盐酸小檗碱对大鼠肾缺血再灌注的保护作用及机制研究
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摘要
目的研究盐酸小檗碱(BBR)预处理对肾脏缺血再灌注的保护作用,并探讨其可能的机制。方法建立大鼠肾脏缺血再灌注(RIRI)损伤模型,随机分为Sham组、RIRI组、RIRI+BBR-1组和RIRI+BBR-2组。72 h后检测大鼠血浆中血尿素氮(BUN)和血清肌酐(Scr)的含量和肾组织中丙二醛(MDA)、白介素-6(IL-6)和白介素-1β(IL-1β)的含量;使用NRK-52E细胞建立缺氧复氧(H/R)模型,MTT法检测细胞损伤情况,测定BBR对NRK-52E细胞中活性氧(ROS)的影响;使用Western Blot方法相关蛋白表达水平变化。结果 RIRI组中BUN、Scr、MDA、IL-6和IL-1β的含量均较Sham组明显上升(q分别=8.06、9.16、7.89、6.48、5.68,P均<0.05)。相比于RIRI组,RIRI+BBR-2组的BUN、Scr、MDA、IL-6和IL-1β的含量都明显降低(q分别=5.68、5.88、6.46、5.45、5.19,P均<0.05),同时RIRI+BBR-1组的MDA的含量也降低(q=4.54,P均<0.05)。H/R明显降低NRK-52E细胞的细胞活力,明显增加细胞中ROS水平(q分别=9.65、18.09,P均<0.05),BBR可逆转H/R导致的细胞活力的降低及ROS水平的升高(q分别=2.95、11.66,P均<0.05)。NAC作为抗氧化剂和BBR发挥相同的作用(q分别=4.26、15.67,P均<0.05)。Baf与BBR同时处理H/R细胞时,结果显示细胞活力比H/R组更低(q=3.87,P<0.05)。H/R明显增加细胞中p-AMPK和LC3Ⅱ的表达,抑制p-mTOR的水平。结论 BBR可降低大鼠肾脏缺血再灌注损伤,其机制与ROS/AMPK/mTOR/LC3通路有关。
Objective To investigate the therapeutic effects of hydrochloric berberine on renal ischemic/reperfusion in rats and its potential mechanism. Methods The rat renal ischemia reperfusion injury model was established and randomly divided into Sham group,RIRI group,RIRI+BBR-1 group and RIRI+BBR-2 group.After 72 hours,automatic biochemical analyzer was used to measure the level of BUN and Scr in plasma,and MDA,IL-6,IL-1β in renal tissue.NRK-52 E cells were used to mimic RIRI condition by hypoxia/reperfusion(H/R).MTT assay was used to detect cell viability to further confirme the therapeutic effects of berberine on ROS in NRK-52 E cells.Western blot was used to measure the protein level. Results The results showed that the levels of BUN,Scr,MDA were significantly increased in RIRI group compared with Sham group,and these parameters were significantly decreased after dealing with BBR(q=8.06,9.16,7.89,6.48,5.68,5.68,5.88,6.46,P<0.05).Meanwhile,BBR reduced the level of IL-6 and IL-1β induced by RIRI as well as the MDA(q=5.45,5.19,4.54,P<0.05).H/R significantly decreased the cell viability of NRK-52 E cells and significantly increased ROS level(q=9.65,18.09,P<0.05),while BBR reversed the decreasing of cell viability and the increasing of ROS level induced by H/R(q=2.95,11.66,P<0.05).NAC played the same role with the BBR(q=4.26,15.67,P<0.05).When Baf and BBR treated H/R cells simultaneously,the results showed that the cell viability was lower than that of H/R group(q= 3.87,P<0.05).H/R significantly increased the expression of p-AMPK and LC3Ⅱ,and inhibited the level of p-mTOR. Conclusion BBR can effectively improve renal function in rats with RIRI,which possibly through ROS/AMPK/mTOR/LC3Ⅱ pathway.
Objective To investigate the therapeutic effects of hydrochloric berberine on renal ischemic/reperfusion in rats and its potential mechanism. Methods The rat renal ischemia reperfusion injury model was established and randomly divided into Sham group,RIRI group,RIRI+BBR-1 group and RIRI+BBR-2 group.After 72 hours,automatic biochemical analyzer was used to measure the level of BUN and Scr in plasma,and MDA,IL-6,IL-1β in renal tissue.NRK-52 E cells were used to mimic RIRI condition by hypoxia/reperfusion(H/R).MTT assay was used to detect cell viability to further confirme the therapeutic effects of berberine on ROS in NRK-52 E cells.Western blot was used to measure the protein level. Results The results showed that the levels of BUN,Scr,MDA were significantly increased in RIRI group compared with Sham group,and these parameters were significantly decreased after dealing with BBR(q=8.06,9.16,7.89,6.48,5.68,5.68,5.88,6.46,P<0.05).Meanwhile,BBR reduced the level of IL-6 and IL-1β induced by RIRI as well as the MDA(q=5.45,5.19,4.54,P<0.05).H/R significantly decreased the cell viability of NRK-52 E cells and significantly increased ROS level(q=9.65,18.09,P<0.05),while BBR reversed the decreasing of cell viability and the increasing of ROS level induced by H/R(q=2.95,11.66,P<0.05).NAC played the same role with the BBR(q=4.26,15.67,P<0.05).When Baf and BBR treated H/R cells simultaneously,the results showed that the cell viability was lower than that of H/R group(q= 3.87,P<0.05).H/R significantly increased the expression of p-AMPK and LC3Ⅱ,and inhibited the level of p-mTOR. Conclusion BBR can effectively improve renal function in rats with RIRI,which possibly through ROS/AMPK/mTOR/LC3Ⅱ pathway.
引文
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13 Cicero AF,Rovati LC,Setnikar I.Eulipidemic effects of berberine administered alone or in combination with other natural cholesterol-lowering agents. A single-blind clinical investigation[J]. Arzneimittelforschung, 2007, 57(1):26-30.
14 Zeng XH,Zeng XJ,Li YY.Efficacy and safety of berberine for congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy[J]. Am J Cardiol,2003,92(2):173-176.
15 Inoue K,Kulsum U,Chowdhury SA,et al.Tumor-specific cytotoxicity and apoptosis-inducing activity of berberines[J].Anticancer Res,2005,25(6B):4053-4059.
16 Ling H,Chen H,Wei M,et al.The Effect of autophagy on inflammation cytokines in renal ischemia/reperfusion injury[J].Inflammation,2016,39(1):347-356.
17 Guan X,Qian Y,Shen Y,et al.Autophagy protects renal tubular cells against ischemia/reperfusion injury in a time-dependent manner[J]. Cell Physiol Biochem,2015,36(1):285-298.
18 Zhang X,Cheng X,Yu L,et al.MCOLN1 is a ROS sensor in lysosomes that regulates autophagy[J].Nat Commun,2016,7:12109.
2 Wu H, Chen G, Wyburn KR, et al.TLR4 activation mediates kidney ischemia/reperfusion injury[J].J Clin Invest,2007,117(10):2847-2859.
3 Nezu M, Souma T, Yu L, et al.Transcription factor Nrf2 hyperactivation in early-phase renal ischemia-reperfusion injury prevents tubular damage progression[J].Kidney Int,2017,91(2):387-401.
4 Wang LH, Li XL, Li Q, et al. Berberine alleviates ischemic arrhythmias via recovering depressed I(to)and I(Ca)currents in diabetic rats[J].Phytomedicine,2012,19(3-4):206-210.
5 Wang LH,Yu CH,Fu Y,et al.Berberine elicits antiarrhythmic effects via IK1/Kir2.1 in the rat type 2 diabetic myocardial infarction model[J]. Phytother Res,2011,25(1):33-37.
6 Zhao GL,Yu LM,Gao WL,et al.Berberine protects rat heart from ischemia/reperfusion injury via activating JAK2/STAT3 signaling and attenuating endoplasmic reticulum stress[J].Acta Pharmacol Sin,2016,37(3):354-367.
7 de Oliveira JS,Abdalla FH,Dornelles GL,et al.Neuroprotective effects of berberine on recognition memory impairment,oxidative stress,and damage to the purinergic system in rats submitted to intracerebroventricular injection of streptozotocin[J].Psychopharmacology(Berl),2018.
8 Shen Y,Zhao Y,Wang L,et al.MicroRNA-194 overexpression protects against hypoxia/reperfusion-induced HK-2 cell injury through direct targeting Rheb[J].J Cell Biochem,2018.
9 Nogueira MA,Coelho AM,Sampietre SN,et al.Beneficial effects of adenosine triphosphate-sensitive K+channel opener on liver ischemia/reperfusion injury[J].World J Gastroenterol,2014,20(41):15319-15326.
10 Yu HH,Kim KJ,Cha JD,et al.Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus[J].J Med Food,2005,8(4):454-461.
11 Choi BH,Ahn IS,Kim YH,et al.Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte[J].Exp Mol Med,2006,38(6):599-605.
12 Zhou L,Yang Y,Wang X,et al.Berberine stimulates glucose transport through a mechanism distinct from insulin[J].Metabolism,2007,56(3):405-412.
13 Cicero AF,Rovati LC,Setnikar I.Eulipidemic effects of berberine administered alone or in combination with other natural cholesterol-lowering agents. A single-blind clinical investigation[J]. Arzneimittelforschung, 2007, 57(1):26-30.
14 Zeng XH,Zeng XJ,Li YY.Efficacy and safety of berberine for congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy[J]. Am J Cardiol,2003,92(2):173-176.
15 Inoue K,Kulsum U,Chowdhury SA,et al.Tumor-specific cytotoxicity and apoptosis-inducing activity of berberines[J].Anticancer Res,2005,25(6B):4053-4059.
16 Ling H,Chen H,Wei M,et al.The Effect of autophagy on inflammation cytokines in renal ischemia/reperfusion injury[J].Inflammation,2016,39(1):347-356.
17 Guan X,Qian Y,Shen Y,et al.Autophagy protects renal tubular cells against ischemia/reperfusion injury in a time-dependent manner[J]. Cell Physiol Biochem,2015,36(1):285-298.
18 Zhang X,Cheng X,Yu L,et al.MCOLN1 is a ROS sensor in lysosomes that regulates autophagy[J].Nat Commun,2016,7:12109.