大蒜素对高糖环境人肾小管上皮细胞转分化和凋亡的抑制作用及其机制研究
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摘要
研究背景
糖尿病肾病是糖尿病常见和严重的慢性并发症之一,也是导致终末期肾病的主要原因之一。糖尿病肾病的发病机制尚未完全阐明。近年研究表明,肾小管间质病变是糖尿病肾病发生发展的关键因素之一,进一步深入研究肾小管间质病变的分子机制、寻找有效治疗的新靶点,对预防和延缓糖尿病肾病的发生发展有重要意义。
肾小管间质纤维化的过程包括肾小管的萎缩和消失、肌成纤维细胞的聚积和细胞外基质的沉积。目前认为肾间质细胞外基质主要由肌成纤维细胞产生,但有证据表明,在一些病理条件下,肾小管上皮细胞可发生上皮细胞-肌成纤维细胞转分化(Tubular epithelial myofibroblast transdifferentiation, TEMT),即肾小管上皮细胞丧失原有的上皮细胞表型特征,获得肌成纤维细胞的表型,大量表达肌成纤维细胞的标志性蛋白α-平滑肌肌动蛋白(a-SMA)和波形蛋白(Vimentin)。已有众多的研究表明,肾小管上皮细胞转分化直接参与肾间质纤维化的进程,是肾间质纤维化发生、发展的的中心环节,已成为肾纤维化乃至慢性肾功能衰竭的重要机制之一
TGF-β1是具有多种重要生物学作用的生长因子,也是目前已知的诱导肾小管上皮细胞转分化作用最强的生长因子,除Smads通路外,尚可通过不同信号传导途径产生多种生物学效应,在肾小管间质纤维化中发挥了核心作用。MAPK信号通路是非Smads依赖途径中的重要通路之一,它是广泛存在于细胞内的信号转导途径,由丝氨酸／苏氨酸蛋白激酶组成。在MAPK家族中,细胞外调节激酶(ERK)是最早被发现的成员,它包括两种异构体ERK1和ERK2。有研究提示,ERK信号通路是细胞信号中传递丝裂原信号的关键激酶,与肾小管上皮细胞转分化和肾脏纤维化密切相关。关于高糖环境下人近端肾小管上皮细胞发生转分化是否与TGF-β1／ERK信号通路有关,目前较少报道。
细胞凋亡与增殖的动态平衡是多细胞生物维系结构完整和内环境稳定的基本生物学特性。随着对细胞凋亡的基因调控、信号转导及其与疾病关系的深入研究,表明细胞凋亡密切参与了糖尿病肾病的发生机制。研究发现,在糖尿病肾病早期即出现肾小管上皮细胞的凋亡,后者是导致肾小管功能障碍的重要原因之一,也是糖尿病肾病进展为慢性肾功能衰竭的重要机制之一。有研究显示,肾小管间质纤维化形成中存在明显的肾小管上皮细胞凋亡现象,病变早期凋亡细胞数随着肾间质纤维化的加重呈明显增高趋势。因此在糖尿病肾病致肾小管间质纤维化的发生发展过程中,肾小管上皮细胞凋亡是高血糖损伤肾小管上皮细胞的重要病理变化形式之一
大蒜素,是大蒜的主要有效成分。现代化学和药理学研究发现大蒜素具有抗菌、抗病毒、抗氧化、抗肿瘤、降血脂、提高机体免疫力等多方面的作用。近年研究表明,大蒜素有抗肺、肝及心肌纤维化作用,主要与其抑制成纤维细胞增殖,抑制促纤维化因子的分泌及减少细胞外基质的合成等有关。另有研究发现,大蒜素可抑制创伤／失血性休克大鼠重要脏器的细胞凋亡。但大蒜素是否具有抑制高糖诱导肾小管上皮细胞转分化和凋亡的作用,目前尚未明确。
研究目的
1.观察大蒜素对高糖环境下人近端肾小管上皮细胞转分化的作用,并初步探讨大蒜素抑制肾小管上皮细胞转分化的机制。
2.观察大蒜素对高糖环境下人近端肾小管上皮细胞凋亡及凋亡相关蛋白Bcl-2和Bax表达的影响,并初步探讨大蒜素抑制肾小管上皮细胞凋亡的机制。
实验方法
1.以人肾小管上皮细胞株HK-2细胞为研究对象,按实验条件分为4组,以DMEM为培养基,即正常糖对照组(低糖5.5mmol/l DMEM)、高糖组(高糖25mmol/l DMEM)、大蒜素组(高糖组加不同终浓度的大蒜素,剂量分别为2.5、5、10、20ug/ml)和ERK抑制剂组(高糖组加20umol/l PD98059),分别培养24h、48h。采用免疫细胞化学法、荧光免疫细胞化学法检测各组细胞内a-SMA、Vimentin和Collagen I的相对表达量,Real-time PCR法检测各组细胞内TGF-β1 mRNA的相对表达,Western Blot法检测各组细胞TGF-β1. p-ERK1/2(磷酸化的ERK1/2)蛋白的相对表达。
2.以人肾小管上皮细胞株HK-2细胞为研究对象,按实验条件分为3组,以DMEM为培养基,即正常糖对照组(低糖5.5mmol/l DMEM)、高糖组(高糖：25mmol/l DMEM)和大蒜素组(高糖组加不同终浓度的大蒜素,剂量分别为2.5、5、10、20ug/ml),细胞培养24h、48h。采用流式细胞术观察各组细胞的凋亡率,采用免疫细胞化学法检测HK-2细胞内Bcl-2和Bax蛋白的相对表达量。
3.应用SPSS 13.0统计软件对数据进行分析,计量资料用均数±标准差((?)±S)表示,各组间比较采用单因素方差分析(one-way ANOVA),计数资料用率(%)表示,各组间比较采用χ2检验,p<0.05有统计学意义。
结果
1.大蒜素对HK-2细胞形态学变化的影响
正常糖对照组生长24h后,形成融合的单层细胞,呈椭圆形或者多边形贴壁细胞,生长48h后细胞间紧密连接,具有典型的上皮细胞铺路石样形态学特征。高糖组生长24h后,细胞形态改变不明显,生长48h后,可见部分细胞发生明显的形态学改变,表现为成纤维细胞样外观：细胞拉长、肥大,呈长梭形,由椭圆形贴壁生长变为长梭形,丧失其铺路石样生长方式。大蒜素组(H+A20ug/ml)干预48小时后,细胞形态与N组无明显差异,光镜下多数细胞保持正常上皮,仅少数发生形态变化。PD98059组细胞生长48h后,受高糖刺激而出现的形态学改变有一定程度的改善。
2.大蒜素对HK-2细胞α-SMA、Vimentin、Collagen I表达的影响
细胞培养48h,高糖组α-SMA、Vimentin、Collagen I表达的积分光密度值(IOD)较正常糖对照组明显增加,均有统计学意义(p<0.01); PD98059组α-SMA、Vimentin和Collagen I表达的IOD较高糖组均明显减少(p<0.01),且与正常糖对照组无明显差异(p>0.05)；高糖环境下经不同浓度的大蒜素(2.5、5、10、20ug/mL)干预后,a-SMA. Vimentin. Collagen I表达的IOD明显下降,并呈浓度依赖性,尤以20ug/mL更为明显(p<0.01),此组与正常糖对照组和PD98059组比较无显著差别(p>0.05)。
3.大蒜素对HK-2细胞TGF-β1mRNA表达的影响
细胞培养48h时,与正常糖对照组比较,高糖组HK-2细胞TGF-β1mRNA表达明显上调(p<0.05)；PD98059组可逆转高糖刺激下的TGF-β1 mRNA表达(p<0.05),但仍高于正常糖对照组(p<0.05)；大蒜素可明显下调高糖刺激下HK-2细胞TGF-β1mRNA的表达,并呈浓度依赖效应,尤其在l0ug/ml、20ug/ml时(p<0.05),同时与PD98059组比较无显著性差异,且20ug／ml时与正常糖对照组比较亦无显著性差异。
4.大蒜素对HK-2细胞TGF-β1、p-ERK 1／2蛋白表达的影响
细胞培养48h时,正常糖对照组HK-2细胞可见少量TGF-β1、p-ERK1/2蛋白的表达；高糖组HK-2细胞TGF-β1, p-ERK1/2的蛋白表达较正常糖对照组明显上调p<0.05)；与高糖组比较,PD98059组TGF-β1、p-ERK 1/2蛋白表达均明显下调(p<0.05),而TGF-β1蛋白与正常糖对照组比较无显著性差异(p>0.05)；与高糖组比较,经不同浓度大蒜素(2.5、5、10、20ug／mL)干预48小时后,HK-2细胞TGF-β1、p-ERK 1／2蛋白的表达水平均呈药物浓度依赖性下调(p<0.05),尤以20ug／mL浓度作用最强,抑制率分别为55.7％(TGF-β1,p<0.05)和37.7％(p-ERK1/2,p<0.05),且与正常糖对照组比较,p-ERK 1／2蛋白表达亦明显下调,而TGF-β1蛋白表达差异无显著性,但两者均仍高于PD98059组(p<0.05)。
5.大蒜素对HK-2细胞凋亡率的影响
细胞培养24h,正常糖对照组只有少量细胞发生凋亡,凋亡率仅为(6.56±1.09)%,而高糖组凋亡率为(16.25±0.58)%,两组间有显著性差异(p<0.01)；大蒜素干预组(2.5.5.10.20ug/mL)的细胞凋亡率分别为(12.54±2.21)%、(9.21±2.85)%、(8.53±2.15)%、(6.81±0.92)%,均较高糖组有明显下降(2.5ug/mL时p<0.05,5、10、20ug／mL时p<0.01),且大蒜素浓度为5、10、20ug／ml时与正常糖对照组比较无显著性差异(p>0.05)。细胞培养48h,正常糖对照组细胞亦只有少量细胞发生凋亡,凋亡率仅为(6.82±2.38)%,高糖组升高为(23.53±3.21)%；而大蒜素干预组(2.5.5.10.20ug/mL)的细胞凋亡率分别为(14.43±1.85)%、(11.68±3.20)%、(10.19±1.93)%、(7.99±2.00)%,与高糖组比较均有明显下降(p<0.01)且大蒜素浓度为10、20ug／mL时与正常糖对照组比较无显著性差异(p>0.05)。
6.大蒜素对HK-2细胞Bcl-2、Bax蛋白表达的影响
细胞培养48h,高糖组Bcl-2蛋白表达的积分光密度值(IOD)为(14.08±2.14),较正常糖对照组的IOD(31.97±4.29)明显降低(p<0.01),不同浓度的大蒜素组的Bcl-2蛋白表达的IOD高于高糖组,并随着浓度的增高而升高(5 ug/mL时p<0.05,10. 20ug/mL p＜0.01),但仍低于正常糖对照组(p<0.05)；而Bax蛋白则正好相反,高糖组Bax蛋白表达的IOD为(109.83±14.13),较正常糖对照组的IOD(29.68±3.30)明显升高(p<0.01),不同浓度大蒜素组的Bax蛋白表达的IOD明显低于高糖组,并随着浓度的增高而逐渐降低(均p<0.01),但仍高于正常糖对照组(p<0.01)。Bcl-2/Bax的比值在高糖组明显低于正常糖对照组(p<0.01),经大蒜素干预后其比值较高糖组有升高(5 ug/mL时(p<0.05,10、20ug／mLp<0.01),并呈浓度依赖性,但仍明显低于正常糖对照组(p<0.01)
结论
1.高糖可诱导HK-2细胞转分化为肌成纤维细胞,上调TGF-β1和细胞外基质成分Collagen I的表达。
2. ERK1/2信号通路抑制剂PD98059可明显抑制高糖环境下HK-2细胞的表型转分化,下调TGF-β1和Collagen I的表达,阻断ERK 1／2信号通路是抑制TEMT、防治肾间质纤维化的重要干预靶点。
3.大蒜素可通过抑制ERK 1／2信号通路的激活而抑制高糖状态下HK-2细胞向肌成纤维细胞转分化,下调TGF-β1和Collagen I的表达,其抑制作用与PD98059相似。
4.高糖诱导肾小管上皮细胞发生凋亡。大蒜素对高糖诱导的肾小管上皮细胞凋亡具有明显的抑制作用,且存在量效关系。
5.大蒜素调节Bcl-2和Bax蛋白的表达,上调Bcl-2/Bax比值是其抑制高糖诱导肾小管上皮细胞凋亡的机制之一
Background
Diabetic nephropathy(DN) is one of the most common and serious chronic diabetic complications. It is one of the main reasons leading to end-stage renal failure. The pathogenesis of diabetic nephropathy has not yet fully understood. Recent studies showed that, tubulointerstitial lesions is one of the key factors in the development of diabetic nephropathy. So it has great significance to take further in-depth study on tubulointerstitial molecular mechanism to find a new target for the effective treatment, prevention and delay the process of diabetic nephropathy.
The process of tubulointerstitial fibrosis includes the atrophy and loss of tubular, myofibroblasts accumulation and extracellular matrix deposition. It's currently considered that the extracellular matrix in renal interstitial fibroblasts is produced mainly by muscle cells. Growing evidence showed that in some pathological conditions, renal tubular epithelial cells can occur in epithelial cells-myofibroblast transdifferentiation(TEMT), by losing its original phenotype and obtaining the phenotype of myofibroblasts, which express a much of a-smooth muscle actin (α-SMA) and Vimentin. Numerous research studies have found that tubular epithelial myofibroblast transdifferentiation, which was the central link of the occurrence and development of renal interstitial fibrosis, involved directly in the process of renal interstitial fibrosis, and had become one of the great mechanisms of renal fibrosis and even chronic renal failure.
TGF-β1 is a growth factor which has a variety of important biological effects. It is also known to be the most potent growth factor, which induces tubular epithelial myofibroblast transdifferentiation. TGF-β1 plays a central role in renal tubular interstitial fibrosis by producing a variety of biological effects via different signal transduction pathways includes Smads. MAPK signaling pathway, a non-Smads dependent pathway, is one of the most important signal transduction pathways which is widely found in cells, composed of the serine/threonine protein kinase. In the MAPK family, extracellular regulated kinase (ERK) is the first member to be found, which consists of two isomers, ERK1 and ERK2. Some studies suggest that, ERK signaling pathway is the key mitogen kinase of cell signals which transfer mitogen signals, and it is closely related to tubular epithelial myofibroblast transdifferentiation and renal fibrosis. Whether human proximal tubular epithelial myofibroblast transdifferentiation in high glucose is related to TGF-β1/ERK signaling pathway or not, is not well known.
Dynamic equilibrium of apoptosis and proliferation is the basic biological properties of maintaining structural integrity of multi-cellular organisms and environment stability. With the studies on gene regulation, signal transduction and its relationship with diseases, it is showed that apoptosis has been closely involved in the pathogenesis of diabetic nephropathy. Studies found that in the early stage of diabetic nephropathy, apoptosis of tubular epithelial cell has occured, which is one of the major reasons leading to tubular dysfunction and is one of the most important mechanisms of the progress of diabetic nephropathy with chronic renal failure. Studies have shown that the apoptosis of renal tubular epithelial cells is obvious in renal interstitial fibrosis, and that apoptotic cells increased significantly with the aggravation of renal interstitial fibrosis in early lesions. Therefore, the apoptosis of tubular epithelial cell is one of the most important pathological change forms of damages on renal tubular epithelial cells by high blood glucose, during the development process in diabetic renal tubular interstitial fibrosis.
Allicin is the main active ingredient of garlic. Modern chemistry and pharmacology studies found that Allicin has many effects such as antibacterial, antiviral, antioxidant, anti-tumor, and so on. Recent studies show that Allicin is known as anti-lung, liver and cardiac fibrosis, mainly associated with the inhibition of fibroblast proliferation, secretion of promoting fibrosis factors and reduction of the synthesis of extracellular matrix, and also Allicin can inhibit apoptosis in vital organs in rats with trauma/hemorrhagic shock. However, whether Allicin could inhibit tubular epithelial myofibroblast transdifferentiation and apoptosis induced by high glucose or not, is not clear.
Objectives
1. To observe the influence of Allicin on human proximal tubular epithelial myofibroblast transdifferentiation in high glucose and study its mechanism preliminary.
2. To observe the influence of Allicin on apoptosis and its gene expression of human proximal tubular epithelial cell in high glucose and study its mechanism preliminary.
Methods
1. Human proximal renal tubular epithelial cells (HK-2) were divided into four groups according to the experimental conditions, with DMEM as the medium:Group Normal Glucose Control (Group N), cultured with glucose (5.5mmol/L) DMEM; Group High Glucose (Group H), cultured with high glucose (25mmol/L) DMEM; Groups Allicin, cultured with high glucose (25mmol/L) DMEM, with different concentrations of Allicin(2.5,5,10 and 20ug/mL); Group H+PD98059, cultured with 25mmol/L glucose DMEM+ERK inhibitor (PD98059)20umol/L. Each group cells were cultured 24h,48h. The integral optical denisity expression of a-SMA, Vimentin and Collagen I were detected by immunocytochemistry. The mRNA expression of TGF-β1 and the protein expression of TGF-β1, p-ERK1/2 were assessed by Real-time PCR and Western Blot method, respectively.
2. Human proximal renal tubular epithelial cells (HK-2) were divided into eight groups according to the experimental conditions, with DMEM as the medium:Group Normal Glucose Control (Group N), cultured with glucose (5.5mmol/L) DMEM; Group High Glucose (Group H), cultured with high glucose (25mmol/L) DMEM; Groups Allicin, cultured with high glucose (25mmol/L) DMEM, with different concentrations of Allicin(2.5,5,10 and 20ug/mL); Group H+PD98059, cultured with 25mmol/L glucose DMEM+ERK inhibitor(PD98059)20umol/L. Each group cells were cultured 24h,48h. The apoptosis rate of HK-2 cells in each group was assessed by flow cytometry using, and the protein expression of Bcl-2 and Bax were assessed by immunocytochemistry.
3. All analyses were performed with the SPSS statistical software package13.0. The data were expressed as mean±SD or rate (%) with One-way analysis of variance (ANOVA) orχ2 test for measurement data or Count data respectively, to perform comparisons between the different groups, and a P value<0.05 was considered as statistically significant.
Results
1. Effect of Allicin on the HK-2 Cell Morphological changes
Cell morphology in each group was observed by inverted phase contrast microscope. When cells were cultured 24h, the cell formation of Group N emerges with single-cell fusion, oval or polygonal; the change of cell morphology of Group H was not obvious. After 48h growth, Group N emerge with tight junctions between epithelial cells, with typical cobblestone morphology; some cells of Group H showed obvious morphological changes, with fibroblast-like appearance:cell elongation, hypertrophy, spindle shape, from oval adherent growth into long-spindle, lose their cobblestone growth pattern. Group A (H+20A) With Allicin intervention after 48 hours, the cells have no significant differences with Group N, and most cells maintain normal epithelium, only a few morphological change occur. After 48h of cell growth, the cell morphological changes stimulated by high glucose in Group PD98059 occured to some extently improved.
2. Effect of Allicin on the expression of a-SMA, Vimentin and Collagen I
When cells were cultured 48h, the expression of integral optical density of a-SMA, Vimentin and Collagen I in Group H increased significantly compared with group N (p＜0.01); the expression of integral optical density of a-SMA, Vimentin, Collagen I in Group PD98059 were significantly lower compared with Group H (p<0.01), and had no significant difference with Group N (p>0.05); the expression of integral optical density of a-SMA, Vimentin, Collagen I in Group A decreased significantly with concentration-dependent manner, especially at 20ug/mL compared with group H (p<0.01), and had no significant difference with Group N and Group PD98059 (p>0.05).
3. Effect of Allicin on the expression of TGF-β1mRNA
When cells were cultured 48h, the expression of TGF-β1mRNA in Group H increased significantlycompared with Group N (p<0.05). The expression of TGF-β1mRNA in Group PD98059 also reduced significantly compared with group H (p<0.05), but was higher than that of Group N (p<0.05). The expression of TGF-β1 mRNAin Group A reduced significantly with concentration-dependent manner compared with group H (especially at 10ug/mL and 20ug/mL,p<0.05), and has no significant difference with Group PD98059 (at lOug/mL and 20ug/mL, p>0.05).
4. Effect of Allicin on the expression of TGF-β1, p-ERK 1/2 protein
When cells were cultured 48h, the expression of TGF-β1, p-ERK 1/2 protein in Group H significantly increasedcompared with Group N (p<0.05). The expression of TGF-β1, p-ERK 1/2 protein in Group PD98059 significantly reduced compared with Group H (p<0.05), while the expression of TGF-β1 protein showed no significant difference with that of Group N (p> 0.05). The expression of TGF-β1, p-ERK 1/2 protein in Group A reduced significantly with a concentration-dependent manner compared with Group H(p<0.05), especially at 20ug/mL, inhibition rates with 55.7%,37.7% respectively (p<0.05), and the expression of p-ERK 1/2 protein was significantly decreased (p<0.05) while the expression of TGF-β1 protein showed no significant difference(p>0.05) compared with Group N. The expression of TGF-β1, p-ERK 1/2 protein in Group A was more higher than that of Group PD98059(p<0.05).
5. Effect of Allicin on the apoptosis rate of HK-2
When cells were cultured 24h, the apoptosis rate of Group N was significantly lower than that of Group H ((6.56±1.09)%vs (16.25±0.58)%, (p<0.01)); the apoptosis rates of Group A (at 2.5,5,10,20 ug/ml), ((12.54±2.21)%, (9.21±2.85)%, (8.53±2.15)%, (6.81±0.92)%respectively), were significantly decreased than that of Group H (at 2.5ug/mL,p<0.05, at 5,10,20 ug/mL, p<0.01), but showed no significant difference with Group N (at 5,10,20 ug/ml, p> 0.05). When cell were culture 48h, the apoptosis rate of Group N was significantly lower than that of Group H ((6.82±2.38)%vs (23.53±3.21)%, (p<0.01)); the apoptosis rates of Group A(at 2.5,5,10,20 ug/ml), ((14.43±1.85)%, (11.68±3.20)%, (10.19± 1.93)%, (7.99±2.00)% respectively), were significantly decreased than that of Group H (p<0.01), but showed no significant difference with Group N (at 5,10,20 ug/mL,p＞0.05).
6. Effect of Allicin on the expression of Bcl-2、Bax
When cell were culture 48h, the expression of integral optical density of Bcl-2 protein in Group H decreased significantly (p<0.01) compared with group N. The expression of integral optical density of Bcl-2 protein in Group A increased significantly with concentration-dependent manner(at 5 ug/ml, p<0.05, at 10,20 ug/mL, p<0.01) compared with group H, but still lower than that of Group N (p<0.05). The expression of integral optical density of Bax protein in Group H increased significantly (p<0.01) compared with group N. The expression of integral optical density of Bax protein in Group A decreased significantly with concentration-dependent manner (p<0.01) compared with group H, but still higher than that of Group N(p<0.01). Bcl-2/Bax ratio was significantly lower than that of Group H (p＜0.01) compared with Group N; Bcl-2/Bax ratio in Group A increased significantly with concentration-dependent manner (at 5 ug/mL, p<0.05, at10, 20ug/mL,p<0.01) compared with Group H, but were still significantly lower than Group N(p<0.01).
Conclusions
1. High glucose can induce renal tubular epithelial transdifferentiation and increase the expression of TGF-β1 and CollagenⅠ.
2. PD98059, the inhibitor of ERK 1/2, can inhibit renal tubular epithelial transdifferentiation induced by high glucose, and decrease the expression of TGF-β1 and Collagen I. Blocking ERK 1/2 signaling pathway may be the important intervention target for the prevention and treatment of renal interstitial fibrosis.
3. Allicin can inhibit renal tubular epithelial transdifferentiation and reduce the expression of TGF-β1 and Collagen I induced by high glucose, which is similar to PD98059, and might be related with inhibiting the activation of ERK 1/2.
4. High glucose can induce apoptosis of renal tubular epithelial cells. Allicin can significantly inhibit apoptosis of renal tubular epithelial cells induced by high glucose, with significant concentration-dependence.
5. Allicin can regulate the expression of Bcl-2 and Bax protein and increase the Bcl-2/Bax ratio, which is one of the mechanism for Allicin to inhibit the apoptosis of renal tubular epithelial cells induced by high glucose.
糖尿病肾病是糖尿病常见和严重的慢性并发症之一,也是导致终末期肾病的主要原因之一。糖尿病肾病的发病机制尚未完全阐明。近年研究表明,肾小管间质病变是糖尿病肾病发生发展的关键因素之一,进一步深入研究肾小管间质病变的分子机制、寻找有效治疗的新靶点,对预防和延缓糖尿病肾病的发生发展有重要意义。
肾小管间质纤维化的过程包括肾小管的萎缩和消失、肌成纤维细胞的聚积和细胞外基质的沉积。目前认为肾间质细胞外基质主要由肌成纤维细胞产生,但有证据表明,在一些病理条件下,肾小管上皮细胞可发生上皮细胞-肌成纤维细胞转分化(Tubular epithelial myofibroblast transdifferentiation, TEMT),即肾小管上皮细胞丧失原有的上皮细胞表型特征,获得肌成纤维细胞的表型,大量表达肌成纤维细胞的标志性蛋白α-平滑肌肌动蛋白(a-SMA)和波形蛋白(Vimentin)。已有众多的研究表明,肾小管上皮细胞转分化直接参与肾间质纤维化的进程,是肾间质纤维化发生、发展的的中心环节,已成为肾纤维化乃至慢性肾功能衰竭的重要机制之一
TGF-β1是具有多种重要生物学作用的生长因子,也是目前已知的诱导肾小管上皮细胞转分化作用最强的生长因子,除Smads通路外,尚可通过不同信号传导途径产生多种生物学效应,在肾小管间质纤维化中发挥了核心作用。MAPK信号通路是非Smads依赖途径中的重要通路之一,它是广泛存在于细胞内的信号转导途径,由丝氨酸／苏氨酸蛋白激酶组成。在MAPK家族中,细胞外调节激酶(ERK)是最早被发现的成员,它包括两种异构体ERK1和ERK2。有研究提示,ERK信号通路是细胞信号中传递丝裂原信号的关键激酶,与肾小管上皮细胞转分化和肾脏纤维化密切相关。关于高糖环境下人近端肾小管上皮细胞发生转分化是否与TGF-β1／ERK信号通路有关,目前较少报道。
细胞凋亡与增殖的动态平衡是多细胞生物维系结构完整和内环境稳定的基本生物学特性。随着对细胞凋亡的基因调控、信号转导及其与疾病关系的深入研究,表明细胞凋亡密切参与了糖尿病肾病的发生机制。研究发现,在糖尿病肾病早期即出现肾小管上皮细胞的凋亡,后者是导致肾小管功能障碍的重要原因之一,也是糖尿病肾病进展为慢性肾功能衰竭的重要机制之一。有研究显示,肾小管间质纤维化形成中存在明显的肾小管上皮细胞凋亡现象,病变早期凋亡细胞数随着肾间质纤维化的加重呈明显增高趋势。因此在糖尿病肾病致肾小管间质纤维化的发生发展过程中,肾小管上皮细胞凋亡是高血糖损伤肾小管上皮细胞的重要病理变化形式之一
大蒜素,是大蒜的主要有效成分。现代化学和药理学研究发现大蒜素具有抗菌、抗病毒、抗氧化、抗肿瘤、降血脂、提高机体免疫力等多方面的作用。近年研究表明,大蒜素有抗肺、肝及心肌纤维化作用,主要与其抑制成纤维细胞增殖,抑制促纤维化因子的分泌及减少细胞外基质的合成等有关。另有研究发现,大蒜素可抑制创伤／失血性休克大鼠重要脏器的细胞凋亡。但大蒜素是否具有抑制高糖诱导肾小管上皮细胞转分化和凋亡的作用,目前尚未明确。
研究目的
1.观察大蒜素对高糖环境下人近端肾小管上皮细胞转分化的作用,并初步探讨大蒜素抑制肾小管上皮细胞转分化的机制。
2.观察大蒜素对高糖环境下人近端肾小管上皮细胞凋亡及凋亡相关蛋白Bcl-2和Bax表达的影响,并初步探讨大蒜素抑制肾小管上皮细胞凋亡的机制。
实验方法
1.以人肾小管上皮细胞株HK-2细胞为研究对象,按实验条件分为4组,以DMEM为培养基,即正常糖对照组(低糖5.5mmol/l DMEM)、高糖组(高糖25mmol/l DMEM)、大蒜素组(高糖组加不同终浓度的大蒜素,剂量分别为2.5、5、10、20ug/ml)和ERK抑制剂组(高糖组加20umol/l PD98059),分别培养24h、48h。采用免疫细胞化学法、荧光免疫细胞化学法检测各组细胞内a-SMA、Vimentin和Collagen I的相对表达量,Real-time PCR法检测各组细胞内TGF-β1 mRNA的相对表达,Western Blot法检测各组细胞TGF-β1. p-ERK1/2(磷酸化的ERK1/2)蛋白的相对表达。
2.以人肾小管上皮细胞株HK-2细胞为研究对象,按实验条件分为3组,以DMEM为培养基,即正常糖对照组(低糖5.5mmol/l DMEM)、高糖组(高糖：25mmol/l DMEM)和大蒜素组(高糖组加不同终浓度的大蒜素,剂量分别为2.5、5、10、20ug/ml),细胞培养24h、48h。采用流式细胞术观察各组细胞的凋亡率,采用免疫细胞化学法检测HK-2细胞内Bcl-2和Bax蛋白的相对表达量。
3.应用SPSS 13.0统计软件对数据进行分析,计量资料用均数±标准差((?)±S)表示,各组间比较采用单因素方差分析(one-way ANOVA),计数资料用率(%)表示,各组间比较采用χ2检验,p<0.05有统计学意义。
结果
1.大蒜素对HK-2细胞形态学变化的影响
正常糖对照组生长24h后,形成融合的单层细胞,呈椭圆形或者多边形贴壁细胞,生长48h后细胞间紧密连接,具有典型的上皮细胞铺路石样形态学特征。高糖组生长24h后,细胞形态改变不明显,生长48h后,可见部分细胞发生明显的形态学改变,表现为成纤维细胞样外观：细胞拉长、肥大,呈长梭形,由椭圆形贴壁生长变为长梭形,丧失其铺路石样生长方式。大蒜素组(H+A20ug/ml)干预48小时后,细胞形态与N组无明显差异,光镜下多数细胞保持正常上皮,仅少数发生形态变化。PD98059组细胞生长48h后,受高糖刺激而出现的形态学改变有一定程度的改善。
2.大蒜素对HK-2细胞α-SMA、Vimentin、Collagen I表达的影响
细胞培养48h,高糖组α-SMA、Vimentin、Collagen I表达的积分光密度值(IOD)较正常糖对照组明显增加,均有统计学意义(p<0.01); PD98059组α-SMA、Vimentin和Collagen I表达的IOD较高糖组均明显减少(p<0.01),且与正常糖对照组无明显差异(p>0.05)；高糖环境下经不同浓度的大蒜素(2.5、5、10、20ug/mL)干预后,a-SMA. Vimentin. Collagen I表达的IOD明显下降,并呈浓度依赖性,尤以20ug/mL更为明显(p<0.01),此组与正常糖对照组和PD98059组比较无显著差别(p>0.05)。
3.大蒜素对HK-2细胞TGF-β1mRNA表达的影响
细胞培养48h时,与正常糖对照组比较,高糖组HK-2细胞TGF-β1mRNA表达明显上调(p<0.05)；PD98059组可逆转高糖刺激下的TGF-β1 mRNA表达(p<0.05),但仍高于正常糖对照组(p<0.05)；大蒜素可明显下调高糖刺激下HK-2细胞TGF-β1mRNA的表达,并呈浓度依赖效应,尤其在l0ug/ml、20ug/ml时(p<0.05),同时与PD98059组比较无显著性差异,且20ug／ml时与正常糖对照组比较亦无显著性差异。
4.大蒜素对HK-2细胞TGF-β1、p-ERK 1／2蛋白表达的影响
细胞培养48h时,正常糖对照组HK-2细胞可见少量TGF-β1、p-ERK1/2蛋白的表达；高糖组HK-2细胞TGF-β1, p-ERK1/2的蛋白表达较正常糖对照组明显上调p<0.05)；与高糖组比较,PD98059组TGF-β1、p-ERK 1/2蛋白表达均明显下调(p<0.05),而TGF-β1蛋白与正常糖对照组比较无显著性差异(p>0.05)；与高糖组比较,经不同浓度大蒜素(2.5、5、10、20ug／mL)干预48小时后,HK-2细胞TGF-β1、p-ERK 1／2蛋白的表达水平均呈药物浓度依赖性下调(p<0.05),尤以20ug／mL浓度作用最强,抑制率分别为55.7％(TGF-β1,p<0.05)和37.7％(p-ERK1/2,p<0.05),且与正常糖对照组比较,p-ERK 1／2蛋白表达亦明显下调,而TGF-β1蛋白表达差异无显著性,但两者均仍高于PD98059组(p<0.05)。
5.大蒜素对HK-2细胞凋亡率的影响
细胞培养24h,正常糖对照组只有少量细胞发生凋亡,凋亡率仅为(6.56±1.09)%,而高糖组凋亡率为(16.25±0.58)%,两组间有显著性差异(p<0.01)；大蒜素干预组(2.5.5.10.20ug/mL)的细胞凋亡率分别为(12.54±2.21)%、(9.21±2.85)%、(8.53±2.15)%、(6.81±0.92)%,均较高糖组有明显下降(2.5ug/mL时p<0.05,5、10、20ug／mL时p<0.01),且大蒜素浓度为5、10、20ug／ml时与正常糖对照组比较无显著性差异(p>0.05)。细胞培养48h,正常糖对照组细胞亦只有少量细胞发生凋亡,凋亡率仅为(6.82±2.38)%,高糖组升高为(23.53±3.21)%；而大蒜素干预组(2.5.5.10.20ug/mL)的细胞凋亡率分别为(14.43±1.85)%、(11.68±3.20)%、(10.19±1.93)%、(7.99±2.00)%,与高糖组比较均有明显下降(p<0.01)且大蒜素浓度为10、20ug／mL时与正常糖对照组比较无显著性差异(p>0.05)。
6.大蒜素对HK-2细胞Bcl-2、Bax蛋白表达的影响
细胞培养48h,高糖组Bcl-2蛋白表达的积分光密度值(IOD)为(14.08±2.14),较正常糖对照组的IOD(31.97±4.29)明显降低(p<0.01),不同浓度的大蒜素组的Bcl-2蛋白表达的IOD高于高糖组,并随着浓度的增高而升高(5 ug/mL时p<0.05,10. 20ug/mL p＜0.01),但仍低于正常糖对照组(p<0.05)；而Bax蛋白则正好相反,高糖组Bax蛋白表达的IOD为(109.83±14.13),较正常糖对照组的IOD(29.68±3.30)明显升高(p<0.01),不同浓度大蒜素组的Bax蛋白表达的IOD明显低于高糖组,并随着浓度的增高而逐渐降低(均p<0.01),但仍高于正常糖对照组(p<0.01)。Bcl-2/Bax的比值在高糖组明显低于正常糖对照组(p<0.01),经大蒜素干预后其比值较高糖组有升高(5 ug/mL时(p<0.05,10、20ug／mLp<0.01),并呈浓度依赖性,但仍明显低于正常糖对照组(p<0.01)
结论
1.高糖可诱导HK-2细胞转分化为肌成纤维细胞,上调TGF-β1和细胞外基质成分Collagen I的表达。
2. ERK1/2信号通路抑制剂PD98059可明显抑制高糖环境下HK-2细胞的表型转分化,下调TGF-β1和Collagen I的表达,阻断ERK 1／2信号通路是抑制TEMT、防治肾间质纤维化的重要干预靶点。
3.大蒜素可通过抑制ERK 1／2信号通路的激活而抑制高糖状态下HK-2细胞向肌成纤维细胞转分化,下调TGF-β1和Collagen I的表达,其抑制作用与PD98059相似。
4.高糖诱导肾小管上皮细胞发生凋亡。大蒜素对高糖诱导的肾小管上皮细胞凋亡具有明显的抑制作用,且存在量效关系。
5.大蒜素调节Bcl-2和Bax蛋白的表达,上调Bcl-2/Bax比值是其抑制高糖诱导肾小管上皮细胞凋亡的机制之一
Background
Diabetic nephropathy(DN) is one of the most common and serious chronic diabetic complications. It is one of the main reasons leading to end-stage renal failure. The pathogenesis of diabetic nephropathy has not yet fully understood. Recent studies showed that, tubulointerstitial lesions is one of the key factors in the development of diabetic nephropathy. So it has great significance to take further in-depth study on tubulointerstitial molecular mechanism to find a new target for the effective treatment, prevention and delay the process of diabetic nephropathy.
The process of tubulointerstitial fibrosis includes the atrophy and loss of tubular, myofibroblasts accumulation and extracellular matrix deposition. It's currently considered that the extracellular matrix in renal interstitial fibroblasts is produced mainly by muscle cells. Growing evidence showed that in some pathological conditions, renal tubular epithelial cells can occur in epithelial cells-myofibroblast transdifferentiation(TEMT), by losing its original phenotype and obtaining the phenotype of myofibroblasts, which express a much of a-smooth muscle actin (α-SMA) and Vimentin. Numerous research studies have found that tubular epithelial myofibroblast transdifferentiation, which was the central link of the occurrence and development of renal interstitial fibrosis, involved directly in the process of renal interstitial fibrosis, and had become one of the great mechanisms of renal fibrosis and even chronic renal failure.
TGF-β1 is a growth factor which has a variety of important biological effects. It is also known to be the most potent growth factor, which induces tubular epithelial myofibroblast transdifferentiation. TGF-β1 plays a central role in renal tubular interstitial fibrosis by producing a variety of biological effects via different signal transduction pathways includes Smads. MAPK signaling pathway, a non-Smads dependent pathway, is one of the most important signal transduction pathways which is widely found in cells, composed of the serine/threonine protein kinase. In the MAPK family, extracellular regulated kinase (ERK) is the first member to be found, which consists of two isomers, ERK1 and ERK2. Some studies suggest that, ERK signaling pathway is the key mitogen kinase of cell signals which transfer mitogen signals, and it is closely related to tubular epithelial myofibroblast transdifferentiation and renal fibrosis. Whether human proximal tubular epithelial myofibroblast transdifferentiation in high glucose is related to TGF-β1/ERK signaling pathway or not, is not well known.
Dynamic equilibrium of apoptosis and proliferation is the basic biological properties of maintaining structural integrity of multi-cellular organisms and environment stability. With the studies on gene regulation, signal transduction and its relationship with diseases, it is showed that apoptosis has been closely involved in the pathogenesis of diabetic nephropathy. Studies found that in the early stage of diabetic nephropathy, apoptosis of tubular epithelial cell has occured, which is one of the major reasons leading to tubular dysfunction and is one of the most important mechanisms of the progress of diabetic nephropathy with chronic renal failure. Studies have shown that the apoptosis of renal tubular epithelial cells is obvious in renal interstitial fibrosis, and that apoptotic cells increased significantly with the aggravation of renal interstitial fibrosis in early lesions. Therefore, the apoptosis of tubular epithelial cell is one of the most important pathological change forms of damages on renal tubular epithelial cells by high blood glucose, during the development process in diabetic renal tubular interstitial fibrosis.
Allicin is the main active ingredient of garlic. Modern chemistry and pharmacology studies found that Allicin has many effects such as antibacterial, antiviral, antioxidant, anti-tumor, and so on. Recent studies show that Allicin is known as anti-lung, liver and cardiac fibrosis, mainly associated with the inhibition of fibroblast proliferation, secretion of promoting fibrosis factors and reduction of the synthesis of extracellular matrix, and also Allicin can inhibit apoptosis in vital organs in rats with trauma/hemorrhagic shock. However, whether Allicin could inhibit tubular epithelial myofibroblast transdifferentiation and apoptosis induced by high glucose or not, is not clear.
Objectives
1. To observe the influence of Allicin on human proximal tubular epithelial myofibroblast transdifferentiation in high glucose and study its mechanism preliminary.
2. To observe the influence of Allicin on apoptosis and its gene expression of human proximal tubular epithelial cell in high glucose and study its mechanism preliminary.
Methods
1. Human proximal renal tubular epithelial cells (HK-2) were divided into four groups according to the experimental conditions, with DMEM as the medium:Group Normal Glucose Control (Group N), cultured with glucose (5.5mmol/L) DMEM; Group High Glucose (Group H), cultured with high glucose (25mmol/L) DMEM; Groups Allicin, cultured with high glucose (25mmol/L) DMEM, with different concentrations of Allicin(2.5,5,10 and 20ug/mL); Group H+PD98059, cultured with 25mmol/L glucose DMEM+ERK inhibitor (PD98059)20umol/L. Each group cells were cultured 24h,48h. The integral optical denisity expression of a-SMA, Vimentin and Collagen I were detected by immunocytochemistry. The mRNA expression of TGF-β1 and the protein expression of TGF-β1, p-ERK1/2 were assessed by Real-time PCR and Western Blot method, respectively.
2. Human proximal renal tubular epithelial cells (HK-2) were divided into eight groups according to the experimental conditions, with DMEM as the medium:Group Normal Glucose Control (Group N), cultured with glucose (5.5mmol/L) DMEM; Group High Glucose (Group H), cultured with high glucose (25mmol/L) DMEM; Groups Allicin, cultured with high glucose (25mmol/L) DMEM, with different concentrations of Allicin(2.5,5,10 and 20ug/mL); Group H+PD98059, cultured with 25mmol/L glucose DMEM+ERK inhibitor(PD98059)20umol/L. Each group cells were cultured 24h,48h. The apoptosis rate of HK-2 cells in each group was assessed by flow cytometry using, and the protein expression of Bcl-2 and Bax were assessed by immunocytochemistry.
3. All analyses were performed with the SPSS statistical software package13.0. The data were expressed as mean±SD or rate (%) with One-way analysis of variance (ANOVA) orχ2 test for measurement data or Count data respectively, to perform comparisons between the different groups, and a P value<0.05 was considered as statistically significant.
Results
1. Effect of Allicin on the HK-2 Cell Morphological changes
Cell morphology in each group was observed by inverted phase contrast microscope. When cells were cultured 24h, the cell formation of Group N emerges with single-cell fusion, oval or polygonal; the change of cell morphology of Group H was not obvious. After 48h growth, Group N emerge with tight junctions between epithelial cells, with typical cobblestone morphology; some cells of Group H showed obvious morphological changes, with fibroblast-like appearance:cell elongation, hypertrophy, spindle shape, from oval adherent growth into long-spindle, lose their cobblestone growth pattern. Group A (H+20A) With Allicin intervention after 48 hours, the cells have no significant differences with Group N, and most cells maintain normal epithelium, only a few morphological change occur. After 48h of cell growth, the cell morphological changes stimulated by high glucose in Group PD98059 occured to some extently improved.
2. Effect of Allicin on the expression of a-SMA, Vimentin and Collagen I
When cells were cultured 48h, the expression of integral optical density of a-SMA, Vimentin and Collagen I in Group H increased significantly compared with group N (p＜0.01); the expression of integral optical density of a-SMA, Vimentin, Collagen I in Group PD98059 were significantly lower compared with Group H (p<0.01), and had no significant difference with Group N (p>0.05); the expression of integral optical density of a-SMA, Vimentin, Collagen I in Group A decreased significantly with concentration-dependent manner, especially at 20ug/mL compared with group H (p<0.01), and had no significant difference with Group N and Group PD98059 (p>0.05).
3. Effect of Allicin on the expression of TGF-β1mRNA
When cells were cultured 48h, the expression of TGF-β1mRNA in Group H increased significantlycompared with Group N (p<0.05). The expression of TGF-β1mRNA in Group PD98059 also reduced significantly compared with group H (p<0.05), but was higher than that of Group N (p<0.05). The expression of TGF-β1 mRNAin Group A reduced significantly with concentration-dependent manner compared with group H (especially at 10ug/mL and 20ug/mL,p<0.05), and has no significant difference with Group PD98059 (at lOug/mL and 20ug/mL, p>0.05).
4. Effect of Allicin on the expression of TGF-β1, p-ERK 1/2 protein
When cells were cultured 48h, the expression of TGF-β1, p-ERK 1/2 protein in Group H significantly increasedcompared with Group N (p<0.05). The expression of TGF-β1, p-ERK 1/2 protein in Group PD98059 significantly reduced compared with Group H (p<0.05), while the expression of TGF-β1 protein showed no significant difference with that of Group N (p> 0.05). The expression of TGF-β1, p-ERK 1/2 protein in Group A reduced significantly with a concentration-dependent manner compared with Group H(p<0.05), especially at 20ug/mL, inhibition rates with 55.7%,37.7% respectively (p<0.05), and the expression of p-ERK 1/2 protein was significantly decreased (p<0.05) while the expression of TGF-β1 protein showed no significant difference(p>0.05) compared with Group N. The expression of TGF-β1, p-ERK 1/2 protein in Group A was more higher than that of Group PD98059(p<0.05).
5. Effect of Allicin on the apoptosis rate of HK-2
When cells were cultured 24h, the apoptosis rate of Group N was significantly lower than that of Group H ((6.56±1.09)%vs (16.25±0.58)%, (p<0.01)); the apoptosis rates of Group A (at 2.5,5,10,20 ug/ml), ((12.54±2.21)%, (9.21±2.85)%, (8.53±2.15)%, (6.81±0.92)%respectively), were significantly decreased than that of Group H (at 2.5ug/mL,p<0.05, at 5,10,20 ug/mL, p<0.01), but showed no significant difference with Group N (at 5,10,20 ug/ml, p> 0.05). When cell were culture 48h, the apoptosis rate of Group N was significantly lower than that of Group H ((6.82±2.38)%vs (23.53±3.21)%, (p<0.01)); the apoptosis rates of Group A(at 2.5,5,10,20 ug/ml), ((14.43±1.85)%, (11.68±3.20)%, (10.19± 1.93)%, (7.99±2.00)% respectively), were significantly decreased than that of Group H (p<0.01), but showed no significant difference with Group N (at 5,10,20 ug/mL,p＞0.05).
6. Effect of Allicin on the expression of Bcl-2、Bax
When cell were culture 48h, the expression of integral optical density of Bcl-2 protein in Group H decreased significantly (p<0.01) compared with group N. The expression of integral optical density of Bcl-2 protein in Group A increased significantly with concentration-dependent manner(at 5 ug/ml, p<0.05, at 10,20 ug/mL, p<0.01) compared with group H, but still lower than that of Group N (p<0.05). The expression of integral optical density of Bax protein in Group H increased significantly (p<0.01) compared with group N. The expression of integral optical density of Bax protein in Group A decreased significantly with concentration-dependent manner (p<0.01) compared with group H, but still higher than that of Group N(p<0.01). Bcl-2/Bax ratio was significantly lower than that of Group H (p＜0.01) compared with Group N; Bcl-2/Bax ratio in Group A increased significantly with concentration-dependent manner (at 5 ug/mL, p<0.05, at10, 20ug/mL,p<0.01) compared with Group H, but were still significantly lower than Group N(p<0.01).
Conclusions
1. High glucose can induce renal tubular epithelial transdifferentiation and increase the expression of TGF-β1 and CollagenⅠ.
2. PD98059, the inhibitor of ERK 1/2, can inhibit renal tubular epithelial transdifferentiation induced by high glucose, and decrease the expression of TGF-β1 and Collagen I. Blocking ERK 1/2 signaling pathway may be the important intervention target for the prevention and treatment of renal interstitial fibrosis.
3. Allicin can inhibit renal tubular epithelial transdifferentiation and reduce the expression of TGF-β1 and Collagen I induced by high glucose, which is similar to PD98059, and might be related with inhibiting the activation of ERK 1/2.
4. High glucose can induce apoptosis of renal tubular epithelial cells. Allicin can significantly inhibit apoptosis of renal tubular epithelial cells induced by high glucose, with significant concentration-dependence.
5. Allicin can regulate the expression of Bcl-2 and Bax protein and increase the Bcl-2/Bax ratio, which is one of the mechanism for Allicin to inhibit the apoptosis of renal tubular epithelial cells induced by high glucose.
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