大蒜素对膜性肾病大鼠肾损伤的保护作用及机制研究

Protective effect and mechanism of allicin on renal injury in rats with membranous nephropathy

目的探讨大蒜素对膜性肾病大鼠肾损伤的影响及潜在机制。方法将65只雄性Wistar大鼠按随机数字表法分为正常对照组10只和造模组55只,造模组通过尾静脉注射阳离子化牛血清白蛋白(cationized bovine serum albumin,C-BSA)制备膜性肾病模型,取50只成模大鼠按随机数字表法分为模型组、大蒜素低剂量(5 mg/kg)组、大蒜素中剂量(10 mg/kg)组、大蒜素高剂量(20 mg/kg)组和盐酸贝那普利(10 mg/kg)组,每组10只大鼠。各组分别1次/d连续灌胃给药治疗4周后,分光光度法检测24 h尿蛋白定量(24 h urinary total protein,24 h UTP)、血尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,Scr)含量,生化分析法检测血清总蛋白(total protein,TP)、白蛋白(albumin,Alb)含量;酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测血清中白细胞介素(interleukin,IL)1β、IL-6、IL-10、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)含量;通过苏木精-伊红(hematoxylin eosin,HE)染色、过碘酸-雪夫(periodic acid-schiff,PAS)染色、免疫球蛋白(immunoglobulin,Ig)G免疫荧光观察肾组织病理学改变,末端标记法(TdT-mediated dUTP nick-end labeling,TUNEL)染色观察肾细胞凋亡状况;蛋白免疫印迹法检测肾组织Toll样受体4(toll-like receptor 4,TLR4)、核因子κB p65(nuclear factor-κB p65,NF-κB p65)、p-NF-κB p65、B淋巴细胞瘤2(B lymphoblastoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸蛋白酶3、激活型半胱氨酸蛋白酶3表达。结果模型组大鼠24 h UTP(45.07±6.19)mg、BUN(46.45±5.22)mmol/L、Scr(24.90±2.81)μmol/L,正常对照组24hUTP(6.41±0.82)mg、BUN(28.93±3.17)mmol/L、Scr(5.74±0.76)μmol/L,模型组显著高于正常对照组,差异有统计学意义(P<0.05);模型组TP(37.25±4.09)g/L、Alb(18.62±2.03)g/L,正常对照组TP(59.38±7.24)g/L、Alb(30.74±2.60)g/L,模型组显著低于正常对照组,差异有统计学意义(P<0.05);模型组血清IL-1β(638.14±69.22)ng/L、IL-6(115.08±13.61)ng/L、TNF-α(341.75±40.93)ng/L,正常对照组IL-1β(157.42±18.06)ng/L、IL-6(32.61±3.74)ng/L、TNF-α(60.28±6.51)ng/L,模型组显著高于正常对照组,差异有统计学意义(P<0.05);模型组IL-10(24.90±3.01)ng/L,正常对照组IL-10(75.14±9.76)ng/L,模型组显著低于正常对照组,差异有统计学意义(P<0.05);模型组肾组织呈现肾小球体积增大、系膜增生、基底膜增厚,肾小管细胞空泡变性、坏死,肾间质大量炎症细胞浸润,毛细血管袢IgG弥漫性沉积等病理学改变;肾细胞凋亡指数(63.90±8.74)%显著高于正常对照组(4.58±0.52)%,差异有统计学意义(P<0.05);TLR4表达量(0.34±0.06)、Bax表达量(0.27±0.05)及p-NF-κB p65/NF-κB p65比值(0.93±0.18)、激活型半胱氨酸蛋白酶3/半胱氨酸蛋白酶3比值(0.67±0.09)显著高于正常对照组TLR4表达量(0.03±0.01)、Bax表达量(0.03±0.01)及p-NF-κB p65/NF-κB p65比值(0.09±0.03)、激活型半胱氨酸蛋白酶3/半胱氨酸蛋白酶3比值(0.15±0.03),Bcl-2表达量(0.08±0.02)显著低于正常对照组Bcl-2表达量(0.37±0.06),差异有统计学意义(P<0.05)。大蒜素中剂量组、大蒜素高剂量组和盐酸贝那普利组24 h UTP[(25.72±3.49)mg、(13.35±1.60)mg、(23.11±3.57)mg]、BUN[(39.28±4.38)mmol/L、(34.17±3.62)mmol/L、(40.35±4.81)mmol/L]、Scr[(14.61±1.85)μmol/L、(9.05±1.12)μmol/L、(12.72±1.50)μmol/L]均显著低于模型组24 h UTP(45.07±6.19)mg、BUN(46.45±5.22)mmol/L、Scr(24.90±2.81)μmol/L、TP[(46.39±5.02)g/L、(53.47±6.58)g/L、(45.63±5.17)g/L]、Alb[(23.38±2.91)g/L、(27.50±3.04)g/L、(24.08±2.86)g/L]均显著高于模型组TP(37.25±4.09)g/L、Alb(18.62±2.03)g/L,差异均有统计学意义(P<0.05);IL-1β[(349.18±44.06)ng/L、(201.33±26.07)ng/L、(317.92±38.64)ng/L]、IL-6[(73.83±9.17)ng/L、(50.90±5.25)ng/L、(82.76±9.83)ng/L]、TNF-α[(228.14±25.60)ng/L、(101.57±12.94)ng/L、(193.82±23.74)ng/L]均显著低于模型组IL-1β(638.14±69.22)ng/L、IL-6(115.08±13.61)ng/L、TNF-α(341.75±40.93)ng/L、IL-10[(49.26±6.35)ng/L、(62.35±7.71)ng/L、(55.60±6.15)ng/L]显著高于模型组IL-10(24.90±3.01)ng/L,差异均有统计学意义(P<0.05);肾组织病理学改变及细胞凋亡状况较模型组明显改善,凋亡指数[(63.90±8.74)%、(63.90±8.74)%、(63.90±8.74)%]显著低于模型组凋亡指数(63.90±8.74)%,差异有统计学意义(P<0.05);TLR4表达量[(0.24±0.04)、(0.09±0.02)、(0.15±0.03)]、Bax表达量[(0.14±0.02)、(0.08±0.02)、(0.16±0.03)]及p-NF-κB p65/NF-κB p65比值[(0.45±0.07)、(0.21±0.04)、(0.31±0.05)]、激活型半胱氨酸蛋白酶3/半胱氨酸蛋白酶3比值[(0.47±0.07)、(0.20±0.04)、(0.38±0.06)]均显著低于模型组TLR4表达量(0.34±0.06)、Bax表达量(0.27±0.05)、p-NF-κB p65/NF-κB p65比值(0.93±0.18)、激活型半胱氨酸蛋白酶3/半胱氨酸蛋白酶3比值(0.67±0.09),Bcl-2表达量[(0.19±0.04)、(0.30±0.06)、(0.28±0.05)]显著高于模型组Bcl-2表达量(0.08±0.02),差异有统计学意义(P<0.05)。除BUN外,大蒜素低剂量组、大蒜素中剂量组、大蒜素高剂量组对膜性肾病大鼠各检测指标的调控作用呈现剂量依赖性(P<0.05)。除IL-10、Bcl-2外,大蒜素高剂量组对各检测指标的调控作用优于盐酸贝那普利组(P<0.05)。结论大蒜素可能通过抑制TLR4/NF-κB信号通路及其介导的炎症反应和细胞凋亡,减轻膜性肾病大鼠肾损伤,保护肾功能。

Objective To explore the effect of allicin on renal injury in rats with membranous nephropathy(MN)and elucidate its potential mechanism.Methods A total of 65 male Wistar rats were assigned into two groups of normal(n=10)and modeling(n=55).Modeling group received an injection of cationized bovine serum albumin(C-BSA)via tail vein for MN modeling.And 50 modeling rats were randomized into five groups of model,low-dose allicin(5 mg/kg),medium-dose allicin(10 mg/kg),high-dose allicin(20 mg/kg)and nenazepril hydrochloride(10 mg/kg)(n=10 each).After once daily continuous dosing for 4 weeks,24 h urinary total protein(24 h UTP),blood urea nitrogen(BUN)and serum creatinine(Scr)were detected by spectrophotometry;serum levels of total protein(TP)and albumin(Alb)by biochemistry;serum levels of interleukin-1β(IL-1β),IL-6/10 and tumor necrosis factor-alpha(TNF-α)by enzyme-linked immunosorbent assay(ELISA).Renal histopathology was observed after stains of hematoxylin eosin(HE)and periodic acid-Schiff(PAS)and immunoglobulin G(IgG)immunofluorescence.Apoptosis of renal cells was observed through TdT-mediated dUTP nick-end labeling(TUNEL)stain.The expressions of Toll-like receptor 4(TLR4),nuclear factor-κB p65(NF-κB p65),p-NF-κB p65,B lymphoblastoma 2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3 and cleaved caspase-3 in renal tissue were detected by Western blot.Results 24 h UTP(45.07±6.19)mg,BUN(46.45±5.22)mmol/L,Scr(24.90±2.81)μmol/L were significantly higher in model group than 24 h UTP(6.41±0.82)mg,BUN(28.93±3.17)mmol/L and Scr(5.74±0.76)μmol/L in normal control group.The difference was statistically significant(P<0.05);TP(37.25±4.09)g/L and Alb(18.62±2.03)g/L were significantly lower in model group than TP(59.38±7.24)g/L and Alb(30.74±2.60)g/L in normal control group.The difference was statistically significant(P<0.05).Serum levels of IL-1β(638.14±69.22)ng/L,IL-6(115.08±13.61)ng/L and TNF-α(341.75±40.93)ng/L were significantly higher in model group than IL-1β(157.42±18.06)ng/L,IL-6(32.61±3.74)ng/L and TNF-α(60.28±6.51)ng/L in normal control group.Serum level of IL-10 was significantly lower in model group than that in normal control group[(24.90±3.01)vs(75.14±9.76)ng/L].The difference was statistically significant(P<0.05).The pathological changes of renal tissue in model group included greater glomerular volume,mesangial hyperplasia,basement membrane thickening,vacuolar degeneration and necrosis of renal tubule cells,infiltration of inflammatory cells in renal interstitium and diffuse deposition of capillary loop IgG.Apoptotic index was statistically higher in model group than that in normal control group[(63.90±8.74)%vs(4.58±0.52)%].The difference was statistically significant(P<0.05).Expressions of TLR4(0.34±0.06)and Bax(0.27±0.05)and ratios of p-NF-κB p65/NF-κB p65(0.93±0.18)and cleaved caspase-3/caspase-3(0.67±0.09)were statistically higher in model group than those of TLR4(0.03±0.01)and Bax(0.03±0.01)and those of p-NF-κB p65/NF-κB p65(0.09±0.03)and cleaved caspase-3/caspase-3(0.15±0.03)in normal control group.The differences were statistically significant(P<0.05).Expression of Bcl-2 was statistically lower in model group than that in normal control group[(0.08±0.02)vs(0.37±0.06)].The difference was statistically significant(P<0.05).24 h UTP[(25.72±3.49),(13.35±1.60),(23.11±3.57)mg],BUN[(39.28±4.38),(34.17±3.62),(40.35±4.81)mmol/L]and Scr[(14.61±1.85),(9.05±1.12),(12.72±1.50)μmol/L]were statistically lower in medium/high-dose allicin and benazepril hydrochloride groups than 24 h UTP(45.07±6.19),BUN(46.45±5.22)and Scr(24.90±2.81)μmol/L in model group.TP[(46.39±5.02),(53.47±6.58),(45.63±5.17)g/L]and Alb[(23.38±2.91),(27.50±3.04),(24.08±2.86)g/L]were statistically higher than TP(37.25±4.09)g/L and Alb(18.62±2.03)g/L in model group.The differences were statistically significant(P<0.05).IL-1β[(349.18±44.06),(201.33±26.07),(317.92±38.64)ng/L],IL-6[(73.83±9.17),(50.90±5.25),(82.76±9.83)ng/L]and TNF-α[(228.14±25.60),(101.57±12.94),(193.82±23.74)ng/L]were significantly lower than IL-1β[(638.14±69.22),IL-6(115.08±13.61)and TNF-α(341.75±40.93)ng/L]in model group.IL-10 was significantly higher than that in model group[(49.26±6.35),(62.35±7.71),(55.60±6.15)vs(24.90±3.01)ng/L].The difference was statistically significant(P<0.05).Renal histopathological changes and apoptosis improved markedly and apoptotic index was significantly lower than that in model group[(63.90±8.74)%,(63.90±8.74)%,(63.90±8.74)%vs(63.90±8.74)%].The difference was statistically significant(P<0.05).Expressions of TLR4[(0.24±0.04),(0.09±0.02),(0.15±0.03)]and Bax[(0.14±0.02),(0.08±0.02),(0.16±0.03)]and ratios of p-NF-κB p65/NF-κB p65[(0.45±0.07),(0.21±0.04),(0.31±0.05)]and cleaved caspase-3/caspase-3[(0.47±0.07),(0.20±0.04),(0.38±0.06)]were significantly lower than those of TLR4(0.34±0.06)and Bax(0.27±0.05)and those of p-NF-κB p65/NF-κB p65(0.93±0.18)and cleaved caspase-3/caspase-3(0.67±0.09)in model group.Expression of Bcl-2 was significantly higher than that in model group[(0.19±0.04),(0.30±0.06),(0.28±0.05)vs(0.08±0.02)].The difference was statistically significant(P<0.05).Except for BUN,low/medium/high-dose allicin group demonstrated dose-dependent regulatory effects on other detection parameters(P<0.05).Except for IL-10/Bcl-2,regulatory effect of high-dose allicin group was better than that of benazepril hydrochloride group(P<0.05).Conclusion Allicin may reduce renal injury and protect renal function in MN rats through suppressing TLR4/NF-κB signaling pathway of modulating inflammatory responses and apoptosis.