改良地黄散抑制TGF-β1-CTGF信号通路改善肾间质纤维化的研究
Effect of Modifie Drehmanniae Prescription on TGF-β1-CTGF Signaling Pathway to Improve Renal Interstitial Fibrosis
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摘要: 目的 研究改良地黄散对部分输尿管梗阻(PUUO)大鼠肾间质纤维化作用以及探讨其可能的机制。方法 80只SD大鼠,随机均分为对照组、模型组、低剂量组、高剂量组,每组20只。高低剂量组、模型组采用腰大肌管包埋法建立PUUO模型,对照组仅切除左侧肾。高剂量组(2 mL/d)和低剂量组(1 mL/d)予以改良地黄散煎剂灌胃,对照组和模型组予均给予等量生理盐水灌胃。分别于建模后4周、8周每组取10只采集血、尿样本,测定各组大鼠血清肌酐(SCr)、尿素氮(BUN)、24 h尿蛋白定量。测量肾组织大小、肾皮质厚度。制备肾组织切片,通过HE染色、Masson染色,观察各组肾脏病理组织学改变,以及肾脏纤维化情况。采用免疫组织化学法检测各组大鼠转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)、α-平滑肌肌动蛋白(α-SMA)在肾组织的表达情况;采用qPCR检测TGF-β1、CTGF mRNA表达。结果 4周时:模型组相大鼠炎性细胞浸润,局部灶状萎缩坏死,较多胶原纤维化形成。高低剂量组较模型组病理改变不明显,肾间质增宽仅有少许间质纤维化。8周后:模型组淋巴细胞浸润,肾小球坏死,结构彻底改变。间质纤维增生网状成片。高低剂量组较模型组炎性细胞浸润较轻,胶原染色较轻淡,高剂量组变化最小。TGF-β1、CTGF、α-SMA蛋白平均阳性表达率两个药物治疗组与模型组相比,4周时低剂量组均无差异,8周时两个药物治疗组均有显著差异( P <0.01)。比较TGF-β1、CTGF mRNA表达时发现,低剂量组4周时与模型组相比均无差异,8周时高剂量组与模型组相比均有差异( P <0.05)。结论 改良地黄散可能通过影响TGF-β1-CTGF信号通路,下调α-SMA的阳性表达,减轻肾纤维化的病理变化,延缓肾纤维化的发展。Abstract: OBJECTIVE To study the effect of modified Rehmannia Recipe on renal interstitial fibrosis in rats with partial ureteral obstruction (PUUO) and to explore its possible mechanism. METHODS Eighty SD rats were randomly divided into control group, model group, low dose group and high dose group ( n =20). In the model group, the model of PUUO was established by lumbar large muscle tube embedding method. Only the left kidney was removed in the control group.High dose group (2 mL/d) and low dose group (1 mL/d) were treated with Modified rehmannia decoction, the control group and the model group were given the same amount of normal saline. The levels of serum creatinine (SCr), urea nitrogen (BUN) and 24 h urinary protein were determined in each group. Blood samples were collected from each group at 4th week and 8th week after modeling. Measurement of renal tissue size, renal cortical thickness. The renal histopathological changes were observed by staining with HE and Masson, and the renal fibrosis was observed. The expressions of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) in renal tissue were detected by immunohistochemistry. The expression of TGF-β1 and CTGF mRNA was detected by qPCR. RESULTS At 4th week, inflammatory cell infiltration, local focal necrosis and necrosis were observed in the model group, and more collagen fibrosis was formed. Compared with the model group, the pathological changes of the high and low dose group were not obvious, and only a small interstitial fibrosis was widened. After 8 weeks: model group lymphocyte infiltration, glomerular necrosis, the structure completely changed. Interstitial fibrous hyperplasia. In the high and low dose group, the infiltration of inflammatory cells was lighter than that of the model group, the staining of the collagen was lighter and the high dose group had the smallest change. The average positive expression rate of TGF-β1, CTGF and α-SMA protein was not significantly different between the two groups at 4th week compared with the model group. At the 8th week, the two drug treatment groups had Significant difference ( P <0.01). Comparison of TGF-β1, CTGF mRNA expression was found when the low-dose group 4 weeks with model group had no differences as compared to high dose group and the model group were different ( P <0.05) as compared to 8th week. CONCLUSION Modified rhubarb may reduce the expression of TGF-β1-CTGF and down-regulate the expression of α-SMA, reduce the pathological changes of renal fibrosis and delay the development of renal fibrosis.