活性氧调控可溶性环氧化物水解酶表达增高在门静脉高压症大鼠体内的实验研究

目的 明确活性氧（reactive oxygen species,ROS）是否可在肝硬化门静脉高压症大鼠肝外环境中调控可溶性环氧化物水解酶（soluble epoxide hydrolase,sEH）表达,验证sEH对这一疾病模型肠系膜血管中肌源性反应的影响。方法 利用多道生物分析仪分别测定正常对照组大鼠、实验对照组大鼠（四氯化碳处理）及NAPDH抑制剂夹竹桃麻素（apocynin,Apo）干预的处理组大鼠门静脉压力;免疫印迹分析3组大鼠肠系膜动脉组织sEH、ROCK及p-moesin蛋白表达水平的变化;血管灌流系统测定各组大鼠肠系膜动脉血管的肌源性反应。多组间均数比较采用单因素方差分析,两两比较采用LSD-t检验。结果 （1）正常对照组、实验对照组和Apo处理组大鼠平均门静脉压力分别为（6.5±0.9）mmHg（1 mm Hg=0．133 kPa）、（15.9± 1.6）mmHg和（10.6±1.2）mm Hg,肝硬化门静脉高压症大鼠经Apo处理后,门静脉压力显著降低（P＜0.05）。 （2）与正常对照组比较,实验对照组大鼠肠系膜组织sEH蛋白表达水平明显增高（P＜0.05）,p-moesin表达显著降低（P＜0.01）,但Apo处理组sEH蛋白表达水平显著降低（P＜0.05）,p-moesin表达回复,但仍与正常对照组存在显著差异（P＜0.05）。（3）肌源性收缩在实验对照组大鼠显著降低（P＜0.05）,经过Apo干预后,肌源性收缩改善,血管恢复收缩活性。结论 四氯化碳致肝硬化门静脉高压症大鼠肝外环境sEH表达增高,与活性氧含量相关。使用NAPDH特异性抑制剂去除活性氧后,sEH表达减低,血管肌源性反应恢复,提示在肝硬化门静脉高压症中限制sEH作用可作为缓解氧化应激外的另一治疗方向。     

Interleukin 10 knockout increases renal fibrosis of ischemia-reperfusion injury model mice

Objective To study the effect of interleukin (IL)-10 knockout (IL-10-/-) on renal repair after renal ischemia-reperfusion injury in mice. Methods Eighteen IL-10-/- mice (KO) aged 8-10 weeks and 18 C57BL/6 wild type mice (WT) aged 8-10 weeks were divided into control group (Sham) and renal ischemia-reperfusion injury (IRI) group. The renal tissue morphology change was observed by Hematoxylin and eosin (HE) staining and Masson staining. The expressions of IL-18, Ki67 and TGF-β1 were detected by immunohistochemistry. The expression of TGF-beta1 and IL-18 were detected by Western blotting. Results Compared with that in WT-IRI group, in KO-IRI group renal pathological damage was more severe, renal interstitial fibrosis was visible, Ki67 expression of renal tubular epithelial cells decreased distinctly (P＜0.01), the expression of TGF-beta1 increased significantly (P＜0.01). Conclusion Repair slows down significantly after kidney ischemia-reperfusion injury and fibrosis occurs gradually in IL-10-/- mice, eventually progressing to chronic kidney disease.     

大黄蒽醌提取物缓解小鼠肾组织纤维化作用的实验研究

目的 通过观察大黄蒽醌提取物抑制小鼠病变肾组织纤维化的作用，探讨大黄治疗肾脏病的作用机制。方法 采用左侧输尿管结扎的方法建立单侧输尿管梗阻(UUO)雄性CD-1小鼠动物模型。用形态学半定量方法评价组织学病变；酸水解-比色法测定肾组织胶原的含量；蛋白印迹技术检测胶原α表达的水平。以α-平滑肌肌动蛋白(α-SMA)作为上皮细胞转分化的观察指标。结果 大黄提取物(50 mg/kg体重)能够显著地减少肾间质的病变，降低肾组织中胶原的聚积，与对照组相比两者差异均有统计学意义。大黄提取物(25、50 mg/kg体重)能够降低病变肾组织中胶原α的表达水平及减少梗阻肾组织中α-SMA的表达，抑制上皮细胞转分化。结论 大黄蒽醌化合物能够改善肾脏的纤维化，其作用可能与降低肾组织内胶原的沉积以及抑制上皮细胞转分化有关。     

罗格列酮对高血压大鼠肾间质纤维化的影响及其机制

目的探讨罗格列酮改善高血压肾间质纤维化（RIF）的机制。方法高血压大鼠随机分为阳性对照组（2K1C组）和罗格列酮治疗组（RGL组），另设假手术组（SHAM组），比较三组大鼠的尾动脉收缩压（SBP）、尿β-微球蛋白（β2-MG）、血肌酐（SCr）、尿素氮（BUN）、肾组织纤维化半定量评分以及BMP7、CTGF的表达及肾脏病理改变。结果①治疗前，2K1C组及RGL组的SBP与尿β2-MG均较SHAM组明显增加。自第4周起，RGL组SBP和尿β2-MG较2K1C组也明显降低。②2K1C组及RGL组RIF程度较SHAM组明显加重，RGL组较2K1C组减轻。③与SHAM组比较，2K1C组及RGL组BMP-7表达减少，CTGF的表达显著增加。④RGL组较2K1C组BMP7表达升高，CTGF的表达减少。⑤BMP-7及CTGF与RIF程度分别呈负相关和正相关。结论罗格列酮可能通过上调高血压大鼠肾问质BMP-7的表达、下调CTGF的表达，发挥对肾脏的保护作用。     

Fluorofenidone reduces renal fibrosis in diabetic kidney disease mice

Objective To investigate the effects of fluorofenidone (AKF-PD) on diabetic kidney disease in db/db mice and its possible mechanisms. Methods (1) Fifty-six mice aged 8 weeks (half male and half female), including 42 db/db mice and 14 wild-type mice were studied. Forty-two db/db mice randomly were divided into model group (mock-treated diabetic db/db mice), AKF-PD (250 mg?kg-1?d-1) treatment group and losartan (20 mg?kg-1?d-1) treatment group. Wild-type mice and model mice were treated with vehicle (0.5% sodium carboxymethylcellulose), while the treatment groups received either AKF-PD or losartan. After 18 weeks, the blood glucose and urinary albumin were measured, the pathological changes of kidney were observed by PAS staining. The protein expressions of type Ⅳ collagen and fibronectin (FN) in kidney tissue were detected by immunohistochemistry. (2) Mouse glomerular mesangial cells (MES-13 cells) were divided into six groups: normal glucose group (5.5 mmol/L glucose), hypertonic group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), AKF-PD group (25.0 mmol/L glucose+400 mg/L AKF-PD) and losartan group (25.0 mmol/L glucose+2 μmol/L losartan). After 72 h treatment, the expressions of type Ⅰ collagen, type Ⅳ collagen and transforming growth factor-β1 (TGF-β1) mRNA were detected by real-time PCR, and the content of TGF-β1 protein in the culture supernatant was detected by ELISA. Results (1) Compared with the wild type mice, model mice had increased weight, blood glucose and glomerulosclerosis index (all P＜0.01), accompanied with heavy albuminuria, glomerular hypertrophy, mesangial area expansion and deposition of collagen type Ⅳ and FN (all P＜0.01). Compared with model mice, in AKF-PD and losartan groups 24 h urinary albumin and glomerulosclerosis index decreased (all P＜0.01), glomerular hypertrophy and mesangial area expansion alleviated, and the protein expressions of collagen type Ⅳ and FN were inhibited (all P＜0.01). (2) Compared with the normal glucose group, the mRNA expressions of type Ⅰ collagen and type Ⅳ collagen increased in high glucose group, meanwhile the mRNA and protein expressions of TGF-β1 increased (all P＜0.01). In AKF-PD and losartan groups the expressions of type Ⅰ collagen, type Ⅳ collagen and TGF-β1 were inhibited as compared with high glucose group (all P＜0.05). Conclusion Fluorofenidone may play an anti-fibrotic effect in db/db mice by reducing the expression of TGF-β1 and inhibiting collagen synthesis in glomerular mesangial cells.     

蛋白酶激活受体2在小鼠肾间质纤维化中的表达及其意义

目的 观察单侧输尿管梗阻(UUO)小鼠模型中蛋白酶激活受体2(PAR-2)在肾小管间质中的表达部位、动态变化及其与肾间质纤维化的关系。方法 制备UUO小鼠模型,采用RT-PCR方法检测UUO术后第1、3、5、7、10、14、21天肾组织中PAR-2mRNA的表达;免疫组化方法检测PAR-2蛋白的表达。分析它与肾间质相对面积、α-平滑肌肌动蛋白(SMA)表达水平的关系。结果 UUO术后第1天肾小管间质可见少量PAR-2mRNA和蛋白的表达,第7-14天表达显著增加,其后维持在较高水平。PAR-2表达量与肾间质相对面积、α-SMA表达量成正相关。PAR-2表达主要定位于肾小管上皮细胞(尤其为近曲小管),也可见于肾毛细血管袢、间质浸润细胞和成纤维细胞。结论 PAR-2在肾小管上皮细胞和肾间质的表达可能参与了肾间质纤维化发生发展的过程。     

斯伐他汀对大鼠实验性肾间质纤维化的影响及其机制探讨

目的：研究羟甲基戊二酰辅酶A（HMG-CoA)还原酶抑制剂斯伐他汀（simvastatin)对大鼠肾间质成纤维细胞（RFB）和5/6肾切除动物模型的影响及其可能机制。方法：通过原代大鼠肾间质成纤维细胞培养和建立大鼠5/6肾切除慢性肾功能衰竭模型，应用甲基噻唑基四唑（MTT）比色法、放射性免疫组织化学，半定量逆转录-聚合酶链反应（RT-PCR）等技术，观察simvastatin对动物模型的防治作用及对RFB的影响。结果:simvastatin治疗组大鼠血脂、血肌酐、尿酸及体重明显下降，与模型对照组比较差异有显著性意义（P＜0.05)，血尿素氮变化不明显。治疗12周后，光镜观察治疗组大鼠肾间质纤维化的程度降低。simvastatin可抑制RFB的增殖活性（A值），减少RFB层粘连蛋白（LN）的合成，使c-fos mRNA的表达逐渐下降，呈一定的剂量依赖性，各浓度加药组与正常对照组比较差异有显著性意义（P＜0.05)。结论：simvastatin可通过抑制细胞增殖，减少细胞外基质的合成以及阻断与细胞增殖有关的c-fos依赖性有丝分裂途径等机制对实验性肾间质纤维化有一定的防治作用。     

The effect of adding carboxymethylcellulose and alginate to hyaluronic acid on reducing epidural fibrosis in a lumbar laminectomized rat model

Objective:This study aimed to compare the anti-epidural fibrosis and anti-inflammation effects of hyaluronic acid (HA)-carboxy­methylcellulose (CMC)-alginate hydrogel, pure HA, and normal saline using a lumbar laminectomized rat model.Methods:Thirty lumbar laminectomized adult rats were randomly assigned to three groups. The control group received normal saline, the HCA group received HA-CMC-alginate gel, and the HA group received pure HA gel soaked over the dura of the laminectomized area before closing the surgical wound. All rats were housed for eight weeks, then epidural fibrosis (EF) was histologically graded. In addition, the fibroblast and inflammatory cell density were computerized for evaluation.Results:The mean fibroblast densities were 32.03 × 10² ± 488, 13.22 × 10² ± 200, and 14.52 × 10² ± 368 cell/mm² in the control, HCA, and HA groups, respectively. The mean inflammatory cell density was 30.74 × 10² ± 459, 5.90 × 10² ± 129, and 11.08 × 10² ± 282 cell/mm² in the control, HCA, and HA groups, respectively. The mean fibroblast and inflammatory cell densities in the HCA and HA groups were significantly lower than in the control group (P < 0.05). The HCA group had a significantly lower inflammatory cell density than the HA group (P < 0.05). The fibrous adherence grading of HCA and HA was significantly lower than the control (P < 0.05).Conclusion:HA-CMC-alginate gel and HA hydrogels seem to have a better preventative effect on EF than no treatment (control). HA-CMC-alginate can exhibit a better anti-inflammatory effect than HA. HA-CMC-alginate can be effective in reducing EF and inflammation after lumbar laminectomy.     

冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化的抑制作用及机制

目的 探讨冬虫夏草菌粉对5/6肾大部切除术大鼠肾脏纤维化的抑制作用及其可能机制.方法 30只雄性SD大鼠随机分为3组:假手术组(Sham组,n=10)、5/6肾大部切除模型组(SNx组,n=10)以及5/6肾大部切除+冬虫夏草菌粉干预组(CS组,n=10).术前及术后4、8、12周分别检测大鼠体质量、尿蛋白量变化,并于术后第12周末处死大鼠,检测血尿素氮、肌酐变化,取肾组织切片行HE、Masson染色观察肾脏病理变化,免疫组化观察转化生长因子β1(TGF-β1)及其Ⅰ型受体(TβR Ⅰ)、Ⅱ型受体(TβR Ⅱ)的表达,免疫荧光观察E-c adherin、α-SMA的表达,Western印迹法检测肾脏组织TGF-β1、TβR Ⅰ、TβR Ⅱ、磷酸化(p)Smad2/3、Smad7、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)的表达.结果 术后CS组大鼠的体质量高于SNx组,尿蛋白量及血尿素氮、血肌酐低于SNx组.肾脏组织病理分析显示,CS组肾小球硬化、肾小管间质损伤程度均显著低于SNx组(均P＜0.01).CS组TGF-β1、TβR Ⅰ、TβR Ⅱ、p-Smad2/3蛋白表达量均显著低于SNx组(均P＜0.05),E-cadherin蛋白表达量显著高于SNx组(P＜0.05),α-SMA蛋白表达量显著低于SNx组(P＜0.05),Smad7蛋白表达量显著高于SNx组(P＜0.05).结论 冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化具有明显的抑制作用,其机制可能是与其抑制TGF-β1及其下游信号通路以及抑制EMT的发生有关.     

miR-210 agonist alleviates renal inflammatory response and fibrosis in diabetic kidney disease rats

Objective To investigate the protective effect and mechanism of microRNA-210 agonist (agomiR-210) on kidney in diabetic kidney disease (DKD) rats. Methods Thirty-six 5-week-old male SD rats were divided into normal control (NC) group, agomiR-NC control group, agomiR-210 control group, DKD model group, DKD+agomiR-NC group and DKD+agomiR-210 group, with 6 rats in each group. Diabetic rats were established by a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ), then were fed for 12 consecutive weeks to construct DKD model rats. During 2nd-4th week of continuous feeding, the rats in DKD+agomiR-210 group were injected with 20 nmol/kg agomiR-210 via tail vein twice a week. Blood glucose levels, 24 h urine albumin (Alb) and 24 h urine microalbumin (MAU) contents were measured regularly. At the end of the 12th week, the rats were sacrificed, and renal tissues were collected. The renal histopathological changes were assessed by HE, PAS and Masson staining methods. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in renal tissues were detected by RT-qPCR and Western blot. The distributions and expressions of α-smooth muscle actin (α-SMA), typeⅠ collagen (Col-Ⅰ), type Ⅳ collagen (Col-Ⅳ) and fibronectin (FN) in renal tissues were detected by immunohistochemical method. The protein expression levels of phospho(p)-Smad3 and p-NF-κB p65 in renal tissues were detected by Western blot and immunohistochemical methods. Results Compared with DKD model group, the renal pathological damages in DKD+agomiR-210 group were improved, the blood glucose level, glycogen deposition and collagen accumulation were significantly decreased (all P＜0.05), the urinary excretions of Alb and MAU were significantly reduced (all P＜0.01), and the expressions of TNF-α, IL-1β, IL-6, α-SMA, Col-Ⅰ, Col-Ⅳ, FN, p-Smad3 and p-NF-κB p65 in renal tissues were significantly decreased (all P＜0.01). Conclusion AgomiR-210 can alleviate renal pathological changes and urinary Alb and MAU excretion in rats with DKD, which may be related to its inhibition of Smad3 and NF-κB activity.     

Inhibition of Src kinase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of Src kinase in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), sham operation+PP2 group (n=8), UUO operation group (n=8) and UUO operation+PP2 group (n=8). The mice were injected 2 mg/kg PP2 by intraperitoneal everyday after surgery in sham+PP2 group and UUO+PP2 group. PP2 dissolved in 1% DMSO (formulated with normal saline). Sham and UUO group were given equal 1% DMSO. The mice were sacrificed at 7th day. Renal collagen was observed with Sirius red stain. The activities of Src, protein kinase B (PKB, AKT), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and the protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting. The expression of collagen I (COLⅠ) was detected by immunohistochemistry and the expressions of matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6) were measured by ELISA. Results Compared with sham mice, UUO mice on 7th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of COLⅠ and FN, and activities of AKT, ERK and p38 MAPK (all P＜0.05). Their renal expressions of α-SMA, TGF-β1, MMP-9, TIMP-1, MCP-1 and IL-6 were also raised (all P＜0.05). Compared with those in UUO group, in UUO+PP2 group the activities of Src, AKT, p38 MAPK and ERK, and expressions of TGF-β1, MCP-1 and IL-6 decreased (all P＜0.05). Additionally, expressions of COLⅠ, FN and α-SMA, collagen deposition and renal fibrosis receded in UUO+PP2 group (all P＜0.05). However, the expressions of MMP-9 and TIMP-1 were not influenced by PP2 treatment. Conclusions Src kinase promotes myofibroblasts accumulation and inflammatory reaction through activating its downstream signaling pathway in the progressing of renal interstitial fibrosis.     

冠状动脉搭桥术后血浆可溶性Fas及其配体的变化

目的比较在心肺转流（CPB）或非CPB下冠状动脉搭桥术（cABG）后可溶性Fas（sFas）和可溶性Fas配体（sFasL）的血浆浓度变化。方法19例病人分别在CPB（CPB组，n=9）或非CPB（非CPB组，n=10）下行择期cABG。术前、术毕和术后取血测定白细胞介素-6（IL-6）、中性粒细胞弹性蛋白酶、sFas、sFasL血浆浓度。结果术毕、术后4h的IL-6和术毕、术后4、12h的中性粒细胞弹性蛋白酶非CPB组明显低于CPB组（P〈0．05或P〈0．01）。CPB组的sFas在术后4、12h明显升高（P〈0．05或P〈0．01），非CPB组在术后12h明显升高（P〈0．01），术后24h恢复至术前水平。两组的sFasL在术毕和术后4、12h均明显升高（P〈0．05或P〈0．01），术后24h恢复至术前水平；其中术后12h非CPB组明显低于CPB组（P〈0．05）。结论CPB或非CPB下行cABG均导致sFas和sFasL血浆浓度升高，但CPB的应用使sFasL血浆浓度升高得更多。sFasL血浆浓度可反映机体炎性反应的程度。     

灌注固定和浸润固定制作肾纤维化模型积水肾标本效果比较

目的 比较灌注固定和浸润固定制作肾纤维化模型积水肾标本的效果.方法 单侧输尿管结扎法成功制作小鼠肾纤维化模型后,分别采用灌注固定及浸润固定制作肾标本,HE染色观察肾组织病理学改变.结果与结论 浸润固定法制作的肾脏标本组织器官的结构更加清晰,能较好地显示早期肾纤维化的病理改变,是值得推广的动物实验技术.     

巨噬细胞在移植肾纤维化中的作用研究进展

缺血-再灌注损伤、排斥反应、钙调磷酸酶抑制剂造成的肾毒性等因素会在肾移植术后使肾细胞外基质过度积聚,逐渐造成移植肾纤维化,最终导致肾衰竭。近年来,巨噬细胞在移植肾纤维化中的作用机制逐渐受到关注,有研究表明哺乳动物雷帕霉素靶蛋白抑制剂等药物可以通过巨噬细胞途径减缓肾移植术后移植肾纤维化。本文就移植肾纤维化的主要病因及病理生理学机制、不同巨噬细胞在移植肾纤维化进展中的作用、外周募集巨噬细胞和肾驻留巨噬细胞对肾损伤区域的浸润、巨噬细胞对肌成纤维细胞的诱导作用及巨噬细胞相关的移植肾纤维化潜在治疗方案进行综述,以期为巨噬细胞在移植肾纤维化中的研究提供参考。     

DJ-1癌基因在肾间质纤维化中表达的实验研究

目的 观察DJ-1癌基因在肾纤维化过程中表达水平、细胞内定位及高表达 DJ-1基因对人肾小管上皮细胞E钙黏蛋白（E-cadherin）、波形蛋白（vimentin）蛋白表达和β连环素（β-catenin）酪氨酸磷酸化水平的影响。 方法 体外实验以人肾小管上皮细胞为研究对象,10 μg/L TGF-β1刺激72 h诱导人肾小管上皮细胞转分化;Western印迹法检测正常组和TGF-β1干预组细胞内E-cadherin、vimentin和DJ-1蛋白表达;RT-PCR法检测两组细胞内DJ-1 mRNA表达水平;应用激光共聚焦显微镜观察DJ-1在细胞内的定位。体内实验以SD大鼠为研究对象,5/6肾切除法制作慢性肾纤维化模型;常规检测BUN和Scr水平;Masson染色检测肾组织纤维化水平;免疫组化法检测肾组织内DJ-1蛋白的表达和分布。脂质体法介导含野生型DJ-1基因的重组真核表达质粒pEGFP-N1-DJ-1或空载体转染人肾小管上皮细胞,倒置荧光显微镜和Western 印迹法鉴定转染效率后,Western 印迹法检测正常组、pEGFP-N1-DJ-1转染组和空载体转染组细胞内E-cadherin、vimentin蛋白的表达及各组β-catenin酪氨酸磷酸化水平。 结果 正常组细胞表达E-cadherin和DJ-1,几乎不表达vimentin;TGF-β1干预组细胞表达vimentin,表达较少E-cadherin,DJ-1 mRNA和蛋白表达均较正常组细胞显著增多（P < 0.05）。DJ-1在正常细胞内大多分布在细胞质,部分分布在细胞核;在转分化细胞内胞质和胞核内表达均有增加,且胞核内增加更显著。假手术组大鼠肾功能正常,肾组织内未见纤维组织,DJ-1主要表达于肾小管,肾小球内几乎没有表达。模型组大鼠肾功能不全,肾组织内可见明显纤维化结构,肾小管内DJ-1表达明显增加。pEGFP-N1-DJ-1转染组较正常组和空载体转染组细胞内DJ-1和vimentin蛋白表达明显增加且β-catenin酪氨酸磷酸化水平升高, 而 E-cadherin蛋白表达减少。 结论 DJ-1基因高表达可能促进了肾间质纤维化。     

大剂量螺内酯对自发性高血压大鼠肾脏纤维化的影响

目的 观察大剂量螺内酯对自发性高血压大鼠（SHR）肾脏纤维化的影响。 方法 8周龄的雄性SHR 24只随机分为低剂量和大剂量螺内酯干预组[分别为20和100 mg&#8226;kg-1&#8226;d-1螺内酯灌胃]和高血压对照组，同时设同源正常对照组京都大鼠（WKY）8只。干预8周，检测收缩压、尿蛋白、血白蛋白、钾、钠、Scr和肾组织及血浆醛固酮水平。肾组织切片分别行HE和Masson染色，以评价肾小球损伤及肾小球内胶原沉积情况。免疫组化SABC法检测肾组织TGF-β1和醛固酮受体蛋白表达。RT-PCR检测肾组织TGF-β1和醛固酮受体mRNA水平。 结果 与高血压组大鼠相比，低剂量螺内酯干预后，尿蛋白减少（P < 0.05），血白蛋白升高（P < 0.05），血浆和肾组织醛固酮水平降低，但差异无统计学意义；大剂量螺内酯干预后，血压没有显著改变，尿蛋白显著升高[（27.3±4.5）比（24.5±3.2） mg/d， P < 0.05]，血白蛋白显著减少[（20.2±4.2）比（22.7±3.5） g/L, P < 0.05]，血浆和肾组织醛固酮水平显著升高[肾组织（28.3±1.5）比（22.2±0.6） ng/g， P < 0.05]。与高血压组比较，低剂量螺内酯干预后，蛋白管型增多、管周炎性细胞浸润均减少（P < 0.05）；大剂量螺内酯干预后，蛋白管型、小管扩张加重，管周炎性细胞浸润明显增多（P < 0.05），肾小球内胶原形成亦明显增多（P < 0.05）。与高血压组大鼠比较，低剂量螺内酯干预后，肾组织醛固酮受体mRNA和蛋白表达均无显著改变，TGF-β1 mRNA和蛋白的表达显著减少（P < 0.05）；大剂量螺内酯干预后，肾组织醛固酮受体及TGF-β1 mRNA和蛋白的表达均显著升高(P < 0.05)。 结论 大剂量螺内酯可以加重高血压肾脏纤维化，可能是通过上调醛固酮及其受体表达实现的。     

Effects of pirfenidone on renal fibrosis in mice with diabetic nephropathy and its mechanisms

Objective To investigate effects of pirfenidone (PFD) on diabetic nephropathy model in db/db mice and to explore its possible mechanisms. Methods (1) Wild-type mice were as the normal control group, and db/db mice were divided into model group and PFD group, with 6 mice in each group. In the PFD group mice were administered continuously by 250 mg?kg-1?d-1 PFD for 18 weeks, and mice in the other two groups were administered by 0.5% sodium carboxymethyl cellulose. Blood glucose and 24 h urinary albumin were measured. The pathological changes of renal tissue were evaluated by PAS staining, PASM staining, Masson staining and Sirius red staining. The expression of collagen type Ⅳ in kidney tissues was detected by immunohistochemistry. (2) Mouse mesangial cells (SV40 MES-13 cells) were cultured as research objects. They were divided into control group, hyperosmolar group, high glucose (HG) group, and 50, 100, 200, 400, 800, 1600 mg/L PFD+HG group. BrdU cell proliferation test was used to evaluate cell proliferation rate. Cells were divided into control group, hyperosmolar group, HG group and PFD+HG group. The mRNA expressions of α-smooth muscle actin (α-SMA), collagen type Ⅰ, collagen type Ⅳ, transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) were detected by real-time PCR. Results (1) Compared with normal control group, the model mice had higher weight, blood glucose and 24 h urinary albumin, accompanied with glomerular hypertrophy, mesangial area expansion, tubulointerstitial fibrosis and deposition of collagen type Ⅳ (all P＜0.05). Compared with those in model group, in PFD group 24 h urinary albumin decreased, glomerular hypertrophy, mesangial area expansion and tubulointerstitial fibrosis alleviated, and the protein expression of collagen type Ⅳ inhibited (all P＜0.05). (2) Compared with those in HG group, MES-13 cell proliferation rates of 100, 200, 400, 800, 1600 mg/L PFD+HG groups decreased (all P＜0.05), and the mRNA expressions of α-SMA, collagen type Ⅰ, collagen type Ⅳ, TGF-β1, IL-1β, IL-6 and MCP-1 reduced in 400 mg/L PFD+HG group (all P＜0.05). Conclusions PFD can inhibit high glucose-induced proliferation and activation of glomerular mesangial cells, decrease the expression of TGF-β1 and proinflammatory factors, as well as reduce the synthesis of collagen, which improve renal fibrosis of db/db mice.     

支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及其机制探讨

Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO), and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy. Methods jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups: jlp+/+- and jlp-/--sham-operated groups(jlp-/--sham group and jlp+/+-sham group), jlp+/+- and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp-/--UUO group and jlp+/+-UUO group). Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining.The expression of JLP in jlp+/+renal tissue was assayed by immunohistochemistry staining, immunofluorescence and Western blotting. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), collagen Ⅰ(COL-Ⅰ), collagen Ⅲ(COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups. Besides, the α-SMA, COL-Ⅰ, COL-Ⅲ, TGF-β1, p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups. Results The expression of JLP was mainly demonstrated in the renal tubules of mice. A large amount of collagen deposition was observed in the renal interstitial area in jlp-/--UUO group compared to jlp+/+-UUO group. Similarly, the expression of α-SMA, COL-Ⅰ, COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp-/-- UUO-operated groups. Meanwhile, Western blotting showed that the expression of α-SMA, COL-Ⅰ, COL-Ⅲ, and TGF-β1 protein was obviously higher in jlp-/--UUO group. Moreover, the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/--UUO group. Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.     

肾清饮对腺嘌呤致大鼠肾间质纤维化的防治作用

目的观察肾清饮对大鼠肾问质纤维化的影响,并探讨其机制。方法将56只大鼠随机分为正常对照组（C组）、模型组（M组）和药物干预组,药物干预组包括肾清饮预防组（S组）、肾清饮低剂量组（SL组）、肾清饮高剂量组（SH组）、苯那普利组（B组）、肾清饮＋苯那普利组（S＋B组）,每组8只。对M组和药物干预组大鼠采用腺嘌呤灌胃法建立肾间质纤维化动物模型。M组给予腺嘌呤灌胃,第4～6周给予蒸馏水灌胃;S组前3周同时给予腺嘌呤及肾清饮（0．4ml／d）灌胃,每毫升肾清饮含生药2．4g,第4～6周给予肾清饮（0．4ml／d）灌胃;SL组和SH组前3周给予腺嘌呤灌胃,第4～6周给予肾清饮灌胃,剂量分别为0．4ml／d和1．2ml／d;B组前3周给予腺嘌呤灌胃,第4～6周给予盐酸苯那普利灌胃,剂量为10mg·kg^-1·d^-1;S＋B组前3周给予腺嘌呤灌胃,第4～6周同时给予盐酸贝那普利（10mg·kg^-1·d^-1）及肾清饮（0．4ml／d）灌胃。比较各组大鼠血肌酐（SCr）、尿素氮（BUN）、24h尿蛋白定量、血浆内皮素1（ET-1）水平、肾组织病理学表现、问质纤维化指数评分及肾组织Ⅰ型胶原、Ⅲ型胶原和转化生长因子β1（TGF-β1）mRNA表达。结果与C组相比,M组SCr、BUN、24h尿蛋白定量和血浆ET-1水平及肾组织Ⅰ型胶原、Ⅲ型胶原和转化生长因子B1（TGF-β1）mRNA表达显著升高（P〈0．01）;与M组相比,各药物干预组SCr、BUN、24h尿蛋白定量和血浆ET-1水平及肾组织Ⅰ型胶原、Ⅲ型胶原和TGF-β1 mRNA表达显著降低（P〈0．01）,肾间质纤维化程度减轻（P〈0．05）;各药物干预组之间相比,s组SCr、BUN、24h尿蛋白定量和血浆ET-1水平及肾组织Ⅰ型胶原、Ⅲ型胶原和TGF-131mRNA表达降低最显著（P〈0．01）。结论肾清饮可延缓肾间质纤维化,减少尿蛋白,改善肾功能。