytes were monitored by flow cytometry and NF-kappa B activity was investigated by electrophoresis mobility shift assay (EMSA). RESULTS: The nuclear expression and DNA binding activity of lung tissue and splenic lymphocytes in asthmatic rats were all significantly higher than those in the control (all P < 0.05), so was the proliferation rate of splenic lymphocytes (P < 0.05), while the apoptosis rate was much lower than that of normal control (P < 0.05). Administration of PDTC could reduce the up-regulated expression and activity of NF-kappa B, the proliferation of splenic lymphocytes lowered, while the apoptosis increased. NF-kappa B activity showed an obviously positive correlation with proliferation of splenic lymphocytes (r = 0.89, P < 0.05) and a significantly negative correlation with apoptosis rate (r = -0.54, P < 0.05). After asthmatic rats had been treated with triptolide in vivo, the NF-kappa B nuclear expression and activity in airway and splenic lymphocytes, as well as the proliferation rate of splenic lymphocytes all lowered significantly (all P < 0.05), the apoptosis rate increased significantly (P < 0.05), at the same time, the inflammatory cell infiltration and high reactivity of airway were significantly alleviated (both P < 0.05). There were obviously positive correlation between the amount of airway eosinophils and reactivity with activity of NF-kappa B (r = 0.79 and r = 0.68, P < 0.05), which indicated that the effect of triptolide was not significantly different from that of dexamethasone (P > 0.05). CONCLUSION: (1) NF-kappa B participates the formation of airway inflammation and hyper-reactivity in asthmatic rats by positive regulation on proliferation and negative regulation on apoptosis of lymphocytes. (2) Triptolide reduces airway inflammation by way of inhibiting NF-kappa B, and further inhibiting the proliferation of lymphocytes, so that to give full play of the role of anti-asthmatic airway inflammatory agents. Whether the molecular mechanism of triptolide in inhibiting NF-kappa B simulates that of glucocorticoid needs further studying.

[Effect of Shen-Mai injection and aminophylline on diaphragmatic muscle cell apoptosis and related gene expression in rats with chronic hypoxia].

OBJECTIVE: To investigate the effect of Shen-Mai injection (SMI) and aminophylline on diaphragmatic muscle cell apoptosis and the Fas/FasL expression in chronic hypoxic rats. METHODS: Seventy-five male Wistar rats were randomly divided into three equal groups, control group (A group), SMI group (B group) and aminophylline group (C group). Then each group was further divided into five subgroups of pre-hypoxia, hypoxia 1 w, 2 w, 3 w and 4 w groups (5 rats each). The concentration of oxygen was (10 +/- 3)%, 7 d/w, 8 h/d for all groups, but only B group and C group received SMI (2 ml/d) and aminophylline (10 mg/kg) respectively. Apoptosis and Fas/FasL expression of diaphragmatic muscle cells were examined by the streptavidin biotin-peroxidase complex (SABC) immunohistochemistry techniques and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and Dunnett-t test was employed to compare the effects of SMI and aminophylline. RESULTS: (1) Fas, FasL expression in normal diaphragmatic muscle cells was very low with a positive rate of (2.77 +/- 0.45)% and (2.32 +/- 0.61)%. After hypoxia, the positive rates increased with the time of hypoxia time. SMI showed an inhibition on diaphragmatic muscle cell Fas and FasL expression;after hypoxia 1 w, 2 w, 3 w and 4 w, Fas expression [(6.36 +/- 4.17)%, (9.77 +/- 4.12)%, (18.02 +/- 6.91)% and (21.09 +/- 8.09)%] and FasL expression [(5.32 +/- 6.16)%, (9.58 +/- 3.79)%, (12.01 +/- 8.71)%, (19.43 +/- 10.31)%] in B group were different from those in A group respectively (all P < 0.05). But aminophylline did not show such an effect, the expression of Fas [(10.87 +/- 3.62)%, (24.13 +/- 3.79)%, (35.39 +/- 9.02)%, (39.56 +/- 10.12)%] and FasL [(9.37 +/- 4.07)%, (20.16 +/- 4.88)%, (31.81 +/- 7.07)%, (35.51 +/- 9.13)%] were not significantly different from those in A group respectively (all P > 0.05). (2) Diaphragmatic muscle cell apoptosis was very low in normal rats with a rate of (0.93 +/- 0.29)%, which also increased after hypoxia and the increase was associated with the time of hypoxia. Apoptosis rate was decreased by the administration of SMI, the rates of B group were (5.01 +/- 3.71)%, (9.37 +/- 3.12)%, (14.66 +/- 8.76)%, (18.16 +/- 7.02)%, respectively. Except for the first week, the differences of other weeks were all statistically significant when compared with A groups (all P < 0.05). But the effect of aminophylline was different, as compared to A group, only the apoptosis rate in hypoxia 4 w [(30.92 +/- 11.13)%] of C group being statistically significant different (P < 0.05). CONCLUSIONS: Fas and FasL participated in diaphragmatic muscle cell apoptosis in rats with chronic hypoxia. SMI showed a definite effect on the Fas and FasL protein expression and decreased diaphragmatic muscle cell apoptosis, which contributed to the therapeutic effect on diaphragmatic fatigue caused by hypoxia.