黄芪桂枝五物汤对糖尿病周围神经病变大鼠模型AGEs/RAGE/NF-κB信号通路的影响

Effect of Huangqi Guizhi Wuwutang on AGEs/RAGE/NF-κB Signaling Pathway in Diabetic Peripheral Neuropathy Rats

目的:研究黄芪桂枝五物汤对糖尿病周围神经病变大鼠模型晚期糖基化终产物(AGEs)/晚期糖基化终产物受体(RAGE)/核转录因子-κB(NF-κB)信号通路的影响,探讨黄芪桂枝五物汤减轻糖尿病周围神经病变的作用机制。方法:以高脂饮食加腹腔注射链脲佐菌素(STZ)诱导法,制作糖尿病周围神经病变大鼠模型。造模成功后,按照随机数字表法分成模型组、黄芪桂枝五物汤高、中、低剂量组、甲钴胺组,共5组,另设正常组,自第5周开始黄芪桂枝五物汤干预,采用黄芪桂枝五物汤高、中、低剂量组(19.40,4.85,2.43 g·kg^-1·d^-1)连续灌胃12周;甲钴胺组采用甲钴胺0.25 mg·kg^-1·d^-1灌胃。实验结束后,采用酶联免疫吸附测定(ELISA)试剂盒检测糖尿病周围神经病变大鼠血清白细胞介素-1β(IL-1β),肿瘤坏死因子-α(TNF-α)含量;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测坐骨神经组织中RAGE,NF-κB p65 mRNA含量;采用蛋白免疫印迹法(Western blot)检测坐骨神经组织中RAGE,NF-κB,磷酸化NF-κB p65(p-NF-κB p65)蛋白的表达变化。结果:与正常组比较,模型组大鼠血清IL-1β,TNF-α含量,坐骨神经组织RAGE,NF-κB p65 mRNA含量,RAGE,NF-κB p65及p-NF-κB p65蛋白表达显著增加(P<0.01),p-NF-κB p65/NF-κB p65升高,p-NF-κB p65蛋白表达显著升高(P<0.01)。经黄芪桂枝五物汤干预后,与模型组比较,各给药组血清IL-1β,TNF-α含量,RAGE,NF-κB p65 mRNA含量,RAGE,NF-κB p65及p-NF-κB p65蛋白表达量均显著降低(P<0.01),黄芪桂枝五物汤高剂量组p-NF-κB p65/NF-κB p65显著降低(P<0.01),且呈剂量相关性,随黄芪桂枝五物剂量增加而效果明显。结论:黄芪桂枝五物汤可减轻糖尿病周围神经病变,其机制可能与阻断AGEs/RAGE/NF-κB信号通路中组织细胞表面RAGE的表达、抑制NF-κB激活及其引发TNF-α触发的氧化应激和过度炎症反应,从而避免细胞受损和功能紊乱有关。

Objective: To study the effect of Huangqi Guizhi Wuwutang on receptor of advanced glycation end products(AGEs)/advanced glycation endproducts(RAGE)/nuclear transcription factor-kappa B p65(NF-κB p65) signaling pathway in the diabetic peripheral neuropathy rats through an animal modeling experiment, and discuss the mechanism of Huangqi Guizhi Wuwutang in alleviating diabetic peripheral neuropathy. Method: Rat model of diabetic peripheral neuropathy was established by high-fat diet and intraperitoneal injection with streptozotocin(STZ). After successful modeling,Huangqi Guizhi Wuwutang intervention began in the fifth week. The patients in high-dose group(19. 40 g ·kg^-1· d^-1),middle-dose group(4. 85 g·kg^-1·d^-1)and low-dose group(2. 43 g·kg^-1·d^-1)were given by gavage continuously for 12 weeks. The western medicine control group was given 25 mg·kg^-1·d^-1 by gavage. After the experiment,serum interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) was used to detect RAGE and NF-κB p65 mRNA expressions in sciatic nerve tissue. The expressions of RAGE,NF-κB and phosphorylation(p)-NF-κB p65 proteins in sciatic nerve tissues were detected by Western blot(WB). Result: Compared with the normal group,serum IL-1β and TNF-α protein levels,RAGE mRNA and NF-κB p65 mRNA levels,RAGE protein,NF-κB p65 protein and p-NF-κB p65 protein levels were significantly increased in the model group(P<0. 01),the ratio of p-NF-κB p65 to NF-κB p65 was increased,and the phosphorylation of NF-κB p65 was enhanced(P<0. 01). After the intervention of Huangqi Guizhi Wuwutang,compared with the model group,serum IL-1β and TNF-α protein levels,RAGE and NF-κB p65 mRNA levels,RAGE protein,NF-κB p65 protein and p-NF-κB p65 protein levels were all decreased(P<0. 01),the ratio of p-NF-κB p65 to NF-κB p65 was decreased in high-dose group(P<0. 01). The effect was obvious with the increase of dose of astragalus cassia twig. Conclusion: Huangqi Guizhi Wuwutang can alleviate diabetic peripheral neuropathy,and its mechanism may be related to blocking the expression of RAGE on tissue cell surface in AGEs/RAGE/NF-κB signaling pathway,inhibiting the activation of NF-κB and inducing TNF-α triggered oxidative stress and excessive inflammatory response,so as to avoid cell damage and dysfunction.