连翘苷元对老年大鼠氧化应激和肾功能的改善作用
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摘要
目的研究连翘苷元对老年大鼠氧化应激和肾功能的影响及相关机制。方法将15只SD雌性老年大鼠(24月龄)随机分成3组,每组5只,老年对照组(Aged rats),老年连翘苷元低剂量组[Aged rats+FA(20 mg/kg)],老年连翘高剂量组[Aged rats+FA(100 mg/kg)]。另取5只12周龄SD雌性大鼠为青年对照组(Young rats)。连翘苷元低、高剂量组大鼠灌胃给药60 d。考马斯亮蓝检测24 h尿液总蛋白并分析血清肌酐和尿素氮水平。HE染色和TUNEL染色检测肾组织病理损伤情况。Western Blot检测Caspase-3和Caspase-9的表达。检测氧化应激标记物超氧化物歧化酶(SOD)、丙二醛(MDA)和还原型谷胱甘肽(GSH)的水平。ELISA检测血清中炎症因子IL-18和IL-1β水平。Western Blot检测核因子E2相关因子(Nrf 2)、血红素氧合酶1(HO-1)和还原型辅酶/醌氧化还原酶(NQO-1)的表达。结果老年对照组与青年对照组相比,尿中蛋白含量显著提升,血清中肌酐和尿素氮含量显著上升;肾组织细胞排列不规则,出现大量炎性细胞浸润,凋亡细胞比率显著提高;Caspase-3和Caspase-9的表达水平显著上调;SOD活性显著降低,MDA含量显著上升,GSH含量显著下降;血清中IL-18和IL-1β含量显著升高;Nrf 2、HO-1和NQO-1的表达显著下调。连翘苷元低、高剂量组与老年对照组相比,尿液中蛋白含量显著降低,血清肌酐和尿素氮含量显著下降;肾组织细胞排列趋向规则,炎性细胞浸润明显减弱,凋亡细胞比率显著下降;Caspase-3和Caspase-9的表达水平显著下调;SOD活性显著提高,MDA含量显著降低,GSH含量显著上升;血清中IL-18和IL-1β含量显著降低;Nrf 2、HO-1和NQO-1的表达显著上调。结论连翘苷元抑制老年大鼠氧化应激反应,改善老年大鼠肾功能,其作用机制可能与Nrf 2-ARE通路激活相关。
Objective To investigate the effects and related mechanisms of forsythoside on oxidative stress and renal function in aged rats. Methods Fifteen SD female aged rats(24 months old) were randomly divided into three groups with 5 rats in each: the old control group(Aged rats), the low dose forsythia group [Aged rats + FA(20 mg/kg)] and the high dose forsythia group [Aged rats + FA(100 mg/kg)].Another 5 female SD rats(12 weeks old) served as the young control group(Young rats). Rats in low and high dose forsythiaside groups were administered for 60 days by gavage. Coomassie brilliant blue was used to detect the total protein of 24 h urine. And the levels of serum creatinine and urea nitrogen were detected by related kits. Renal injury was detected by HE staining and TUNEL staining. Western Blot was used to detect the expression of Caspase-3 and Caspase-9. The levels of SOD, MDA and GSH were detected by related kits.ELISA was used to detect the levels of inflammatory cytokines IL-18 and IL-1 beta in serum. Results Compared with the young control group, the content of protein in urine increased significantly in the aged control group. The content of creatinine and urea nitrogen in the serum increased significantly. The arrangement of renal tissue cells was irregular. A large number of inflammatory cells were infiltrated. The ratio of apoptotic cells increased significantly. The expression level of Caspase-3 and Caspase-9 increased significantly. The activity of SOD decreased significantly. MDA content increased significantly. GSH content decreased significantly. Serum IL-18 and IL-1 beta increased significantly. The expression of Nrf 2, HO-1 and NQO-1 decreased significantly(P<0.01). In the low and high dose group, the content of protein in urine decreased significantly. The content of creatinine and urea nitrogen in the serum decreased significantly. The arrangement of renal tissue cells tended to be regular. The infiltration of inflammatory cells decreased significantly. And the ratio of apoptotic cells decreased significantly. The expression level of Caspase-3 and Caspase-9 decreased significantly. The activity of SOD increased significantly. The content of MDA decreased significantly. The content of GSH increased significantly. The contents of IL-18 and IL-1 beta in serum decreased significantly. The expression of Nrf 2, HO-1 and NQO-1 increased significantly(P<0.01). Conclusions Forsythin inhibits oxidative stress and improves renal function in aged rats,and the mechanism may be related to the activation of Nrf 2-ARE pathway.
Objective To investigate the effects and related mechanisms of forsythoside on oxidative stress and renal function in aged rats. Methods Fifteen SD female aged rats(24 months old) were randomly divided into three groups with 5 rats in each: the old control group(Aged rats), the low dose forsythia group [Aged rats + FA(20 mg/kg)] and the high dose forsythia group [Aged rats + FA(100 mg/kg)].Another 5 female SD rats(12 weeks old) served as the young control group(Young rats). Rats in low and high dose forsythiaside groups were administered for 60 days by gavage. Coomassie brilliant blue was used to detect the total protein of 24 h urine. And the levels of serum creatinine and urea nitrogen were detected by related kits. Renal injury was detected by HE staining and TUNEL staining. Western Blot was used to detect the expression of Caspase-3 and Caspase-9. The levels of SOD, MDA and GSH were detected by related kits.ELISA was used to detect the levels of inflammatory cytokines IL-18 and IL-1 beta in serum. Results Compared with the young control group, the content of protein in urine increased significantly in the aged control group. The content of creatinine and urea nitrogen in the serum increased significantly. The arrangement of renal tissue cells was irregular. A large number of inflammatory cells were infiltrated. The ratio of apoptotic cells increased significantly. The expression level of Caspase-3 and Caspase-9 increased significantly. The activity of SOD decreased significantly. MDA content increased significantly. GSH content decreased significantly. Serum IL-18 and IL-1 beta increased significantly. The expression of Nrf 2, HO-1 and NQO-1 decreased significantly(P<0.01). In the low and high dose group, the content of protein in urine decreased significantly. The content of creatinine and urea nitrogen in the serum decreased significantly. The arrangement of renal tissue cells tended to be regular. The infiltration of inflammatory cells decreased significantly. And the ratio of apoptotic cells decreased significantly. The expression level of Caspase-3 and Caspase-9 decreased significantly. The activity of SOD increased significantly. The content of MDA decreased significantly. The content of GSH increased significantly. The contents of IL-18 and IL-1 beta in serum decreased significantly. The expression of Nrf 2, HO-1 and NQO-1 increased significantly(P<0.01). Conclusions Forsythin inhibits oxidative stress and improves renal function in aged rats,and the mechanism may be related to the activation of Nrf 2-ARE pathway.
引文
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[15]Valentijn FA,Falke LL,Nguyen TQ,et al.Cellular senescence in the aging and diseased kidney[J].J Cell Commun Signal,2018,12(1):69-82.
[16]Pan CW,Zhou GY,Chen WL,et al.Protective effect of forsythiaside a on lipopolysaccharide/d-galactosamine-induced liver injury[J].In Immunopharmacol,2015,26(1):80-85.
[17]Brentnall M,Rodriguez-Menocal L,DE Guevara RL,et al.Caspase-9caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis[J].BMC Cell Biol,2013,14(1):32-38.
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[19]Choudhary GS,Alharbi S,Almasan A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219(414):1-9.
[20]D'Mello SR,Kuan CY,Flavell RA,et al.Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium[J].J Neurosci Res,2015,59(1):24-31.
[21]Huang C,Lin Y,Su H,et al.Forsythiaside protects against hydrogen peroxide-induced oxidative stress and apoptosis in pc12 cell[J].Neurochem Res,2015,40(1):27-35.
[22]Shin HS,Park SY,Song HG,et al.The androgenic alopecia protective effects of forsythiaside-a and the molecular regulation in a mouse model[J].Phytother Res,2015,29(6):870-876.
[23]Trachootham D,Lu W,Ogasawara MA,et al.Redox regulation of cell survival[J].Antioxid Redox Signal,2008,10(8):1343-1374.
[24]Rahman K.Studies on free radicals,antioxidants,and co-factors[J].Clin Interv Aging,2007,2(2):219-236.
[25]Powers SK,Jackson MJ.Exercise-induced oxidative stress:cellular mechanisms and impact on muscle force production[J].Physiol Rev,2008,88(4):1243-1276.
[26]Lu T,Piao XL,Zhang Q,et al.Protective effects of forsythia suspensa extract against oxidative stress induced by diquat in rats[J].Food Chem Toxicol,2010,48(2):764-770.
[27]周建,沈瑞明,郑跃芳,等.创伤性颅脑损伤患者il-1β、il-6、il-10水平变化及临床意义[J].医学分子生物学杂志,2017,14(5):287-290.
[28]Grivennikov SI,Greten FR,Karin M.Immunity,inflammation,and cancer[J].Cell,2010,140(6):883-899.
[29]Qian J,Ma X,Xun Y,et al.Protective effect of forsythiaside a on ovainduced asthma in mice[J].Eur J Pharmacol,2017,812(1):250-255.
[30]Cheng L,Li F,Ma R,et al.Forsythiaside inhibits cigarette smokeinduced lung inflammation by activation of nrf2 and inhibition of nf-κb[J].Int Immunopharmacol,2015,28(1):494-499.
[31]Mahmoud AM,Alexander MY,Tutar Y,et al.Oxidative stress in metabolic disorders and drug-induced injury:the potential role of nrf2and ppars activators[J].Oxid Med Cell Longev,2017,2017:2508909.
[32]曹娟,郑伟,朱柯斌,等.Musashi-1、cox-2、nrf-2在子宫腺肌病中的表达及意义[J].医学分子生物学杂志,2017,14(6):337-341.
[33]Wallace DC.A mitochondrial paradigm of metabolic and degenerative diseases,aging,and cancer:a dawn for evolutionary medicine[J].Annu Rev Genet,2005,39:359-407.
[34]Itoh K,Chiba T,Takahashi S,et al.An nrf2/small maf heterodimer mediates the induction of phase ii detoxifying enzyme genes through antioxidant response elements[J].Biochem Biophys Res Commun,1997,236(2):313-322.
[35]Wang Y,Zhao H,Lin C,et al.Forsythiaside a exhibits antiinflammatory effects in lps-stimulated bv2 microglia cells through activation of nrf2/ho-1 signaling pathway[J].Neurochem Res,2016,41(4):659-665.
[2]Maw TT,Fried L.Chronic kidney disease in the elderly[J].Clin Geriatr Med,2013,29(3):611-624.
[3]Campbell KH,O'Hare AM.Kidney disease in the elderly:update on recent literature[J].Curr Opin Nephrol Hypertens,2008,17(3):298-303.
[4]Winearls CG,Glassock RJ.Classification of chronic kidney disease in the elderly:pitfalls and errors[J].Nephron Clin Pract,2011,119(Suppl.1):c2-c4.
[5]Stevens LA,Levey AS.Chronic kidney disease in the elderly--how to assess risk[J].N Engl J Med,2005,352(20):2122-2124.
[6]Piao XL,Jang MH,Cui J,et al.Lignans from the fruits of forsythia suspensa[J].Bioorg Med Chem Lett,2008,18(6):1980-1984.
[7]Lu T,Piao XL,Zhang Q,et al.Protective effects of forsythia suspensa extract against oxidative stress induced by diquat in rats[J].Food Chem Toxicol,2010,48(2):764-770.
[8]Nishibe S,Okabe K,Tsukamoto H,et al.The structure of forsythiaside isolated from forsythia suspensa[J].Chem Pharm Bull,2008,30(3)1048-1050.
[9]Jiao J,Gai QY,Fu YJ,et al.Application of white-rot fungi treated fructus forsythiae shell residue as a low-cost biosorbent to enrich forsythiaside and phillygenin[J].Chem Eng Sci,2012,74(22):244-255.
[10]Iizuka T,Nagai M.Vasorelaxant effects of forsythiaside from the fruits of forsythia suspensa[J].Yakugaku Zasshi,2005,125(2):219-224.
[11]Smith GL,Shlipak MG,Havranek EP,et al.Serum urea nitrogen creatinine,and estimators of renal function:mortality in older patients with cardiovascular disease[J].Arch Intern Med,2006,166(10):1134-1142.
[12]Perrone RD,Madias NE,Levey AS.Serum creatinine as an index of renal function:new insights into old concepts[J].Clin Chem,1992,38(10):1933-1953.
[13]Ruilope LM,van Veldhuisen DJ,Ritz E,et al.Renal function:the cinderella of cardiovascular risk profile[J].J Am Coll Cardiol,2001,38(7):1782-1787.
[14]Macedo E.Blood urea nitrogen beyond estimation of renal function[J]Crit Care Med,2011,39(2):405-406.
[15]Valentijn FA,Falke LL,Nguyen TQ,et al.Cellular senescence in the aging and diseased kidney[J].J Cell Commun Signal,2018,12(1):69-82.
[16]Pan CW,Zhou GY,Chen WL,et al.Protective effect of forsythiaside a on lipopolysaccharide/d-galactosamine-induced liver injury[J].In Immunopharmacol,2015,26(1):80-85.
[17]Brentnall M,Rodriguez-Menocal L,DE Guevara RL,et al.Caspase-9caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis[J].BMC Cell Biol,2013,14(1):32-38.
[18]Mcilwain DR,Berger T,Mak TW.Caspase functions in cell death and disease[J].Cold Spring Harb Perspect Biol,2013,5(4):a008656.
[19]Choudhary GS,Alharbi S,Almasan A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219(414):1-9.
[20]D'Mello SR,Kuan CY,Flavell RA,et al.Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium[J].J Neurosci Res,2015,59(1):24-31.
[21]Huang C,Lin Y,Su H,et al.Forsythiaside protects against hydrogen peroxide-induced oxidative stress and apoptosis in pc12 cell[J].Neurochem Res,2015,40(1):27-35.
[22]Shin HS,Park SY,Song HG,et al.The androgenic alopecia protective effects of forsythiaside-a and the molecular regulation in a mouse model[J].Phytother Res,2015,29(6):870-876.
[23]Trachootham D,Lu W,Ogasawara MA,et al.Redox regulation of cell survival[J].Antioxid Redox Signal,2008,10(8):1343-1374.
[24]Rahman K.Studies on free radicals,antioxidants,and co-factors[J].Clin Interv Aging,2007,2(2):219-236.
[25]Powers SK,Jackson MJ.Exercise-induced oxidative stress:cellular mechanisms and impact on muscle force production[J].Physiol Rev,2008,88(4):1243-1276.
[26]Lu T,Piao XL,Zhang Q,et al.Protective effects of forsythia suspensa extract against oxidative stress induced by diquat in rats[J].Food Chem Toxicol,2010,48(2):764-770.
[27]周建,沈瑞明,郑跃芳,等.创伤性颅脑损伤患者il-1β、il-6、il-10水平变化及临床意义[J].医学分子生物学杂志,2017,14(5):287-290.
[28]Grivennikov SI,Greten FR,Karin M.Immunity,inflammation,and cancer[J].Cell,2010,140(6):883-899.
[29]Qian J,Ma X,Xun Y,et al.Protective effect of forsythiaside a on ovainduced asthma in mice[J].Eur J Pharmacol,2017,812(1):250-255.
[30]Cheng L,Li F,Ma R,et al.Forsythiaside inhibits cigarette smokeinduced lung inflammation by activation of nrf2 and inhibition of nf-κb[J].Int Immunopharmacol,2015,28(1):494-499.
[31]Mahmoud AM,Alexander MY,Tutar Y,et al.Oxidative stress in metabolic disorders and drug-induced injury:the potential role of nrf2and ppars activators[J].Oxid Med Cell Longev,2017,2017:2508909.
[32]曹娟,郑伟,朱柯斌,等.Musashi-1、cox-2、nrf-2在子宫腺肌病中的表达及意义[J].医学分子生物学杂志,2017,14(6):337-341.
[33]Wallace DC.A mitochondrial paradigm of metabolic and degenerative diseases,aging,and cancer:a dawn for evolutionary medicine[J].Annu Rev Genet,2005,39:359-407.
[34]Itoh K,Chiba T,Takahashi S,et al.An nrf2/small maf heterodimer mediates the induction of phase ii detoxifying enzyme genes through antioxidant response elements[J].Biochem Biophys Res Commun,1997,236(2):313-322.
[35]Wang Y,Zhao H,Lin C,et al.Forsythiaside a exhibits antiinflammatory effects in lps-stimulated bv2 microglia cells through activation of nrf2/ho-1 signaling pathway[J].Neurochem Res,2016,41(4):659-665.