丹参酮ⅡA联合右美托咪定通过AMPK/ULK1自噬通路减少肾缺血再灌注损伤作用的研究

Effects of TanshinoneⅡA Combined with Dexmetomidine in Regulating Autophagy Levels Against Renal Ischemia Reperfusion Injury by AMPK/ULK1 Pathway

目的:探讨丹参酮ⅡA联合右美托咪定通过AMPK/ULK1通路对肾缺血再灌注后细胞自噬与凋亡的影响。方法:选取健康Wistar雄性大鼠50只,随机均分为五组,即A组(丹参酮ⅡA+右美托咪定)、B组(右美托咪定)、C组(丹参酮ⅡA)、D组(缺血再灌注模型组)和E组(假手术组)。采用夹闭双侧肾蒂45 min建立肾缺血再灌注模型。在缺血前30 min,A组大鼠腹腔注射丹参酮提取物+右美托咪定25μg/kg,B组大鼠腹腔注射右美托咪定25μg/kg,C组大鼠腹腔注射丹参注射液,D、E组大鼠给予等量0.9%氯化钠溶液。再灌注24 h后,采用苏木精-伊红染色法观察肾组织病理变化;采用免疫印迹法测定AMPK、ULK1的表达;取血标本测定血清肿瘤坏死因子α(TNF-α)、白细胞介素(IL)6和IL-10水平。结果:A组大鼠肾组织病理学改变较D组减轻。E组大鼠细胞凋亡不明显;D组大鼠可见大量的凋亡细胞和严重的组织损伤;A组大鼠的组织损伤较轻,仅见散在的凋亡细胞;B、C组大鼠组织损伤轻微,可见少量的凋亡细胞。与D组比较,A、B和C组大鼠的凋亡指数明显降低,差异均有统计学意义(P<0.05)。与D组比较,A组大鼠肾组织中的AMPK、ULK1表达明显减少,差异均有统计学意义(P<0.05)。与B、C和D组比较,A组大鼠的IL-6、TNF-α水平明显降低,而IL-10水平明显升高,差异均有统计学意义(P<0.05)。结论:肾缺血再灌注时,丹参酮ⅡA联合右美托咪定能够减轻组织的炎症反应,减少肾脏细胞凋亡,缓解组织损伤,可能是通过AMPK/ULK1通路抑制自噬水平来实现的。

OBJECTIVE:To probe into the effects of tanshinoneⅡA combined with dexmedetomidine on autophagy and apoptosis after renal ischemia reperfusion through AMPK/ULK1 pathway.METHODS:Fifty healthy male Wistar rats were randomly divided into five groups:group A(tanshinoneⅡA+dexmedetomidine),group B(dexmedetomidine),group C(tanshinoneⅡA),group D(ischemia reperfusion model)and group E(sham surgery group).The renal ischemia-reperfusion model was established by clamping the bilateral renal pedicles for 45 min.Before ischemia of 30 min,rats in group A were intraperitoneally injected with tanshinone extraction+25μg/kg dexmedetomidine,rats in group B were injected intraperitoneally with 25μg/kg dexmedetomidine,rats in group C were injected intraperitoneally with Danshen injection,rats in group D and E were given the same amount of 0.9%sodium chloride solution.After reperfusion of 24 h,the pathological changes of renal tissue were observed by HE staining method;the expression of AMPK and ULK1 were determined by Western blot;blood samples were taken to determine serum tumor necrosis factorα(TNF-α),interleukin(IL)6 and IL-10.RESULTS:The histopathological changes of rats in group A were lighter than those in group D.Apoptosis was not obvious in group E,a large number of apoptotic cells and severe tissue damages were observed in group D,and only scattered apoptotic cells were seen in group A with mild tissue damages.The tissue damages of rats in groups B and C were slight,and a small number of apoptotic cells were visible.Compared with group D,apoptosis index of rats in groups A,B and C decreased significantly,with statistically significant difference(P<0.05).Compared with group D,the expression of AMPK and ULK1 in renal tissues of rats in group A rats decreased significantly,and the differences were statistically significant(P<0.05).Compared with group B,C and D,the levels of IL-6 and TNF-αin group A decreased significantly,while the levels of IL-10 increased significantly,with statistically significant difference(P<0.05).CONCLUSIONS:During renal ischemia reperfusion,tanshinoneⅡA combined with dexmedetomidine can reduce the tissue inflammation,decrease the renal cell apoptosis and alleviate the tissue damage,which may be achieved through inhibition of autophagy levels by AMPK/ULK1 pathway.