益气活血化瘀汤对膜性肾病大鼠肾脏组织Nephrin mRNA和Podocin mRNA表达的影响
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摘要
目的观察益气活血化瘀汤对膜性肾病大鼠肾脏组织Nephrin mRNA、Podocin mRNA表达的影响。方法 SD大鼠随机分为正常对照组和造模组,其中造模组采用抗近端小管刷状缘抗原(抗Fx1A)血清一次性尾静脉注射诱导被动型海曼(Heymann)肾炎模型,造模成功后将造模组随机分为模型组、洛丁新组及益气活血化瘀汤低、中、高剂量组,每组6只。每组按相应剂量灌胃4周,每周收取大鼠24 h尿量,检测24 h尿蛋白定量(UTP)。4周末处死大鼠,心脏取血,检测血肌酐(Scr)、尿素氮(BUN)、血清总蛋白(TP)、血清白蛋白(ALB)、总胆固醇(TC)、高密度脂蛋白(HDL-c)、三酰甘油(TG)水平,取肾脏行Masson、PASM染色观测肾脏组织病理改变,免疫荧光法观察Ig G、C3改变,实时荧光PCR检测Nephrin mRNA、Podocin mRNA的表达。结果与正常对照组比较,其余5组UTP均升高(P<0.05),Nephrin mRNA、Podocin mRNA表达量均减少(P<0.01);与模型组比较,给药组UTP均减少(P<0.05),Nephrin和Podocin mRNA表达量均增加(P<0.01),TP、ALB有所升高但差异无统计学意义(P>0.05),Scr、BUN、TC、HDL-c、TG差异无统计学意义(P>0.05);与洛丁新组比较,益气活血化瘀汤低、中、高剂量组UTP有所减少,但差异无统计学意义(P>0.05),各生化指标差异无统计学意义(P>0.05),益气活血化瘀汤低、中、高剂量组肾脏组织Nephrin mRNA、Podocin mRNA表达均有所增加,但差异无统计学意义(P>0.05);益气活血化瘀汤各组之间各指标差异无统计学意义(P>0.05)。结论益气活血化瘀汤可明显减少膜性肾病大鼠的尿蛋白程度,减少肾脏病理损伤,上调肾脏组织Nephrin mRNA、Podocin mRNA的表达,延缓膜性肾病的进展。
Objective To observe the effects of Yiqi Huoxue Huayu Decoction on the expressions of Nephrin mRNA and Podocin mRNA in the renal tissue of rats with membranous nephropathy. Methods SD rats were randomly divided into the normal control group and modeling group. The passive Heymann nephritis model was established by one-time tail intravenous injection of anti Fx1 A serum. After successful molding,the modeling group was randomly divided into the model group,lotensin group,Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose. Each group was treated with the corresponding dosage by intragastric administration for 4 weeks,and the 24 h urine volume of rats was collected every week to detect the 24 h urinary protein quantification( UTP). The rats were sacrificed at the end of four weeks,the blood were collected from the heart to detect the serum creatinine( Scr),blood urea nitrogen( BUN),serum total protein( TP),serum albumin( ALB),total cholesterol( TC),high-density lipoprotein( HDL-c) and triglyceride( TG),the kidney tissue was collected to observe the pathological changes after Masson and PASM staining. The Ig G and C3 changes were observed by immunofluorescence,and the expressions of Nephrin mRNA and Podocin mRNA were determined by real-time fluorescence PCR. Results Compared with the normal control group,the UTP level was increased( P<0.05) and the mRNA expressions of Nephrin and Podocin were decreased( P<0.01) in the other five groups. Compared with the model group,the UTP level was reduced( P<0.05) and the mRNA expressions of Nephrin and Podocin were increased( P<0.01) in the drug-treatment groups. The levels of TP and ALB were increased in the drug-treatment groups but with no statistically significant differences( P>0.05),and there were no statistically significant differences on the levels of Scr,BUN,TC,HDL-c and TG between the model group and drug-treatment groups( P>0.05). Compared with the lotensin group,the UTP level was reduced in the Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose,but with no statistically significant differences( P>0.05). There were no statistically significant differences on the biochemical indexes between lotensin group and Yiqi Huoxue Huayu Decoction groups( P>0.05). Compared with the lotensin group,the expressions of Nephrin mRNA and Podocin mRNA in the renal tissue were increased in the Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose,but with no statistically significant differences( P>0.05). There were no statistically significant differences on all the indexes among the Yiqi Huoxue Huayu Decoction groups with different doses( P>0.05).Conclusion Yiqi Huoxue Huayu Decoction can significantly reduce the urinary protein level in rats with membranous nephropathy, decrease the renal pathological damage,up-regulate the expressions of Nephrin mRNA and Podocin mRNA in renal tissue and delay the progress of membranous nephropathy.
Objective To observe the effects of Yiqi Huoxue Huayu Decoction on the expressions of Nephrin mRNA and Podocin mRNA in the renal tissue of rats with membranous nephropathy. Methods SD rats were randomly divided into the normal control group and modeling group. The passive Heymann nephritis model was established by one-time tail intravenous injection of anti Fx1 A serum. After successful molding,the modeling group was randomly divided into the model group,lotensin group,Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose. Each group was treated with the corresponding dosage by intragastric administration for 4 weeks,and the 24 h urine volume of rats was collected every week to detect the 24 h urinary protein quantification( UTP). The rats were sacrificed at the end of four weeks,the blood were collected from the heart to detect the serum creatinine( Scr),blood urea nitrogen( BUN),serum total protein( TP),serum albumin( ALB),total cholesterol( TC),high-density lipoprotein( HDL-c) and triglyceride( TG),the kidney tissue was collected to observe the pathological changes after Masson and PASM staining. The Ig G and C3 changes were observed by immunofluorescence,and the expressions of Nephrin mRNA and Podocin mRNA were determined by real-time fluorescence PCR. Results Compared with the normal control group,the UTP level was increased( P<0.05) and the mRNA expressions of Nephrin and Podocin were decreased( P<0.01) in the other five groups. Compared with the model group,the UTP level was reduced( P<0.05) and the mRNA expressions of Nephrin and Podocin were increased( P<0.01) in the drug-treatment groups. The levels of TP and ALB were increased in the drug-treatment groups but with no statistically significant differences( P>0.05),and there were no statistically significant differences on the levels of Scr,BUN,TC,HDL-c and TG between the model group and drug-treatment groups( P>0.05). Compared with the lotensin group,the UTP level was reduced in the Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose,but with no statistically significant differences( P>0.05). There were no statistically significant differences on the biochemical indexes between lotensin group and Yiqi Huoxue Huayu Decoction groups( P>0.05). Compared with the lotensin group,the expressions of Nephrin mRNA and Podocin mRNA in the renal tissue were increased in the Yiqi Huoxue Huayu Decoction groups with low-,middle-and high-dose,but with no statistically significant differences( P>0.05). There were no statistically significant differences on all the indexes among the Yiqi Huoxue Huayu Decoction groups with different doses( P>0.05).Conclusion Yiqi Huoxue Huayu Decoction can significantly reduce the urinary protein level in rats with membranous nephropathy, decrease the renal pathological damage,up-regulate the expressions of Nephrin mRNA and Podocin mRNA in renal tissue and delay the progress of membranous nephropathy.
引文
[1]EL-HUSSEINI A,SAXON D,JENNINGS S,et al.Idiopathic membranous nephropathy:diagnostic and therapeutic challenges[J].Am J Nephrol,2016,43(2):65-70.
[2]周飞,李春国,商明,等.肾脏疾病患者262例肾活检病理类型及流行病学特点[J].中国社区医师,2018,34(2):127-128.
[3]PAN X,XU J,REN H,et a1.Changing spectrum of biopsy-proven primary glomerular diseases over the past 15 years:a single-center study in China[J].Contrib Nephrol,2013(181):22-30.
[4]OBANA M,NAKANISHI K,SAKO M,et al.Segmental membranous glomerulonephritis in children:comparison with global membranous glomerulonephritis[J].Clin J Am Soc Nephrol,2006,1(4):723-729.
[5]WANG P R,KITAMURA H,SHIMIZU A,et al.Glomerular damage in experimental proliferative glomerulonephritis under glomerular capillary hypertension[J].Kidney Blood Press Res,2015,40(2):188-199.
[6]丁磊,汪宏.特发性膜性肾病的诊治进展[J].家庭医药,2017(10):228-229.
[7]刘学永,何聪芹,王聪舟,等.膜性肾病的中医诊治进展[J].临床医药文献杂志,2017,4(76):15039-15041.
[8]张晓丹,邓跃毅.中医医家辨治膜性肾病经验探析[J].现代中西医结合杂志,2017,26(1):112-114.
[9]佘阿敏,盛笑梅.膜性肾病的中医药研究进展[J].长春中医药大学学报,2016,32(1):205-208.
[10]武亚丹,程锦国.程锦国运用中满分消丸治疗膜性肾病经验介绍[J].新中医,2018,50(1):188-190.
[11]CUNNINGHAM P N,HACK B K,REN G,et al.Glomerular complement regulation is overwhelmed in passive Heymann nephritis[J].Kidney Int,2001,60(3):900-909.
[12]TSUKADA Y,ONO K,MAEZAWA A,et al.A major pathogenic antigen of Heymann nephritis is present exclusively in the renal proximal tubule brush border--studies with a monoclonal antibody against pronasedigested tubular antigen[J].Clin Exp Immunol,1994,96(2):303-310.
[13]KERJASCHKI D,FARQUHAR M G.The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border[J].Proc Natl Acad Sci U S A,1982,79(18):5557-5561.
[14]张英,白香妮.肾功能相关检测指标在慢性肾病临床筛查中的作用分析[J].医学临床研究,2017,34(11):2243-2244.
[15]TIAN X,KIM J J,MONKLEY S M,et al.Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance[J].J Clin Invest,2014,124(3):1098-1113.
[16]FUKUSUMI Y,MIYAUCHI N,HASHIMOTO T,et al.Therapeutic target for nephrotic syndrome:Identificationof novel slit diaphragm associated molecules[J].World J Nephrol,2014,3(3):77-84.
[17]程鹏,曹灵.足细胞与膜性肾病[J].实用医学杂志,2010,26(6):1063-1064.
[18]GDEL M,OSTENDORF B N,BAUMER J,et al.A novel domain regulating degradation of the glomerular slit diaphragm protein podocin in cell culture systems[J].PLo S One,2013,8(2):e57078.
[19]NAKATSUE T,KOIKE H,HAN G D,et al.Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy[J].Kidney Int,2005,67(6):2239-2253.
[20]达展云,范亚平,沈良兰,等.Nephrin和Podocin在3种膜性肾病表达中的差异及其意义[J].中国现代医学杂志,2009,19(15):2381-2385.
[2]周飞,李春国,商明,等.肾脏疾病患者262例肾活检病理类型及流行病学特点[J].中国社区医师,2018,34(2):127-128.
[3]PAN X,XU J,REN H,et a1.Changing spectrum of biopsy-proven primary glomerular diseases over the past 15 years:a single-center study in China[J].Contrib Nephrol,2013(181):22-30.
[4]OBANA M,NAKANISHI K,SAKO M,et al.Segmental membranous glomerulonephritis in children:comparison with global membranous glomerulonephritis[J].Clin J Am Soc Nephrol,2006,1(4):723-729.
[5]WANG P R,KITAMURA H,SHIMIZU A,et al.Glomerular damage in experimental proliferative glomerulonephritis under glomerular capillary hypertension[J].Kidney Blood Press Res,2015,40(2):188-199.
[6]丁磊,汪宏.特发性膜性肾病的诊治进展[J].家庭医药,2017(10):228-229.
[7]刘学永,何聪芹,王聪舟,等.膜性肾病的中医诊治进展[J].临床医药文献杂志,2017,4(76):15039-15041.
[8]张晓丹,邓跃毅.中医医家辨治膜性肾病经验探析[J].现代中西医结合杂志,2017,26(1):112-114.
[9]佘阿敏,盛笑梅.膜性肾病的中医药研究进展[J].长春中医药大学学报,2016,32(1):205-208.
[10]武亚丹,程锦国.程锦国运用中满分消丸治疗膜性肾病经验介绍[J].新中医,2018,50(1):188-190.
[11]CUNNINGHAM P N,HACK B K,REN G,et al.Glomerular complement regulation is overwhelmed in passive Heymann nephritis[J].Kidney Int,2001,60(3):900-909.
[12]TSUKADA Y,ONO K,MAEZAWA A,et al.A major pathogenic antigen of Heymann nephritis is present exclusively in the renal proximal tubule brush border--studies with a monoclonal antibody against pronasedigested tubular antigen[J].Clin Exp Immunol,1994,96(2):303-310.
[13]KERJASCHKI D,FARQUHAR M G.The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border[J].Proc Natl Acad Sci U S A,1982,79(18):5557-5561.
[14]张英,白香妮.肾功能相关检测指标在慢性肾病临床筛查中的作用分析[J].医学临床研究,2017,34(11):2243-2244.
[15]TIAN X,KIM J J,MONKLEY S M,et al.Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance[J].J Clin Invest,2014,124(3):1098-1113.
[16]FUKUSUMI Y,MIYAUCHI N,HASHIMOTO T,et al.Therapeutic target for nephrotic syndrome:Identificationof novel slit diaphragm associated molecules[J].World J Nephrol,2014,3(3):77-84.
[17]程鹏,曹灵.足细胞与膜性肾病[J].实用医学杂志,2010,26(6):1063-1064.
[18]GDEL M,OSTENDORF B N,BAUMER J,et al.A novel domain regulating degradation of the glomerular slit diaphragm protein podocin in cell culture systems[J].PLo S One,2013,8(2):e57078.
[19]NAKATSUE T,KOIKE H,HAN G D,et al.Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy[J].Kidney Int,2005,67(6):2239-2253.
[20]达展云,范亚平,沈良兰,等.Nephrin和Podocin在3种膜性肾病表达中的差异及其意义[J].中国现代医学杂志,2009,19(15):2381-2385.