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Aim To explore the mechanism by which calpain-1 promotes hypoxia-induced pulmonary hypertension pulmonary artery endothelial cell apoptosis through endoplasmic reticulum stress. Methods C57BL/6 wild-type (WT) and calpain-1 gene knockout mice (KO) were reared in a hypoxic chamber (10% O
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Objective: To explore the influences of pulchinenoside on the proliferation and migration of oral squamous cell carcino-ma cells and the expression of calpain 1 ( calpain 1). Methods: The human oral squamous cell carcinoma CAL27 cells were treated with pulchinenoside at the concentrations of 0, 1. 0,2. 0, 4. 0, 8. 0, 12. 0 mg·L-1. MTT method was used to detect the effect of pul-chinenoside on the proliferation of CAL27 cells; the effects of pulchinenoside on the proliferation and migration of CAL27 cells were de-tected by cell scratch test and Transwell experiment; the expression changes of calpain 1, E-cadherin and N-cadherin protein in CAL27 cells were detected by Western blot. Results: MTT results showed that, compared with the control group, the inhibitory rate of pul-chinenoside for the proliferation of CAL27 cells increased significantly (P<0. 05) in a time- and dose-dependent manner. The light microscope results showed that the CAL27 cells morphology changed from the long spindle shape to the cobblestone like epithelioid after the 48-hour treatment with 12. 0 mg·L-1pulchinenoside. Cell scratch test results showed that with the increase of pulchinenoside con-centration, the CAL27 cell migration ability gradually weakened. Transwell experimental results show that with the increase of pul-chinenoside concentration, the CAL27 cell migration and invasion abilities decreased gradually (P <0.05). Western blot results showed that with the increase of the concentration of pulchinenoside, the expression levels of calpain 1 and N-cadherin protein in CAL27 cells decreased gradually (P<0. 05), the expression level of E-cadherin protein increased gradually (P>0. 05). Conclusion:Pulchinenoside can inhibit the proliferation, migration and invasion of oral squamous cell carcinoma CAL27 cells, and its mechanism may be related to the down-regulation of calpain 1 and N-cadherin expressions and up-regulation of E-cadherin expression.
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Objective To investigate the effects of N-butylphthalide (NBP) on cognitive function in acute severe carbon monoxide (CO) poisoning rats and its mechanism. Methods 120 health Sprague-Dawley (SD) rats were randomly divided into three groups (n = 40): normal control group (NC group), CO poisoning group (CO group) and NBP treatment group (NBP group). The acute severe CO poisoning model was established in a hyperbaric oxygen chamber by intoxicated with 1 000 ×10-6CO for 40 minutes, followed with 3 000 ×10-6CO for another 20 minutes, and then received hyperbaric oxygen therapy 1.5 hours once a day until sacrificed. Rats in NBP group were administered orally NBP 60 mg/kg for 2 times daily until death. NC group and CO group were treated with equal amount of pure olive oil. Four rats in each group were taken from 1, 3, 7, 14, 30 days after model setup, respectively. The cognitive function score was assessed by Morris water maze test. The changes in ultrastructure of hippocampus were observed under transmission electron microscope. The expressions of calpain 1 and Ca2+/calmodulin dependent protein kinase Ⅱ(CaMK Ⅱ) in hippocampus of brain tissue were detected by immunofluorescence staining, and the localization of the two target proteins in neurons was observed by immunofluorescence double staining. Results Compared with NC group, the escape latency at 1 day after poisoning in CO group was significantly prolonged (s: 55.6±3.2 vs. 44.5±3.5, P < 0.05), and the times of the platform crossing was significantly decreased (times: 1.3±0.8 vs. 6.6±1.2, P < 0.05);the ultrastructure of hippocampus was obviously injured; the protein expressions of calpain 1 and CaMK Ⅱ in brain tissue were significantly increased at 1 day after CO poisoning [calpain 1 (A value): 41.24±5.21 vs. 6.44±1.13, CaMK Ⅱ (A value): 56.19±5.04 vs. 9.84±1.53, both P < 0.05], and the protein expression of calpain 1 reached the peak at 3 days (A value: 59.34±6.11), the protein expression of CaMK Ⅱ reached the peak at 1 day (A value:56.19±5.04). Compared with CO group, the cognitive function was significantly improved in NBP group in the late stage of poisoning [7-30 days, escape latency (s): 40.3±1.9 vs. 49.1±3.1 at 7 days, 30.1±2.9 vs. 39.4±3.1 at 30 days;times of the platform crossing (times): 2.8±1.0 vs. 1.0±0.9 at 14 days, 3.2±0.8 vs. 1.0±0.9 at 30 days, all P < 0.05];the degree of injury of hippocampal neuron was relatively slight; the protein expression of calpain 1 in brain tissue was significantly decreased from 3 days after CO poisoning (A value: 39.63±3.03 vs. 59.34±6.11, P < 0.05), and the protein expression of CaMK Ⅱ was significantly decreased from 1 day after CO poisoning (A value: 42.22±3.84 vs. 56.19±5.04, P < 0.05). Immunofluorescence double staining suggested that calpain 1 and CaMK Ⅱ protein could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed that the expression of calpain 1 and CaMK Ⅱ was positively correlated (R 2= 0.852, P = 0.002). Conclusions NBP treatment could maintain ultrastructure integrity of hippocampus, balance the expression levels of calpain 1 and CaMK Ⅱproteins, and significantly improve cognitive impairment induced by CO poisoning, thus play a protective role against hippocampus damage in rats with acute severe CO poisoning.
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We previously demonstrated that the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 gp41 induced cell death in human neuronal cells. Present study was conducted to further elucidate the pathogenic mechanisms involved in HIV-1 gp41-induced neurodegeneration in AIDS patients with cognitive deficits. The effect of LLP-1 on activation of calpain-1, a calcium-activated cysteine protease, which has been implicated in neuronal degeneration and death, was monitored by the proteolysis of spectrin in rat organotypic hippocampal slice cultures. Protease specific spectrin breakdown products revealed that LLP-1 generated~150/145-kDa fragments characteristic of calpain-1 activation in hippocampus undergoing cell death as evidenced by LDH release. This spectrin cleavage pattern was further confirmed by in vitro calpain-1 proteolysis. Futhermore, calpectin and MDL28170, inhibitors of calpain activity, blocked calpain-1-mediated spectrin cleavage. Spectrin cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin. Among pharmacological agents tested, apocynin, NADPH oxidase inhibitor, ameliorated the LLP-1-induced spectrin. Given the role of spectrin essential for synapse stabilization, LLP-1-induced spectrin cleavage as occurs with the activation of calpain-1 may be an important effector in LLP-1mediated cell injury in hippocampus, which is primarily linked to cognitive dysfunction.