大黄丹参对高脂饮食诱发的胰腺纤维化大鼠影响的研究

Effect of rhubarb and salvia miltiorrhiza on pancreatic fibrosis induced by high fat diet in rats

目的研究大黄丹参对高脂饮食诱导的胰腺纤维化大鼠氧化应激、内质网应激相关因子的影响。方法将40只雄性清洁级SD大鼠,采用随机数表法均分成对照组、模型组、大黄丹参低、中、高剂量组,每组8只;对照组喂以普通饲料,其余4组均喂以高脂饲料连续10周建立胰腺纤维化模型。造模同时,各组以相应药物灌胃,对照组与模型组生理盐水灌胃,造模结束后取材。光镜、电镜观察胰腺形态学和超微结构变化。生化检测血清过氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)、总胆固醇(TC)、甘油三酯(TG)含量。实时荧光定量核酸扩增检测(PCR)及蛋白质印迹法(Western blot)检测胰腺C-Jun氨基酸末端激酶(JNK)、葡萄糖调节蛋白(GRP)及半胱氨酸门冬氨酸特异性蛋白酶-12(Caspase-12)信使RNA(mRNA)及蛋白表达。组间数据比较采用单因素方差分析。结果模型组胰腺形态学和超微结构均可见内质网肿胀、细胞间隙变窄等病理变化,大黄丹参各剂量组均可不同程度改善以上病理变化。大黄丹参中、高剂量组SOD[(24.71±2.35)、(33.80±3.21)U/ml]、GSH含量[(61.41±5.57)、(101.34±17.32)gGSH/L]含量高于模型组(12.77±3.17、32.05±5.59,P<0.01),差异有统计学意义,大黄丹参低、中、高剂量组MDA[(7.10±0.61)、(6.13±0.63)、(5.47±0.57)μmol/L]、TG[(0.43±0.08)、(0.38±0.06)、(0.22±0.03)mmol/L]含量低于模型组(8.27±1.12、0.69±0.13,P<0.01),大黄丹参中、高剂量组TC[(0.58±0.19)、(0.39±0.15)mmol/L]含量低于模型组(1.18±0.44,P<0.05)。大黄丹参中、高剂量组胰腺组织JNK蛋白及mRNA(0.74±0.09、0.69±0.10;0.33±0.04、0.25±0.02)低于模型组(0.99±0.12,1.00±0.00,P<0.05);大黄丹参中、高剂量组P-JNK蛋白(0.50±0.06、0.52±0.13)低于模型组(1.13±0.11,P<0.01);大黄丹参中、高剂量组胰腺组织GRP蛋白及mRNA(0.67±0.13、0.38±0.14;0.51±0.02、0.26±0.02)低于模型组(1.09±0.18、1.00±0.00,P<0.05),大黄丹参中、高剂量组胰腺组织Caspase-12 mRNA(0.68±0.08、0.36±0.06)低于模型组(1.00±0.00,P<0.01);大黄丹参高剂量组Caspase-12蛋白(0.41±0.10)低于模型组(0.95±0.13,P<0.01),差异均有统计学意义。结论大黄丹参可通过降低氧化应激,维持内质网应激稳态来抑制胰腺纤维化。

Objective To study the effects of rhubarb and salvia miltiorrhiza on the pancreatic fibrosis in rats induced by high-fat whether via C-Jun N-terminal kinase(JNK),glucose responsive protein(GRP)and cysteinyl aspartate specific proteinase-12(Caspase-12)mRNA and protein regulation.Methods A total of 40 male specific pathogen free(SPF)SD rats were randomly divided into control group,model group,radix etrhizomarhei and salvia miltiorrhiza low,medium and high dose groups(n=8 each).The control group was fed on ordinary feed,and the animals in the rest of four groups were fed on high fat forage for 10 weeks to induce pancreatic fibrosis.At the same time,rhubarb and salvia miltiorrhiza low,medium and high dose groups were gavaged with the corresponding concentrations of drugs respectively.The control group and model group received oral saline.The pancreatic tissues were harvested and subjected to hematoxylin-eosin staining,and examined under the electron microscopy.Serum superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA),total cholesterol(TC)and triglyceride(TG)contents were determined by biochemical method.The expression of JNK,GRP,Caspase-12 mRNA and protein was detected by real time PCR and Western blotting respectively.One-way analysis of variance(ANOVA)was used for data comparison between groups.Results There were pathological changes such as swollen endoplasmic reticulum and narrowed intercellular space in the gross morphology and ultrastructure of pancreas in model group.Different doses of rhubarb and salvia miltiorrhiza could improve the gross morphological and ultrastructural changes of pancreas to varying degrees.The levels of SOD(24.71±2.35,33.80±3.21)and GSH(61.41±5.57,101.34±17.32)in rhubarb and salvia miltiorrhiza medium and high dose groups were higher than those in model group(12.77±3.17,32.05±5.59,P<0.01).The levels of MDA(7.10±0.61,6.13±0.63,5.47±0.57)and TG(0.43±0.08,0.38±0.06,0.22±0.03)in rhubarb and salvia miltiorrhiza low,medium and high dose groups were lower than those in model group(8.27±1.12,0.69±0.13,P<0.01).The TC levels in rhubarb and salvia miltiorrhiza medium and high dose groups(0.58±0.19,0.39±0.15)were lower than those in model group(1.18±0.44,P<0.05).JNK protein and mRNA expression in pancreatic tissue of rhubarb and salvia miltiorrhiza medium and high dose groups(0.74±0.09,0.69±0.10;0.33±0.04,0.25±0.02)was lower than the model group(0.99±0.12,1.00±0.00,P<0.05).The P-JNK protein expression in rhubarb and salvia miltiorrhiza medium and high dose groups(0.50±0.06,0.52±0.13)were lower than those in model group(1.13±0.11,P<0.01).The GRP protein and mRNA expression levels in pancreatic tissue of rhubarb and salvia miltiorrhiza middle and high dose groups were(0.67±0.13,0.38±0.14;0.51±0.02,0.26±0.02)were lower than in the model group(1.09±0.18,1.00±0.00,P<0.05).The Caspase-12 mRNA expression in pancreatic tissue of rhubarb and salvia miltiorrhiza middle and high dose groups(0.68±0.08,0.36±0.06)was lower than that of the model group(1.00±0.00,P<0.01).The Caspase-12 protein expression in the rhubarb and salvia miltiorrhiza high-dose group(0.41±0.10)was lower than that in the model group(0.95±0.13,P<0.01).Conclusion Rhubarb and salvia miltiorrhiza can inhibit pancreatic fibrosis by reducing oxidative stress and maintaining endoplasmic reticulum stress homeostasis.