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免疫球蛋白A肾病(IgAN)是世界上最常见的肾小球肾炎形式之一,也是终末期肾病的主要原因[1]。25%~30%的IgAN病人需要在20~25年内进行肾脏替代治疗,另外20%~40%的病人在发病后20年内会发生终末期肾病[2-3]。其以血尿和蛋白尿为主要临床表现,病理特征包括肾小球系膜细胞增生并伴有以IgA为主的免疫复合物的沉积[4]。肾移植和透析的成本已成为病人和社会的巨大财务负担[5]。因此,开发用于治疗IgAN的有效药物是十分重要的。雷公藤甲素(triptolide, TR)是中药雷公藤的主要活性成分之一,具有抗炎、抗凋亡及免疫调节等作用[6],对大鼠肾缺血再灌注损伤、糖尿病肾损伤及慢性肾衰均具有保护作用[7-9]。并且其对于IgAN及系统性红斑狼疮等自身免疫性疾病的治疗效果也较为突出,但是其具体治疗机制目前尚不明确。NLRP3是近几年发现的重要的炎症复合体之一,其能触发Caspase-1、白细胞介素(IL)-1β和IL-18的成熟以执行炎症反应[10-11]。并且有研究[12-13]已经证实,IL-1β和IL-18是NLRP3炎症小体激活的两个关键标志物,在IgAN病人血清和尿液中表达升高,提示NLRP3炎症小体可能通过调节炎症在IgAN中发挥重要作用。本研究通过建立IgAN大鼠模型,观察雷公藤甲素对IgAN大鼠的治疗作用,并且从NLRP3炎症小体方面探讨其可能的作用机制。
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与对照组相比,模型组血清中Scr、BUN及尿液中24 h TUP含量明显升高(P < 0.05~P < 0.01);经不同剂量雷公藤甲素治疗后,血清中Scr、BUN及尿液中24 h TUP含量均明显降低(P < 0.05~P < 0.01);但各组大鼠血清中TP、ALT及AST水平无明显变化(P>0.05)(见表 1)。
指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 BUN/(mmol/L) 3.91±0.53 19.87±2.99** 12.37±2.65△ 11.52±2.01△△ 20.21 < 0.01 0.003 Scr/(μmol/L) 41.02±3.58 66.83±11.02* 45.76±5.68△ 41.59±10.03△ 6.48 < 0.05 3.622 TP/(g/L) 59.03±8.22 60.52±10.57 59.63±7.86 63.19±10.28 0.12 >0.05 0.004 ALT/(U/L) 39.98±6.35 43.17±4.97 45.91±6.04 42.81±3.72 0.61 >0.05 0.005 AST/(U/L) 88.04±10.85 91.48±10.35 87.59±9.28 85.64±7.96 0.19 >0.05 0.008 24 h UTP/(mg/24 h) 1.98±0.14 46.92±9.95** 29.91±5.69△△ 24.68±3.73△△ 28.40 < 0.01 8.368 与对照组比较* P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 表 1 生化指标及24 h UTP含量变化的比较(x±s;ni=10)
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与对照组相比,模型组IgAN大鼠血清中炎症因子TNF-α、IL-17A、IFN-γ和IL-4水平明显升高(P < 0.05~P < 0.01);雷公藤甲素低、高剂量组均能不同程度的降低大鼠血清中TNF-α、IL-17A、IFN-γ和IL-4水平(P < 0.05~P < 0.01)(见表 2)。对照组少见IgA免疫球蛋白的沉积,而模型组肾小球IgA免疫球蛋白沉积明显(P < 0.01),雷公藤甲素治疗组IgA免疫球蛋白的沉积情况明显减轻(P < 0.01)(见图 1)。
指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 TNF-α/(ng/mL) 101.76±20.01 291.33±49.60** 199.62±25.54△ 168.68±21.05△△ 19.05 < 0.01 2.523 IL-17A/(ng/mL) 7.58±1.45 28.04±4.50* 16.12±4.58△ 13.16±3.25△ 14.58 < 0.05 0.062 IFN-γ/(ng/mL) 8.43±3.00 19.26±12.98** 12.51±3.20△ 11.77±4.65△ 4.75 < 0.05 4.732 IL-4/(ng/mL) 3.63±0.61 11.20±1.31** 6.33±0.64△△ 5.83±1.12△△ 32.74 < 0.01 5.019 IgA相对荧光强度 100±21.54 280.73±42.39** 171.69±41.02△△ 165.07±32.52△△ 13.64 < 0.01 0.000 与对照组比较* P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 表 2 肾组织中炎症因子水平的比较(x±s;ni=10)
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与对照组相比,模型组肾组织中IL-1β、Caspase-1、IL-8及NLRP3蛋白的表达明显升高(P < 0.05~P < 0.01);雷公藤甲素治疗后,肾组织中IL-1β、Caspase-1、IL-18及NLRP3蛋白的表达明显降低(P < 0.05~P < 0.01)(见图 2、表 3)。
蛋白指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 IL-1β 1.00±0.36 1.97±0.50* 1.32±0.21△ 1.36±0.15△△ 4.47 < 0.05 0.003 Caspase-1 1.00±0.19 2.91±0.33** 1.99±0.38△△ 1.83±1.23△△ 22.35 < 0.01 0.008 IL-18 1.00±0.17 2.32±0.12** 1.71±0.13△△ 1.76±1.13△△ 41.95 < 0.01 0.187 NLRP3 1.00±0.21 2.53±0.55** 1.90±0.29△△ 1.76±0.28△△ 9.24 < 0.01 1.732 与对照组比较*P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 表 3 肾组织中IL-1β、Caspase-1、IL-18及NLRP3蛋白的相对表达(x±s;ni=10)
雷公藤甲素对IgA肾病大鼠的肾保护作用及对NLRP3炎症小体的影响
Effect of Triptolide on renal protection and NLRP3 inflammatome in IgA nephropathy rats
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摘要:
目的探讨雷公藤甲素对IgA肾病(IgAN)大鼠的肾保护作用及其与NLRP3炎症小体的关系。 方法40只SPF级SD雄性大鼠随机分为对照组、模型组和雷公藤甲素低、高剂量组。采用口服牛γ-球蛋白(BGG)8周及尾静脉注射BGG方法建立IgAN大鼠模型。于造模完成后灌胃给予低、高剂量雷公藤甲素治疗8周。观察大鼠血清中肌酐(Scr)、尿素氮(BUN)、总蛋白(TP)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)及24 h尿蛋白(24 h TUP)水平;血清中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-17A、γ干扰素(IFN-γ)及IL-4水平;肾组织中IgA沉积情况;肾脏中IL-1β、Caspase-1、IL-18及NLRP3的表达情况。 结果与模型组相比,雷公藤甲素组血清Scr、BUN及尿液中24 h TUP含量明显降低(P < 0.05~P < 0.01);雷公藤甲素能降低血清中TNF-α、IL-17A、IFN-γ和L-4水平(P < 0.05~P < 0.01),减轻IgA的沉积(P < 0.01),抑制IL-1β、Caspase-1、IL-18及NLRP3的表达(P < 0.05~P < 0.01)。 结论雷公藤甲素对IgA肾病大鼠肾脏具有保护作用,其作用机制可能是通过抑制NLRP3炎症小体的激活,进而抑制炎症反应。 Abstract:ObjectiveTo investigate the protective effects of triptolide on kidney of IgA nephropathy(IgAN)rats and its relationship with NLRP3 inflammasome. MethodsForty SPF SD male rats were randomly divided into the control group, model group, low-dose triptolide group and high-dose triptolide groups.The IgAN rat model was established by oral bovine γ-globulin(BGG)for 8 weeks and intravenous injection of BGG, and treated with intragastric administration combined with low- and high-dose triptolide for 8 weeks.The levels of creatinine(Scr), urea nitrogen(BUN), total protein(TP), albumin, alanine aminotransferase(ALT), aspartate aminotransferase(AST), 24 h urinary protein(24 h TUP), TNF-α, IL-17A, IFN-γ and IL-4 were detected in four groups.The deposition of IgA and expression levels of IL-1β, Caspase-1, IL-18 and NLRP3 in renal tissue were observed. ResultsCompared with the model group, the levels of serum Scr and BUN, and 24 h TUP significantly decreased in triptolide group(P < 0.05 to P < 0.01).The triptolide could decrease the levels of serum TNF-α, IL-17A, IFN-γ, and IL-4(P < 0.05 to P < 0.01), reduce the deposition of IgA(P < 0.01), and inhibit the expression of IL-1β, Caspase-1, IL-18 and NLRP3(P < 0.05 to P < 0.01). ConclusionsThe protective effects of triptolide on IgA nephropathy may be related to the inhibition of NLRP3 inflammasome for suppressing the renal inflammatory response. -
Key words:
- triptolide /
- IgA nephropathy /
- inflammation /
- NLRP3 inflammasome
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表 1 生化指标及24 h UTP含量变化的比较(x±s;ni=10)
指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 BUN/(mmol/L) 3.91±0.53 19.87±2.99** 12.37±2.65△ 11.52±2.01△△ 20.21 < 0.01 0.003 Scr/(μmol/L) 41.02±3.58 66.83±11.02* 45.76±5.68△ 41.59±10.03△ 6.48 < 0.05 3.622 TP/(g/L) 59.03±8.22 60.52±10.57 59.63±7.86 63.19±10.28 0.12 >0.05 0.004 ALT/(U/L) 39.98±6.35 43.17±4.97 45.91±6.04 42.81±3.72 0.61 >0.05 0.005 AST/(U/L) 88.04±10.85 91.48±10.35 87.59±9.28 85.64±7.96 0.19 >0.05 0.008 24 h UTP/(mg/24 h) 1.98±0.14 46.92±9.95** 29.91±5.69△△ 24.68±3.73△△ 28.40 < 0.01 8.368 与对照组比较* P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 表 2 肾组织中炎症因子水平的比较(x±s;ni=10)
指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 TNF-α/(ng/mL) 101.76±20.01 291.33±49.60** 199.62±25.54△ 168.68±21.05△△ 19.05 < 0.01 2.523 IL-17A/(ng/mL) 7.58±1.45 28.04±4.50* 16.12±4.58△ 13.16±3.25△ 14.58 < 0.05 0.062 IFN-γ/(ng/mL) 8.43±3.00 19.26±12.98** 12.51±3.20△ 11.77±4.65△ 4.75 < 0.05 4.732 IL-4/(ng/mL) 3.63±0.61 11.20±1.31** 6.33±0.64△△ 5.83±1.12△△ 32.74 < 0.01 5.019 IgA相对荧光强度 100±21.54 280.73±42.39** 171.69±41.02△△ 165.07±32.52△△ 13.64 < 0.01 0.000 与对照组比较* P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 表 3 肾组织中IL-1β、Caspase-1、IL-18及NLRP3蛋白的相对表达(x±s;ni=10)
蛋白指标 对照组 模型组 低剂量组 高剂量组 F P MS组内 IL-1β 1.00±0.36 1.97±0.50* 1.32±0.21△ 1.36±0.15△△ 4.47 < 0.05 0.003 Caspase-1 1.00±0.19 2.91±0.33** 1.99±0.38△△ 1.83±1.23△△ 22.35 < 0.01 0.008 IL-18 1.00±0.17 2.32±0.12** 1.71±0.13△△ 1.76±1.13△△ 41.95 < 0.01 0.187 NLRP3 1.00±0.21 2.53±0.55** 1.90±0.29△△ 1.76±0.28△△ 9.24 < 0.01 1.732 与对照组比较*P < 0.05,**P < 0.01;与模型组比较△P < 0.05,△△P < 0.01 -
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