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Objective To investigate the effects of diazoxide pretreatment on the expression of hypoxia-inducible factor-1α (HIF-1α) mRNA and p53 mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R).Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates ( 100 μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 ×106/ml and randomly divided into 4 groups ( n = 24 each): normal control group (group C), H/R group, H/R +diazoxide pretreatment group (group DZ) and H/R + diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in DZ and DZ + 5-HD groups respectively. The cell vitality, apoptotic rate and expression of HIF-1α mRNA and p53 mRNA were detected at the end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of HIF- 1α mRNA and p53 mRNA up-regulated in H/R group ( P ＜ 0.01). Compared with group H/R, the cell vitality was significantly increased, apoptotic rate decreased, the expression of HIF-1α mRNA up-regulated and the expression of p53 mRNA down-regulated in group DZ ( P ＜ 0.05 or 0.01 ). 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above (P ＜ 0.05 ). Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through up-regulating the expression of HIF-1α and down-regulating the expression of p53, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.
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ObjectiveTo investigate the effects of diazoxide pretreatment on expression of phosphatidylinositol 3-kinase(PI3K) mRNA and protein serine/threonine kinase(Akt) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R).MethodsThe SD rat myocardial microvascular endothelial cells were cultured.The cells were seeded in 96-well plates ( 100μd/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into 4 groups ( n =25 each):normal control group (group C),H/R group,diazoxide pretreatment group (group DZ) and diazoxide pretreatment + 5-hydroxydecanoate (5-HD,a mitochondrial ATP-sensitive potassium channel blocker) group (group DZ + 5-HD).The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation.Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in groups DZ and DZ + 5-HD respectively.The cell viability,apoptotic rate and expression of PI3K mRNA and Akt mRNA were detected at the end of reoxygenation.ResultsCompared with group C,the cell viability was significantly decreased,while the apoptotic rate increased and expression of PI3K mRNA and Akt mRNA up-regulated in group H/R (P ＜0.05 or 0.01).Compared with group H/R,the cell viability was significantly increased,while the apoptotic rate decreased and expression of PI3K mRNA and Akt mRNA up-regulated in group DZ (P ＜ 0.05 or 0.01).5-HD could inhibit diazoxide pretreatment-induced changes mentioned above (P ＜ 0.05 or 0.01 ).ConclusionDiazoxide pretreatment can reduce H/R injury by promoting PI3K gene and Akt gene transcription through activation of mitochondrial ATP-sensitive potassium channels in rat myocardial microvascular endothelial cells.
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Objective To investigate the effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine (FKN) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R). Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates (100μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into4 groups (n = 24 each): Ⅰ normal control group (group C), Ⅱ H/R group, Ⅲ diazoxide pretreatment group (group DZ), Ⅳ diazoxide pretreatment + mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) group (group DZ + 5-HD). The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100μmol/L were added to the cultured medium 2 h before hypoxia in group DZ and DZ + 5-HD respectively. The cell vitality, apoptotic rate and expression of NF-κB mRNA and FKN mRNA were detected at end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of NF-κB mRNA and FKN mRNA up-regulated in H/R group. Compared with group H/R, the cell vitality was significantly increased,apoptotic rate decreased and the expression of NF-κB mRNA and FKN mRNA down-regulated in group DZ. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through down-regulating the expression of NFκB and FKN, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.
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AIM: To investigate the relationship between cytokine level and nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: All New Zealand rabbits were divided randomly into three groups: (1) control group, (2) extracapsular cataract extraction group (ECCE), (3) extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation of all experimental rabbit eyes, including cornea edema and anterior chamber exudation were observed via zoom-photo slitlamp microscope 1,3,7,14,30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying, and for NO-2／NO-3 and cytokine assay, including interleukin-2(IL-2), tumour necrosis factor-α(TNF-α). Statistics were taken by SPSS softwear. RESULTS: (1) Total WBC in aqueous humor were higher and anterior chamber exudation were more severe in ECCE+IOL group than that in ECCE group and control group. (2) The level of IL-2 and TNF-α and the content of NO-2／NO-3 in aqueous humor of ECCE+IOL group were higher than that in ECCE group and control group 1-14 d postoperation respectively, and it increased to peak value at 3-7 d postoperation and decreased gradually after two weeks postoperation. (3) The change regularity of IL-2, TNF-α and NO-2／NO-3 in each group were basically similar, i.e. when the level of cytokine (IL-2 and TNF-α) was normal, the content of NO-2／NO-3 was normal too, when the level of cytokine (IL-2 and TNF-α) increased, the content of NO-2／NO-3 increased too. CONCLUSION: The intraocular inflammation after ECCE+IOL was more severe than that after simple ECCE. NO, IL-2 and TNF-α play an important role in intraocular inflammation after intraocular lens implantation. The changes of IL-2 and TNF-α level was closely related with NO content in aqueous humor.
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AIM: To investigate the relationship between the level of interleukin-2 (IL-2), tumour necrosis factor-?(TNF-?) and nitric oxide (NO) in aqueous humor after intraocular lens implantation. METHODS: New Zealand rabbits were divided randomly into three groups: (1)control group; (2)extracapsular cataract extraction group (ECCE); (3)extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation in all experimental rabbit eyes was observed via zoom-photo slit-lamp microscope on 1, 3, 7, 14 d and 30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell(WBC) counting and classifying , and for determining IL-2, TNF-? and NO 2 -/NO 3 - contents.RESULTS: (1)The level of IL-2 and TNF-? and NO 2 - / NO 3 - in aqueous humor of ECCE+IOL group were higher than that in ECCE and control at 1 to 14 days postoperation, respectively, it increased to peak value at 3 to 7 days postoperation and decreased gradually two weeks postoperation; (2) The changes in IL-2, TNF-? and NO 2 -/NO 3 - in each group were basically similer; (3) The changes of IL-2 and TNF-? level were closely related with NO content in aqueous humor( r=0.69, P
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AIM: To investigate the effect of nitric oxide (NO) in ocular inflammation after intraocular lens implantation. METHODS: All New Zealand rabbits were divided randomly into three groups: 1. control group, 2. L-arginine (L-Arg) group, 3. N-nitro-L-arginine (L-NNA) group. Extracapsular cataract extraction (ECCE) and posterior chamber intraocular lens implantation (IOL) were operated in all animals of each groups. The inflammatory response of all rabbit eyes, including cornea edema and anterior chamber exudation were investigated 1, 3, 7, 14, 30 days afteroperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying, as well as for NO- 2/NO- 3 measurement. RESULTS:NO- 2/NO- 3 contents, total WBC and anterior chamber exudation in aqueous humor of L-Arg group were higher than that in control group. While that of L-NNA group were lower than that in control group.CONCLUSION:NO plays a role in intraocular inflammatory response after intraocular lens implantation. L-NNA, a nitric oxide synthase exhibitor, decreased NO contents, therefore it may reduce intraocular inflammatory response after intraocular lens implantation.