化瘀涤痰通路中药调控肾上腺髓质素抑制肾小管上皮细胞表型转化的研究
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摘要
慢性肾衰竭在我国近年来的发病率有显著增高的趋势，是严重影响人类健康的疾病之一。目前已有大量的临床及实验结果表明肾间质纤维化在慢性肾脏疾病进展中具有重要作用，故有效地抑制肾间质纤维化可减缓肾脏疾病的进展。肾间质纤维化是多种慢性肾脏疾病进展至慢性肾衰竭的共同病理改变，由多种因素诱导而成，细胞增殖及表型转化均参与这一过程并发挥着重要的作用。肾小管上皮细胞表型转化（tubularepithelial-myofibroblast transdifferentiation，TEMT）的概念由Strutz于1994年提出，并已被证实为肾间质纤维化的核心病理改变，其标志物为α-平滑肌肌动蛋白（α-SMA）。TEMT的发生机制较为复杂，与多种因素相关，单一途径阻断难以获得理想效果，故寻找具有多种生理效应、多途径抑制TEMT的药物对减缓其进展有重要作用。肾上腺髓质素（adrenomedullin，ADM）是1993年发现的一种生物活性多肽，已证实该物质分布于体内多个重要脏器及组织中，肾脏是ADM表达及分布较多的器官之一。ADM具有拮抗转化生长因子β1（Transforming growth factor-β1，TGF-β1)、血管紧张素II（AngiotensinⅡ，AngⅡ）、抗氧化、抗炎等多种生理作用，可以拮抗多种脏器纤维化。TEMT进程中，最为重要的发生机制是转化生长因子-β的诱导作用，故推论ADM可通过调控TGF-β、抑制TEMT从而减轻肾脏损伤，其具体作用机制有待进一步探索。ADM尚未用于临床，中药抑制肾间质纤维化的作用是否与其相关？前期实验研究已证实化瘀涤痰通络中药——肾络通能通过抑制TGF-β1拮抗TEMT的进展，是否通过调控ADM而发挥作用尚不明确。本实验研究采用ADM基因部分敲除小鼠（+/-），以单侧输尿管梗阻（UUO）方法复制肾间质纤维化模型，以醛固酮受体阻断剂依普利酮为对照药物，观察肾络通对野生及ADM基因敲除小鼠肾组织中α-SMA、TGF-β1、Ⅲ型胶原（collagen type Ⅲ，Col Ⅲ）及ADM表达的影响，探讨化瘀涤痰通络中药抑制TEMT拮抗肾间质纤维化的作用机制及其与ADM的关系，为临床提供治疗思路和实验依据。
第一部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠肾功能及肾脏组织形态学的影响
方法：野生及ADM基因敲除小鼠各20只均随机分为四组，即野生小鼠分为假手术组（WT-Sham group）、模型组（WT-UUO group）、依普利酮组（WT-UUOE group）及中药组（WT-UUOS group）四组；基因敲除小鼠同样分为假手术组（AMKO-Sham group）、模型组（AMKO-UUOgroup）、依普利酮组（AMKO-UUOE group）及中药组（AMKO-UUOSgroup）四组。除假手术组游离左侧输尿管但不结扎外，其余各组均行左侧输尿管结扎术。中药组给予化瘀涤痰通络中药肾络通27.6g/(kg.d)经口灌服；依普利酮组给予依普利酮混匀于饲料中按100mg/(kg.d)剂量食入。假手术组及模型组经口灌服与中药汤剂等剂量的生理盐水。观察一般情况，给药10天。于实验结束时，称重，断头取血，分离血清，分别测定血肌酐（SCr）、尿素氮（BUN）水平；摘取左侧肾脏并称重，计算各组肾重/体重比值，进行肾脏组织HE、Masson染色，观察组织病理学改变。
数据资料以均数±标准差（±s）表示，使用SPSS16.0for windows统计软件进行统计，组间比较采用单因素方差分析。显著性差异水平以0.05和0.01为标准。
结果:
1实验小鼠肾重/体重比值测定结果
野生及基因敲除小鼠的模型组、依普利酮组及中药组显著高于相应的假手术组（P均＜0.01），但各组之间差异无统计学意义（P均>0.05）。（见Table1-1）
2小鼠肾组织病理形态学检测结果
HE染色显示，野生及基因敲除小鼠中，假手术组未见明显异常。模型组可见肾小管扩张，肾小管上皮细胞水肿，部分坏死、脱落，炎性细胞在肾间质区广泛浸润。Masson染色显示，野生及基因敲除小鼠中，假手术组肾脏结构未见明显异常，小管基底膜完整，肾间质可见少量胶原成分。模型组肾组织间质胶原成分沉积显著增多，并呈条带状或灶状分布，且基因敲除小鼠重于野生小鼠。依普利酮组及中药组间质胶原成分较模型组明显减少，其中基因敲除小鼠较野生小鼠损伤为重。
3肾功能检测结果
Scr及BUN测定结果显示，野生及基因敲除中各组小鼠的Scr及BUN值虽有差异，但无统计学意义（P均>0.05）。
第二部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠α-SMA的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏α-SMAmRNA及蛋白的表达。统计学方法同第一部分。
结果：
1肾组织α-SMA mRAN检测结果采用RT-PCR测定α-SMA mRNA在肾组织中的表达，结果显示，野生及基因敲除中的假手术组小鼠呈弱表达，模型组表达较相应的假手术组显著增强（P＜0.01；P＜0.01），且基因敲除模型组显著高于野生小鼠模型组（P＜0.05）。依普利酮组及中药组表达下调（P＜0.01；P＜0.01）。
2肾组织α-SMA蛋白检测结果Western Blot结果显示，野生及基因敲除小鼠中的假手术组肾组织α-SMA蛋白表达较弱，模型组表达明显增强，显著高于相应的假手术组（P＜0.01；P＜0.01），且基因敲除模型组显著高于野生小鼠模型组（P＜0.05）。依普利酮组及中药组表达明显低于相应的模型组（P＜0.01；P＜0.01），中药组与依普利酮组无明显差异（P均>0.05）。
3肾组织α-SMA免疫组化检测光镜下野生及基因敲除小鼠的假手术组仅在血管平滑肌细胞表达，肾小球、肾小管及间质未见表达；模型组中α-SMA表达明显增多，多见于皮髓交界及皮质区的小管间质部分。基因敲除小鼠模型组表达尤其明显。依普利酮组及中药组α-SMA表达较模型组明显减少。但基因敲除小鼠表达较野生小鼠更为明显。第三部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠TGF-β1、Col III的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏TGF-β1、Col Ⅲ mRNA及蛋白的表达，并用天狼猩红染色法测定肾脏胶原组织表达。统计学方法同第一部分。
结果：
1肾组织TGF-β1、Col Ⅲ mRNA检测
1.1TGF-β1mRNA RT-PCR测定结果显示，野生及基因敲除中的假手术组小鼠呈弱表达，模型组显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组表达更为明显，显著高于野生小鼠模型组（P＜0.05）；依普利酮组及中药组明显低于模型组（P＜0.05；P＜0.05）。
1.2Col Ⅲ mRNART-PCR测定结果显示，野生及基因敲除中小鼠的假手术组呈极弱表达，模型组显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组显著高于野生小鼠模型组（P＜0.01）；依普利酮组及中药组明显低于相应模型组小鼠（P＜0.01; P＜0.01）。野生及基因敲除小鼠之间比较，基因敲除小鼠中药组及依普利酮组表达均明显高于野生型同组小鼠（P＜0.05；P＜0.05）。
2肾组织中TGF-β1及Col Ⅲ蛋白检测
2.1TGF-β1Western Blot测定野生及基因敲除小鼠假手术组TGF-β1蛋白少量表达，模型组表达显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组表达显著高于野生小鼠模型组（P＜0.01）。依普利酮组及中药组表达明显低于相应模型组（P＜0.01；P＜0.01）。
2.2Col Ⅲ Western Blot测定结果显示，野生及基因敲除小鼠假手术组少量表达，模型组显著高于相应的假手术组（P＜0.01；P＜0.01），基因敲除模型组显著高于野生小鼠模型组（P＜0.01）。野生及基因敲除小鼠依普利酮组及中药组明显低于相应模型组（P＜0.05；P＜0.01）。野生及基因敲除两型之间比较，基因敲除小鼠中药组与依普利酮组均明显高于野生型的同组小鼠（P＜0.05；P＜0.05）。
3肾组织TGF-β1及Col Ⅲ免疫组化检测
3.1TGF-β1免疫组化检测野生及基因敲除小鼠的假手术组肾脏TGF-β1呈弱表达，可见于肾小球及肾小管上皮细胞；模型组TGF-β1表达明显增强，其中基因敲除小鼠尤其显著；依普利酮组及中药组表达较模型组明显减少，但基因敲除小鼠较野生小鼠表达明显。
3.2Col Ⅲ免疫组化检测野生及基因敲除小鼠的假手术组肾脏可见少量表达，可见于肾小球及肾小管间质；模型组Col Ⅲ表达明显增多，以间质最为明显，其中基因敲除小鼠甚于野生小鼠；依普利酮组及中药组表达较相应模型组明显减少，但基因敲除小鼠较野生小鼠表达明显。
4肾脏胶原组织天狼猩红染色测定假手术组肾间质可见胶原组织表达于肾小球、肾小管及间质，野生及基因敲除小鼠间无明显差异；模型组表达明显增多，主要见于肾小管间质，皮质及髓质表达均明显增强，ADM基因敲除小鼠表达尤为显著；依普利酮组及中药组表达较相应模型组明显减弱，但两组中ADM基因敲除小鼠较野生小鼠表达明显。
第四部分化瘀涤痰通络中药对肾间质纤维化小鼠ADM表达的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏ADM mRNA及蛋白的表达。统计学方法同第一部分。
结果：
1肾组织ADM mRNA检测野生及基因敲除小鼠中假手术组均有表达，但基因敲除小鼠的表达明显低于野生小鼠（P＜0.01）。模型组显著低于相应假手术组（P＜0.01；P＜0.01），其中基因敲除模型组明显低于野生小鼠模型组（P＜0.01）。依普利酮组及中药组ADM表达明显高于相应模型组（P均＜0.05）。
2肾组织ADM蛋白检测野生小鼠假手术组ADM表达明显，基因敲除型假手术组表达减弱，与野生型有显著差异（P＜0.01）；模型组ADM表达显著低于相应假手术组（P＜0.01；P＜0.01），基因敲除模型组明显低于野生小鼠模型组（P＜0.01）。依普利酮组及中药组ADM表达明显高于相应模型组（P＜0.05；P＜0.05）。
3肾组织ADM免疫组化检测野生小鼠假手术组ADM表达较为广泛，在皮质及髓质均有表达，且以髓质表达为明显，基因敲除小鼠假手术组表达较野生小鼠假手术组明显减弱；两模型组ADM表达明显减少，髓质部减少更为明显，且基因敲除小鼠较野生小鼠表达显著减少，在髓质部几无表达；依普利酮及中药组ADM表达较相应模型组增多，其中野生小鼠表达较基因敲除小鼠为多。
结论：
1结扎单侧输尿管复制肾间质纤维化实验动物模型，肾间质胶原成分显著增多，其中ADM基因敲除UUO小鼠较野生小鼠更为明显。依普利酮及中药可抑制胶原沉积，抑制作用对野生小鼠更为显著。
2肾间质纤维化实验小鼠肾脏TEMT标志物α-SMA表达较假手术组明显增多，其中基因敲除小鼠较野生小鼠为甚；化瘀涤痰通络中药及依普利酮可下调α-SMA在mRNA及蛋白水平的表达。
3化瘀涤痰通络中药可下调UUO肾间质纤维化模型小鼠肾组织中TGF-β1、Col Ⅲm RNA及蛋白的表达，减少ECM沉积。
4肾间质纤维化实验小鼠肾脏ADM表达较假手术组显著降低，化瘀涤痰通络中药可上调其表达，对野生小鼠ADM的上调作用显著强于基因敲除小鼠。
5化瘀涤痰通络中药可上调UUO肾间质纤维化模型小鼠的ADM表达，下调TGF-β1水平，抑制TEMT，减轻肾间质纤维化程度。这可能是中医药防治肾间质纤维化的作用靶点之一。
Chronic renal failure (CRF) is one of the most serious diseases in China andthe incidence of CRF has been increasing in recent years. A large number ofclinical and animal experimental results showed that the renal interstitialfibrosis (RIF) plays an important role in chronic kidney diseases and CRF canbe slow down by preventing RIF. RIF is a common pathological process ofchronic kidney diseases and it can be induced by a variety of factors, in whichcell proliferation and myofibroblast transdifferentiation are involved mainly.Tubular epithelial-myofibroblast transdifferentiation (TEMT) was put forwardby Strutz in1994and was proved to be a core pathological change of RIF andthe α-smooth muscle actin (α-SMA) was considered to be the marker ofTEMT. TEMT also has a complex mechanism related to various factors andit is hard to be prevented effectively by single pathway. However, it isnecessary to searched for the pharmaceuticals with more physiological effectsand more action pathways. Discovered in1993, adrenomedullin (ADM) is akind of biologically active polypeptide and distribute in many vital organs andtissues of the body with expressing more in kidney. ADM has variousphysiological effects including inhibiting the expression of transforminggrowth factor beta1(TGF-β1) and angiotensin II (AngⅡ), anti-oxidation andanti-inflammatory. The TGF-β1is the key cytokine in the process of TEMTand it is therefore believed that ADM can inhibit TEMT by down-regulatingthe expression of TGF-β1and then can delay the process of RIF. However, itsspecific mechanism remains to be explored. Meanwhile, it has been proved inour former experiments that the Chinese herbs can antagonize TEMT byinhibiting the expression of TGF-β1but it’s still not clear whether Chineseherbs are in the way of regulating ADM in the process. To figure out the mechanism of Chinese herbs on antagonizing TEMT and the correlation toAMD, this study was designed to observe Chinese herbs’ effects on theexpression of α-SMA, TGF-β1, Col Ⅲand ADM in adrenomedullinknockout(AMKO) mice with unilateral ureteral obstruction(UUO), whileeplerenone, the aldosterone receptor blocker, was used as the comparator.
PartⅠ: Effect of Chinese herbs on renal function and pathomorphologyin TEMT In adrenomedullin knockout mice with UUO
Methods: Both of wild and adrenomedullin knockout (AMKO) mice weredivided into four groups respectively. They are WT-Sham group, WT-UUOgroup, WT-UUOE group, WT-UUOS group, AMKO-Sham group,AMKO-UUO group, AMKO-UUOE group and AMKO-UUOS group. Exceptthe mice in two Sham groups, the others in the rest groups were ligated theirleft ureters under ethyl ether anesthesia. The mice in two UUOS groups weregiven Chinese herbs27.6g/(kg.d) by oral gavage and two UUOE groups weregiven eplerenone100mg/(kg.d) in their forage. The others got the samevolume of saline. At the10day after surgery, all mice were sacrificed andspecimens of blood were got to detect the contents of Blood Urea Nitrogen(BUN) and Serum Creatinine (SCr). And the left kidneys were cut to bemeasured the index of kidney weight/body weight (KW/BW) and parts of leftkidneys of mice were prepared respectively for HE and Masson staining.
The data were expressed as mean±SD. SPSS16.0software was adoptedand one way ANOVA was applying to comparison among many groups. Allresults are considered significant at P<0.05and P<0.01.
Results:
1Index of kidney weight/body weight
The Index of KW/BW of the mice in UUO，UUOE and UUOS groups wassignificantly higher than that of two Sham groups respectively (P<0.01) butthere was no obvious difference among them (P<0.05).
2The result of pathomorphology
HE staining showed that no abnormalities in two Sham groups. In thekidneys of UUO，UUOE and UUOS groups, the necrosis and detachment of the renal tubules epithelial cells were observed with a large of inflammatorycells infiltrated in renal interstitial space. Mssson staining showed that therewas no abnormality in two Sham groups. There was many collagen depositedin renal interstitium just like strip-shaped and spotty-shaped in the two UUOgroups and the AMKO-UUO group was more serious than that of WT-UUOone.
3The result of renal function
There was no significant difference of Scr in all the8groups. Neither wasBUN.(P>0.05)
Part Ⅱ: Effect of Chinese herbs on the express of α-SMA in TEMT inadrenomedullin knockout mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days. The expressions of α-SMA inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.
Results:
1The results of RT-PCR Compared with corresponding Sham groups, theexpression of α-SMA mRNA was enhanced obviously in UUO groups(P<0.01; P<0.01), and the AMKO-UUO was higher than that of WT-UUO onesignificantly(P<0.05). The high expression of α-SMA mRNA wasdown-regulated respectively in both UUOE and UUOS groups (P<0.01;P<0.01).
2The Results of Western Blot A little of expression of α-SMA protein inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of α-SMA in WT-UUO and AMKO-UUO groups was enhancedremarkably (P<0.01; P<0.01), and the AMKO-UUO was higher than that ofWT-UUO one significantly (P<0.05). Compared with corresponding UUOgroups, the high expression of α-SMA was significantly decreased in UUOEand UUOS groups (P<0.01; P<0.01) and there was no significant differencebetween the UUOE and UUOS groups (P>0.05; P>0.05).
3The Results of Immunohistochemistry showed that there was a little expression of α-SMA only in vascular smooth muscle cells in two Shamgroups and it was remarkably increased in WT-UUO and AMKO-UUO groupswith the expanding scope of tubulointerstitium in cortical andcorticomedullary area. And the AMKO-UUO was more serious than that ofWT-UUO one. The expression of α-SMA in UUOE and UUOS groups wasreduced significantly.
Part Ⅲ: Effect of Chinese herbs on the express of TGF-β1and Col Ⅲ inTEMT in adrenomedullin knockout mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to Part Ⅰ,10days. The expressions of TGF-β1and ColⅢ in kidneys were detected by RT-PCR, Western Blot andimmunohistochemistry respectively. In addition, the expression of Collagentissues was detected by sirius red staining.
Results:
1The expression of TGF-β1mRNA and Col III mRNA detected by RT-PCR
1.1The result of TGF-β1mRNA detected by RT-PCR showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO one (P<0.05). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.05; P<0.05).
1.2The result of Col III mRNA detected by RT-PCR showed that littleexpression of Col III in WT-Sham and AMKO-Sham groups was observed andit was increased obviously in UUO groups (P<0.01; P<0.01)with expressingmore in AMKO-UUO one (P<0.01). Compared with corresponding UUOgroups, the expression was reduced in both UUOE groups and UUOS groupssignificantly (P<0.01; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice (P<0.05; P<0.05).
2The expression of TGF-β1and Col III protein detected by Western Blot
2.1The result of TGF-β1protein detected by Western Blot showed that a little expression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.01; P<0.01).
2.2The result of Col III protein detected by Western Blot showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE groups and UUOSgroups significantly (P<0.05; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice.(P<0.05; P<0.05)
3The expression of TGF-β1and Col III detected by immunohistochemistry
3.1The result of TGF-β1detected by immunohistochemistry demonstrated thatthere was weak expression in glomerular and tubular epithelial cells in twoSham groups, and the expression was remarkably increased in WT-UUO andAMKO-UUO groups. The AMKO-UUO group expressed more than that ofWT-UUO ones. Compared with corresponding UUO groups the expression ofTGF-β1in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS groups.
3.2The result of Col III detected by immunohistochemistry demonstrated thatthere was modest expression in glomerular and interstitial in two Sham groupsand the expression was remarkably increased in WT-UUO and AMKO-UUOgroups, especially in tubulointerstitium. The mice in the AMKO-UUO groupexpressed more than WT-UUO one. Compared with corresponding UUOgroups the expression of Col III in UUOE and UUOS groups was reducedwith expressing more in AMKO-UUOE and AMKO-UUOS groups.
4The result of collagen tissues detected by sirius red staining demonstratedthat there was modest expression in glomerular, tubular and interstitial in twoSham groups and there was no obviously difference between them. The expression was remarkably increased in WT-UUO and AMKO-UUO groupmainly in renal tubulointerstitium with expression more in AMKO-UUOgroup. Compared with corresponding UUO groups the expression of collagentissues in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS ones.
Part Ⅳ: Effect of Chinese herbs on the express of ADMin renalinterstitial fibrosis mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days.The expressions of ADM inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.
Results:
1The Results of RT-PCR The expression of ADM in AMKO-Sham groupwas weaker than that of WT-Sham group significantly(P<0.01). Comparedwith corresponding Sham groups, the expression of ADM mRNA was reducedobviously in UUO groups (P<0.01; P<0.01) and the AMKO-UUO was weakerthan that of WT-UUO one significantly (P<0.01). The low expression of ADMmRNA was up-regulated respectively in both UUOE and UUOS groups(P<0.05).
2The Results of Western Blot There was some expression of ADM inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of ADM in AMKO-Sham group is weaker than that of WT-Shamgroup(P<0.01). The expression in WT-UUO and AMKO-UUO groups wasreduced remarkably (P<0.01; P<0.01) and the AMKO-UUO was lower thanthat of WT-UUO one significantly (P<0.01). Compared with correspondingUUO groups the low expression of ADM was significantly up-regulated in theUUOE and UUOS groups (P<0.01; P<0.01).
3The Results of Immunohistochemistry In WT-Sham and AMKO-Shamgroups, there was some expression in cortical and corticomedullary area,especially in medullary. And the expression in AMKO-Sham group is weakerthan that of WT-Sham one. The expression was remarkably reduced in WT-UUO and AMKO-UUO groups and AMKO-UUO expressed less thanWT-UUO group. There was increasing expression of ADM in UUOE andUUOS groups than that of corresponding UUO groups respectively withexpressing more in wild mice.
Conclusions:
1The animal models of renal interstitial fibrosis were established byunilateral ureteral obstruction. The expression of collagen tissues in the kidneywere increased remarkably in UUO mice, especially in adrenomedullinknockout mice. Chinese herbs and eplerenone can inhibit collagen depositionin renal interstitial and the effect was more significant in wild-type mice withUUO.
2The expression of α-SMA, which is the marker of TEMT, was strongerobviously in UUO mice, especially in adrenomedullin knockout mice. Chineseherbs and eplerenone can down-regulate the expression level of α-SMAmRNA and protein.
3Chinese herbs have the function of de-gradating collagen tissues andreducing the deposition of ECM by down-regulating the expression of TGF-β1and Col Ⅲ mRNA and protein.
4Compared with Sham groups, The expression of ADM was significantlyreduced in UUO groups of wild type and adrenomedullin Knockout mice andChinese herbs can effectively up-regulated its expression level, especially inwide mice.
5Chinese herbs can inhabit TEMT and antagonize the progress of RIF bydown-regulating the expression of TGF-β1and Col Ⅲ while the expressionof ADM was up-regulated. It may be the one of the targets of the mechanismin Chinese herbs on RIF prevention.
第一部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠肾功能及肾脏组织形态学的影响
方法：野生及ADM基因敲除小鼠各20只均随机分为四组，即野生小鼠分为假手术组（WT-Sham group）、模型组（WT-UUO group）、依普利酮组（WT-UUOE group）及中药组（WT-UUOS group）四组；基因敲除小鼠同样分为假手术组（AMKO-Sham group）、模型组（AMKO-UUOgroup）、依普利酮组（AMKO-UUOE group）及中药组（AMKO-UUOSgroup）四组。除假手术组游离左侧输尿管但不结扎外，其余各组均行左侧输尿管结扎术。中药组给予化瘀涤痰通络中药肾络通27.6g/(kg.d)经口灌服；依普利酮组给予依普利酮混匀于饲料中按100mg/(kg.d)剂量食入。假手术组及模型组经口灌服与中药汤剂等剂量的生理盐水。观察一般情况，给药10天。于实验结束时，称重，断头取血，分离血清，分别测定血肌酐（SCr）、尿素氮（BUN）水平；摘取左侧肾脏并称重，计算各组肾重/体重比值，进行肾脏组织HE、Masson染色，观察组织病理学改变。
数据资料以均数±标准差（±s）表示，使用SPSS16.0for windows统计软件进行统计，组间比较采用单因素方差分析。显著性差异水平以0.05和0.01为标准。
结果:
1实验小鼠肾重/体重比值测定结果
野生及基因敲除小鼠的模型组、依普利酮组及中药组显著高于相应的假手术组（P均＜0.01），但各组之间差异无统计学意义（P均>0.05）。（见Table1-1）
2小鼠肾组织病理形态学检测结果
HE染色显示，野生及基因敲除小鼠中，假手术组未见明显异常。模型组可见肾小管扩张，肾小管上皮细胞水肿，部分坏死、脱落，炎性细胞在肾间质区广泛浸润。Masson染色显示，野生及基因敲除小鼠中，假手术组肾脏结构未见明显异常，小管基底膜完整，肾间质可见少量胶原成分。模型组肾组织间质胶原成分沉积显著增多，并呈条带状或灶状分布，且基因敲除小鼠重于野生小鼠。依普利酮组及中药组间质胶原成分较模型组明显减少，其中基因敲除小鼠较野生小鼠损伤为重。
3肾功能检测结果
Scr及BUN测定结果显示，野生及基因敲除中各组小鼠的Scr及BUN值虽有差异，但无统计学意义（P均>0.05）。
第二部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠α-SMA的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏α-SMAmRNA及蛋白的表达。统计学方法同第一部分。
结果：
1肾组织α-SMA mRAN检测结果采用RT-PCR测定α-SMA mRNA在肾组织中的表达，结果显示，野生及基因敲除中的假手术组小鼠呈弱表达，模型组表达较相应的假手术组显著增强（P＜0.01；P＜0.01），且基因敲除模型组显著高于野生小鼠模型组（P＜0.05）。依普利酮组及中药组表达下调（P＜0.01；P＜0.01）。
2肾组织α-SMA蛋白检测结果Western Blot结果显示，野生及基因敲除小鼠中的假手术组肾组织α-SMA蛋白表达较弱，模型组表达明显增强，显著高于相应的假手术组（P＜0.01；P＜0.01），且基因敲除模型组显著高于野生小鼠模型组（P＜0.05）。依普利酮组及中药组表达明显低于相应的模型组（P＜0.01；P＜0.01），中药组与依普利酮组无明显差异（P均>0.05）。
3肾组织α-SMA免疫组化检测光镜下野生及基因敲除小鼠的假手术组仅在血管平滑肌细胞表达，肾小球、肾小管及间质未见表达；模型组中α-SMA表达明显增多，多见于皮髓交界及皮质区的小管间质部分。基因敲除小鼠模型组表达尤其明显。依普利酮组及中药组α-SMA表达较模型组明显减少。但基因敲除小鼠表达较野生小鼠更为明显。第三部分化瘀涤痰通络中药对ADM基因敲除肾间质纤维化小鼠TGF-β1、Col III的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏TGF-β1、Col Ⅲ mRNA及蛋白的表达，并用天狼猩红染色法测定肾脏胶原组织表达。统计学方法同第一部分。
结果：
1肾组织TGF-β1、Col Ⅲ mRNA检测
1.1TGF-β1mRNA RT-PCR测定结果显示，野生及基因敲除中的假手术组小鼠呈弱表达，模型组显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组表达更为明显，显著高于野生小鼠模型组（P＜0.05）；依普利酮组及中药组明显低于模型组（P＜0.05；P＜0.05）。
1.2Col Ⅲ mRNART-PCR测定结果显示，野生及基因敲除中小鼠的假手术组呈极弱表达，模型组显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组显著高于野生小鼠模型组（P＜0.01）；依普利酮组及中药组明显低于相应模型组小鼠（P＜0.01; P＜0.01）。野生及基因敲除小鼠之间比较，基因敲除小鼠中药组及依普利酮组表达均明显高于野生型同组小鼠（P＜0.05；P＜0.05）。
2肾组织中TGF-β1及Col Ⅲ蛋白检测
2.1TGF-β1Western Blot测定野生及基因敲除小鼠假手术组TGF-β1蛋白少量表达，模型组表达显著高于假手术组（P＜0.01；P＜0.01），基因敲除模型组表达显著高于野生小鼠模型组（P＜0.01）。依普利酮组及中药组表达明显低于相应模型组（P＜0.01；P＜0.01）。
2.2Col Ⅲ Western Blot测定结果显示，野生及基因敲除小鼠假手术组少量表达，模型组显著高于相应的假手术组（P＜0.01；P＜0.01），基因敲除模型组显著高于野生小鼠模型组（P＜0.01）。野生及基因敲除小鼠依普利酮组及中药组明显低于相应模型组（P＜0.05；P＜0.01）。野生及基因敲除两型之间比较，基因敲除小鼠中药组与依普利酮组均明显高于野生型的同组小鼠（P＜0.05；P＜0.05）。
3肾组织TGF-β1及Col Ⅲ免疫组化检测
3.1TGF-β1免疫组化检测野生及基因敲除小鼠的假手术组肾脏TGF-β1呈弱表达，可见于肾小球及肾小管上皮细胞；模型组TGF-β1表达明显增强，其中基因敲除小鼠尤其显著；依普利酮组及中药组表达较模型组明显减少，但基因敲除小鼠较野生小鼠表达明显。
3.2Col Ⅲ免疫组化检测野生及基因敲除小鼠的假手术组肾脏可见少量表达，可见于肾小球及肾小管间质；模型组Col Ⅲ表达明显增多，以间质最为明显，其中基因敲除小鼠甚于野生小鼠；依普利酮组及中药组表达较相应模型组明显减少，但基因敲除小鼠较野生小鼠表达明显。
4肾脏胶原组织天狼猩红染色测定假手术组肾间质可见胶原组织表达于肾小球、肾小管及间质，野生及基因敲除小鼠间无明显差异；模型组表达明显增多，主要见于肾小管间质，皮质及髓质表达均明显增强，ADM基因敲除小鼠表达尤为显著；依普利酮组及中药组表达较相应模型组明显减弱，但两组中ADM基因敲除小鼠较野生小鼠表达明显。
第四部分化瘀涤痰通络中药对肾间质纤维化小鼠ADM表达的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏，采用RT-PCR、Western-Blot、免疫组化法检测各组小鼠肾脏ADM mRNA及蛋白的表达。统计学方法同第一部分。
结果：
1肾组织ADM mRNA检测野生及基因敲除小鼠中假手术组均有表达，但基因敲除小鼠的表达明显低于野生小鼠（P＜0.01）。模型组显著低于相应假手术组（P＜0.01；P＜0.01），其中基因敲除模型组明显低于野生小鼠模型组（P＜0.01）。依普利酮组及中药组ADM表达明显高于相应模型组（P均＜0.05）。
2肾组织ADM蛋白检测野生小鼠假手术组ADM表达明显，基因敲除型假手术组表达减弱，与野生型有显著差异（P＜0.01）；模型组ADM表达显著低于相应假手术组（P＜0.01；P＜0.01），基因敲除模型组明显低于野生小鼠模型组（P＜0.01）。依普利酮组及中药组ADM表达明显高于相应模型组（P＜0.05；P＜0.05）。
3肾组织ADM免疫组化检测野生小鼠假手术组ADM表达较为广泛，在皮质及髓质均有表达，且以髓质表达为明显，基因敲除小鼠假手术组表达较野生小鼠假手术组明显减弱；两模型组ADM表达明显减少，髓质部减少更为明显，且基因敲除小鼠较野生小鼠表达显著减少，在髓质部几无表达；依普利酮及中药组ADM表达较相应模型组增多，其中野生小鼠表达较基因敲除小鼠为多。
结论：
1结扎单侧输尿管复制肾间质纤维化实验动物模型，肾间质胶原成分显著增多，其中ADM基因敲除UUO小鼠较野生小鼠更为明显。依普利酮及中药可抑制胶原沉积，抑制作用对野生小鼠更为显著。
2肾间质纤维化实验小鼠肾脏TEMT标志物α-SMA表达较假手术组明显增多，其中基因敲除小鼠较野生小鼠为甚；化瘀涤痰通络中药及依普利酮可下调α-SMA在mRNA及蛋白水平的表达。
3化瘀涤痰通络中药可下调UUO肾间质纤维化模型小鼠肾组织中TGF-β1、Col Ⅲm RNA及蛋白的表达，减少ECM沉积。
4肾间质纤维化实验小鼠肾脏ADM表达较假手术组显著降低，化瘀涤痰通络中药可上调其表达，对野生小鼠ADM的上调作用显著强于基因敲除小鼠。
5化瘀涤痰通络中药可上调UUO肾间质纤维化模型小鼠的ADM表达，下调TGF-β1水平，抑制TEMT，减轻肾间质纤维化程度。这可能是中医药防治肾间质纤维化的作用靶点之一。
Chronic renal failure (CRF) is one of the most serious diseases in China andthe incidence of CRF has been increasing in recent years. A large number ofclinical and animal experimental results showed that the renal interstitialfibrosis (RIF) plays an important role in chronic kidney diseases and CRF canbe slow down by preventing RIF. RIF is a common pathological process ofchronic kidney diseases and it can be induced by a variety of factors, in whichcell proliferation and myofibroblast transdifferentiation are involved mainly.Tubular epithelial-myofibroblast transdifferentiation (TEMT) was put forwardby Strutz in1994and was proved to be a core pathological change of RIF andthe α-smooth muscle actin (α-SMA) was considered to be the marker ofTEMT. TEMT also has a complex mechanism related to various factors andit is hard to be prevented effectively by single pathway. However, it isnecessary to searched for the pharmaceuticals with more physiological effectsand more action pathways. Discovered in1993, adrenomedullin (ADM) is akind of biologically active polypeptide and distribute in many vital organs andtissues of the body with expressing more in kidney. ADM has variousphysiological effects including inhibiting the expression of transforminggrowth factor beta1(TGF-β1) and angiotensin II (AngⅡ), anti-oxidation andanti-inflammatory. The TGF-β1is the key cytokine in the process of TEMTand it is therefore believed that ADM can inhibit TEMT by down-regulatingthe expression of TGF-β1and then can delay the process of RIF. However, itsspecific mechanism remains to be explored. Meanwhile, it has been proved inour former experiments that the Chinese herbs can antagonize TEMT byinhibiting the expression of TGF-β1but it’s still not clear whether Chineseherbs are in the way of regulating ADM in the process. To figure out the mechanism of Chinese herbs on antagonizing TEMT and the correlation toAMD, this study was designed to observe Chinese herbs’ effects on theexpression of α-SMA, TGF-β1, Col Ⅲand ADM in adrenomedullinknockout(AMKO) mice with unilateral ureteral obstruction(UUO), whileeplerenone, the aldosterone receptor blocker, was used as the comparator.
PartⅠ: Effect of Chinese herbs on renal function and pathomorphologyin TEMT In adrenomedullin knockout mice with UUO
Methods: Both of wild and adrenomedullin knockout (AMKO) mice weredivided into four groups respectively. They are WT-Sham group, WT-UUOgroup, WT-UUOE group, WT-UUOS group, AMKO-Sham group,AMKO-UUO group, AMKO-UUOE group and AMKO-UUOS group. Exceptthe mice in two Sham groups, the others in the rest groups were ligated theirleft ureters under ethyl ether anesthesia. The mice in two UUOS groups weregiven Chinese herbs27.6g/(kg.d) by oral gavage and two UUOE groups weregiven eplerenone100mg/(kg.d) in their forage. The others got the samevolume of saline. At the10day after surgery, all mice were sacrificed andspecimens of blood were got to detect the contents of Blood Urea Nitrogen(BUN) and Serum Creatinine (SCr). And the left kidneys were cut to bemeasured the index of kidney weight/body weight (KW/BW) and parts of leftkidneys of mice were prepared respectively for HE and Masson staining.
The data were expressed as mean±SD. SPSS16.0software was adoptedand one way ANOVA was applying to comparison among many groups. Allresults are considered significant at P<0.05and P<0.01.
Results:
1Index of kidney weight/body weight
The Index of KW/BW of the mice in UUO，UUOE and UUOS groups wassignificantly higher than that of two Sham groups respectively (P<0.01) butthere was no obvious difference among them (P<0.05).
2The result of pathomorphology
HE staining showed that no abnormalities in two Sham groups. In thekidneys of UUO，UUOE and UUOS groups, the necrosis and detachment of the renal tubules epithelial cells were observed with a large of inflammatorycells infiltrated in renal interstitial space. Mssson staining showed that therewas no abnormality in two Sham groups. There was many collagen depositedin renal interstitium just like strip-shaped and spotty-shaped in the two UUOgroups and the AMKO-UUO group was more serious than that of WT-UUOone.
3The result of renal function
There was no significant difference of Scr in all the8groups. Neither wasBUN.(P>0.05)
Part Ⅱ: Effect of Chinese herbs on the express of α-SMA in TEMT inadrenomedullin knockout mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days. The expressions of α-SMA inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.
Results:
1The results of RT-PCR Compared with corresponding Sham groups, theexpression of α-SMA mRNA was enhanced obviously in UUO groups(P<0.01; P<0.01), and the AMKO-UUO was higher than that of WT-UUO onesignificantly(P<0.05). The high expression of α-SMA mRNA wasdown-regulated respectively in both UUOE and UUOS groups (P<0.01;P<0.01).
2The Results of Western Blot A little of expression of α-SMA protein inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of α-SMA in WT-UUO and AMKO-UUO groups was enhancedremarkably (P<0.01; P<0.01), and the AMKO-UUO was higher than that ofWT-UUO one significantly (P<0.05). Compared with corresponding UUOgroups, the high expression of α-SMA was significantly decreased in UUOEand UUOS groups (P<0.01; P<0.01) and there was no significant differencebetween the UUOE and UUOS groups (P>0.05; P>0.05).
3The Results of Immunohistochemistry showed that there was a little expression of α-SMA only in vascular smooth muscle cells in two Shamgroups and it was remarkably increased in WT-UUO and AMKO-UUO groupswith the expanding scope of tubulointerstitium in cortical andcorticomedullary area. And the AMKO-UUO was more serious than that ofWT-UUO one. The expression of α-SMA in UUOE and UUOS groups wasreduced significantly.
Part Ⅲ: Effect of Chinese herbs on the express of TGF-β1and Col Ⅲ inTEMT in adrenomedullin knockout mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to Part Ⅰ,10days. The expressions of TGF-β1and ColⅢ in kidneys were detected by RT-PCR, Western Blot andimmunohistochemistry respectively. In addition, the expression of Collagentissues was detected by sirius red staining.
Results:
1The expression of TGF-β1mRNA and Col III mRNA detected by RT-PCR
1.1The result of TGF-β1mRNA detected by RT-PCR showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO one (P<0.05). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.05; P<0.05).
1.2The result of Col III mRNA detected by RT-PCR showed that littleexpression of Col III in WT-Sham and AMKO-Sham groups was observed andit was increased obviously in UUO groups (P<0.01; P<0.01)with expressingmore in AMKO-UUO one (P<0.01). Compared with corresponding UUOgroups, the expression was reduced in both UUOE groups and UUOS groupssignificantly (P<0.01; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice (P<0.05; P<0.05).
2The expression of TGF-β1and Col III protein detected by Western Blot
2.1The result of TGF-β1protein detected by Western Blot showed that a little expression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE and UUOS groupssignificantly (P<0.01; P<0.01).
2.2The result of Col III protein detected by Western Blot showed that a littleexpression of TGF-β1in WT-Sham and AMKO-Sham groups was observedand it was increased obviously in UUO groups(P<0.01; P<0.01)withexpressing more in AMKO-UUO(P<0.01). Compared with correspondingUUO groups, the expression was reduced in both UUOE groups and UUOSgroups significantly (P<0.05; P<0.01). And there was more expression inAMKO-UUOE and AMKO-UUOS groups than corresponding groups of wildtype mice.(P<0.05; P<0.05)
3The expression of TGF-β1and Col III detected by immunohistochemistry
3.1The result of TGF-β1detected by immunohistochemistry demonstrated thatthere was weak expression in glomerular and tubular epithelial cells in twoSham groups, and the expression was remarkably increased in WT-UUO andAMKO-UUO groups. The AMKO-UUO group expressed more than that ofWT-UUO ones. Compared with corresponding UUO groups the expression ofTGF-β1in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS groups.
3.2The result of Col III detected by immunohistochemistry demonstrated thatthere was modest expression in glomerular and interstitial in two Sham groupsand the expression was remarkably increased in WT-UUO and AMKO-UUOgroups, especially in tubulointerstitium. The mice in the AMKO-UUO groupexpressed more than WT-UUO one. Compared with corresponding UUOgroups the expression of Col III in UUOE and UUOS groups was reducedwith expressing more in AMKO-UUOE and AMKO-UUOS groups.
4The result of collagen tissues detected by sirius red staining demonstratedthat there was modest expression in glomerular, tubular and interstitial in twoSham groups and there was no obviously difference between them. The expression was remarkably increased in WT-UUO and AMKO-UUO groupmainly in renal tubulointerstitium with expression more in AMKO-UUOgroup. Compared with corresponding UUO groups the expression of collagentissues in UUOE and UUOS groups was reduced with expressing more inAMKO-UUOE and AMKO-UUOS ones.
Part Ⅳ: Effect of Chinese herbs on the express of ADMin renalinterstitial fibrosis mice with UUO
The methods of animal grouping，model establishment, medication andstatistics were the same to PartⅠ,10days.The expressions of ADM inkidneys were detected by RT-PCR, Western Blot and immunohistochemistryrespectively.
Results:
1The Results of RT-PCR The expression of ADM in AMKO-Sham groupwas weaker than that of WT-Sham group significantly(P<0.01). Comparedwith corresponding Sham groups, the expression of ADM mRNA was reducedobviously in UUO groups (P<0.01; P<0.01) and the AMKO-UUO was weakerthan that of WT-UUO one significantly (P<0.01). The low expression of ADMmRNA was up-regulated respectively in both UUOE and UUOS groups(P<0.05).
2The Results of Western Blot There was some expression of ADM inkidney tissues was observed in WT-Sham and AMKO-Sham groups，while theexpression of ADM in AMKO-Sham group is weaker than that of WT-Shamgroup(P<0.01). The expression in WT-UUO and AMKO-UUO groups wasreduced remarkably (P<0.01; P<0.01) and the AMKO-UUO was lower thanthat of WT-UUO one significantly (P<0.01). Compared with correspondingUUO groups the low expression of ADM was significantly up-regulated in theUUOE and UUOS groups (P<0.01; P<0.01).
3The Results of Immunohistochemistry In WT-Sham and AMKO-Shamgroups, there was some expression in cortical and corticomedullary area,especially in medullary. And the expression in AMKO-Sham group is weakerthan that of WT-Sham one. The expression was remarkably reduced in WT-UUO and AMKO-UUO groups and AMKO-UUO expressed less thanWT-UUO group. There was increasing expression of ADM in UUOE andUUOS groups than that of corresponding UUO groups respectively withexpressing more in wild mice.
Conclusions:
1The animal models of renal interstitial fibrosis were established byunilateral ureteral obstruction. The expression of collagen tissues in the kidneywere increased remarkably in UUO mice, especially in adrenomedullinknockout mice. Chinese herbs and eplerenone can inhibit collagen depositionin renal interstitial and the effect was more significant in wild-type mice withUUO.
2The expression of α-SMA, which is the marker of TEMT, was strongerobviously in UUO mice, especially in adrenomedullin knockout mice. Chineseherbs and eplerenone can down-regulate the expression level of α-SMAmRNA and protein.
3Chinese herbs have the function of de-gradating collagen tissues andreducing the deposition of ECM by down-regulating the expression of TGF-β1and Col Ⅲ mRNA and protein.
4Compared with Sham groups, The expression of ADM was significantlyreduced in UUO groups of wild type and adrenomedullin Knockout mice andChinese herbs can effectively up-regulated its expression level, especially inwide mice.
5Chinese herbs can inhabit TEMT and antagonize the progress of RIF bydown-regulating the expression of TGF-β1and Col Ⅲ while the expressionof ADM was up-regulated. It may be the one of the targets of the mechanismin Chinese herbs on RIF prevention.
引文
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