Intermedin对糖尿病大鼠心肌缺血再灌注炎性细胞因子的影响

The Effects of Intermedin on Inflammatory Response Induced by Myocardium Ischemia Reperfusion in Diabetic Rats

目的观察Intermedin对糖尿病大鼠心肌缺血再灌注后炎性细胞因子的影响,从而进一步探讨Intermedin对糖尿病心肌缺血再灌注损伤保护作用的机制。方法健康雄性SD大鼠74只,给予适应性饲养一周后,将74只雄性SD大鼠随机分为糖尿病组(50只)和非糖尿病组(24只)。非糖尿病组给予枸橼酸缓冲液腹腔注射,糖尿病组建立糖尿病大鼠模型,通过腹腔注射链脲佐菌素复制I型糖尿病大鼠模型。然后阻断大鼠冠状动脉左前降支30 min制备心肌缺血再灌注损伤模型。将成功造模的大鼠随机分为5个实验组:对照组(NS)、缺血再灌注组(NIR)、糖尿病组(DS)、糖尿病缺血再灌注组(DIR)和应用Intermedin干预的治疗组(IMD)。应用ELISA法测定血清中炎性细胞因子TNF-α、IL-6和IL-1β的含量。应用RT-PCR法检测心肌组织中TNF-α、IL-1β和IL-6的m RNA的表达。免疫组织化学S-P法和Western-blot检测心肌组织NF-κB的表达。结果与各自对照组和糖尿病组比较,NIR组与DIR组大鼠血清中TNF-α、IL-6、IL-1β含量升高(P<0.05)。但是同DIR组比较,IMD组大鼠血清中TNF-α、IL-6、IL-1β含量则明显降低。(P<0.05)。NIR组和DIR组大鼠心肌组织中TNF-α、IL-6、IL-1βm RNA表达与各自对照组和糖尿病组比较均明显增强(P<0.05)。IMD组TNF-α、IL-6、IL-1βm RNA表达比DIR组明显减弱(P<0.05)。免疫组织化学结果显示,当心肌缺血再灌注后其心肌组织中可观察到核移位的现象,即在NIR组和DIR组中NF-κB活化增强,阳性颗粒出现在胞浆和胞核。而在对照组和糖尿病组大鼠心肌组织中观察到NF-κB呈低表达状态,并且阳性表达出现在胞浆中。同DIR组相比较,IMD组NF-κB的表达活性将低,阳性表达的细胞数明显减少(P<0.05)。Western blot结果显示:分别与对照组和糖尿病组相比较,NIR组和DIR组NF-κB表达量均显著增加(P<0.05),而IMD组NF-κB表达显著低于DIR组(P<0.05)。结论 Intermedin对糖尿病大鼠心肌缺血再灌注具有保护作用,其部分保护作用机制可能与intermedin可以抑制NF-κB活化,从而减少炎性因子如TNF-α、IL-1β和IL-6的表达,减轻炎症反应有关。

Aim To observe the change of activation of myocardial NF-κB and its downstream inflammatory factors such as tumor necrosisfactor-alpha( TNF-α),whiteinterleukin-1β( IL-1β) and whiteinterleukin-6( IL-6) after myocardium ischemia reperfusion in diabetic rat and its mechanism. Methods 74 healthy male Sprague-Dawley rats were randomly divided into diabetics group( n = 50) and non-diabetics group( n = 24). Experimental diabetes was induced in the animals by a single intraperitoneal administration of STZ dissolved in 0. 1 mol / L citrate buffer( p H 4. 5) at a dose of 55 mg / kg. Normal rats received an equal volume of citrate buffer. Three days post-STZ injection,tail vein blood glucose samples were collected and measured with Onetouchglucometer( Johnson & Johnson,USA). The rats were considered diabetic and used for the study only if their glucose levels were greater than 15 mmol / L. Rats were housed 8 weeks after vehicle or STZ injection.Animals were anesthetized intraperitoneally with pento-barbital sodium( 50 mg / kg) followed by a tracheotomy and an artificial ventilation. The left femoral vein was cann-ulated for administration of drugs. A fourth-intercostal spacethoracotomy was performed,and the pericardium was excised to expose the heart. The artery was occluded for 30 min by tightening the ligature. The rats in non-diabetic group were randomly reassigned into sham-operated group( NS),I / R group( NIR),the rats in diabetic group were randomly reassigned into sham-operated group( DS),I / R group( DIR) and intermedin groups( n =12). Determinate the content of tumor necrosis factor alpha in( TNF-α),interleukin 6( IL-6) and interleukin 1 beta( IL-1β) in serum with ELISA method. Determinate m RNA expression of TNF-α,IL-6 and IL-1β in myocardial tissue with real time fluorescent quantitative PCR. The translocation of NF-κB in the cardiomyocytes was detected by immunohistochemistry and the protein expression of NF-κB was determined with westernblot. Results In NIR and DIR group,the content of TNF-α,IL-1β and IL-6 in serum was significantly higher than that in their respective control groups( P < 0. 05). In DS and DIR group,the above indexes were significantly higher than those in NS and NIR group( P < 0. 05). The content of TNF-α,IL-6 and IL-1β in serum of the rats in group IMD is lower than that of DIR group( P < 0. 05). After ischemia and reperfusion,the m RNA expression of TNF-α,IL-6,and IL-1β in myocardial tissues in NIR group and DIR were significantly enhanced than that of their respective control groups,NS group and DS group( P < 0. 05). In IMD group,the expression of m RNA of IL-6,TNF-α and IL-1β decreased obviously than that of DIR group( P < 0. 05). Immunohistochemical results show: NF-κB in myocardial tissues showed low expression state in NS group and DS group,and the positive expression appeared in the cytoplasm. In NIR and DIR group,the NF-κB activated,enhanced expression,positive granules appeared in cytoplasm and nucleus,that is the nuclear translocation. NF-κB activity of IMD group is lower than that of the DI / R group.The positive number of nuclei expression is significantly less( P < 0. 05). After ischemia reperfusion,the NF-κB cell shift from the plasma to the nucleus is limited. The Western blot results show: the NF-κB expression of the NIR and the DIR group were significantly increased after ischemia and reperfusion compared with NS and DS group( P < 0. 05). The protein expression of NF-κB in IMD group was significantly lower than that in DIR group( P < 0. 05). Conclusion Intermedin on diabetic rats plays a protective role in myocardial ischemia reperfusion,some of its protection mechanism may be related to Intermedin which can inhibit the nf-kappa B activation,thus decrease the inflammatory factors such as: TNF alpha,beta IL-1,and the expression of IL-6,to reduce inflammation reactions.