虾青素对Ⅲ度烧伤大鼠急性肾损伤的作用及机制

Effect and mechanism of astaxanthin on acute kidney injury in rats with full-thickness burns

目的探讨虾青素对Ⅲ度烧伤大鼠急性肾损伤的作用及机制。方法取48只8~10周龄雄性SD大鼠,按随机数字表法分为假伤组、单纯烧伤组、烧伤+溶剂组、烧伤+低剂量虾青素组、烧伤+中剂量虾青素组、烧伤+高剂量虾青素组,每组8只。假伤组大鼠背部皮肤浸入20℃的温水中15 s模拟致伤;其他5组大鼠背部皮肤浸入100℃沸水中15 s,造成30%体表总面积Ⅲ度烧伤。伤后即刻及伤后6 h除假伤组外的5组大鼠进行液体复苏。伤后30 min,假伤组和单纯烧伤组大鼠按1 mL/kg尾静脉注射生理盐水,烧伤+溶剂组大鼠按1 mL/kg尾静脉注射虾青素溶剂,烧伤+低剂量虾青素组、烧伤+中剂量虾青素组、烧伤+高剂量虾青素组大鼠分别按5、10、20 mg/kg尾静脉注射5、10、20 mg/mL的用虾青素溶剂溶解的虾青素溶液。伤后48 h,取肾脏组织,苏木精-伊红染色行组织病理学观察和肾小管损伤评分;取静脉血,血生化分析仪检测血肌酐水平,酶联免疫吸附测定法检测血尿素氮水平。取伤后48 h肾脏组织,采用实时荧光定量反转录PCR法检测髓过氧化物酶(MPO)、白细胞介素1β(IL-1β)和IL-6的mRNA表达,蛋白质印迹法检测Toll样受体4(TLR4)、磷酸化核因子κB(p-NF-κB)p65及血红素加氧酶1(HO-1)的蛋白表达,免疫荧光法检测HO-1的表达。对数据行Kruskal-Wallis H检验、Dunn-Sidák校正、单因素方差分析、Bonferroni法。结果(1)伤后48 h,假伤组大鼠肾组织中无炎症细胞浸润和细胞变性坏死,肾小管结构完整;烧伤各组大鼠肾小管内呈现出上皮细胞与基底膜分离、管腔内空泡细胞和裂解蛋白聚集等损伤表现,烧伤+高剂量虾青素组大鼠肾组织损伤程度较单纯烧伤组明显降低。(2)伤后48 h,与假伤组比较,单纯烧伤组、烧伤+溶剂组、烧伤+低剂量虾青素组和烧伤+中剂量虾青素组大鼠肾小管损伤评分显著升高(P<0.05或P<0.01);与单纯烧伤组和烧伤+溶剂组比较,烧伤+中剂量虾青素组和烧伤+高剂量虾青素组大鼠肾小管损伤评分显著降低(P<0.05或P<0.01);与烧伤+低剂量虾青素组比较,烧伤+高剂量虾青素组大鼠肾小管损伤评分显著降低(P<0.01)。(3)伤后48 h,假伤组大鼠血肌酐水平为(2.42±0.06)mg/L,显著低于单纯烧伤组、烧伤+溶剂组、烧伤+低剂量虾青素组和烧伤+中剂量虾青素组的(6.11±0.11)、(6.48±0.08)、(5.79±0.09)、(4.03±0.12)mg/L(P<0.05或P<0.01);假伤组大鼠血尿素氮水平为(21.9±1.3)mmol/L,显著低于单纯烧伤组和烧伤+溶剂组的(32.1±7.4)、(30.2±4.8)mmol/L,P<0.05或P<0.01。伤后48 h,与单纯烧伤组和烧伤+溶剂组比较,烧伤+中剂量虾青素组大鼠血肌酐和血尿素氮水平[(16.0±2.9)mmol/L]、烧伤+高剂量虾青素组大鼠血肌酐[(3.02±0.08)mg/L]和血尿素氮水平[(14.5±2.9)mmol/L]及烧伤+低剂量虾青素组大鼠血尿素氮水平[(22.8±5.5)mmol/L]显著降低(P<0.05或P<0.01)。伤后48 h,与烧伤+低剂量虾青素组比较,烧伤+高剂量虾青素组大鼠血肌酐和血尿素氮水平及烧伤+中剂量虾青素组大鼠血肌酐水平明显降低(P<0.05或P<0.01)。(4)伤后48 h,与假伤组比较,单纯烧伤组、烧伤+溶剂组、烧伤+低剂量虾青素组、烧伤+中剂量虾青素组大鼠肾组织中MPO、IL-1β和IL-6 mRNA表达及烧伤+高剂量虾青素组大鼠肾组织中的IL-1β和IL-6 mRNA表达显著升高(P<0.01);与单纯烧伤组和烧伤+溶剂组比较,烧伤+低剂量虾青素组、烧伤+中剂量虾青素组和烧伤+高剂量虾青素组大鼠肾组织中MPO、IL-1β和IL-6 mRNA表达显著降低(P<0.01);与烧伤+低剂量虾青素组比较,烧伤+中剂量虾青素组和烧伤+高剂量虾青素组大鼠肾组织中MPO、IL-1β和IL-6 mRNA表达显著降低(P<0.01);与烧伤+中剂量虾青素组比较,烧伤+高剂量虾青素组大鼠肾组织中MPO、IL-1β和IL-6 mRNA表达显著降低(P<0.01)。(5)伤后48 h,与假伤组比较,单纯烧伤组、烧伤+溶剂组、烧伤+低剂量虾青素组和烧伤+中剂量虾青素组大鼠肾组织中TLR4和p-NF-κB p65蛋白表达及烧伤+高剂量虾青素组大鼠肾组织中TLR4蛋白表达显著升高(P<0.01)。与单纯烧伤组比较,烧伤+低剂量虾青素组、烧伤+中剂量虾青素组和烧伤+高剂量虾青素组的大鼠肾组织中TLR4和p-NF-κB p65蛋白表达显著降低(P<0.01)。(6)蛋白质印迹法结合免疫荧光法检测结果显示,伤后48 h,与假伤组比较,烧伤+溶剂组、烧伤+低剂量虾青素组、烧伤+中剂量虾青素组和烧伤+高剂量虾青素组大鼠肾组织中的HO-1蛋白表达显著升高(P<0.01);与单纯烧伤组比较,烧伤+中剂量虾青素组和烧伤+高剂量虾青素组大鼠肾组织中HO-1蛋白表达显著升高(P<0.01)。结论虾青素可缓解肾组织结构损伤和功能减退,并调节损伤相关炎症因子释放,从而实现对烧伤后大鼠急性肾损伤的保护。HO-1/TLR4/NF-κB信号通路是虾青素实现抗炎性肾保护作用的主要调节机制。

Objective To explore the effect and mechanism of astaxanthin on acute kidney injury in rats with full-thickness burns.Methods Forty-eight male Sprague Dawley rats of 8 to 10 weeks were divided into sham injury group,simple burn group,burn+vehicle group,burn+low-dose astaxanthin group,burn+medium-dose astaxanthin group,and burn+high-dose astaxanthin group according to the random number table,with 8 rats in each group.The back skin of rats in sham injury group were immersed in warm water of 20℃for 15 s to simulate burn injury,and the back skin of rats in the other 5 groups were immersed in boiled water of 100℃for 15 s to inflict full-thickness burn of 30%total body surface area.Fluid resuscitation was performed in rats in the 5 groups except of sham injury group immediately and 6 h after injury.At 30 min after injury,the rats in sham injury group and simple burn group were injected with 1 mL/kg normal saline via tail vein,rats in burn+vehicle group were injected with 1 mL/kg astaxanthin solvent via tail vein,and rats in burn+low-dose astaxanthin group,burn+medium-dose astaxanthin group,and burn+high-dose astaxanthin group were respectively injected with 5,10,20 mg/kg astaxanthin solution of 5,10,20 mg/mL via tail vein.The renal tissue was collected at post injury hour(PIH)48,and hematoxylin eosin staining was used for histopathological observation and renal tubular injury score.At PIH 48,the venous blood was collected for detecting serum creatinine level through blood biochemical analyzer,and blood urea nitrogen(BUN)level was detected by enzyme-linked immunosorbent assay.The renal tissue was collected to detect the mRNA expressions of myeloperoxidase(MPO),interleukin-1β(IL-1β),and IL-6 by real-time fluorescent quantitative reverse transcription polymerase chain reaction method,and the protein expressions of Toll like receptor 4(TLR4),phosphorylated nuclear factor kappa B(p-NF-кB)p65,and heme oxygenase 1(HO-1)were detected by Western blotting.Besides,the expression of HO-1 in renal tissue was detected by immunofluorescence method.Data were statistically analyzed with Kruskal-Wallis H test,Dunn-Sidák correction,one-way analysis of variance,and Bonferroni method.Results(1)At PIH 48,there were no inflammatory cell infiltrating and degeneration or necrosis of cells in renal tissue of rats in sham injury group,and the structure of renal tubules was intact.The renal tubules of burn rats in each group showed injury manifestation of separation between epithelial cell and basement membrane,and vacuole cells and lysate protein aggregation.The injury degree of renal tissue of rats in burn+high-dose astaxanthin group was obviously decreased compared with that in simple burn group.(2)At PIH 48,compared with that of sham injury group,the renal tubular damage scores of rats in simple burn group,burn+vehicle group,burn+low-dose astaxanthin group,and burn+medium-dose astaxanthin group were significantly increased(P<0.05 or P<0.01).Compared with those of simple burn group and burn+vehicle group,the renal tubular damage scores of rats in burn+medium-dose astaxanthin group and burn+high-dose astaxanthin group were significantly decreased(P<0.05 or P<0.01).Compared with that of burn+low-dose astaxanthin group,the renal tubular damage score of rats in burn+high-dose astaxanthin group was significantly decreased(P<0.01).(3)At PIH 48,the level of serum creatinine of rats in sham injury group was(2.42±0.06)mg/L,which was significantly lower than(6.11±0.11),(6.48±0.08),(5.79±0.09),(4.03±0.12)mg/L of simple burn group,burn+vehicle group,burn+low-dose astaxanthin group,and burn+medium-dose astaxanthin group(P<0.05 or P<0.01).The level of BUN of rats was(21.9±1.3)mmol/L in sham injury group,significantly lower than(32.1±7.4)mmol/L of simple burn group and(30.2±4.8)mmol/L of burn+vehicle group(P<0.05 or P<0.01).At PIH 48,compared with those of simple burn group and burn+vehicle group,the levels of serum creatinine and BUN of(16.0±2.9)mmol/L in burn+medium-dose astaxanthin group,serum creatinine of(3.02±0.08)mg/L and BUN of(14.5±2.9)mmol/L in burn+high-dose astaxanthin group,and serum creatinine of(22.8±5.5)mmol/L of rats in burn+low-dose astaxanthin group were significantly decreased(P<0.05 or P<0.01).At PIH 48,compared with those of burn+low-dose astaxanthin group,the levels of serum creatinine and BUN of burn+high-dose astaxanthin group and serum creatinine of burn+medium-dose group were obviously decreased(P<0.05 or P<0.01).(4)At PIH 48,compared with those of sham injury group,the mRNA expressions of MPO,IL-1β,and IL-6 in renal tissue of rats in simple burn group,burn+vehicle group,burn+low-dose astaxanthin group,and burn+medium dose astaxanthin group,and the mRNA expressions of IL-1βand IL-6 in renal tissue of rats in burn+high-dose astaxanthin group were obviously increased(P<0.01).Compared with those of simple burn group and burn+vehicle group,the mRNA expressions of MPO,IL-1β,and IL-6 in renal tissue of rats were significantly decreased in burn+low-dose astaxanthin group,burn+medium-dose astaxanthin group,and burn+high-dose astaxanthin group(P<0.01).Compared with those of burn+low-dose astaxanthin group,the mRNA expressions of MPO,IL-1β,and IL-6 in renal tissue of rats were significantly decreased in burn+medium-dose astaxanthin group and burn+high-dose astaxanthin group(P<0.01).The mRNA expressions of MPO,IL-1β,and IL-6 in renal tissue of rats in burn+high-dose astaxanthin group were significantly decreased compared with those of burn+medium-dose astaxanthin group(P<0.01).(5)At PIH 48 h,compared with those of sham injury group,the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in simple burn group,burn+vehicle group,burn+low-dose astaxanthin group,and burn+high-dose astaxanthin group were obviously increased(P<0.01).Compared with those of simple burn group,the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in burn+low-dose astaxanthin group,burn+medium dose astaxanthin group,and burn+high-dose astaxanthin group were significantly decreased(P<0.01).(6)The results of Western blotting combined with immunofluorescence method showed that compared with that of sham injury group,the protein expression of HO-1 in renal tissue of rats in burn+vehicle group,burn+low-dose astaxanthin group,burn+medium-dose astaxanthin group,and burn+high-dose astaxanthin group were significantly increased at PIH 48(P<0.01),and the protein expression of HO-1 in renal tissue of rats in burn+medium-dose astaxanthin group and burn+high-dose astaxanthin group was significantly increased compared with that of simple burn group(P<0.01).Conclusions Astaxanthin can attenuate the structural damage and functional decline of renal tissue and regulate the release of injury-related inflammatory factors,thus to protect the rats from acute kidney injury after burn.The HO-1/TLR4/NF-кB signaling pathway is the main regulatory mechanism of astaxanthin to achieve anti-inflammation-based renoprotection.