| DN is a part of micovasclor complication and one of the common reason of (ESRD). The researchers keep working on the reason and treatment of DN. The traditional Chinese herbs play an important role in preventing DN because of its advantage. Professor Zhao Yuyong invented an effective traditional medicine, Shenluotong, according to the tradional Chinese medicine theory and his many years of clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and the blood, dispersing blood stasis and dredging collateral and is common to most of DN patients. The clinical data showed that the prescription had good effect on DN. But the mechanism is still unknown. It is known that the podocytes adhere to the outside of (GBM), which synthesize and secret collagen α5 (IV), the main component of GBM, as well as gelatinases (MMP2, MMP9), degradating type-IV collagen which constitutes the structure and function of the GBM. When the nephrons were injured, the podocytes lose the balance of poducing and degenerating type-IV collagen, promote the development of glomerulosclerosis, So the effect of podocyte in developing DN induced much more attention. In 1997, the podocyte cells were incubated successfully, making it possible for researching the mechamism of podocytes in DN development. Objective:This study aim to assess the mechanism of high glucose on the production of gelatinase and collagen α5 (IV) in cultured podocytes and the signaling pathway, investigate the mechanism of Shen Luo Tong on treating DN to improve the research about Chinese traditional medicine. Part I High glucose increased the production of MMP9 and type-IV collagen in podocyte Methods: The immortalized mouse podocyte cell line was used, and the cells were treated with normal concentration of D-glucose (NG group), high concentration of D-glucose (HG group), and mannitol (MN group). The culture medium supernatants were collected every day for ten days and the activity of MMP9 and MMP2 was detected by gelatin zymography, the level of MMP9 mRNA was detected by RT-PCR at day 0, 2, 5. The level of collagen α5 (IV) protein in culture supernatant was detected by Western blot analysis. Result: 1. The effect of high glucose on MMP9 activity in the supernatant: The MMP9 activity of the supernatant in HG group increased at 2nd day as compared with NG group, reached peak at 3rd day(144.2±18.7% to NG, p=0.006), then declined from 5th day, back to base level at 7th day (76.6±16.4% to NG,p=0.218) and kept at base level until 10th day. Both groups of NG and MN, did not affect the activity of MMP9, the activity of MMP2 was not affected in three group. 2. The effect of high glucose on the MMP9mRNA: The level of MMP9mRNA was stimulated with HG at 2nd day as compared with NG group (199.8±40.2% to NG, p=0.003), but the base level of MMP9mRNA was observed at 5th day (90.9±8.8% to NG, p=0.411). The changes of MMP9mRNA were parallel with the activity of MMP9. 3. The effect of high glucose on collagen α5 (IV) protein in the supernatant: There was quite high base level of collagen α5 (IV) protein in the supernatant of NG group. High glucose induced a decrease of collagen α5 (IV ) protein level at 2nd day, reached the minimum at 3rd day as compared with NG group (41.9±25.5% to NG,p=0.047), then back to the base level form 5th day. It was no significant change on the level of collagen α5 (IV) protein in the supernatant in NG and MN groups. 4. The line regression analysis between the MMP9 activity and Collagen IV α5 protein in HG group: When the MMP9 activity of the supernatant in 7 days was used as X, the level of collagen α5 (IV) protein was used as Y,regression analysis showed that both had a stronger negative correlation (r= -0.577, p<0.006). Part II High glucose regulates the production of MMP9 in podocytes through ERK signal pathway and upregulation of Ets-1 Protein Methods: 1. Western blot analysis detect the activation of mitogen-acivated protein kinase (MAPKs, including ERK1/2, p38, JNK) signaling pathway in podocytes:The cells were divided into three groups (same as the part I ). The activation of MAPKs signaling pathway in podocytes were detected by Western blot analysis, when cells was stimulated at 0min, 30min, 1h, 6h, 12h,24h,48h. 2. Western blot analysis detect the level of a transcription factor (Ets-1) protein: The cells were divided into three groups (same as the part I ), Ets-1 protein were detected by Western blot analysis, when the cells were stimulated for 0d,2d,5d. 3. Western blot analysis detect the level of Ets-1 in nuclear and cytoplasm: Podocytes were incubated with HG,MN,NG for 1h, and were collected, lysesd, divided into the cytoplasmic and nuclear protein, which was used Western blot analysis for detecting Ets-1. 4. PD98059 blocked the effect of high glucose: Pre-treatment with PD98059, a specific inhibitors of ERK for 30 min, the podocytes were divided into three groups as before. the activity of MMP9 and MMP2 was detected by gelatin zymography, the level of MMP9 mRNA was detected by RT-PCR, The level of collagen α5 (IV) in culture supernatant, Ets-1 protein and Ets-1 activity was detected by Western blot analysis. Results: 1. The activation of MAPKs signaling pathway in HG group: Phosphorylation of ERK1/2 in podocytes was occurred as early as 30 min after high glucose treatment, reached the peak level at 6h, keep activation at24h, then back to the base level at 48h. The activation of p38 and JNK were undetectable in our experiments. 2. The effect of high glucose on transcription factor Ets-1 protein: The level of Ets-1 was stimulated by HG at 2nd day as compared with that of NG(180.8±33.1% to NG, p=0.000), but back base level of Ets-1 at 5th day in HG. It was no significant change on the level of Ets-1 protein in NG and MN groups. 3. The effect of high glucose on activity of Ets-1: There was quite low level of Ets-1activation in cytoplasm proteins in the supernatant of HG,MN,NG groups. High glucose induced an prominent increase of Ets-1 activity in nuclear protein as compared with NG, MN group (259.5±68.3% to NG,P=0.001), showing that the activity of Ets-1 in nuclear was up-regulated by HG. It was not sinificant change on the level of Ets-1 nuclear protein in NG and MN groups. The loading control of cytoplasm protein, GAPDH was expressed in cytoplasm protein similarly in three groups, no expression in nuclear protein. The loading control of nuclear protein, histone H4, was expressed in nuclear protein similarly in three groups, no expression in cytoplasm protein. The above results showed the division of nuclear and cytoplasm protein was very pure. 4. PD98059 blocked the effect of high glucose: Pre-treatment with PD98059, a specific inhibitors of ERK activation for 30 min could abolish the HG-stimulated increases on MMP9 activity (187.6±2.9% VS 91.6±10.5%,p=0.001)and MMP9mRNA(205.9 ±50.2% vs113.2±24.2%,p=0.006), and the decreases of collagen α5 (IV)(52.8±3.4% VS 101.0±29.1%,p=0.02 ) 5. PD98059 blocked the influence of high glucose on the level of Ets-1 and the activity of Ets-1: Pre-treatment with PD98059 for 30 min could abolish the HG-stimulated increases in Ets-1 level (82.6±28.4% to NG, p=0.000) and Ets-1activity. Part III Regulation of Shen Luo Tong on MMP9 production induced by high glucose in podocyteMethods: 1. Preparement of the mice serum with Shen Luo Tong:Shenloutong was composed of membranous Acuiellaria baicalensis, Salvia miltiorrhiza, Ligusticum wallichii. Bombyx batryticatus, Zaocys and Radix et Rhizoma Rhei, etc. SD rats, weighed 180g—220g, were randomly divided into two groups, blank control group and treatment group. The treatment groups were feed with traditional medicine, and the blank group was feed with equal quantity of distilled water. The thigh artery blood was taken after end treatment 1h. The serum was divided and penetrated through 0.22μm filter after antibiotic treatment. 2. Treatment of podocytes by the serum with Shen Luo Tong:The podocytes were divided into three dosage of rat serum, low (1.25%), medium (2.5%), high (5%) respectively. 3. The activity of MMP9 and MMP2 was detected by gelatin zymography, MMP9mRNA level was detected by RT-PCR, the level of collagen IV α5 protein in culture supernatant was detected by Western blot analysis. Result: 1. The regulation of Shenluotong on MMP9, MMP2 activity induced by high glucose: Activity of MMP9 in Shenluotong groups ware lower than that of blank control and glucose treatment group significantly, especially in Shenluotong high group. The treatment of 5% Shenluotong decreased the activity of MMP9 activity on 3rd day, than that of high glucose treatment group (53.7±7.6% to HG, p=0.004), but no effect on MMP2 activity. 2. The regulation of Shenluotong on MMP9 mRNA expression induced by high glucose: The expression of MMP9mRNA in Shenluotong groups was lower significantly than that of blank control and glucose treatment group, especially in Shenluotong high group. The treatment of 5% Shenluotong can down-regulate the expression of MMP9mRNA increased by HG (43.3±10.8% to HG, p=0.003). 3. The regulation of Shenluotong on the collagen α5 (IV) protein expression:...
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