【作者】 谢林；

【导师】 王怡；

【作者基本信息】 上海中医药大学 ， 中医内科学， 2019， 硕士

【摘要】 目的:通过体内动物实验研究二丁二仙汤及其君药淫羊藿苷防治肾间质纤维化的作用及机制。方法:10周龄雄性C57BL/6小鼠随机分为7组,假手术组(SHAM)、模型组(UUO)、模型+二丁二仙汤组(EDEX+UUO)、模型+雌二醇组(E2+UUO)、模型+淫羊藿苷组(ICA+UUO)、模型+GPR30拮抗剂组(G-15+UUO)、模型+GPR30拮抗剂+淫羊藿苷组(G-15+ICA+UUO)。单侧输尿管梗阻模型的制作,小鼠逐层打开腹腔后,游离左侧输尿管,本模型采用4-0丝线结扎,结扎左肾下级水平,关闭腹腔并逐层缝合。本模型通过手术结扎一侧输尿管引使肾脏引流系统梗阻,从而引起慢性肾损伤,进而导致肾间质纤维化。G-15给药方式均予以皮下植入微渗透压泵(Alzet Minipump model 1007D),给药速率为200μg/kg/d,维持7天。治疗7天后,各组小鼠进行心脏灌注,予以眼球采血,提取血清,检测雌二醇水平、两侧肾脏摘除,称取重量,固定后进行Masson’s、HE、PAS染色,光镜下观察肾脏病理情况。荧光定量PCR检测c-Myc,FN,Ⅰ型胶原,GPR30 m RNA表达变化。Western Blot方法检测总β-catenin、磷酸化β-catenin(ser-552)、GPR30、c-Myc蛋白表达变化。免疫荧光检测a-SMA、Ⅰ型胶原,免疫组化检测GPR30。结果:(1)实验前后小鼠体重无明显差异(P>0.05)。ICA+UUO组与G-15+UUO组比较,血清雌二醇水平提高(P<0.05),G-15+ICA+UUO组与G-15+UUO组比较,血清雌二醇水平增高(P<0.05)。(2)肾脏组织病理染色,Masson’s染色显示,与假手术组比较,模型组的间质纤维程度明显,间质蓝色胶原面积显著增多,光镜视野下大量肾小管萎缩、扩张,同时间质伴有大量单核细胞的浸润,肾小球存在部分性代偿增大,二丁二仙汤组和淫羊藿苷组与模型组相比,肾间质纤维化的程度有不同程度的改善,大体表现为皮髓质界限较模型组清晰,另外有部分小管的上皮细胞仍维持相对完整的结构,肾小管扩张程度较模型组轻,间质内蓝色胶原面积以及单核细胞浸润情况较模型组好转。PAS染色提示,模型组小鼠有蛋白管型形成,肾小管扩张,刷状缘的脱落,间质纤维化,肾小球硬化,EREX+UUO及ICA+UUO组肾脏病理明显改善。肾脏病理损伤评分也显示UUO组肾小管及间质损伤,小动脉硬化,肾小球硬化程度较EREX+UUO及ICA+UUO组纤维化程度高。HE染色显示模型组大量炎性细胞浸润,EREX+UUO及ICA+UUO组程度相对较轻。(3)免疫荧光显示:在假手术组α-SMA只在血管上表达,而在UUO组及G-15+UUO及G-15+ICA+UUO组,间质表达增多。a-SMA及I型胶原肾组织免疫荧光也显示G-15干预UUO后,纤维化加重,二丁二仙汤组和淫羊藿苷组肾纤维化程度较之UUO较轻。(4)肾脏组织GPR30免疫组化染色,可见棕黄色染色,发现与SHAM组相比,UUO组GPR30表达量明显下降。(5)实时定量PCR和Western Blot结果提示UUO组小鼠纤维化指标FN、c-Myc、总β-catenin、磷酸化β-catenin(ser-552)表达比SHAM组明显增加。GPR30阻断剂组小鼠FN、Ⅰ型胶原、c-Myc、总β-catenin、磷酸化β-catenin(ser-552)表达升高。ICA+UUO、EDEX+UUO组与UUO组比较,小鼠纤维化表达表达降低。同时EDEX+UUO组与ICA+UUO组与UUO组比较,小鼠GPR30水平表达增高。(6)对GPR30进行免疫组化染色我们发现,在假手术组中,GPR30主要大量表达在肾小管上皮细胞,而在肾脏梗阻的情况下,肾小管受打击后,肾小管上皮细胞GPR30表达明显下降,而使用G-15(GPR30拮抗剂)可使UUO纤维化程度加重。同时予α-SMA和I型胶原肾组织免疫荧光染色也提示,G15干预UUO后,纤维化加重,EDEX+UUO组与ICA+UUO组可改善肾纤维化,而q PCR结果亦提示在G15可明显加重FN m RNA表达。结论:1、二丁二仙汤和淫羊藿苷能提高UUO肾纤维化模型小鼠雌二醇水平,减少肾组织FN,Ⅰ型胶原,a-SMA、cmyc等纤维化成分的聚积,降低肾脏病理损伤的程度,从而保护肾脏功能,提高GPR30的表达,调控Wnt/β-catenin信号通路,延缓肾纤维化的进程。2、GPR30拮抗剂可协同加重纤维化进展,提示GPR30可能对肾脏纤维化有重要保护作用,阻断其表达后,可引起纤维化加重。与此同时淫羊藿苷对肾纤维化有一定保护作用,可能从GPR30起作用,干预肾脏纤维化。3、EREX+UUO及ICA+UUO组干预,小鼠蛋白免疫印迹有明显趋势,但无统计学意义,提示药物作用时间的可能较短,有待进一步的研究。更多还原

【Abstract】 Objective: To study the effect and mechanism of Erdingerxian Decoction and Icariin on preventing and treating renal interstitial fibrosis in vivo.Methods: Ten-week-old male C57BL/6 mice were randomly divided into seven groups:sham-operated group(SHAM),model group(UUO),model + Erdingerxian Decoction group(EDEX + UUO),model + estradiol group(E2 + UUO),model + icariin group(ICA + UUO),model + GPR30 antagonist group(G-15 + UUO),model + GPR30 antagonist + icariin group(G-15 +ICA+ UUO).Unilateral Ureteral Obstruction(UUO)model was made.After opening the abdominal cavity layer by layer,the left ureter was free.The model was ligated with 4-0 silk thread,the lower level of the left kidney was ligated,the abdominal cavity was closed and sutured layer by layer.In this model,the kidney drainage system was obstructed by surgical ligation of one side of the ureter,which resulted in chronic renal injury and renal interstitial fibrosis.G-15 were subcutaneously implanted with a microosmotic pump(Alzet Minipump model 1007D)at a rate of 200 ug/kg/d for 7 days.After 7 days of treatment,the mice in each group were perfused with heart,blood was taken from heart,serum was extracted,estradiol level was detected,kidneys were removed on both sides,weight was weighed,Masson’s,HE and PAS staining were performed after fixation,and pathological changes of kidneys were observed under light microscope.Fluorescence quantitative PCR was used to detect the expression of c-Myc,FN,collagen type I and GPR30.Western Blot method was used to detect the expression of total beta-catenin,phosphorylated beta-catenin(ser-552),GPR30 and c-Myc protein.Immunofluorescence was used to detect a-SMA,type I collagen,immunohistochemistry was used to detect GPR30.Result:(1)There was no significant difference in the weight of mice before and after the experiment(P > 0.05).Compared with G-15+UUO group,serum estradiol level increased in ICA+UUO group(P < 0.05).Compared with G-15+ICA+UUO group,serum estradiol level increased in G-15+ICA+UUO group(P < 0.05).(2)Kidney histopathological staining and Masson’s staining showed that compared with sham-operated group,the degree of interstitial fibrosis in model group was obvious,the area of interstitial blue collagen increased significantly,a large number of renal tubules atrophied and dilated under light microscopy,accompanied by a large number of monocytes infiltration in interstitium,partial compensatory enlargement of glomerulus,Erdingerxian Decoction group and icariin group and model group were similar.Compared with the model group,the degree of renal interstitial fibrosis was improved in varying degrees.Generally speaking,the boundary of cortex and medulla was clearer than that of the model group.In addition,some epithelial cells of tubules remained relatively intact.The degree of tubular dilation was lighter than that of the model group,and the area of blue collagen in the interstitium and the infiltration of monocytes were better than that of the model group.PAS staining showed that there were protein tubular formation,renal tubular dilatation,brush edge shedding,interstitial fibrosis,glomerulosclerosis,EREX + UUO and ICA + UUO groups,and renal pathology improved significantly.Renal pathological damage score also showed that the degree of tubular and interstitial injury,arteriosclerosis and glomerulosclerosis in UUO group was higher than that in EREX + UUO and ICA + UUO group.HE staining showed that a large number of inflammatory cells infiltrated in the model group,and the degree of EREX + UUO and ICA + UUO groups was relatively mild.(3)Immunofluorescence showed that in sham-operated group,the expression of alpha-SMA was only in blood vessels,but in UUO group,G-15+UUO group and G-15+ICA+UUO group,the expression of interstitial was increased.Immunofluorescence of a-SMA and collagen I also showed that the fibrosis was aggravated after G-15 intervention with UUO.The degree of renal fibrosis in Erdingerxian Decoction group and icariin group was lighter than that in UUO group.(4)Immunohistochemical staining of GPR30 in kidney tissue showed brown-yellow staining.Compared with SHAM group,the expression of GPR30 in UUO group decreased significantly.(5)Real-time quantitative PCR and Western Blot showed that the expression of FN,c-Myc,total beta-catenin and phosphorylated beta-catenin(ser-552)in UUO groupwas significantly higher than that in SHAM group.The expression of FN,collagen type I,c-Myc,total beta-catenin and phosphorylated beta-catenin(ser-552)increased in GPR30 blocker group.Compared with UUO group,ICA +UUO,EDEX +UUO group decreased the expression of fibrosis in mice.Meanwhile,the expression of GPR30 in EDEX + UUO group was higher than that in ICA + UUO group and UUO group.(6)Immunohistochemical staining of GPR30 showed that in sham-operated group,GPR30 was mainly expressed in renal tubular epithelial cells.In the case of renal obstruction,the expression of GPR30 in renal tubular epithelial cells was significantly decreased after the renal tubule was hit,and the use of G-15(GPR30 antagonist)could aggravate the fibrosis of UUO.At the same time,immunofluorescence staining of renal tissue with alpha-SMA and type I collagen also indicated that the fibrosis was aggravated after G15 intervention with UUO.EDEX+UUO group and ICA+UUO group could improve renal fibrosis,while q PCR results also indicated that FN expression could be significantly increased in G15.Conclusion: 1.Erdianxian Decoction and icariin can increase the estradiol level of UUO renal fibrosis model mice,reduce the accumulation of fibrosis components such as FN,type I collagen,a-SMA,cmyc,and reduce the degree of renal pathological damage,thereby protecting renal function,increasing the expression of GPR30,regulating Wnt/beta-catenin signaling pathway,and delaying the process of renal fibrosis.2.GPR30 antagonists can synergistically aggravate the progress of fibrosis,suggesting that GPR30 may have an important protective effect on renal fibrosis,and blocking its expression may lead to aggravation of fibrosis.Meanwhile,icariin has a protective effect on renal fibrosis,possibly from GPR30,and interfering with renal fibrosis.3.EREX+UUO and ICA+UUO groups showed a significant trend of protein immunoblotting in mice,but there was no statistical significance,suggesting that the action time of drugs may be short,and further research is needed.更多还原