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施他宁缓解急性胰腺炎胰腺腺泡细胞线粒体损伤的研究

Study on the effect of stilamin in alleviating mitochondrial injury of pancreatic acinar cells in acute pancreatitis

摘要目的:探讨施他宁对AP时胰腺腺泡细胞线粒体损伤的影响及其可能机制。方法:将24只C57BL/6小鼠随机分为对照组、AP组、AP+施他宁治疗组(施他宁组)。AP组采用腹腔注射左旋精氨酸的方法制备AP模型,施他宁组在制模后2 h腹腔注射0.4 mg/kg施他宁,对照组腹腔注射等体积生理盐水。常规行胰腺组织病理学检查;ELISA法检测血清IL-6、IL-10、TNF-α水平,生物化学法检测血清淀粉酶、脂肪酶水平以及胰腺组织反映氧化应激反应的超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)水平;Mitotracker红色荧光标记法检测胰腺腺泡细胞内线粒体数量,蛋白质印迹法检测反映线粒体数量的ND-3蛋白表达量;免疫组织化学染色检测反映线粒体功能的线粒体融合蛋白(Mfn-2)和线粒体转录因子A(TFAM)表达量。结果:与AP组比较,施他宁组小鼠胰腺病理评分显著降低[(2.07±0.50)分比(3.93±0.64)分],血清淀粉酶及脂肪酶水平显著下降[(1 493±172)U/L比(1 832±86)U/L,(225.4±83.2)U/L比671.0±164.5)U/L]分;血清IL-6、TNF-α水平显著下降,而IL-10水平显著升高[(99.09±39.65)ng/L比(358.60±139.22)ng/L,(22.75±11.24)ng/L比(40.83±1.62)ng/L,(15.12±5.03)ng/L比(9.92±8.73)ng/L];Mitotracker荧光染色密度及ND-3、Mfn-2、TFAM蛋白表达量均显著增加(71.67±17.62比40.00±10.15,0.45±0.16比0.11±0.05,78%比54%,86%比47%),MDA、SOD、GSH水平显著升高[(5.00±1.73)nmol/mg蛋白比(7.33±2.08)nmol/mg蛋白,(17.33±3.21)U/mg蛋白比(8.67±2.07)U/mg蛋白,(131.33±20.55)U/mg蛋白比(77.33±29.69)U/mg蛋白],差异均具有统计学意义( P值均<0.05)。 结论:施他宁可能通过保护受损的胰腺腺泡细胞内线粒体数目及其功能,降低机体氧化应激水平,进而缓解AP小鼠胰腺组织的损伤及炎症反应程度。

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abstractsObjective:To investigate the effect of stilamin on mitochondrial injury of acute pancreatitis (AP)-related acinar cells and the possible mechanism.Methods:24 C57BL/6 mice were randomly divided into control group, AP group and AP+ stilamin treatment group (Stilamin group). AP model was prepared by intraperitoneal injection of l-arginine in AP group, 0.4 mg/kg stilamin was intraperitoneally injected at 2 h after modeling in stilamin group, and control group received intraperitoneal injection with saline in the same volume. Histopathological examination of pancreatic tissue was performed routinely. Serum levels of IL-6, IL-10 and TNF- α were detected by ELISA, serum levels of amylase and lipase, and serum levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) reflecting oxidative stress were detected by biochemistry method, respectively. Mitotracker red fluorescent labeling was used to detect the number of mitochondria in pancreatic acinar cells, and western blot was used to detect the ND-3 protein expression reflecting the number of mitochondria. The expression levels of mitochondrial fusion protein (Mfn-2) and mitochondrial transcription factor A (TFAM) reflecting mitochondrial function were determined by immunohistochemical staining.Results:Compared with AP group, the pancreatic pathology scores of mice in Stilamin group were significantly decreased [(2.07±0.50) vs (3.93±0.64)], serum amylase and lipase levels were significantly decreased [(1 493±172)U/L vs (1 832±86)U/L, (225.4±83.2)U/L vs (671.0±164.5)U/L]; serum IL-6 and TNF-α levels were greatly decreased, while IL-10 levels were obviously increased [(99.09±39.65)ng/L vs (358.60±139.22)ng/L, (22.75±11.24)ng/L vs (40.83±1.62)ng/L, (15.12±5.03)ng/L vs (9.92±8.73)ng/L]. Mitotracker staining density and expression levels of ND-3, Mfn-2 and Tfam were increased [(71.67±17.62) vs (40.00±10.15), (0.45±0.16) vs (0.11±0.05), 78% vs 54%, 86% vs 47%], and MDA, SOD, and GSH levels were increased [(5.00±1.73)nmol/mg vs (7.33±2.08)nmol/mg protein, (17.33±3.21)U/mg vs (8.67±2.07)] U/mg protein. The ratio of (131.33±20.55)U/mg to (77.33±29.69)U/mg protein was statistically significant (all P<0.05). Conclusions:By protecting the mitochondria number and function of damaged pancreatic acinar cells and reducing the level of oxidative stress, stilamin could alleviate the level of pancreatic tissue damage and inflammatory response in mice with acute pancreatitis.

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