Losartan Alleviates Renal Fibrosis by Down-Regulating HIF-1α and Up-Regulating MMP-9/TIMP-1 in Rats with 5/6 Nephrectomy

Background: This study investigated the effects of losartan intervention on the expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in renal fibrosis in rats with 5/6 nephrectomy. Methods: Sprague Dawley rats were randomly divided into three groups. Rats in the losartan group were gavaged with losartan (33.3 mg/kg/day) from 1 week after 5/6 nephrectomy, and those in the sham group and the model group only received an equal volume of saline solution by gavage. Rats were sacrificed at the ends of the 4, 8 and 12 weeks, respectively. Urinary N-acetyl-glucosaminidase (NAG), 24-h urinary protein, serum cystatin C, blood urea nitrogen (BUN), and serum creatinine (Scr) levels were assessed. Kidney tissues were observed under light and electron microscope. The expressions of HIF-1α, transforming growth factor-β1 (TGF-β1), MMP-9, and TIMP-1 were determined by immunohistochemistry and Western blotting. Results: Twenty-four hour urinary protein, urinary NAG, serum cystatin C, BUN, and Scr levels in the model group were significantly higher than those in the sham group (p < 0.05), but losartan treatment improved these changes. The apparent glomerular sclerosis and tubulointerstitial fibrosis were also found in the model group, which were ameliorated by losartan. The expressions of HIF-1α, TGF-β1, MMP-9, and TIMP-1 were elevated and MMp-9/TIMP-1 ratio was lowered in the model group (p < 0.05), but losartan increased the expression of MMP-9 and MMp-9/TIMP-1 ratio (p < 0.05) and lessened the expressions of HIF-1α, TGF-β1, and TIMP-1 (p < 0.05). Conclusion: Losartan may ameliorate renal fibrosis partly by down-regulating HIF-1α and up-regulating MMP-9/TIMP-1 in rats with 5/6 nephrectomy.     

miRNA let-7al抑制前列腺癌PC-3细胞中IGF1R的表达

miRNAlet-7al表达减少和IGF1R信号通路激活都与前列腺癌的发生发展密切相关,两者之间在肿瘤中的表达关系尚未明确。我们最近研究发现,在前列腺癌PC-3细胞中,let-7al可直接靶向调控IGFIR的表达,抑制细胞增殖。首先通过TargetScan分析,发现IGF1R是let-7al的靶基因之一,在它的3’非翻译区存在3个let-7al的靶点（T1、T2、T3）。采用实时定量PCR、免疫印迹和荧光素酶报告基因检测技术检测Pc．3细胞中let-7al对IGF1R基因表达的影响。结果显示,let-7al可直接作用于IGF1R mRNA的3’非翻译区中的T1和T2靶点,靶向抑fNIGFIR基因的表达。进一步采用RT-PCR、荧光素酶报告基因检测、MTT、流式细胞术和Hoechst 33342染色技术,检测let-7al导致的IGF1R表达下调对IGF1R信号通路的影响。结果表明,let-7al下调IGF1R表达可使E1k1活化减少、其靶基因c-los表达降低、细胞增殖抑制、凋亡增强和细胞周期阻滞;反之,抑制let-7al可使IGF1R表达上调,伴有E1k1活性增强、c-fos表达增加、细胞增殖加快。以上结果提示,miRNA let-7a有可能成为前列腺癌治疗的新靶点。     

Allopurinol Protective Effect of Renal Ischemia by Downregulating TNF-α, IL-1β, and IL-6 Response

Allopurinol is a well-known antioxidant that protects tissue against ischemia and reperfusion injury, blocking purine catabolism, and possibly reducing TNF-α and other cytokines. It also plays a significant role in reducing the inflammatory processes by inhibiting chemotaxis and other inflammatory mediators. The objective of this study was to define the role of allopurinol regarding kidney ischemic injury particularly as to its effect on inflammatory molecules such as TNF-α, IL-1β, and IL-6 response. One hundred and twenty five rats were subjected to warm renal ischemia. Five more animals were included as sham. Animal survival and plasma levels of lipid peroxidation, myeloperoxidase, lactate dehydrogenase, glutathione, urea, creatinine, and cytokines were determined. Inflammatory parameters (TNF-α, IL-1β, and IL-6) were measured in all groups by quantitative immunosorbent assay. Further, immunohistological and histopathological studies were carried out on animals treated prior to, or following reperfusion with 10 and 50 mg/kg of Allopurinol. The statistical analysis included ANOVA and Fisher test as well as χ² test. Significance was reached at a p < 0.05. The results of this study indicated that Allopurinol protected against kidney ischemia–reperfusion injury since significantly better results of survival, biochemical analysis, and histopathological testing were observed in treated animals as compared to ischemic controls. In conclusion, Allopurinol protected ischemic kidneys through a mechanism associated with downregulation of TNF-α, IL-1 β, and IL-6, in addition to other well-known effects such as decreased lipid peroxidation and neutrophil activity. It also increased antioxidant capacity and diminished endogenous peroxidase stain in renal ischemic tissue. Therefore, this experiment showed an effectiveness of allopurinol protection against proteomic and morphological damage.     

肝细胞癌中Smad7和TGFβRⅠ的表达及与其临床病理的关系

目的　研究Smad7、TGFβRⅠ蛋白在肝细胞癌 (hepatocellularcarcinoma ,HCC)中的表达及与其临床病理的关系 ,探讨Smad7在HCC发生、发展中的作用。方法　应用SP免疫组化染色法 ,检测 62例HCC中Smad7、TGFβRⅠ蛋白表达情况 ,并分析它们与HCC临床病理的关系。 结果　 62例HCC组织中Smad7、TGFβRⅠ蛋白的阳性表达率分别为 85 .48%、2 9.0 3 % ,Smad7表达明显高于正常肝组织 ,而TGFβRⅠ表达明显低于正常肝组织 (P <0 .0 5 ) ,两者在HCC中表达呈负性相关。且Smad7蛋白表达与肿瘤的临床分期及有无瘤栓、包膜完整性、复发时间长短、血AFP、肿瘤结节数及有无转移有相关性。结论　Smad7与HCC发生、发展密切相关。     

All-Trans Retinoic Acid Attenuates the Renal Interstitial Fibrosis Lesion in Rats but Not By Transforming Growth Factor-β1/Smad3 Signaling Pathway

All-trans retinoic acid (ATRA) is an important therapeutic agent for prevention of the renal diseases. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway is a key signaling pathway which takes part in the progression of renal interstitial fibrosis (RIF). This investigation was performed to study the effect of ATRA in RIF rats and its effect on the TGF-β1/Smad3 signaling pathway. Sixty Wistar male rats were divided into three groups at random: sham operation group (SHO), model group subjected to unilateral ureteral obstruction (GU), model group treated with ATRA (GA), n = 20, respectively. RIF index, protein expression of TGF-β1, collagen-IV (Col-IV) and fibronectin (FN) in renal interstitium, and mRNA and protein expressions of Smad3 in renal tissue were detected at 14-day and 28-day after surgery. The RIF index was markedly elevated in group GU than in SHO group (p < 0.01), and the RIF index of GA group was alleviated when compared with that in GU group (p < 0.01). Compared with in group SHO, the mRNA/protein expression of Smad3 in renal tissue was significantly increased in group GU (p < 0.01). However, the mRNA and protein expressions of Smad3 in renal tissue in GA group were not markedly alleviated by ATRA treatment when compared with those in GU (each p > 0.05). Protein expressions of TGF-β1, Col-IV, and FN in GU group were markedly increased than those in SHO group (each p < 0.01), and their expressions in GA group were markedly down-regulated by ATRA treatment than those of GU group (all p < 0.01). The protein expression of Smad3 was positively correlated with RIF index, protein expression of TGF-β1, Col-IV or FN (each p < 0.01). In conclusion, ATRA treatment can alleviate the RIF progression in UUO rats. However, ATRA cannot affect the signaling pathway of TGF-β1/Smad3 in the progression of RIF.     

Exogenous bFGF or TGFβ1 accelerates healing of reconstructed dura by CO2 laser soldering in minipigs

This study aims to explore the probable mechanism of better result of dural reconstruction by CO₂ laser soldering and the effect of exogenous basic fibroblast growth factor (bFGF) or transforming growth factor-beta1(TGFβ₁) on wound healing. In part I of the study, ten minipigs were randomized into two equal groups, and the dural defects were reconstructed by conventional fibrin glue (FG) bonding (group I _a) or by CO₂ laser soldering (group I_b). In part II, 36 minipigs were randomized into three equal groups, and the dural defect was reconstructed by CO₂ laser soldering; then exogenous bFGF or TGFβ₁ was administered in group II_b and group II_c, respectively, while group II_a served as control group. The dural specimens were harvested at 1st week postoperatively in part I; and at 1st, 2nd, 3rd, and 4th week postoperatively in part II, they were examined for healing condition and subjected to hematoxylin–eosin (HE) staining and immunohistochemical (IHC) staining with antibodies against bFGF and TGFβ₁. In part I, group I_b showed higher fibroblast cell density than group I_a (P?<?0.05). The optical density (OD) for IHC staining with antibodies against bFGF of group I_b was significantly higher than that of group I_a (P?<?0.05), and for IHC staining with antibodies against TGFβ₁, group I_b showed positive staining while group I_a was negative. In part II, administering exogenous bFGF or TGFβ₁ made a left shift of fibroblast cell number–time curve compared with control group. For specimens' IHC staining with antibodies against bFGF, the OD of group II_b was higher than that of group II_a in the corresponding time. For specimens' IHC staining with antibodies against TGFβ₁, the OD of groups II_b and II_c was both higher than that of group II_a (P?<?0.05 and P?<?0.01, respectively). In conclusion, CO₂ laser may trigger fibroblast proliferation through stimulating the secretion of bFGF and TGFβ₁. Topically administering exogenous bFGF or TGFβ₁ could accelerate the healing of the reconstructed dura by enhancing secretion of bFGF and/or TGFβ₁ and promoting the process of fibroblast gathering–degrading.     

Distribution of intracellular and extracellular expression of transforming growth factor—β1（TGF—β1） in human testis and their association with spermatogenesis

AIM: Spermatogenic dysfunction may result from thickening of seminiferous tubular basement membrane (BM) with tubular sclerosis. Transforming growth factor beta1 (TGF-beta1) plays an important role in fibrogenesis. The intracellular and extracellular expression of TGF-beta1 in the testis were immunohistochemically determined, using LC antibody (LC) for intracellular TGF-beta1 and CC antibody (CC) for extracellular TGF-beta1. METHODS: Twenty-three testicular biopsy specimens were obtained from varicocele and five from Sertoli-cell-only (SCO) patients, and five from normal volunteers. The relative area involved by the expression of TGF-beta1 for CC or LC (TGF-beta1 index for CC or LC) was examined, and semen parameters and serum hormonal levels and TGF-beta1 were analyzed. The Johnson score (JS), the BM thickness, and the tubular diameter were also determined. RESULTS: Immunoreactivity for CC was hardly detected. That for LC was detected in the Sertoli and germ cells. The TGF-beta1 index for LC was significantly higher in the varicoceles than in the normal testes. Interestingly, that for LC was significantly higher in the varicoceles than in the SCO. The level of serum TGF-beta1 was significantly higher in varicoceles than in the normal testes. CONCLUSION: The distribution of the intracellular and extracellular expression of TGF-beta1 in human testis was demonstrated. It suggests that TGF-beta1 is related to fibrosis of seminiferous tubules and may lead to spermatogenic disruption.     

Study of Transforming Growth Factor-β1 Gene,mRNA, and Protein in Japanese Renal Transplant Recipients

Background

Transforming growth factor (TGF)-β1 may contribute to chronic allograft nephropathy and graft loss; however, the exact molecular mechanism remains unclear. Therefore, we assess the relationship between TGF-β1 gene polymorphisms, expression, and development of allograft nephropathy.

Methods

We studied 135 renal transplant recipients at our hospital. TGF-β1 gene polymorphisms (codons 10 and 25) were determined from peripheral blood leukocyte DNA. Plasma TGF-β1 mRNA was measured by real-time polymerase chain reaction and TGF-β1 protein levels were assessed by enzyme-linked immunosorbent assay. The relationship between TGF-β1 genotyping, expression, and rejection and results of renal biopsy were evaluated.

Results

The genotype frequency of transplant recipients was 49.6%, 30.4%, and 20.0% for C/T, C/C and T/T at codon 10, 100% for G/G at codon 25, respectively. According to the criteria of Banff ‘97 classification, 24 cases were classified as acute rejection and whose genotypes were 16, 3, and 5 cases for C/T, C/C and T/T at codon 10. Plasma mRNA expression was elevated in 14 cases and decreased in 8 cases after acute rejection. We measured 267 specimens of TGF-β1 protein and there was no relation between amount of TGF-β1 protein and mRNA.

Conclusion

Our results suggest that the relationship between plasma TGF-β1 expression and the development of allograft nephropathy remains uncertain. Frequency of allograft rejection differ with TGF-β1 codon 10 genotypes and the high-risk genotype was different from the reports of other countries.     

microRNA-196b、microRNA-217与TGFβR1蛋白在胰腺导管腺癌组织中的表达及其意义

目的检测胰腺导管腺癌组织中微小RNA(microRNA,miR)-196b、miR-217与转化生长因子β受体1(transforming growth factorβreceptor 1,TGFβR1)蛋白的表达水平,并探讨其临床意义。方法前瞻性收集2014年3月到2017年3月期间在重庆医科大学附属第二医院行胰腺癌根治术的30例胰腺导管腺癌患者的组织标本(包括胰腺导管腺癌组织及其癌旁组织),利用实时荧光定量聚合酶链反应法检测miR-196b和miR-217的表达水平,采用Western blotting法检测TGFβR1蛋白的表达水平。结果胰腺导管腺癌组织中miR-196b和TGFβR1蛋白的表达水平均高于癌旁组织(P0.001),而miR-217的表达水平低于癌旁组织(P=0.001)。胰腺导管腺癌组织中miR-196b的表达水平与TGFβR1蛋白的表达水平呈正相关(r=0.803,P0.001),而miR-217的表达水平与TGFβR1蛋白的表达水平呈负相关(r=–0.839,P0.001)。结论胰腺导管腺癌组织中TGFβR1蛋白的表达可能同时受miR-196b和miR-217的双向调控,这种双向调控机制可能是制约胰腺导管腺癌发生和发展的重要机制之一,也可能是治疗胰腺导管腺癌的潜在靶点。     

山药多糖通过miR-98-5p/TGFβR1分子轴调控肝癌细胞凋亡的机制研究

目的探究山药多糖(CYPS)通过miR-98-5p/TGFβR1分子轴调控肝癌(HCC)细胞凋亡的分子机制。方法qPCR检测miR-98-5p在不同HCC细胞系中的表达情况,使用不同浓度的CYPS处理Huh-7细胞,CCK-8检测细胞增殖活力,Annexin V-FITC/PI流式细胞术检测细胞凋亡,Western blotting检测TGFβR1和细胞凋亡相关蛋白Caspase-3、Caspase-8、Bcl-2、Bax的表达,双荧光素酶报告基因系统验证miR-98-5p与TGFβR1的靶向关系。结果与人正常肝细胞HL-7702相比,miR-98-5p在HCC细胞系中表达升高(均P<0.05),且在Huh-7细胞中的表达高于其他HCC细胞(P<0.05);CYPS处理可明显抑制Huh-7细胞的增殖活力并诱导细胞凋亡(均P<0.05),且10-3 kg/L CYPS对细胞增殖活力的抑制作用比其他浓度明显。双荧光素酶报告基因实验证实,miR-98-5p靶向调控TGFβ1。与10-3 kg/L CYPS组相比,miR-98-5p mimics可下调10-3 kg/L CYPS对细胞增殖活力(P<0.05)的抑制作用和对细胞凋亡(P<0.01)的促进作用,CYPS+miR-98-5p mimics+pcDNA-TGFβR1组中细胞增殖活力与CYPS+miR-98-5p mimics组相比明显降低(P<0.05),细胞凋亡水平明显升高(P<0.01)。结论CYPS可下调miR-98-5p,促进TGFβR1的表达,抑制Huh-7细胞增殖并诱导细胞凋亡。     

肾衰泻浊丸对慢性肾衰竭大鼠BMP-7／Smads／TGF—β1信号转导通路的影响

目的：通过实验研究观察肾衰泻浊丸对腺嘌呤致慢性肾衰竭（CRF）大鼠BMP-7／Smads／TGF-β1岛信号转导通路的影响,探讨肾衰泻浊九延缓CRF进展及抗肾间质纤维化的机制。方法：采用腺嘌呤致CRF大鼠模型,观察实验大鼠治疗后血清BUN、Scr的变化及肾脏病理变化;采用免疫组织化学法检测肾组织中TGF-β1,Smad6及BMP-7蛋白表达;采用WesternBlot法检测肾组织内的Smad6、BMP-7蛋白的表达,采用半定量RT—PCR检测肾组织内的TGF-β1 mRNA的表达水平。结果：肾衰泻浊丸能够明显降低CRF大鼠血清BUN、Scr水平,与对照组比较差异有统计学意义;能够减轻肾小管间质损伤;能够降低肾组织中TGF-β1蛋白的表达,增加Smad6,BMP-7蛋白的表达,与对照组比较差异有统计学意义（P〈0．05）;能够下调CRF大鼠肾组织中TGF—β1 mRNA的表达,与对照组比较差异有统计学意义（P〈0．05）。结论：肾衰泻浊丸可能是通过升高Smad6,BMP-7蛋白的表达,降低TGF-β1的蛋白表达,从而抑制了TGF-β1信号向细胞核内转导的通路;肾衰泻浊丸通过影响BMP-7／Smads／TGF-β1信号通路的转导而减轻肾间质纤维化可能是其延缓CRF进展的机制之一。