注射用丹参多酚酸盐通过调节NF-κB/IκB和p38 MAPK信号通路防治胆汁淤积性肝损伤
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摘要
目的探讨注射用丹参多酚酸盐对α-萘异硫氰酸酯(ANIT)诱导的大鼠胆汁淤积性肝损伤的治疗作用及其机制。方法将40只成年雄性SD大鼠随机均分为4组,即正常组(对照组)、胆汁淤积性肝损伤组(模型组)、注射用丹参多酚酸盐低剂量组(20 mg·kg~(-1),ip,qd)和注射用丹参多酚酸盐高剂量组(40 mg·kg~(-1),ip,qd)。对照组大鼠连续7 d给予等量生理盐水(ip),第5日时给予等量花生油(ig);模型组大鼠连续7 d给予等量生理盐水(ip),第5日时灌胃ANIT(75 mg·kg~(-1),每15 mg ANIT溶于1 mL花生油);低剂量组和高剂量组大鼠连续7 d给予丹参多酚酸盐,第5日灌胃ANIT(75mg·kg~(-1))。第7日给药后禁食24 h处死各组大鼠,称量体重和肝重;检测血清谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、γ-谷氨酰基转移酶(γ-GT)、总胆红素(TBIL)和总胆汁酸(TBA)。HE染色考察肝组织病理学改变,Western blot检测肝脏组织中NF-κB/IκB和p38 MAPK信号通路相关蛋白的表达;Real-time PCR检测肝脏组织中IL~(-1)β、IL-6、TGF-β和TNF-α基因的表达水平。结果与模型组相比,注射用丹参多酚酸盐能够显著降低胆汁淤积性肝损伤大鼠血清AST、ALT、ALP、γ-GT、TBIL以及TBA水平(P均<0.05),并且显著改善大鼠肝脏组织病理学结构。与此同时,与模型组相比,丹参多酚酸盐给药组的大鼠肝脏中核蛋白NF-κB的表达降低,细胞质中NF-κB和IκBα表达增多,p38蛋白的磷酸化水平降低,炎症因子IL~(-1)β、IL-6、TGF-β和TNF-α的m RNA表达水平也降低(P <0.05)。结论注射用丹参多酚酸盐可改善大鼠胆汁淤积性肝损伤,其机制可能与抑制NF-κB/IκB和p38 MAPK信号通路有关。
Objective To investigate the therapeutic effect and potential mechanisms of salvianolate on cholestatic liver injury induced by naphthalene isothiocyanate(ANIT) in rats. Methods Totally 40 male Sprague-Dawley(SD) rats were randomly divided into a normal group(the control group), a cholestatic liver injury group(the model group), a salvianolate low-dose group(20 mg·kg~(-1), ip, qd) and a salvianolate highdose group(40 mg·kg~(-1), ip, qd). Rats in the control group were given normal saline(ip) for 7 consecutive days and on the 5 th day the vehicle(peanut oil) was given(ig). The same treatment was provided to the model group, but the rats were given ANIT(75 mg·kg~(-1), ig 15 mg ANIT was dissolved in 1 mL peanut oil) on the5 th day. The low dose and high dose groups were administrated salvianolate for 7 consecutive days and given ANIT(75 mg·kg~(-1), ig) on the 5 th day. The rats in each group were sacrificed on the 7 th day after fasting for24 h, and the body weight and liver weight were measured. AST, ALT, ALP, γ-GT and TBIL were detected by automatic biochemical analyzer. TBA levels were analyzed with Total Bile acid Assay Kit. HE staining was performed to evaluate the changes of morphology in the liver tissue. Western blot was used to determine the expression of signaling related proteins in NF-κ B/Iκ B and p38 MAPK pathways. Real-time PCR was used to detect the mRNA levels of IL~(-1)β, IL-6, TGF-β, and TNF-α. Results Compared with those of the model group, the serum levels of AST, ALT, ALP, γ-GT, TBIL and TBA were lower in the salvianolate treatment groups(P < 0.05). Salvianolate improved the pathological structure of the rat liver tissue. Meanwhile, the relative expression level of NF-κ B in the nucleus, the phosphorylation level of p38 and the mRNA levels of inflammation factors were all decreased in the salvianolate treatment groups as compared with the model group(P< 0.05). Conclusion The therapeutic effect and potential mechanisms of salvianolate on cholestatic liver injury induced by ANIT may be related to the inhibition of NF-κB/IκB and p38 MAPK pathways.
Objective To investigate the therapeutic effect and potential mechanisms of salvianolate on cholestatic liver injury induced by naphthalene isothiocyanate(ANIT) in rats. Methods Totally 40 male Sprague-Dawley(SD) rats were randomly divided into a normal group(the control group), a cholestatic liver injury group(the model group), a salvianolate low-dose group(20 mg·kg~(-1), ip, qd) and a salvianolate highdose group(40 mg·kg~(-1), ip, qd). Rats in the control group were given normal saline(ip) for 7 consecutive days and on the 5 th day the vehicle(peanut oil) was given(ig). The same treatment was provided to the model group, but the rats were given ANIT(75 mg·kg~(-1), ig 15 mg ANIT was dissolved in 1 mL peanut oil) on the5 th day. The low dose and high dose groups were administrated salvianolate for 7 consecutive days and given ANIT(75 mg·kg~(-1), ig) on the 5 th day. The rats in each group were sacrificed on the 7 th day after fasting for24 h, and the body weight and liver weight were measured. AST, ALT, ALP, γ-GT and TBIL were detected by automatic biochemical analyzer. TBA levels were analyzed with Total Bile acid Assay Kit. HE staining was performed to evaluate the changes of morphology in the liver tissue. Western blot was used to determine the expression of signaling related proteins in NF-κ B/Iκ B and p38 MAPK pathways. Real-time PCR was used to detect the mRNA levels of IL~(-1)β, IL-6, TGF-β, and TNF-α. Results Compared with those of the model group, the serum levels of AST, ALT, ALP, γ-GT, TBIL and TBA were lower in the salvianolate treatment groups(P < 0.05). Salvianolate improved the pathological structure of the rat liver tissue. Meanwhile, the relative expression level of NF-κ B in the nucleus, the phosphorylation level of p38 and the mRNA levels of inflammation factors were all decreased in the salvianolate treatment groups as compared with the model group(P< 0.05). Conclusion The therapeutic effect and potential mechanisms of salvianolate on cholestatic liver injury induced by ANIT may be related to the inhibition of NF-κB/IκB and p38 MAPK pathways.
引文
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[17]王蓉,潘沛,王彧杰,等.丹参多酚酸盐对肝纤维化大鼠NF-κB和IκBα表达的影响[J].中国新药与临床杂志,2011,30(1):51-55.
[18]Bailie MB,Pearson JM,Lappin PB,et al. Platelets and alpha-naphthylisothiocyanate-induced liver injury[J]. Toxicol Appl Pharmacol,1994,129(2):207-213.
[19]张文炜,毛子旭,徐列明.丹参酚酸B盐对人肝星状细胞内肌细胞增强因子2的影响[J].自然科学进展,2008,18(5):573-578.
[20]Wang CY,Ma FL,Liu JT,et al. Protective effect of salvianic acid a on acute liver injury induced by carbon tetrachloride in rats[J]. Biol Pharm Bull,2007,30(1):44-47.
[21]Wan JM,Sit WH,Lee CL,et al. Protection of lethal toxicity of endotoxin by Salvia miltiorrhiza BUNGE is via reduction in tumor necrosis factor alpha release and liver injury[J]. Int Immunopharmacol,2006,6(5):750-758.
[22]高玉琪,吴宗贵,梁春,等.丹参多酚酸盐对ApoE基因敲除小鼠血清TNF-α的影响[J].放射免疫学杂志,2008,21(6):547-549.
[23]Jin F,Cheng D,Tao JY,et al. Anti-inflammatory and anti-oxidative effects of corilagin in a rat model of acute cholestasis[J]. BMC Gastroenterol,2013,13(1):79.
[24]Teng H,Chen M,Zou A,et al. Hepatoprotective effects of licochalcone B on carbon tetrachloride-induced liver toxicity in mice[J]. Iran J Basic Med Sci,2016,19(8):910-915.
[25]Zhong W,Qian K,Xiong J,et al. Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-kappaB related signaling[J]. Biomed Pharmacother,2016,83:302-313.
[26]NafeesS,RashidS,AliN,etal.Rutinameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats:role of NFkappaB/MAPK pathway[J].Chem Biol Interact,2015,231:98-107.
[27]王凤华,韩吉春,李德芳,等.霍山石斛水提取物通过介导NF-κB/p65和p38 MAPK减轻小鼠酒精性肝损伤[J].天然产物研究与开发,2017,29(4):569-574.
[2]丁艳,赵雷,贾德胜,等.转录因子κB和早期生长反应因子1在急性肝内胆汁淤积性肝损伤大鼠模型中的表达[J].临床儿科杂志,2008,26(10):879-884.
[3]冯彦.丹参的现代药理研究及临床应用[J].中医临床研究,2017,9(30):46-47.
[4]隋玉玲.丹参多酚酸盐的药理作用与临床应用研究进展[J].临床合理用药杂志,2017,10(23):178-180.
[5]Khamphaya T,Chansela P,Piyachaturawat P,et al. Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats[J]. Eur J Pharmacol,2016,789:254-264.
[6]Fang ZZ,Tanaka N,Lu D,et al. Role of the lipid-regulated NF-κB/IL-6/STAT3 axis in alpha-naphthyl isothiocyanate-induced liver injury[J]. Arch Toxicol,2017,91(5):2235-2244.
[7]Paumgartner G,Beuers U. Ursodeoxycholic acid in cholestatic liver disease:mechanisms of action and therapeutic use revisited[J]. Hepatology,2002,36(3):525-531.
[8]AllenK,JaeschkeH,CoppleBL.Bileacidsinduce inflammatory genes in hepatocytes:a novel mechanism of inflammation during obstructive cholestasis[J]. Am J Pathol,2011,178(1):175-186.
[9]Weerachayaphorn J,Luo Y,Mennone A,et al. Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice[J]. J Hepatol,2014,60(1):160-166.
[10]Kauppinen A,Suuronen T,Ojala J,et al. Antagonistic crosstalk between NF-kappaB and SIRT1 in the regulation of inflammation and metabolic disorders[J]. Cell Signal,2013,25(10):1939-1948.
[11]Wang Y,Wang R,Wang YJ,et al. Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38MAPK,NF-kappa B/Ikappa Balpha,and Bcl-2/Bax signaling[J]. Drug Des Devel Ther,2015,9(1):6303-6317.
[12]Baek HS,Park N,Kwon Y,et al. Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-zeta-nuclear factor-kappaB signaling pathway in prostate cancer cells[J]. Oncotarget,2017,8(43):74263-74275.
[13]Zhao X,Zou X,Li Q,et al. Total flavones of fermentation broth by co-culture of Coprinus comatus and Morchella esculenta induces an anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages cells via the MAPK signaling pathway[J]. Microb Pathog,2018,125:431-437.
[14]Liu CM,Ma JQ,Liu SS,et al. Puerarin protects mouse liver against nickel-induced oxidative stress and inflammation associated with the TLR4/p38/CREB pathway[J].Chem Biol Interact,2016,243:29-34.
[15]马文芳,孙建鹰,赵静.丹参多酚酸盐对急性肝衰竭大鼠的早期治疗作用研究[J].中国急救医学,2012,32(9):811-813.
[16]杨丹红,叶再元,金波,等.丹参多酚酸盐对实验性肝硬化大鼠肝组织TNF-α和IL-6mRNA的表达影响[J].中华中医药学刊,2013,31(6):1297-1300.
[17]王蓉,潘沛,王彧杰,等.丹参多酚酸盐对肝纤维化大鼠NF-κB和IκBα表达的影响[J].中国新药与临床杂志,2011,30(1):51-55.
[18]Bailie MB,Pearson JM,Lappin PB,et al. Platelets and alpha-naphthylisothiocyanate-induced liver injury[J]. Toxicol Appl Pharmacol,1994,129(2):207-213.
[19]张文炜,毛子旭,徐列明.丹参酚酸B盐对人肝星状细胞内肌细胞增强因子2的影响[J].自然科学进展,2008,18(5):573-578.
[20]Wang CY,Ma FL,Liu JT,et al. Protective effect of salvianic acid a on acute liver injury induced by carbon tetrachloride in rats[J]. Biol Pharm Bull,2007,30(1):44-47.
[21]Wan JM,Sit WH,Lee CL,et al. Protection of lethal toxicity of endotoxin by Salvia miltiorrhiza BUNGE is via reduction in tumor necrosis factor alpha release and liver injury[J]. Int Immunopharmacol,2006,6(5):750-758.
[22]高玉琪,吴宗贵,梁春,等.丹参多酚酸盐对ApoE基因敲除小鼠血清TNF-α的影响[J].放射免疫学杂志,2008,21(6):547-549.
[23]Jin F,Cheng D,Tao JY,et al. Anti-inflammatory and anti-oxidative effects of corilagin in a rat model of acute cholestasis[J]. BMC Gastroenterol,2013,13(1):79.
[24]Teng H,Chen M,Zou A,et al. Hepatoprotective effects of licochalcone B on carbon tetrachloride-induced liver toxicity in mice[J]. Iran J Basic Med Sci,2016,19(8):910-915.
[25]Zhong W,Qian K,Xiong J,et al. Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-kappaB related signaling[J]. Biomed Pharmacother,2016,83:302-313.
[26]NafeesS,RashidS,AliN,etal.Rutinameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats:role of NFkappaB/MAPK pathway[J].Chem Biol Interact,2015,231:98-107.
[27]王凤华,韩吉春,李德芳,等.霍山石斛水提取物通过介导NF-κB/p65和p38 MAPK减轻小鼠酒精性肝损伤[J].天然产物研究与开发,2017,29(4):569-574.