电针对内毒素性急性肾损伤大鼠肾小管上皮细胞焦亡的影响

Effect of electroacupuncture on pyroptosis in renal tubular epithelial cells of rats with acute kidney injury induced by endotoxin

目的评价电针对内毒素性急性肾损伤(AKI)大鼠肾小管上皮细胞焦亡的影响。方法健康清洁级SD大鼠24只,雌雄不拘,体重160~182 g,6~8周龄,采用随机数字表法将大鼠分为4组(n=6):对照组(C组)、AKI组、电针+AKI组(EA组)和非穴位电针+AKI组(SEA组)。采用腹腔注射LPS 10 mg/kg的方法制备内毒素血症模型。EA组于造模前5 d(1次/d)、造模当日LPS给药前30 min开始电针刺激,选择双侧足三里及肾俞穴,疏密波,频率15 Hz,刺激强度以大鼠出现轻微肌颤为宜,30 min/次,留针至LPS注射后6 h;SEA组刺激穴位旁开0.5 cm处。于LPS注射后6 h时,经心脏取血,采用全自动生化分析仪检测血清BUN和Cr浓度,采用ELISA法检测血清中性粒细胞白明胶酶脂蛋白(NGAL)、肾损伤分子-1(KIM-1)和IL-6和TNF-α浓度。处死大鼠取左侧肾皮质组织,采用TUNEL法确定肾小管上皮细胞焦亡率;取右侧肾皮质组织采用Western blot法检测caspase-1、IL-1β表达,采用RT-PCR检测caspase-1 mRNA和IL-1βmRNA的表达。结果与C组比较,AKI组血清BUN、Cr、NGAL、KIM-1、TNF-α和IL-6浓度升高,肾小管上皮细胞焦亡率升高,肾皮质caspase-1、IL-1β及其mRNA表达上调(P<0.05)。与AKI组比较,EA组血清BUN、Cr、NGAL、KIM-1、TNF-α和IL-6浓度降低,肾小管上皮细胞焦亡率降低,肾皮质caspase-1、IL-1β及其mRNA表达下调(P<0.05),SEA组上述指标差异无统计学意义(P>0.05)。结论电针减轻大鼠内毒素性AKI的机制可能与抑制肾小管上皮细胞焦亡有关。

Objective To evaluate the effect of electroacupuncture(EA)on pyroptosis in renal tubular epithelial cells of rats with acute kidney injury(AKI)induced by endotoxin.Methods Twenty-four healthy clean-grade Sprague-Dawley rats of either gender,aged 6-8 weeks,weighing 160-182 g,were divided into 4 groups(n=6 each)using a random number table method:control group(group C),group AKI,EA plus AKI group(group EA),sham EA at non-acupoint plus AKI group(group SEA).The model of endotoxemia was established by intraperitoneally injecting 10 mg/kg lipopolysaccharide.Bilateral 30 min EA stimulation of Zusanli and Shenyu(according to atlas of animal acupoint)was performed starting from 5 days before establishing the model(once a day)and at 30 min before lipopolysaccharide administration on the day of establishing the model,with disperse-dense waves,frequency of 15 Hz,and the needle was kept until 6 h after injection of LPS in group EA.EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenyu in group SEA.At 6 h after LPS injection,blood was taken from the heart,and the concentrations of serum blood urea nitrogen(BUN)and creatinine(Cr)were detected by an automatic biochemical analyzer,and the serum concentrations of neutrophil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)by enzyme-linked immunosorbent assay.The rats were then sacrificed,and the left renal cortex was obtained for determination of pyroptosis rate of renal tubular epithelial cells(by TUNEL).The right renal cortex was obtained to detect the expression of caspase-1 and IL-1βby Western blot,and the expression of caspase-1 mRNA and IL-1βmRNA was detected by real-time polymerase chain reaction.Results Compared with group C,the concentrations of BUN,Cr,NGAL,KIM-1,TNF-α,and IL-6 were significantly increased,the pyroptosis rate of renal tubular epithelial cells was increased,the expression of caspase-1 and IL-1βprotein and mRNA in the renal cortex was up-regulated in group AKI(P<0.05).Compared with group AKI,the concentrations of BUN,Cr,NGAL,KIM-1,TNF-α,and IL-6 were significantly decreased,the pyroptosis rate of renal tubular epithelial cells was decreased,the expression of caspase-1 and IL-1βprotein and mRNA in the renal cortex was down-regulated in group SEA(P>0.05).Conclusion The mechanism by which EA reduces AKI may be related to inhibiting pyroptosis in renal tubular epithelial cells of rats.