升降散加减方治疗IgAN大鼠作用机制探讨

Discussion on the mechanism of Shengjiangsan modified formula in the treatment of IgAN rats

目的通过探讨疏通三焦、清热利湿理论指导下的升降散加减方对IgA肾病(immunoglobulin a nephropathy,IgAN)大鼠血清转化生长因子β1(transforming growth factorβ1,TGF-β1)、白细胞介素-6(interleukin-6,IL-6)、半乳糖缺乏-IgA(galactose deficient IgA,Gd-IgA)的影响以了解其可能作用机制。方法将48雄性健康清洁级SD大鼠随机分为正常组12只、造模组36只,将大鼠适应性饲养1周后采用联合牛血清白蛋白(bovine serum albumin,BSA)灌胃+尾静脉注射脂多糖(lipopolysaccharide,LPS)+皮下注射四氯化碳(CCl 4)、蓖麻油的免疫复合的方法建立实验性IgAN大鼠模型,方案如下:口服免疫原BSA剂量较常用剂量增加1倍,剂量600 mg/kg隔天灌胃,持续6周;CCl 4注射方式由既往的腹腔注射改为皮下注射,皮下注射蓖麻油0.5 mL+0.10 mL的CCl 4,每周1次,持续9周,并联合运用LPS,分别于第6周和8周尾静脉注射0.05 mg的LPS,模型制作成功后,将造模组大鼠随机分成中药组、西药组和模型组各12只。中药组每日灌服浓缩至含生药1 g/mL中药水提液,西药组每日灌服1.8 mg/kg盐酸贝那普利,模型组、正常组每日灌服等量蒸馏水,每日1次,共8周。于实验第1周、第9周、第15周、第19周检测24小时尿蛋白定量(24 hours urinary total protein,24 h-UTP),第19周股动脉取血化验血清肌酐(serum creatinine,Scr)、血尿素氮(blood urea nitrogen,BUN)、血清白蛋白(albumin,ALB),并用ELISA法检测血清中TGF-β1、IL-6、Gd-IgA的含量,同时留取肾组织,行光镜、电镜及免疫荧光观察。结果(1)中药组、西药组大鼠精神状态较模型组大鼠精神状态好;(2)西药组与中药组24 h-UTP较模型组明显降低(P<0.05);(3)各组间大鼠血清中Scr、BUN、ALB相比,无明显差异(P>0.05);(4)西药组与中药组较模型组光镜、电镜及免疫荧光表达强度显著下降;(5)西药组与中药组血清TGF-β1、IL-6、Gd-IgA的含量较模型组明显减少(P<0.05)。结论疏通三焦、清热利湿理论指导下的升降散加减方对IgAN大鼠有治疗作用,其作用机制可能与降低血清中TGF-β1、IL-6、Gd-IgA的含量有关。

Objective To investigate the possible mechanism of Shengjiangsan modified formula,under the guidance of the theory of dredging sanjiao and clearing heat and promoting diuresis,on transforming growth factor-β1(TNF-β1),interleukin-6(IL-6) and galactokinase deficiency-IgA(Gd-IgA) in serum of immunoglobulin a nephropathy(IgAN) rats.Methods 48 clean grade male SD rats weighing 150 to 180 g were randomly divided into normal group(12 rats) and modeling group(36 rats).After one week of adaptive feeding,the experimental IgA nephropathy rat model was established by gavage combined with bovine serum albumin(BSA),tail vein injection of lipopolysaccharide(LPS),subcutaneous injection of carbon tetrachloride(CCl4) and castor oil.The protocol is as follows:the dose of oral immunogen BSA is doubled compared with the usual dose,600 mg/kg is given by gavage every other day for 6 weeks;The injection method of CCl4 was changed from the previous intraperitoneal injection to subcutaneous injection,with the subcutaneous injection of 0.5 mL castor oil and 0.10 mL CCl4 once a week for 9 weeks,while 0.05 mg LPS was injected into the tail vein at week 6 and 8,respectively.After successfully modeling,the rats in modeling group were randomly divided into Chinese medicine group,western medicine group and model group,12 rats in each group.Chinese medicine group was administrated daily with Chinese medicine concentrated to 1 g/mL water extract,western medicine group was administrated daily with 1.8 mg/kg Benazepril hydrochloride,model group and the normal group both were administrated with the same amount of distilled water once a day for 8 weeks.In this experiment,The 24 hours urinary total protein(24 h-UTP) was detected at week 1,9,15,and 19.The serum creatinine(Scr) and blood urea nitrogen(BUN) and albumin(ALB) of serum in femoral artery blood were detected at week 19.The contents of TGF-β1,IL-6 and GD-IgA in serum were detected by ELISA,and kidney tissues were retained for observation by light microscopy,electron microscopy and immunofluorescence.Results(1) The mental status of rats in Chinese medicine group and western medicine group was better than in model group.(2) Compared with model group,the 24 h-UTP in western medicine group and Chinese medicine group decreased significantly(P<0.05).(3) There was no significant difference among Scr,BUN and ALB in different groups(P>0.05).(4) Compared with model group,the expression intensity of light microscope,electron microscopy and immunofluorescence in western medicine group and Chinese medicine group decreased significantly(P<0.05).(5) The contents of TGF-β1,IL-6 and Gd-IgA in western medicine group and Chinese medicine group decreased significantly than those in the model group(P<0.05).Conclusion Shengjiangsan modified formula,under the guidance of the theory of dredging sanjiao and clearing heat and promoting diuresis,has a therapeutic effect on IgAN rats,and its mechanism of action may be related to the reduction of the contents of TGF-β1,IL-6 and Gd-IgA in serum.