The Effect And Mechanism Of Allicin On Human Tubular Epithelial-myofibroblast Transdifferentiation And Apoptosis In High Glucose Condition

BackgroundDiabetic 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.Objectives1. 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. Methods1. 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. Results1. Effect of Allicin on the HK-2 Cell Morphological changesCell 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 IWhen 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-β1mRNAWhen 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 proteinWhen 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-2When 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,BaxWhen 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).Conclusions1. 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.