大黄游离蒽醌对脂肪干细胞移植治疗大鼠急性胰腺炎的影响
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摘要
背景:研究表明大黄游离蒽醌能够保护肠黏膜细胞结构和功能的稳定,但尚未有将其与脂肪干细胞联合治疗大鼠急性胰腺炎的报道。目的:观察并探讨大黄游离蒽对脂肪干细胞移植治疗大鼠急性胰腺炎的影响及其机制。方法:体外快速复苏并培养购自中国医学科学院的冻存鼠脂肪干细胞,随后采用MTT比色法测定脂肪干细胞的细胞存活率并行PKH-26标记。将北京维通利华动物实验技术有限公司提供的SD大鼠随机平均分为4组,病理模型组大鼠采用肠壁穿刺逆行胰胆管注射体积分数5%牛黄胆酸钠建立急性胰腺炎病理模型,然后尾静脉注射0.5 mL的L-DMEM完全培养液;大黄游离蒽组建模后给予大黄游离蒽醌200 mg/kg灌胃;脂肪干细胞组建模后尾静脉注射0.5 mL细胞浓度1×10~7 L~(-1)脂肪干细胞;联合治疗组建模后同时给予以上2种干预。各个实验组中所有干预治疗措施每日进行1次且连续治疗3 d。结果与结论:(1)与理模型组的大鼠相比,大黄游离蒽醌组和脂肪干细胞组大鼠血清淀粉酶活性以及白细胞介素6水平显著降低(P <0.05),胰蛋白酶原激活肽水平显著升高(P <0.05);经过脂肪干细胞移植和大黄游离蒽醌灌胃处理联合治疗,上述3指标水平较大黄游离蒽醌组和脂肪干细胞组进一步降低(P<0.05);(2)苏木精-伊红染色结果显示,此2个单独治疗组大鼠胰腺组织中脂肪变性、出血、细胞坏死及炎症细胞浸润等病理变化程度均有明显减轻,联合治疗组中大鼠的病理变化的缓解程度则更为明显;(3)荧光显微镜下观察可见,PKH-26标记的阳性细胞数联合治疗组最多,脂肪干细胞组次之,而在大黄游离蒽组及病理模型组则未见(P <0.05);(4)TUNEL法检测可见,与病理模型组比较,2个单独治疗组中胰腺组织中的细胞凋亡数明显降低,联合治疗组进一步降低(P <0.01);(5)RT-PCR和Western blot法检测显示,病理模型组大鼠胰腺组织转化生长因子β1及Smad2/3基因和蛋白表达最高,联合治疗组最低(P <0.01),而2个单独干预组均较病理模型组显著较低(P <0.05);(6)结果证实,大黄游离蒽干预联合脂肪干细胞移植治疗大鼠急性胰腺炎可以有效改善模型大鼠急性胰腺炎的血液生化指标水平;并可明显地缓解急性胰腺炎模型大鼠炎症反应程度和胰腺组织形态学病变以及胰腺细胞的凋亡,这可能与降低转化生长因子β/Smad信号通路有关。
BACKGROUND: Studies have shown that rhubarb-free anthraquinone can protect the stability of the intestinal mucosa cell structure and function. However, the combination of rhubarb-free anthraquinone with adipose-derived stem cells to treat acute pancreatitis in rats has not yet been reported. OBJECTIVE: To investigate the effects of rhubarb-free anthraquinone on the treatment of acute pancreatitis by fat stem cell transplantation in rats. METHODS: Cryopreserved mouse adipose-derived stem cells(provided by the Chinese Academy of Medical Sciences) were rapidly recovered and cultured in vitro. The survival cells were labeled using PKH-26, and then the cell survival rate of adipose-derived stem cells was determined by MTT colorimetry. Sprague-Dawley rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. were equally randomized into four groups. Animal models of acute pancreatitis were made in all rats through intestinal wall puncture for retrograde administration of 5% sodium taurocholate to the pancreaticobiliary tube, and then the model rats were given tail vein injection of L-DMEM complete medium(0.5 mL) in the model group, intragastric administration of 200 mg/kg rhubarb-free anthraquinone in the rhubarb-free anthraquinone group, tail vein injection of adipose-derived stem cells(1×10~7 cells/L, 0.5 mL) in the cell transplantation group, and intragastric administration of 200 mg/kg rhubarb-free anthraquinone plus tail vein injection of 1×10~7 adipose-derived stem cells(0.5 mL) in the combined treatment group. All interventions in each group were performed once a day, for 3 continuous days. RESULTS AND CONCLUSION:(1) The levels of serum amylase and interleukin-6 were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group compared with the model group(P < 0.05), while the level of trypsinogen activation peptide significantly increased(P < 0.05). In the combined treatment group, the levels of three indicators mentioned above were significantly decreased compared with the rhubarb-free anthraquinone group and cell transplantation group(P < 0.05).(2) The results of hematoxylin-eosin staining showed that the pathological changes in the pancreatic tissue of rats were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, such as fatty degeneration, hemorrhage, cell necrosis and inflammatory cell infiltration. The remission of pathological changes was more obvious in the combined treatment group.(3) The number of positive cells labeled by PKH-26 was the highest in the combined treatment group, followed by the cell transplantation group, while there were no PKH-26-positive cells in the rhubarb-free anthraquinone group and model group(P < 0.05).(4) Compared with the model group, the number of apoptotic cells in the pancreatic tissue was significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, and lowest in the combined treatment group(P < 0.01).(5) The expression of transforming growth factor-β1 and Smad2/3 at gene and protein levels was highest in the model group, followed by the rhubarb-free anthraquinone group and cell transplantation group(P < 0.05), and lowest in the combined treatment group(P < 0.01). To conclude, the combined intervention of rhubarb-free anthraquinone and adipose-derived stem cell transplantation could effectively improve the blood biochemical index level in the rats with acute pancreatitis, significantly relieve the degree of inflammatory response, pancreatic tissue morphology, and apoptosis of pancreatic cells, which may be related to the reduction of the transforming growth factor-β/Smad signal pathway.
BACKGROUND: Studies have shown that rhubarb-free anthraquinone can protect the stability of the intestinal mucosa cell structure and function. However, the combination of rhubarb-free anthraquinone with adipose-derived stem cells to treat acute pancreatitis in rats has not yet been reported. OBJECTIVE: To investigate the effects of rhubarb-free anthraquinone on the treatment of acute pancreatitis by fat stem cell transplantation in rats. METHODS: Cryopreserved mouse adipose-derived stem cells(provided by the Chinese Academy of Medical Sciences) were rapidly recovered and cultured in vitro. The survival cells were labeled using PKH-26, and then the cell survival rate of adipose-derived stem cells was determined by MTT colorimetry. Sprague-Dawley rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. were equally randomized into four groups. Animal models of acute pancreatitis were made in all rats through intestinal wall puncture for retrograde administration of 5% sodium taurocholate to the pancreaticobiliary tube, and then the model rats were given tail vein injection of L-DMEM complete medium(0.5 mL) in the model group, intragastric administration of 200 mg/kg rhubarb-free anthraquinone in the rhubarb-free anthraquinone group, tail vein injection of adipose-derived stem cells(1×10~7 cells/L, 0.5 mL) in the cell transplantation group, and intragastric administration of 200 mg/kg rhubarb-free anthraquinone plus tail vein injection of 1×10~7 adipose-derived stem cells(0.5 mL) in the combined treatment group. All interventions in each group were performed once a day, for 3 continuous days. RESULTS AND CONCLUSION:(1) The levels of serum amylase and interleukin-6 were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group compared with the model group(P < 0.05), while the level of trypsinogen activation peptide significantly increased(P < 0.05). In the combined treatment group, the levels of three indicators mentioned above were significantly decreased compared with the rhubarb-free anthraquinone group and cell transplantation group(P < 0.05).(2) The results of hematoxylin-eosin staining showed that the pathological changes in the pancreatic tissue of rats were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, such as fatty degeneration, hemorrhage, cell necrosis and inflammatory cell infiltration. The remission of pathological changes was more obvious in the combined treatment group.(3) The number of positive cells labeled by PKH-26 was the highest in the combined treatment group, followed by the cell transplantation group, while there were no PKH-26-positive cells in the rhubarb-free anthraquinone group and model group(P < 0.05).(4) Compared with the model group, the number of apoptotic cells in the pancreatic tissue was significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, and lowest in the combined treatment group(P < 0.01).(5) The expression of transforming growth factor-β1 and Smad2/3 at gene and protein levels was highest in the model group, followed by the rhubarb-free anthraquinone group and cell transplantation group(P < 0.05), and lowest in the combined treatment group(P < 0.01). To conclude, the combined intervention of rhubarb-free anthraquinone and adipose-derived stem cell transplantation could effectively improve the blood biochemical index level in the rats with acute pancreatitis, significantly relieve the degree of inflammatory response, pancreatic tissue morphology, and apoptosis of pancreatic cells, which may be related to the reduction of the transforming growth factor-β/Smad signal pathway.
引文
[1]阮佼,张家骏,曹志刚.重视肠道衰竭在重症急性胰腺炎发病中的作用[J].中国急救医学,2015,35(s2):5-6.
[2]磨庆福.重症急性胰腺炎的发病原因及发病机制研究进展[J].胃肠病学和肝病学杂志,2014,23(9):1107-1110.
[3]赵宁.重症急性胰腺炎诊治进展[J].中华急诊医学杂志,2014,23(10):67-68.
[4]龙靓,刘湘,郭小兰,等.2012-2014年我院急性胰腺炎患者营养药物使用情况调查[J].药物流行病学杂志,2015(12):740-743.
[5]刘大晟,罗羽宏,李接兴.重症急性胰腺炎早期手术与延期手术治疗效果Meta分析[J].肝胆胰外科杂志,2014,26(4):289-292.
[6]张芬,杨柳,王浩,等.急性胰腺炎的研究进展[J].现代生物医学进展,2016,16(15):2983-2986.
[7]Tang Y,Gan X,Cheheltani R,et al.Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model.Nanomedicine.2014;10(8):1711-1718.
[8]Atashi F,Modarressi A,Pepper MS.The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review.Stem Cells Dev.2015;24(10):1150-1163.
[9]Jeong H,Yim HW,Cho Y,et al.Efficacy and safety of stem cell therapies for patients with stroke:a systematic review and single arm meta-analysis.Int J Stem Cells.2014;7(2):63.
[10]Bruin JE,Saber N,Braun N,et al.Treating diet-induced diabetes and obesity with human embryonic stem cell-derived pancreatic progenitor cells and antidiabetic drugs.Stem Cell Rep.2015;4(4):605-620.
[11]Morigi M,Coppi PD.Cell therapy for kidney injury:different options and mechanisms-mesenchymal and amniotic fluid stem cells.Nephronexp Nephrol.2014;126(2):59.
[12]Teng M,Sun J,Zhao Z,et al.A brief review:adipose-derived stem cells and their therapeutic potential in cardiovascular diseases.Stem Cell Res Ther.2017;8(1):124.
[13]Tabatabaei RQ,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:a review.World J Stem Cell.2017;9(8):107.
[14]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells attenuate liver ischemia-reperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[15]Chen L,Qin F,Ge M,et al.Application of adipose-derived stem cells in heart disease.J Cardiovasc Transl.2014;7(7):651-663.
[16]庄培涛,谭雪莹,邱建涛,等.人脂肪间充质干细胞对急性坏死性胰腺炎大鼠治疗疗效的观察[J].中华胰腺病杂志,2017,17(2):82-87.
[17]竹林,赵健蕾,彭小航,等.大黄中游离蒽醌类化合物在实验性急性胰腺炎大鼠的组织药理学研究[J].中国中药杂志,2014,39(2):304-308.
[18]刘媛琪,王璐璐,陈立,等.大黄游离蒽醌对重症急性胰腺炎大鼠肝脏的影响[J].时珍国医国药,2015,26(8):1827-1829.
[19]Tohill M,Terenghi G.Stem-cell plasticity and therapy for injuries of the peripheral nervous system.Biotechnol Appl Biochem.2011;40(1):17-24.
[20]陈立,范玲,谭小勇,等.大黄游离蒽醌对重症急性胰腺炎大鼠肠道免疫功能的影响及其作用机制[J].山东医药,2016,56(24):13-16.
[21]刘京伟,陈玲玲,赵安成.血清SAA、CRP、PCT及TAP联合检测在急性重症胰腺炎早期诊治中的临床意义[J].国际检验医学杂志,2016,37(13):1811-1813.
[22]范永熙,单海滨,李兆然.急性胰腺炎患者血清PCT、IL-6和hs-CRP水平变化的临床意义[J].中国现代医学杂志,2014,24(10):46-49.
[23]于宁,王晓倩,张欣.IL-10、TGF-β及其mRNA在急性胰腺炎合并肝、肾损伤中的表达[J].检验医学与临床,2017,14(17):2610-2612.
[24]Zhang J,Ning X,Cui W,et al.Transforming growth factor(TGF)-β-induced microRNA-216a promotes acute pancreatitis via Akt and TGF-βpathway in mice.Digest Dis Sci.2015;60(1):127-35.
[25]Akbarshahi H,Sam A,Chen C,et al.Early activation of pulmonary TGF-β1/Smad2 signaling in mice with acute pancreatitis-associated acute lung injury.Mediators Inflamm.2014;2014(1):148029.
[26]Sakao M,Okazaki K,Uchida K,et al.The role of Smad2/3linker phosphorylation in the regenerative phase of acute pancreatitis.Pancreatology.2016;16(4):S84-S85.
[27]杨芳勇,王丽娜,岳小强,等.中药大黄在急性胰腺炎治疗中的临床应用[J].中华胰腺病杂志,2015,15(3):212-214.
[28]王璐璐,刘媛琪,陈立,等.大黄游离蒽醌对重症急性胰腺炎大鼠肾损伤的影响[J].中药药理与临床,2015,31(2):31-34.
[29]杨永茂,王平,张艳,等.大黄游离蒽醌在正常和重症急性胰腺炎犬体内吸收动力学比较研究[J].中国中西医结合杂志,2012,32(4):494-498.
[30]金佳佳,陈建华,杨伟刚.经皮芒硝超声导入对急性胰腺炎患者炎性因子的影响[J].湖北中医杂志,2018,40(1):6-8.
[31]彭智,邱彬.基因重组干扰素γ对呼吸道合胞病毒毛细支气管炎患儿血清SP-D、TGF-β以及IL-4水平的影响[J].中国医师杂志,2018,20(4):612-614.
[32]许希燕,王巧稚,罗茂,等.阿魏酸联合脂肪间充质干细胞调节TGF-β/Smad信号转导通路对肝星状细胞凋亡的影响研究[J].重庆医学,2018,47(15):1997-2000.
[33]范惠.中医药干预肾纤维化TGF-β1/Smad信号转导通路的体外研究概况[J].泸州医学院学报,2017,40(2):221-224.
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[35]石占利,方堃,孙静,等.大黄素对重症急性胰腺炎大鼠胰腺细胞凋亡的干预作用[J].中华全科医学,2017,15(11):1830-1834.
[2]磨庆福.重症急性胰腺炎的发病原因及发病机制研究进展[J].胃肠病学和肝病学杂志,2014,23(9):1107-1110.
[3]赵宁.重症急性胰腺炎诊治进展[J].中华急诊医学杂志,2014,23(10):67-68.
[4]龙靓,刘湘,郭小兰,等.2012-2014年我院急性胰腺炎患者营养药物使用情况调查[J].药物流行病学杂志,2015(12):740-743.
[5]刘大晟,罗羽宏,李接兴.重症急性胰腺炎早期手术与延期手术治疗效果Meta分析[J].肝胆胰外科杂志,2014,26(4):289-292.
[6]张芬,杨柳,王浩,等.急性胰腺炎的研究进展[J].现代生物医学进展,2016,16(15):2983-2986.
[7]Tang Y,Gan X,Cheheltani R,et al.Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model.Nanomedicine.2014;10(8):1711-1718.
[8]Atashi F,Modarressi A,Pepper MS.The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review.Stem Cells Dev.2015;24(10):1150-1163.
[9]Jeong H,Yim HW,Cho Y,et al.Efficacy and safety of stem cell therapies for patients with stroke:a systematic review and single arm meta-analysis.Int J Stem Cells.2014;7(2):63.
[10]Bruin JE,Saber N,Braun N,et al.Treating diet-induced diabetes and obesity with human embryonic stem cell-derived pancreatic progenitor cells and antidiabetic drugs.Stem Cell Rep.2015;4(4):605-620.
[11]Morigi M,Coppi PD.Cell therapy for kidney injury:different options and mechanisms-mesenchymal and amniotic fluid stem cells.Nephronexp Nephrol.2014;126(2):59.
[12]Teng M,Sun J,Zhao Z,et al.A brief review:adipose-derived stem cells and their therapeutic potential in cardiovascular diseases.Stem Cell Res Ther.2017;8(1):124.
[13]Tabatabaei RQ,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:a review.World J Stem Cell.2017;9(8):107.
[14]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells attenuate liver ischemia-reperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[15]Chen L,Qin F,Ge M,et al.Application of adipose-derived stem cells in heart disease.J Cardiovasc Transl.2014;7(7):651-663.
[16]庄培涛,谭雪莹,邱建涛,等.人脂肪间充质干细胞对急性坏死性胰腺炎大鼠治疗疗效的观察[J].中华胰腺病杂志,2017,17(2):82-87.
[17]竹林,赵健蕾,彭小航,等.大黄中游离蒽醌类化合物在实验性急性胰腺炎大鼠的组织药理学研究[J].中国中药杂志,2014,39(2):304-308.
[18]刘媛琪,王璐璐,陈立,等.大黄游离蒽醌对重症急性胰腺炎大鼠肝脏的影响[J].时珍国医国药,2015,26(8):1827-1829.
[19]Tohill M,Terenghi G.Stem-cell plasticity and therapy for injuries of the peripheral nervous system.Biotechnol Appl Biochem.2011;40(1):17-24.
[20]陈立,范玲,谭小勇,等.大黄游离蒽醌对重症急性胰腺炎大鼠肠道免疫功能的影响及其作用机制[J].山东医药,2016,56(24):13-16.
[21]刘京伟,陈玲玲,赵安成.血清SAA、CRP、PCT及TAP联合检测在急性重症胰腺炎早期诊治中的临床意义[J].国际检验医学杂志,2016,37(13):1811-1813.
[22]范永熙,单海滨,李兆然.急性胰腺炎患者血清PCT、IL-6和hs-CRP水平变化的临床意义[J].中国现代医学杂志,2014,24(10):46-49.
[23]于宁,王晓倩,张欣.IL-10、TGF-β及其mRNA在急性胰腺炎合并肝、肾损伤中的表达[J].检验医学与临床,2017,14(17):2610-2612.
[24]Zhang J,Ning X,Cui W,et al.Transforming growth factor(TGF)-β-induced microRNA-216a promotes acute pancreatitis via Akt and TGF-βpathway in mice.Digest Dis Sci.2015;60(1):127-35.
[25]Akbarshahi H,Sam A,Chen C,et al.Early activation of pulmonary TGF-β1/Smad2 signaling in mice with acute pancreatitis-associated acute lung injury.Mediators Inflamm.2014;2014(1):148029.
[26]Sakao M,Okazaki K,Uchida K,et al.The role of Smad2/3linker phosphorylation in the regenerative phase of acute pancreatitis.Pancreatology.2016;16(4):S84-S85.
[27]杨芳勇,王丽娜,岳小强,等.中药大黄在急性胰腺炎治疗中的临床应用[J].中华胰腺病杂志,2015,15(3):212-214.
[28]王璐璐,刘媛琪,陈立,等.大黄游离蒽醌对重症急性胰腺炎大鼠肾损伤的影响[J].中药药理与临床,2015,31(2):31-34.
[29]杨永茂,王平,张艳,等.大黄游离蒽醌在正常和重症急性胰腺炎犬体内吸收动力学比较研究[J].中国中西医结合杂志,2012,32(4):494-498.
[30]金佳佳,陈建华,杨伟刚.经皮芒硝超声导入对急性胰腺炎患者炎性因子的影响[J].湖北中医杂志,2018,40(1):6-8.
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