López-Neblina Fernando, Toledo-Pereyra Luis H, Toledo Alexander H, Walsh Jon
Trauma, Surgery Research, and Molecular Biology, Borgess Research Institute, Kalamazoo, Michigan 49048, USA.
J Surg Res. 2007 Jun 1;140(1):121-8. doi: 10.1016/j.jss.2006.12.003. Epub 2007 Mar 13.
Dantrolene is a ryanodine receptor and intracellular calcium antagonist. The ryanodine receptor (RyR) Ca(2+) release channel mobilizes Ca(2+) from internal stores to support a variety of cellular functions, including the inflammatory response after ischemia and reperfusion. The pharmacological mechanism of dantrolene is associated with the inhibition of the release of Ca(2+) from the skeletal muscle sarcoplasmic reticulum (SR). We hypothesized that dantrolene could exert a protective effect in our model of liver ischemia and reperfusion by modulating TNF-alpha and IL-10.
Mice subjected to 90 min of partial (70 to 80%) hepatic ischemia and 3 h of reperfusion were divided into five groups (n = 6/group): sham, ischemic control, and the dantrolene 1 mg/kg group studied at three times of administration: 15 min before reperfusion (DAN-PRE), at the time of reperfusion (DAN-RP), and 15 min after reperfusion (DAN-POS). The parameters measured at 3 h of reperfusion included serum liver function tests alanine aminotransferase (ALT) and aspartate aminotransferase (AST), TNF-alpha, and IL-10 in serum and liver histology.
It was demonstrated that the RyR intracellular calcium antagonist dantrolene offered the most significant protection for the ischemic liver when given before reperfusion and at the time of reperfusion. AST significantly differed between the control group and the DAN-PRE and DAN-RP groups (P < 0.05). ALT showed a statistically significant decrease in the DAN-PRE treated group and a decrease, although not significant, in the DAN-RP. Histological examination demonstrated a significant decrease in vacuolization in the same both groups (P < 0.05). Necrosis was significantly diminished when dantrolene was used at the time of reperfusion; congestion decreased in the same groups but without statistical significant difference. The levels of TNF-alpha were significantly decreased in the DAN-RP group. There was a decrease in TNF-alpha in the DAN-PRE group but not statistically significant. IL-10 reflected the protection observed in necrosis and vacuolization in the histopathology with an increment at the time of reperfusion (P < 0.05). DAN-POS did not exert a protective effect in ALT, AST, liver histology, or cytokine response.
For the first time the ryanodine receptor antagonist dantrolene offered significant functional and structural protection of the ischemic liver when given at the time for reperfusion and partial protection when given prereperfusion. RyR inhibition approach down-regulated the expression of TNF-alpha and induced an increment of the protective cytokine IL-10 when administered at the time of reperfusion. There was no protective effect of dantrolene after reperfusion.
丹曲林是一种兰尼碱受体和细胞内钙拮抗剂。兰尼碱受体（RyR）钙释放通道从内部储存库中释放钙离子，以支持多种细胞功能，包括缺血再灌注后的炎症反应。丹曲林的药理机制与抑制骨骼肌肌浆网（SR）中钙离子的释放有关。我们推测，丹曲林可通过调节肿瘤坏死因子-α（TNF-α）和白细胞介素-10（IL-10），对我们的肝脏缺血再灌注模型发挥保护作用。
将经历90分钟部分（70%至80%）肝脏缺血和3小时再灌注的小鼠分为五组（每组n = 6）：假手术组、缺血对照组，以及丹曲林1mg/kg组，该组在三个给药时间点进行研究：再灌注前15分钟（DAN-PRE）、再灌注时（DAN-RP）和再灌注后15分钟（DAN-POS）。在再灌注3小时时测量的参数包括血清肝功能检测指标丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）、血清中的TNF-α和IL-10，以及肝脏组织学。
结果表明，RyR细胞内钙拮抗剂丹曲林在再灌注前和再灌注时给药，对缺血肝脏提供了最显著的保护作用。对照组与DAN-PRE组和DAN-RP组之间的AST有显著差异（P < 0.05）。ALT在DAN-PRE治疗组中显示出统计学上的显著降低，在DAN-RP组中虽有降低但不显著。组织学检查表明，这两组的空泡化均显著减少（P < 0.05）。再灌注时使用丹曲林，坏死显著减少；相同组中的充血减少，但无统计学显著差异。DAN-RP组中的TNF-α水平显著降低。DAN-PRE组中的TNF-α有所降低，但无统计学显著差异。IL-10反映了在组织病理学中观察到的对坏死和空泡化的保护作用，在再灌注时有所增加（P < 0.05）。DAN-POS在ALT、AST、肝脏组织学或细胞因子反应方面未发挥保护作用。
首次发现，兰尼碱受体拮抗剂丹曲林在再灌注时给药，对缺血肝脏提供了显著的功能和结构保护，在再灌注前给药则提供了部分保护。RyR抑制方法在再灌注时给药可下调TNF-α的表达，并诱导保护性细胞因子IL-10增加。丹曲林在再灌注后无保护作用。
