去甲斑蝥素对糖尿病肾病肾小管间质纤维化的作用及机制研究
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摘要
糖尿病肾病(Diabetic nephropathy,DN)已成为终末期肾功能衰竭的重要原因之。DN早期可出现肾小管间质纤维化,且不依赖肾小球病变直接促进肾功能水平下降。阻止DN肾小管间质纤维化已成为当前的研究热点。然而目前尚没有理想的逆转DN肾小管间质纤维化的药物。
去甲斑蝥素(Norcantharidin NCTD)是人工合成的斑蝥素衍生物。本研究所两位博士研究生前期研究发现：NCTD能减轻蛋白负荷大鼠和梗阻性肾肾病大鼠的肾间质纤维化程度；体外实验证实NCTD能抑制白蛋白刺激的肾小管上皮细胞NF-κB和CTGF的表达；阻止TGF-β1介导的肾小管上皮细胞转分化。我们推测NCTD是一种极有前景的抗肾间质纤维化药物。但NCTD对DN肾小管间质纤维化的作用如何尚不清楚。
钙调蛋白磷酸酶(Calcineurin CaN)在肾组织尤其肾小管上皮细胞上表达,研究证实CaN在促进DN肾小球硬化过程中起重要作用,但尚无其在DN肾间质纤维化过程中的研究。据报道NCTD是CaN的抑制剂,NCTD是否通过抑制DN肾小管上皮细胞CaN信号通路,发挥抗DN肾间质纤维化的作用亟待深入研究。
目的：本课题拟通过DN动物模型和高糖刺激的肾小管上皮细胞模型,从三个方面进行研究：1.观察NCTD对2型糖尿病肾病大鼠肾小管间质纤维化的影响；2.观察NCTD对高糖刺激的HK-2细胞细胞外基质和TGF-β1表达的影响；3。研究NCTD对糖尿病肾病肾小管上皮细胞CaN表达的影响,探讨NCTD抗DN肾间质纤维化与其抑制CaN的关系。通过本研究不但可扩大NCTD的适应症,而且为将其开发成为治疗DN,延缓肾功能进展的有效药物提供理论依据。
方法：1.建立2型DN大鼠模型：SD大鼠随机分为正常对照组、模型组和NCTD治疗组。采用高糖高脂饲料喂养联合低剂量STZ注射诱发SD大鼠2型糖尿病肾病模型(STZ溶于0.1mol/L枸橼酸盐缓冲液中,pH值4.5),对照组只注射相同体积的枸橼酸盐缓冲液,72h后测定血糖和尿糖,血糖值≥16.7mmol/L,尿糖值+++～++++者确定为DM模型。成模后,NCTD治疗组予以腹腔注射去甲斑蝥素(0.05mg/kg/d和0.1mg/kg/d)。分别于第3、8周末处死大鼠,取部分肾皮质组织置于4%多聚甲醛固定,观察各组之间肾脏病理学变化。采用免疫组化方法检测FN, ColⅣ, TGF-β1和CaN分布以及表达情况。
2.常规培养HK-2细胞,无血清DMEM培养基同步培养24h,细胞分组：(1)正常糖组(NG, D-Glucose 5.5mmol/L),(2)甘露醇对照组(NG+M, D-Glucose5.5 mmol/L+mannitol 24.5 mmol/L), (3)高糖组1(HG, D-Glucose 30 mmol/L), (4)高糖+NCTD(D-Glucose 30 mmol/L+NCTD2.5,5,mg/L)。台盼蓝排斥实验检测NCDT对高糖刺激的细胞毒性。MTT法检测NCTD对高糖刺激的细胞增殖的影响。收集培养6、24、48h后细胞总RNA及蛋白,用实时定量PCR检测细胞FN、ColⅣ, TGF-pl和CaN mRNA水平的表达,采用间接细胞免疫荧光法检测CaN下游NFATc在细胞分布,采用Western blot检测FN、ColⅣ、TGF-β, CaN和NFATc蛋白蛋白水平的表达。
3.常规培养HK-2细胞,转染CaN siRNA,细胞分5组：(1)正常糖组(NG, D-Glucose 5.5mmol/L),(2)高糖组(HG, D-Glucose 30 mmol/L),(3)高糖+CaNsiRNA组,用lipofectamine 2000转染试剂将带绿色荧光的CaN siRNA转染到细胞内,24h后培养于30mM D-葡萄糖的DMEM中,(4)高糖+CaN siRNA+NCTD组。细胞转染CaN-SiRNA 24h后,用5ug/ml NCTD预处理15min后培养于30mMD-葡萄糖的DMEM中,(5)高糖+NCTD组：用5ug/ml NCTD预处理15min后再用30mMD-葡萄糖的DMEM培养中。转染后在荧光倒置显微镜下观察绿色荧光,检测转染效率。采用Western-blot及实时定量PCR检测CaN蛋白及RNA表达水平,明确CaN siRNA干扰效果。采用Western blot比较NCTD对HK-2表达FN, ColIV及TGF-β1的影响与其抑制CaN的关系。
结果：1.成功构建DN模型：72h测定血糖和尿糖,血糖值>16.7mmol/L,尿糖值+++～+++,确定为糖尿病模型。8周末时,糖尿病大鼠的血糖显著高于正常对照组,且出现了明显的“三多一少”症状,检测尿白蛋白明显增高,Masson染色肾小管间质出现明显的胶原沉积,说明DN大鼠模型建立是成功的。NCTD组与模型组比较,能减小肾小管间质胶原相对面积(P<0.05),下调肾小管区胶原Ⅳ,FN的表达,以及TGF-β1蛋白的表达(P<0.05)。
2.正常大鼠肾小管有少量CaN蛋白表达,8周末糖尿病肾组织CaN蛋白表达增加,NCTD以剂量依赖方式抑制Cano的表达(P<0.05)。
3.台盼蓝实验结果提示,不同浓度NCTD作用72小时后,浓度超过5mg/L的NCDT对高糖环境下的HK-2细胞有明显的毒性。MTT实验结果提示,NCTD2.5mg/L和l5mg/L干预组能抑制HG诱导的HK-2细胞的增殖(P<0.05)5mg/LNCTD的抑制作用更强些。所以,体外实验选择NCTD 2.5mg/L和5mg/L两个浓度组。
4.实时定量PCR和1Western blot结果显示：30mM D-葡萄糖可引起HK-2细胞表达FN, ColⅣ和TGF-β1 mRNA以及蛋白水平的升高(P<0.05),而NCTD可抑制高糖刺激的FN, ColⅣ和TGF-β1的表达(P<0.05)。30mM D-甘露醇对上述指标均无影响(P>0.05)
5.实时定量PCR和Western blot:结果显示：30mMD-葡萄糖可导致HK-2细胞表达CaN mRNA以及蛋白水平的升高,而NCTD在基因以及蛋白水平均可抑制CaN的表达(p<0.05)。免疫荧光发现,CaN下游NFATc在正常对照组中存在于胞浆,高糖刺激后细胞核内开始表达,高糖刺激30min后发生明显的核转位,去甲斑蝥素能在一定程度上抑制核转位的发生,减少高糖刺激后核内NFATc的表达。
6.转染CaN-SiRNA后,高糖刺激后HK-2细胞中CaN mRNA以及蛋白表达均减少,而FN, ColⅣ以及TGF-β1蛋白水平表达都明显增强(P<0.05)。相比于CaN-SiRNA组,NCTD可抑制高糖刺激的FN, ColⅣ和TGF-β1的表达。
结论：1.NCTD能下调HK-2细胞FN, Col IV及TGF-β1的表达,减轻DN早期肾间质纤维化。
2.NCTD能下调DN肾小管上皮细胞CaN的表达,阻断CaN/NFATc信号通路；
3.NCTD抗DN肾小管间质纤维化的作用与其阻断CaN/NFATc信号通路无关。
Objective Diabetic nephropathy (DN) is one of the common causes of chronic renal disease. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past years. It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis and tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy. However, there is no effective drugs to reverse the tubulointerstitial injury and fibrosis.
Norcantharidin (NCTD),a demethylated analog of cantharidin, possesses potential anticancer activity. NCTD not only decreased the targeted gene expression involved in tumor metastasis such as VEGF、FAK and TGF-β1 but also maintained the balance of matrix metalloproteinase 2 and tissue inhibitor metalloproteinase. Our studies previously found that NCTD inhibited the tubulointerstitial lesions which attenuated the development of renal interstitial fibrosis by inhibiting the expressions of NF-κB and CTGF in protein overload nephropathy. NCTD inhibited the expression of fibronectin in tubular epithelial cells induced by albumin. NCTD could attenuate the tubulointerstitial fibrosis and inhibit tubular epithelial-mesenchymal transition in rat model of obstructive nephropathy as well as inhibiting EMT of tubular cells by down-regulating TGF-β1 in vitro. Since it inhibited tubulointerstitial fibrosis in several models, we wondered if NCTD could have a similar effect in DN tubulointerstitial fibrosis.
Calcineurin expression is observed at glomeruli as well as renal tubular epithelial cells and it is up-regulated in DN. It is reported calcineurin iinhibitor can attenuate the glomerular fibrosis, but there is no report about if calcineurin plays a role in tubulointerstitial injury and fibrosis. NCTD is a calcineurin inhibitor and we wonder if NCTD could attenuate the tubulointerstitial fibrosis by down-regulating calcineurin pathway. To investigate the effect of norcantharidin in tubulointersitial firosis in DN and the possible and mechanism, the expression of FN(fibronectin),collagenⅣand TGF-β1 were analysed in STZ-induced diabetic model and in human renal proximal tubular epithelial cell line treated with high glucose and norcantharidin. The expression of calcineurin is also observed in the model. Furthermore, the role of calcineurin was investigated as a potent target of NCTD by small interfering RNA. It is aimed at providing experimentals evidence to develop NCTD as a potential treatment in DN fibrosis.
Methods:1Sprague-Dawley rats were randomly divided into control(n=8), DN(n=9), low dose NCTD (0.05mg/kg/d) groups(n=12), and high dose NCTD(0.1mg/kg/d) groups(n=11).The DN model was induced by injection intra-peritoneally with 30 mg/kg body weight STZ in 0.1mol/L sodium citrate solution(pH 4.5),after high calorie given for two months. The rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was＋＋＋～＋＋＋＋after 72 hours of the injection. NCTD was administered daily after the model was built. Rats were sacrificed at day 21and day 56 after and the kidneys were harvested and subjected to the studies. Renal lesions and the expression of FN,collagenⅣ, TGF-β1 and CaN were detected by HE, Masson and immunohistochemistry respectively. 2 Human kidney proximal tubular epithelial cell line(HK-2)cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth, then the cells were divided into 4 groups:NG group(media containing 5.5mM glucose);mannitol control group(media containing 5.5mM glucose and 24.5mM mannitol);HG group(media containing 30mM glucose);NCTD group(treated with NCTD at varied dose and 30mM glucose). Trypan blue dye exclusive assay and MTT assay were carried out to determin the effective and safe dose of NCTD in high glucose. The cells were collected to extracte total RNA and protein at 6,24 and 48 hours after incubation.The expression of FN,collagenⅣ, TGF-β1 and CaN were examined by realtime-PCR and Western-blot. NFATc(an important downstream molecular of CaN) nuclear translocation was examined by immunofluorescence.
3 Small interfering RNA(siRNA)targeting human calcineurin gene sequences were synthesized to silence calcineurin gene expression. Transient transfection was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Fluorescent microscopy was used to examine GFP expression.. HK-2 cells were divided into 5 groups:NG group, HG group, HG+ CaN-siRNA group, HG+CaN-siRNA+NCTD group and HG+NCTD group. The CaN interference efficiency is deteced by realtime-PCR and Western-blot. FN, collagenIV and TGF-β1 expression was analyzed by Western-blot after the cells were treated for 24h.
Results:1 Extracellular matrix deposition and tubulointerstitial fibrosis were observed in DN rats, while the pathological changes were reduced in that of NCTD-treated group(P<0.05). The expressions of FN, collagenⅣand TGF-β1 were increased in the tubulointerstitial field of DN rats compared with the control. NCTD treatment can reverse the fibrosis to a certein degree(P<0.05).
2 Expression of CaN was detected in normal kidney tubular fields and the expression increased in the kidney tubulointerstitial field in DN rats. NCTD treatment down-reguated its expression in a dose dependent manner(P<0.05).
3 The mRNA and protein expression of FN, collagenⅣincreased in HK-2 cells treated with HG in a time-depedent manner, while that in cells treated by 5μg/mlNCTD was dramatically inhibited(P<0.05). The expression of TGF-β1 in HK-2 treated by HG was also inhibit by NCTD in a time-dependent manner.
4 The mRNA and protein expression of CaN increased in HK-2 cells treated with HG and NCTD inhibited its expression in a dose-dependent manner.5μg/ml NCTD had an impressive inhibiting effect(P<0.01). NCTD can also inhibit NFATc nuclear translocation induced by 30mM glucose in HK-2 cells
5 After CaN-siRNA transfection, green fluorescent protein(GFP)was observed in about 70% HK-2 cells, showing that the transfection is successful. Compared with control group, the CaN mRNA expression was reduced to 48±12% in CaN-siRNA groups (P<0.05).Correspondingly, protein expression of CaN was decreased to 62±15%(P<0.05).Western blot showed that CaN-siRNA increase the expression of FN,collagenⅣand TGF-β1 induced by HG,whereas NCTD combined with CaN siRNA can lower the level of these proteins(p<0.05).
Conclusions:1 NCTD can down-regulate FN, collagen IV and TGF-β1 expression in tubulointerstitial fields and may have an positive effect in attenuating tubulointerstitial fibrogenesis of in the early stage of DN rats. NCTD could significantly decreased the mRNA and protein expression of FN, collagenⅣand TGF-β1 in HK-2 stimulated by 30mM glucose.
2 NCTD can down-regulate CaN/NFATc pathway activated by high glucose in the proximal tubular epithelial cells.
3. The effect of NCTD on tubulointerstitial fibrosis of DN is not by inhibiting CaN.
去甲斑蝥素(Norcantharidin NCTD)是人工合成的斑蝥素衍生物。本研究所两位博士研究生前期研究发现：NCTD能减轻蛋白负荷大鼠和梗阻性肾肾病大鼠的肾间质纤维化程度；体外实验证实NCTD能抑制白蛋白刺激的肾小管上皮细胞NF-κB和CTGF的表达；阻止TGF-β1介导的肾小管上皮细胞转分化。我们推测NCTD是一种极有前景的抗肾间质纤维化药物。但NCTD对DN肾小管间质纤维化的作用如何尚不清楚。
钙调蛋白磷酸酶(Calcineurin CaN)在肾组织尤其肾小管上皮细胞上表达,研究证实CaN在促进DN肾小球硬化过程中起重要作用,但尚无其在DN肾间质纤维化过程中的研究。据报道NCTD是CaN的抑制剂,NCTD是否通过抑制DN肾小管上皮细胞CaN信号通路,发挥抗DN肾间质纤维化的作用亟待深入研究。
目的：本课题拟通过DN动物模型和高糖刺激的肾小管上皮细胞模型,从三个方面进行研究：1.观察NCTD对2型糖尿病肾病大鼠肾小管间质纤维化的影响；2.观察NCTD对高糖刺激的HK-2细胞细胞外基质和TGF-β1表达的影响；3。研究NCTD对糖尿病肾病肾小管上皮细胞CaN表达的影响,探讨NCTD抗DN肾间质纤维化与其抑制CaN的关系。通过本研究不但可扩大NCTD的适应症,而且为将其开发成为治疗DN,延缓肾功能进展的有效药物提供理论依据。
方法：1.建立2型DN大鼠模型：SD大鼠随机分为正常对照组、模型组和NCTD治疗组。采用高糖高脂饲料喂养联合低剂量STZ注射诱发SD大鼠2型糖尿病肾病模型(STZ溶于0.1mol/L枸橼酸盐缓冲液中,pH值4.5),对照组只注射相同体积的枸橼酸盐缓冲液,72h后测定血糖和尿糖,血糖值≥16.7mmol/L,尿糖值+++～++++者确定为DM模型。成模后,NCTD治疗组予以腹腔注射去甲斑蝥素(0.05mg/kg/d和0.1mg/kg/d)。分别于第3、8周末处死大鼠,取部分肾皮质组织置于4%多聚甲醛固定,观察各组之间肾脏病理学变化。采用免疫组化方法检测FN, ColⅣ, TGF-β1和CaN分布以及表达情况。
2.常规培养HK-2细胞,无血清DMEM培养基同步培养24h,细胞分组：(1)正常糖组(NG, D-Glucose 5.5mmol/L),(2)甘露醇对照组(NG+M, D-Glucose5.5 mmol/L+mannitol 24.5 mmol/L), (3)高糖组1(HG, D-Glucose 30 mmol/L), (4)高糖+NCTD(D-Glucose 30 mmol/L+NCTD2.5,5,mg/L)。台盼蓝排斥实验检测NCDT对高糖刺激的细胞毒性。MTT法检测NCTD对高糖刺激的细胞增殖的影响。收集培养6、24、48h后细胞总RNA及蛋白,用实时定量PCR检测细胞FN、ColⅣ, TGF-pl和CaN mRNA水平的表达,采用间接细胞免疫荧光法检测CaN下游NFATc在细胞分布,采用Western blot检测FN、ColⅣ、TGF-β, CaN和NFATc蛋白蛋白水平的表达。
3.常规培养HK-2细胞,转染CaN siRNA,细胞分5组：(1)正常糖组(NG, D-Glucose 5.5mmol/L),(2)高糖组(HG, D-Glucose 30 mmol/L),(3)高糖+CaNsiRNA组,用lipofectamine 2000转染试剂将带绿色荧光的CaN siRNA转染到细胞内,24h后培养于30mM D-葡萄糖的DMEM中,(4)高糖+CaN siRNA+NCTD组。细胞转染CaN-SiRNA 24h后,用5ug/ml NCTD预处理15min后培养于30mMD-葡萄糖的DMEM中,(5)高糖+NCTD组：用5ug/ml NCTD预处理15min后再用30mMD-葡萄糖的DMEM培养中。转染后在荧光倒置显微镜下观察绿色荧光,检测转染效率。采用Western-blot及实时定量PCR检测CaN蛋白及RNA表达水平,明确CaN siRNA干扰效果。采用Western blot比较NCTD对HK-2表达FN, ColIV及TGF-β1的影响与其抑制CaN的关系。
结果：1.成功构建DN模型：72h测定血糖和尿糖,血糖值>16.7mmol/L,尿糖值+++～+++,确定为糖尿病模型。8周末时,糖尿病大鼠的血糖显著高于正常对照组,且出现了明显的“三多一少”症状,检测尿白蛋白明显增高,Masson染色肾小管间质出现明显的胶原沉积,说明DN大鼠模型建立是成功的。NCTD组与模型组比较,能减小肾小管间质胶原相对面积(P<0.05),下调肾小管区胶原Ⅳ,FN的表达,以及TGF-β1蛋白的表达(P<0.05)。
2.正常大鼠肾小管有少量CaN蛋白表达,8周末糖尿病肾组织CaN蛋白表达增加,NCTD以剂量依赖方式抑制Cano的表达(P<0.05)。
3.台盼蓝实验结果提示,不同浓度NCTD作用72小时后,浓度超过5mg/L的NCDT对高糖环境下的HK-2细胞有明显的毒性。MTT实验结果提示,NCTD2.5mg/L和l5mg/L干预组能抑制HG诱导的HK-2细胞的增殖(P<0.05)5mg/LNCTD的抑制作用更强些。所以,体外实验选择NCTD 2.5mg/L和5mg/L两个浓度组。
4.实时定量PCR和1Western blot结果显示：30mM D-葡萄糖可引起HK-2细胞表达FN, ColⅣ和TGF-β1 mRNA以及蛋白水平的升高(P<0.05),而NCTD可抑制高糖刺激的FN, ColⅣ和TGF-β1的表达(P<0.05)。30mM D-甘露醇对上述指标均无影响(P>0.05)
5.实时定量PCR和Western blot:结果显示：30mMD-葡萄糖可导致HK-2细胞表达CaN mRNA以及蛋白水平的升高,而NCTD在基因以及蛋白水平均可抑制CaN的表达(p<0.05)。免疫荧光发现,CaN下游NFATc在正常对照组中存在于胞浆,高糖刺激后细胞核内开始表达,高糖刺激30min后发生明显的核转位,去甲斑蝥素能在一定程度上抑制核转位的发生,减少高糖刺激后核内NFATc的表达。
6.转染CaN-SiRNA后,高糖刺激后HK-2细胞中CaN mRNA以及蛋白表达均减少,而FN, ColⅣ以及TGF-β1蛋白水平表达都明显增强(P<0.05)。相比于CaN-SiRNA组,NCTD可抑制高糖刺激的FN, ColⅣ和TGF-β1的表达。
结论：1.NCTD能下调HK-2细胞FN, Col IV及TGF-β1的表达,减轻DN早期肾间质纤维化。
2.NCTD能下调DN肾小管上皮细胞CaN的表达,阻断CaN/NFATc信号通路；
3.NCTD抗DN肾小管间质纤维化的作用与其阻断CaN/NFATc信号通路无关。
Objective Diabetic nephropathy (DN) is one of the common causes of chronic renal disease. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past years. It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis and tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy. However, there is no effective drugs to reverse the tubulointerstitial injury and fibrosis.
Norcantharidin (NCTD),a demethylated analog of cantharidin, possesses potential anticancer activity. NCTD not only decreased the targeted gene expression involved in tumor metastasis such as VEGF、FAK and TGF-β1 but also maintained the balance of matrix metalloproteinase 2 and tissue inhibitor metalloproteinase. Our studies previously found that NCTD inhibited the tubulointerstitial lesions which attenuated the development of renal interstitial fibrosis by inhibiting the expressions of NF-κB and CTGF in protein overload nephropathy. NCTD inhibited the expression of fibronectin in tubular epithelial cells induced by albumin. NCTD could attenuate the tubulointerstitial fibrosis and inhibit tubular epithelial-mesenchymal transition in rat model of obstructive nephropathy as well as inhibiting EMT of tubular cells by down-regulating TGF-β1 in vitro. Since it inhibited tubulointerstitial fibrosis in several models, we wondered if NCTD could have a similar effect in DN tubulointerstitial fibrosis.
Calcineurin expression is observed at glomeruli as well as renal tubular epithelial cells and it is up-regulated in DN. It is reported calcineurin iinhibitor can attenuate the glomerular fibrosis, but there is no report about if calcineurin plays a role in tubulointerstitial injury and fibrosis. NCTD is a calcineurin inhibitor and we wonder if NCTD could attenuate the tubulointerstitial fibrosis by down-regulating calcineurin pathway. To investigate the effect of norcantharidin in tubulointersitial firosis in DN and the possible and mechanism, the expression of FN(fibronectin),collagenⅣand TGF-β1 were analysed in STZ-induced diabetic model and in human renal proximal tubular epithelial cell line treated with high glucose and norcantharidin. The expression of calcineurin is also observed in the model. Furthermore, the role of calcineurin was investigated as a potent target of NCTD by small interfering RNA. It is aimed at providing experimentals evidence to develop NCTD as a potential treatment in DN fibrosis.
Methods:1Sprague-Dawley rats were randomly divided into control(n=8), DN(n=9), low dose NCTD (0.05mg/kg/d) groups(n=12), and high dose NCTD(0.1mg/kg/d) groups(n=11).The DN model was induced by injection intra-peritoneally with 30 mg/kg body weight STZ in 0.1mol/L sodium citrate solution(pH 4.5),after high calorie given for two months. The rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was＋＋＋～＋＋＋＋after 72 hours of the injection. NCTD was administered daily after the model was built. Rats were sacrificed at day 21and day 56 after and the kidneys were harvested and subjected to the studies. Renal lesions and the expression of FN,collagenⅣ, TGF-β1 and CaN were detected by HE, Masson and immunohistochemistry respectively. 2 Human kidney proximal tubular epithelial cell line(HK-2)cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth, then the cells were divided into 4 groups:NG group(media containing 5.5mM glucose);mannitol control group(media containing 5.5mM glucose and 24.5mM mannitol);HG group(media containing 30mM glucose);NCTD group(treated with NCTD at varied dose and 30mM glucose). Trypan blue dye exclusive assay and MTT assay were carried out to determin the effective and safe dose of NCTD in high glucose. The cells were collected to extracte total RNA and protein at 6,24 and 48 hours after incubation.The expression of FN,collagenⅣ, TGF-β1 and CaN were examined by realtime-PCR and Western-blot. NFATc(an important downstream molecular of CaN) nuclear translocation was examined by immunofluorescence.
3 Small interfering RNA(siRNA)targeting human calcineurin gene sequences were synthesized to silence calcineurin gene expression. Transient transfection was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Fluorescent microscopy was used to examine GFP expression.. HK-2 cells were divided into 5 groups:NG group, HG group, HG+ CaN-siRNA group, HG+CaN-siRNA+NCTD group and HG+NCTD group. The CaN interference efficiency is deteced by realtime-PCR and Western-blot. FN, collagenIV and TGF-β1 expression was analyzed by Western-blot after the cells were treated for 24h.
Results:1 Extracellular matrix deposition and tubulointerstitial fibrosis were observed in DN rats, while the pathological changes were reduced in that of NCTD-treated group(P<0.05). The expressions of FN, collagenⅣand TGF-β1 were increased in the tubulointerstitial field of DN rats compared with the control. NCTD treatment can reverse the fibrosis to a certein degree(P<0.05).
2 Expression of CaN was detected in normal kidney tubular fields and the expression increased in the kidney tubulointerstitial field in DN rats. NCTD treatment down-reguated its expression in a dose dependent manner(P<0.05).
3 The mRNA and protein expression of FN, collagenⅣincreased in HK-2 cells treated with HG in a time-depedent manner, while that in cells treated by 5μg/mlNCTD was dramatically inhibited(P<0.05). The expression of TGF-β1 in HK-2 treated by HG was also inhibit by NCTD in a time-dependent manner.
4 The mRNA and protein expression of CaN increased in HK-2 cells treated with HG and NCTD inhibited its expression in a dose-dependent manner.5μg/ml NCTD had an impressive inhibiting effect(P<0.01). NCTD can also inhibit NFATc nuclear translocation induced by 30mM glucose in HK-2 cells
5 After CaN-siRNA transfection, green fluorescent protein(GFP)was observed in about 70% HK-2 cells, showing that the transfection is successful. Compared with control group, the CaN mRNA expression was reduced to 48±12% in CaN-siRNA groups (P<0.05).Correspondingly, protein expression of CaN was decreased to 62±15%(P<0.05).Western blot showed that CaN-siRNA increase the expression of FN,collagenⅣand TGF-β1 induced by HG,whereas NCTD combined with CaN siRNA can lower the level of these proteins(p<0.05).
Conclusions:1 NCTD can down-regulate FN, collagen IV and TGF-β1 expression in tubulointerstitial fields and may have an positive effect in attenuating tubulointerstitial fibrogenesis of in the early stage of DN rats. NCTD could significantly decreased the mRNA and protein expression of FN, collagenⅣand TGF-β1 in HK-2 stimulated by 30mM glucose.
2 NCTD can down-regulate CaN/NFATc pathway activated by high glucose in the proximal tubular epithelial cells.
3. The effect of NCTD on tubulointerstitial fibrosis of DN is not by inhibiting CaN.
引文
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