温肾健脾活血泄浊方对慢性肾脏病大鼠肾脏转化生长因子-β1-Smad2/3信号通路的作用

Effect of warming kidney and strengthening spleen and promoting blood circulation and eliminating turbidity decoction on TGF-β1-Smad2/3 signaling pathway in kidney tissue of rats with chronic kidney disease

目的探讨金洪元教授温肾健脾活血泄浊方对慢性肾脏病(chronic kidney disease,CKD)实验大鼠的肾脏组织内转化生长因子(transforming growth factor,TGF)-β1-Smad2/3信号通路的作用。方法将56只大鼠随机分为空白组13只,其余43只大鼠灌服腺嘌呤溶液制备CKD大鼠模型,经造模鉴定后,取10只空白组大鼠作为空白对照组,40只造模成功大鼠随机分为模型组、中药低、中、高剂量组,每组10只。中药低、中、高剂量组分别给予的灌服浓度为9.18 g/(kg·d)、18.36 g/(kg·d)、36.72 g/(kg·d),空白对照组及模型组大鼠均灌服等体积生理盐水,连续灌服4周。经干预后,观察大鼠一般情况;运用酶联免疫吸附法检测大鼠尿液24小时尿蛋白定量、肌酐、TGF-β1及血清TGF-β1、胱抑素-C及肌酐定量水平;苏木精—伊红染色、马松(Masson)染色观察大鼠肾脏病理改变;蛋白质免疫印迹法检测大鼠肾组织内TGF-β1、Smad2、Smad3、p-Smad2、p-Smad3、Smad7的蛋白表达水平。结果各组大鼠经温肾健脾活血泄浊方干预后,与模型组相比,中药低、中、高剂量组大鼠尿液中TGF-β1、肌酐、24小时尿蛋白定量水平显著下降(P<0.05);血清中TGF-β1、胱抑素-C及肌酐定量显著降低,且中药低、中、高剂量组大鼠血清TGF-β1、胱抑素-C及肌酐定量水平均随着中药剂量增加而降低,具有负相关性,差异有统计学意义(P<0.05)。与模型组比较,中药低、中、高剂量组大鼠肾组织中TGF-β1、Smad2、Smad3蛋白定量显著降低,且随着中药剂量增加而降低,但差异无统计学意义;Smad7随中药剂量增加而升高,且差异有统计学意义(P<0.05),具有正相关性。苏木精—伊红染色结果示:中药各剂量组大鼠肾小管病变程度较模型组显著降低,纤维化面积较模型组明显减少;中药低、中、高剂量组较模型组大鼠肾组织结构紊乱程度降低,肾间质炎性细胞浸润减少,纤维组织减少;中药中、高剂量组较中药低剂量组大鼠肾组织炎性细胞浸润稍显减少,肾组织结构稍显整齐。马松染色结果示:中药低、中、高剂量组较模型组大鼠肾脏间质纤维组织增生程度均降低,纤维化比例减少,但组间肾脏间质纤维组织增生程度差异不明显。结论温肾健脾活血泄浊方可有效逆转CKD大鼠肾组织损伤,并且可能是通过TGF-β1-Smad2/3通路下调TGF-β1的水平来发挥作用,进而改善CKD大鼠肾组织纤维化。

Objective To investigate the effect of Professor Jin Hongyuan’s warming kidney and strengthening spleen and promoting blood circulation and eliminating turbidity decoction on transforming growth factor in kidney tissue of rats with chronic kidney disease(CKD)and to discuss the role of TGF-β1-Smad2/3 signaling pathway.Methods 56 rats were randomly divided into the blank group with 13 rats,and the remaining 43 rats were given adenine solution to prepare CKD rat model.After modeling identification,10 rats in the blank group were selected as the blank control group,and 40 rats with successful modeling were randomly divided into the model group,low,medium and high dose groups of traditional Chinese medicine with 10 rats in each group.The concentrations of the low,medium and high dose groups were administrated by gavage in the dose of 9.18 g/(kg•d),18.36 g/(kg•d)and 36.72 g/(kg•d),respectively.The rats in the blank control group and the model group were given equal volume of normal saline for 4 weeks.After intervention,the general condition of rats was observed.The quantitative levels of urine protein,creatinine,TGF-β1 and serum TGF-β1,cystatin C and creatinine in 24 hours were detected by enzyme-linked immunosorbent assay.The pathological changes of kidney were observed by hematoxylin-eosin and Masson staining.The protein expression levels of TGF-β1,Smad2,Smad3,p-Smad2,p-Smad3 and Smad7 in rat renal tissue were detected by Western blot.Results Compared with the model group,the levels of TGF-β1,creatinine and 24-hour urinary protein in urine of rats in the low,medium and high dose groups of traditional Chinese medicine were significantly decreased(P<0.05).The quantitative levels of TGF-β1,cystatin C and creatinine in serum were significantly decreased,and the quantitative levels of TGF-β1,cystatin C and creatinine in serum of rats in the high,medium and low dose groups of traditional Chinese medicine were decreased with the increase of dose,with a negative correlation,and the difference was statistically significant(P<0.05).Compared with the model group,the protein quantity of TGF-β1,Smad2 and Smad3 in renal tissue of rats in the low,medium and high dose groups of traditional Chinese medicine was significantly decreased,and decreased with the increase of Chinese medicine dose,but the difference was not statistically significant.Smad7 increased with the increase of Chinese medicine dose,and the difference was statistically significant(P<0.05),with a positive correlation.The results of hematoxylin-eosin staining showed that the degree of renal tubule lesion and fibrosis area of rats in each dose group were significantly reduced than those in the model group.Compared with the model group,the renal tissue structure of rats in the low,medium and high dose groups of traditional Chinese medicine were decreased,the inflammatory cell infiltration of renal interstitial decreased and the fibrous tissue decreased.Compared with the low dose group of traditional Chinese medicine,the inflammatory cell infiltration of renal tissue was slightly reduced in the medium and high dose groups of traditional Chinese medicine,and the structure of renal tissue was slightly orderly.The results of masson staining showed that compared with the model group,the proliferation degree of renal interstitial fiber tissue was reduced in the three groups of low,medium and high doses groups of traditional Chinese medicine,and the proportion of fibrosis was reduced,but the difference of renal interstitial fiber tissue proliferation degree was not obvious among the groups.Conclusion Warming the kidney and strengthening the spleen and activating the blood to relieve turbidness can effectively reverse the renal tissue injury in rats with CKD,and it may play a role by down-regulating the level of TGF-β1 through the TGF-β1-SMad2/3 pathway,and then improve the renal tissue fibrosis in rats with CKD.