针灸对糖尿病造影剂肾病大鼠肾脏细胞凋亡基因Fas/FasL的影响
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摘要
目的:通过观察针灸对糖尿病造影剂肾病大鼠肾脏细胞凋亡基因Fas/FasL的影响,探讨针灸对糖尿病造影剂肾病的保护机制。方法:雄性SD大鼠随机分为对照组、模型组、针刺组、艾灸组、联合组,每组8只。采用腹腔注射链脲佐菌素法制备糖尿病造影剂肾病大鼠模型。针刺组、艾灸组及联合组分别给予针刺、艾灸及针刺+艾灸联合治疗,3组均选取"三阴交""肾俞""脾俞",每日1次,连续7d。用HE染色法观察大鼠肾脏病理形态,醋酸铀-柠檬酸双染色法观察大鼠肾脏超微结构,脲酶法测定大鼠血清尿素氮(BUN)含量,苦味酸法测定大鼠血清肌酐(Scr)含量,氧化还原法检测大鼠肾脏组织一氧化氮合酶(NOS)活性,黄嘌呤氧化酶法检测大鼠肾脏组织超氧化物歧化酶(SOD)活性,硫代巴比妥酸法检测大鼠肾脏组织丙二醛(MDA)含量,比色法检测大鼠肾脏组织总抗氧化能力(T-AOC),Western blot法及荧光定量PCR法检测大鼠肾脏组织Fas、FasL蛋白及mRNA表达水平。结果:光学显微镜下,对照组大鼠肾小球、肾小管及间质无明显病理改变;模型组大鼠肾小球呈分叶状,肾小管上皮细胞多有局部坏死、空泡样变性。电镜下模型组大鼠肾小球血管袢基底膜增厚,肾小管上皮细胞胞质见大量空泡,线粒体严重肿胀,线粒体嵴排列紊乱、断裂或消失,可见凋亡样改变的细胞。针刺组和艾灸组肾脏病理改变基本同模型组;联合组大鼠肾脏病理改变有所改善。与对照组比较,模型组大鼠血清BUN、Scr含量明显升高(P<0.01);与模型组比较,艾灸组大鼠血清BUN含量明显降低(P<0.05),针刺组、联合组大鼠血清BUN、Scr含量明显降低(P<0.05);与针刺组比较,联合组大鼠血清BUN含量明显降低(P<0.05);与艾灸组比较,联合组大鼠血清BUN、Scr含量明显降低(P<0.05)。与对照组比较,模型组大鼠肾脏NOS、SOD、T-AOC活性明显降低(P<0.05)、MDA含量明显升高(P<0.01);与模型组比较,针刺组、艾灸组大鼠肾脏NOS活性明显升高(P<0.01,P<0.05)、MDA含量明显降低(P<0.05),联合组大鼠肾脏NOS、SOD、T-AOC活性明显升高(P<0.01,P<0.05)、MDA含量明显降低(P<0.05);与针刺组和艾灸组比较,联合组大鼠肾脏NOS、SOD、T-AOC活性明显升高(P<0.05)。与对照组比较,模型组大鼠肾脏Fas和FasL蛋白及mRNA表达水平升高(P<0.01,P<0.05);与模型组比较,艾灸组大鼠肾脏FasL蛋白及mRNA表达水平均降低(P<0.05),针刺组和联合组大鼠肾脏Fas和FasL蛋白及mRNA表达水平均降低(P<0.05,P<0.01);与针刺组和艾灸组比较,联合组大鼠肾脏Fas和FasL蛋白及mRNA表达水平均明显降低(P<0.05)。结论:针刺、艾灸均可降低糖尿病造影剂肾病大鼠肾脏的氧化应激反应,下调Fas/FasL表达,减少肾细胞凋亡,减轻肾损伤;针刺联合艾灸具有协同增效作用。
Objective To observe the effect of acupuncture and moxibustion on renal Fas and Fas ligand(FasL expression in contrast-induced nephropathy(CIN)diabetic rats,so as to explore its protective mechanisms.Methods A total of 40 diabetic male SD rats were randomly divided into control,model,acupuncture,moxibustion and combined treatment(acupuncture combined with moxibustion)groups(n=8 rats in each group).The diabetic model was established by i.p.injection of Streptozotocin(60 mg/kg),followed by regular raising for 9 weeks.Then,the manual acupuncture,moxibustion or acupuncture plus moxibustion at "Sanyinjiao"(SP6)"Shenshu"(BL23)and"Pishu"(BL20)was given to the diabetic rats,once daily for 7 days.On the 8 th day,these diabetic rats received i.p.injection of compound Meglumine Diatrizoate Injection(10 mL/kg)for establishing CIN model.Then,the venous blood specimens were collected for detecting contents of serum urine nitrogen(BUN,with urease method)and creatinine(Scr,with picric acid method).The kindey tissues were exteriorized for detecting nitricoxide synthase(NOS,with oxidation-reduction method),malondialdehyde(MDA,with thiobarbituric acid method),superoxide dismutase(SOD,with xanthine oxidase technique)and total antioxidant capacity(T-AOC,with colorimetric technique).The expression levels of Fas and FasL in the kidney tissues were detected by quantitative real-time PCR and Western blot separately.The histopathological and ultrastructural changes of the kidney were observed under microscope(after H.E.staining)and transmission electron microscope,respectively.Results Following modeling and compared with the control group,the BUN,Scr and renal MDA contents,and Fas and FasL mRNA and protein expression levels were significantly increased(P<0.01,P<0.05),and renal NOS,SOD and T-AOC levels considerably decreased in the model group(P<0.05).After the interventions and compared with the model group,serum BUN and Scr contents and renal Fas mRNA and protein expression levels in both acupuncture group and combined treatment group,BUN in the moxibustion group,and renal MDA content and FasL mRNA and protein expression levels in the 3 treatment groups were obviously down-regulated(P<0.05,P<0.01),while renal NOS in the 3 treatment groups,and SOD and T-AOC levels in the combined treatment group were considerably up-regulated(P<0.01,P<0.05).The effects of the combined treatment were significantly superior to those of simple acupuncure and simple moxibustion in down-regulating serum BUN contents,and Fas and FasL mRNA and protein expression levels,and in up-regulating renal NOS,SOD and T-AOC activity(P<0.05).No significant differences were found between the acupuncture and moxibustion groups in down-regulating serum BUN,Scr and renal MDA contents,and Fas and FasL mRNA and protein levels,as well as in up-regulating NOS content(P>0.05).H.E.staining showed lobular changes of glomeruli,many local necrosis and vascular degeneration in the model group,and transmission electron microscope displayed thickened basement membrane of the glomerular blood loop,many vacuoles in the cytoplasm of renal tubular epithelial cells,severely swollen mitochondria with disordered,broken or disappeared cristae,and some apoptotic cells in the model group,which were relatively milder in the combined treatment group.Conclusion Joint application of acupuncture and moxibustion can reduce the oxidative stress and renal injury in CIN diabetic rats,which may be associated with its function in down-regulating the expression of renal Fas and FasL genes and proteins.Acupuncture and moxibustion has a synergistic effect.
Objective To observe the effect of acupuncture and moxibustion on renal Fas and Fas ligand(FasL expression in contrast-induced nephropathy(CIN)diabetic rats,so as to explore its protective mechanisms.Methods A total of 40 diabetic male SD rats were randomly divided into control,model,acupuncture,moxibustion and combined treatment(acupuncture combined with moxibustion)groups(n=8 rats in each group).The diabetic model was established by i.p.injection of Streptozotocin(60 mg/kg),followed by regular raising for 9 weeks.Then,the manual acupuncture,moxibustion or acupuncture plus moxibustion at "Sanyinjiao"(SP6)"Shenshu"(BL23)and"Pishu"(BL20)was given to the diabetic rats,once daily for 7 days.On the 8 th day,these diabetic rats received i.p.injection of compound Meglumine Diatrizoate Injection(10 mL/kg)for establishing CIN model.Then,the venous blood specimens were collected for detecting contents of serum urine nitrogen(BUN,with urease method)and creatinine(Scr,with picric acid method).The kindey tissues were exteriorized for detecting nitricoxide synthase(NOS,with oxidation-reduction method),malondialdehyde(MDA,with thiobarbituric acid method),superoxide dismutase(SOD,with xanthine oxidase technique)and total antioxidant capacity(T-AOC,with colorimetric technique).The expression levels of Fas and FasL in the kidney tissues were detected by quantitative real-time PCR and Western blot separately.The histopathological and ultrastructural changes of the kidney were observed under microscope(after H.E.staining)and transmission electron microscope,respectively.Results Following modeling and compared with the control group,the BUN,Scr and renal MDA contents,and Fas and FasL mRNA and protein expression levels were significantly increased(P<0.01,P<0.05),and renal NOS,SOD and T-AOC levels considerably decreased in the model group(P<0.05).After the interventions and compared with the model group,serum BUN and Scr contents and renal Fas mRNA and protein expression levels in both acupuncture group and combined treatment group,BUN in the moxibustion group,and renal MDA content and FasL mRNA and protein expression levels in the 3 treatment groups were obviously down-regulated(P<0.05,P<0.01),while renal NOS in the 3 treatment groups,and SOD and T-AOC levels in the combined treatment group were considerably up-regulated(P<0.01,P<0.05).The effects of the combined treatment were significantly superior to those of simple acupuncure and simple moxibustion in down-regulating serum BUN contents,and Fas and FasL mRNA and protein expression levels,and in up-regulating renal NOS,SOD and T-AOC activity(P<0.05).No significant differences were found between the acupuncture and moxibustion groups in down-regulating serum BUN,Scr and renal MDA contents,and Fas and FasL mRNA and protein levels,as well as in up-regulating NOS content(P>0.05).H.E.staining showed lobular changes of glomeruli,many local necrosis and vascular degeneration in the model group,and transmission electron microscope displayed thickened basement membrane of the glomerular blood loop,many vacuoles in the cytoplasm of renal tubular epithelial cells,severely swollen mitochondria with disordered,broken or disappeared cristae,and some apoptotic cells in the model group,which were relatively milder in the combined treatment group.Conclusion Joint application of acupuncture and moxibustion can reduce the oxidative stress and renal injury in CIN diabetic rats,which may be associated with its function in down-regulating the expression of renal Fas and FasL genes and proteins.Acupuncture and moxibustion has a synergistic effect.
引文
[1]SPAGNOLI V,AZZALINIL,TADROS V X,et al.Contrast-induced nephropathy:an update[J].Ann Cardiol Angeiol(Paris),2016,65(2):87-94.
[2]王漫,张智龙,吉学群,等.调理脾胃针法对糖尿病肾病患者氧化应激水平的影响[J].针刺研究,2015,40(5):409-414.
[3]李林,孙玉霞,马家怡,等.不同取穴不同灸时温和灸对局灶硬化性肾炎大鼠肾功能的影响[J].针刺研究,2016,41(6):521-527.
[4]赵凯,许蔚,陈卓阳.针刺治疗对2型糖尿病患者对比剂肾病的预防作用[J].中国中医基础医学杂志,2018,24(3):382-384.
[5]焦占全.糖尿病大鼠造影剂急性肾损害机制及普罗布考保护作用的实验研究[D].天津:天津医科大学,2012.
[6]ARI E,YILMAZ Y,KEDRAH A E.Protective effect of the vasopressin agonist terlipressin in a rat model of contrast-induced nephropathy[J].Am J Nephrol,2011,33(3):269-276.
[7]郭义.实验针灸学[M].北京:中国中医药出版社,2008:414.
[8]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[9]ZHAO K,LI Y J,GAO S,et al.Effect of Dongchongxiacao(Cordyceps)therapy on contrast-induced nephropathy in patients with type 2diabetes and renal insufficiency undergoing coronary angiography[J].J Tradit Chin Med,2015,35(4):422-427.
[10]ZHAO K,LI Y J,ZHANG H.Role of Dongchongxiacao(Cordyceps)in prevention of contrast-induced nephropathy in patients with stable angina pectoris[J].J Tradit Chin Med,2013,33(3):283-286.
[11]KANDA T,FUKUSATO T,MATSUDA M,et al.Gadolinium-based contrast agent accumulates in the brain evenin subjects without severe renal dysfunction:evaluation of autopsy brain specimens with inductively coupled plasma mass spectroscopy[J].Radiology,2015,276(1):228-232.
[12]LIU T Q,FANG Y,LIU S P,et al.Limb ischemic preconditioning protects against contrast-induced acute kidney injury in rats via phosphorylation of GSK-3β[J].Free Radic Biol Med,2015,81:170-182.
[13]褚芹,王琳,刘国真,等.针刺对糖尿病肾病血液流变学影响的临床随机对照研究[J].针刺研究,2007,32(5):335-337.
[14]朱庆均,肖丽,杨军,等.基于Fas/FasL途径的金匮肾气丸对肾阳虚模型睾丸细胞凋亡的影响[J].时珍国医国药,2017,28(2):272-276.
[15]PAN Y,CHEN Y C,LI Q N,et al.The synthesis and evaluation of novel hydroxyl substituted chalcone analogs with in vitro anti-free radicals pharmacological activity and in vivo anti-oxidation activity in a free radical-injury Alzheimer’s model[J].Molecules,2013,18(2):1693-1703.
[16]NAGHIZADEH B,MANSOURI M T,GHORBANZADEHB,et al.Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats[J].Phytomedicine,2013,20(6):537-542.
[17]李丹,李庆羚,马强,等.艾灸对慢性心力衰竭大鼠心室质量指数及心肌组织凋亡相关蛋白表达水平的影响[J].针刺研究,2018,43(2):80-85.
[18]戈立秀,赵凯,高巧营,等.山莨菪碱联合普罗布考对糖尿病大鼠造影剂肾损伤凋亡基因FAS/FASL表达的影响[J].中国实验诊断学,2018,22(6):1049-1053.
[2]王漫,张智龙,吉学群,等.调理脾胃针法对糖尿病肾病患者氧化应激水平的影响[J].针刺研究,2015,40(5):409-414.
[3]李林,孙玉霞,马家怡,等.不同取穴不同灸时温和灸对局灶硬化性肾炎大鼠肾功能的影响[J].针刺研究,2016,41(6):521-527.
[4]赵凯,许蔚,陈卓阳.针刺治疗对2型糖尿病患者对比剂肾病的预防作用[J].中国中医基础医学杂志,2018,24(3):382-384.
[5]焦占全.糖尿病大鼠造影剂急性肾损害机制及普罗布考保护作用的实验研究[D].天津:天津医科大学,2012.
[6]ARI E,YILMAZ Y,KEDRAH A E.Protective effect of the vasopressin agonist terlipressin in a rat model of contrast-induced nephropathy[J].Am J Nephrol,2011,33(3):269-276.
[7]郭义.实验针灸学[M].北京:中国中医药出版社,2008:414.
[8]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[9]ZHAO K,LI Y J,GAO S,et al.Effect of Dongchongxiacao(Cordyceps)therapy on contrast-induced nephropathy in patients with type 2diabetes and renal insufficiency undergoing coronary angiography[J].J Tradit Chin Med,2015,35(4):422-427.
[10]ZHAO K,LI Y J,ZHANG H.Role of Dongchongxiacao(Cordyceps)in prevention of contrast-induced nephropathy in patients with stable angina pectoris[J].J Tradit Chin Med,2013,33(3):283-286.
[11]KANDA T,FUKUSATO T,MATSUDA M,et al.Gadolinium-based contrast agent accumulates in the brain evenin subjects without severe renal dysfunction:evaluation of autopsy brain specimens with inductively coupled plasma mass spectroscopy[J].Radiology,2015,276(1):228-232.
[12]LIU T Q,FANG Y,LIU S P,et al.Limb ischemic preconditioning protects against contrast-induced acute kidney injury in rats via phosphorylation of GSK-3β[J].Free Radic Biol Med,2015,81:170-182.
[13]褚芹,王琳,刘国真,等.针刺对糖尿病肾病血液流变学影响的临床随机对照研究[J].针刺研究,2007,32(5):335-337.
[14]朱庆均,肖丽,杨军,等.基于Fas/FasL途径的金匮肾气丸对肾阳虚模型睾丸细胞凋亡的影响[J].时珍国医国药,2017,28(2):272-276.
[15]PAN Y,CHEN Y C,LI Q N,et al.The synthesis and evaluation of novel hydroxyl substituted chalcone analogs with in vitro anti-free radicals pharmacological activity and in vivo anti-oxidation activity in a free radical-injury Alzheimer’s model[J].Molecules,2013,18(2):1693-1703.
[16]NAGHIZADEH B,MANSOURI M T,GHORBANZADEHB,et al.Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats[J].Phytomedicine,2013,20(6):537-542.
[17]李丹,李庆羚,马强,等.艾灸对慢性心力衰竭大鼠心室质量指数及心肌组织凋亡相关蛋白表达水平的影响[J].针刺研究,2018,43(2):80-85.
[18]戈立秀,赵凯,高巧营,等.山莨菪碱联合普罗布考对糖尿病大鼠造影剂肾损伤凋亡基因FAS/FASL表达的影响[J].中国实验诊断学,2018,22(6):1049-1053.
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