灯盏细辛基于Psmb8/NF-κB轴抑制糖尿病肾病大鼠炎症反应

Inhibitory effect of erigeron breviscapus on inflammation in diabetic nephropathy rats by Psmb8/NF-κB axis

目的探讨灯盏细辛(erigeron breviscapus,EB)是否通过介导β型蛋白酶体亚基8(proteasome subunitβtype 8,Psmb8)及核因子-κB(nuclear factor-κB,NF-κB)轴抑制糖尿病肾病大鼠炎症反应。方法随机选10只SPF雄性SD大鼠作为正常组,剩余大鼠用高脂饮食联合链脲佐菌素(streptozotocin,STZ)构建糖尿病肾病(diabetic nephropathy,DN)大鼠模型。成模大鼠共41只,随机剔除1只,将剩余大鼠随机分为模型组、EB低剂量组、EB中剂量组和EB高剂量组,每组10只。其中EB低剂量、EB中剂量和EB高剂量组分别灌胃EB 20、40、80 mg·kg^(-1)·d^(-1),正常组和模型组大鼠给予等量生理盐水,持续给药8周。检测各组大鼠空腹血糖(fasting blood glucose,FBG)、肾脏指数(renal mass index,KI);用全自动生化分析仪检测24 h尿蛋白(urine protein,UPro)、血清肌酐(serum creatinine,Scr)和血尿素氮(blood urea nitrogen,BUN)水平;HE染色观察大鼠肾组织病理形态变化;用酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18)的含量;用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测Psmb8和NF-κB p65 mRNA相对表达量;用蛋白印迹(Western blotting)法检测Psmb8、NF-κB p65及p-NF-κB p65蛋白的相对表达量。结果与正常组比较,模型组大鼠FBG、KI升高,体质量下降(P<0.05),肾小球形态见明显病理改变,间质水肿并伴有大量炎性细胞浸润,血清TNF-α、IL-1β及IL-18含量升高,肾组织中Psmb8和NF-κB p65 mRNA及Psmb8、NF-κB p65和p-NF-κB p65蛋白的相对表达量升高(P<0.05);与模型组比较,EB低、中、高剂量组大鼠FBG和KI下降,体质量升高(P<0.05),肾组织炎性病理情况见不同程度减轻,血清TNF-α、IL-1β及IL-18含量降低,肾组织中Psmb8和NF-κB p65 mRNA及Psmb8、NF-κB p65和pNF-κB p65蛋白的相对表达量降低(P<0.05);且EB对DN大鼠的炎症抑制作用呈剂量依赖性。结论EB可抑制DN大鼠炎症反应,其作用机制可能与抑制Psmb8/NF-κB轴激活有关。

Objective To investigate whether erigeron breviscapus(EB)can mediate proteasome subunitβtype 8(Psmb8)/nuclear factor-κB(NF-κB)axis to inhibit inflammation in diabetic nephropathy(DN)rats.Methods Ten SPF male SD rats were randomly selected as the normal group,and the rest rats were treated with streptozotocin(STZ)to construct diabetic nephropathy(DN)rat model.There were 41 model rats,After one rat was randomly eliminated,the remaining rats were randomly divided into model group,EB low-dose group,EB medium-dose group and EB high-dose group,with 10 rats in each group.The EB low,medium and high dose groups were given EB 20,40 and 80 mg·kg^(-1)·d^(-1),respectively.The normal group and model group were given the same amount of normal saline for 8 weeks.Fasting blood glucose(FBG)and renal mass index(KI)were detected.The levels of 24-hour urine protein(UPro),serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by automatic biochemical analyzer.HE staining was used to observe the pathological changes of renal tissues.The serum levels of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β)and interleukin-18(IL-18)were determined by ELISA.The mRNA relative expression levels of Psmb8 and NF-κB P65 were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The relative protein expression levels of Psmb8,NF-κB p65 and p-NF-κB p65 were detected by Western blotting.Results Compared with normal group,FBG,KI increased,body weight decreased in model group(P<0.05),the glomerular morphology showed obvious pathological changes,interstitial edema accompanied by a large numbers of inflammatory cell infiltration,and the serum TNF-α,IL-1βand IL-18 contents increased.The mRNA expression of Psmb8 and NF-κB p65 and the relative protein expression of Psmb8,NF-κB P65 and p-NF-κB p65 were increased in renal tissues(P<0.05).Compared with model group,the FBG and KI were decreased and body weight was increased in low,medium and high dose EB groups(P<0.05),the inflammatory pathology of renal tissue was alleviated to varying degrees,and the contents of TNF-α,IL-1βand IL-18 in serum were decreased.The mRNA expression of Psmb8 and NF-κB p65 and the relative protein expression of Psmb8,NF-κB p65 and p-NF-κB p65 were decreased in renal tissue(P<0.05).The effect of EB on DN rats was dose-dependent.Conclusion EB can inhibit inflammatory response in DN rats,and its mechanism may be related to inhibiting the activation of Psmb8/NF-κB axis.