| BackgroundDiabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). The characteristic pathological changes of DN include mesangial extracellular matrix (ECM) accumulation, glomerulosclerosis and tubule interstitial fibrosis, which eventually lead to the progressive increase of urinary protein/albumin excretion and decline of renal function. The pathogenesis of DN is complicated. Multiple mechanisms contribute to the development and outcomes of diabetic nephropathy, such as the interactions among hyperglycemia induced metabolic and hemodynamic changes, increased production of advanced glycation end products (AGEs), enhanced polyol pathway, activation of protein kinase C (PKC), and oxidative stress. How to prevent and treat DN effectively has been concerned considerably by clinicians.Mesangial cells (MCs) is one of the most important intrinsic glomerular cells, maintaining structural integrity of the glomerular microvascular bed and mesangial matrix homeostasis. In response to metabolic or hemodynamic injury induced by hyperglycemia, MCs undergo hypertrophy, proliferation with excessive production of ECM, such as collagen and fibronectin (FN). MCs may also respond to injury that primarily involves podocytes and endothelial cells or to abnormalities of the glomerular basement membrane, to produce growth factors, chemokines and cytokines. These factors exert autocrine and paracrine effects on MCs or on other glomerular cells, facilitating the progress of glomerulosclerosis.Transforming growth factor-β1(TGFβ1) is one of the crucial mediators of renal fibrosis. MCs are able to secrete TGFβ1, and express the receptor of TGFβ1. Thus, TGFβ1exerts direct effects on MCs through autocrine and paracrine way, promoting the glomerulosclerosis and the renal fibrosis. In the diabetic state, a variety of profibrosis factors can induce the TGFβ1synthesis and activation by MCs, in turn leading Smads protein phosphorylation, to promote ECM generation and inhibit ECM degradation.Mitogen activated protein kinase (MAPK) pathway is another important signaling pathway playing roles in pathogenesis of DN. MAPK pathway mainly includes extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38, transducting extracellular signals into cells. Hyperglycemia, AGEs and the activation of PKC and oxidative stress may induce MAPKs activation. Once be activated, MAPKs phosphorylate their downstream transcription factors, regulating expression of the target genes. MAPK pathway may have direct effects, or interact with TGFβ1/Smads pathway involving in the pathogenesis of DN.The development of diabetes involves metabolic, endocrine, and hemodynamic abnormalities which can promote a state of chronic inflammation. In the kidney, this can lead to the accumulation of kidney macrophages, promoting the development of renal injury, kidney fibrosis and the renal function decline. Recent evidence has highlighted the production of monocyte chemoattractant protein-1(MCP-1) by diabetic kidneys as a major factor influencing macrophage accumulation in this disease. High levels of glucose have been shown to stimulate MCP-1production by mesangial cells through ways of the activation of PKC, increased levels of oxidative stress, and the activation of the transcription factor nuclear factor-icB (NFκB). Selective inhibition of MCP-1has been proven to be an effective reduction of kidney macrophages infiltration and TGFβ1expression, preventing the progress of glomerulosclerosis in animal models.Prostacyclin (PGI2), belonging to the prostaglandin (PG) family, is an end product derived from the sequential metabolism of arachidonic acid via cyclooxygenase-2(COX-2) and prostacyclin synthase (PGIS). It has been well known that PGI2has the ability to inhibit platelet aggregation and has powerful vasodilatory effects via relaxation of smooth muscle since first reported in1976. There are two recognized receptors that mediate the effects of PGI2:1) binding to the cell surface G protein-coupled IP receptor and activate cAMP/PKA second messenger system,2) binding to the nuclear peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and alter target gene transcription. The character of PGI2is labile, and its half life is only4min at37degrees C. Therefore, many synthetic stable PGI2analogues are very useful to help us to study the biological effects of PGI2in vivo and in vitro.Beraprost sodium (BPS) is the only one stable PGI2analogue taken as a tablet. In vitro studies, BPS, which inhibited the stretch-induced FN overexpression through the inhibition of the stretch-induced activation of MAPKs in cAMP/PKA dependent manner, may work protectively against the injury from glomerular hypertension in MCs. It also has been shown that BPS inhibited high glucose-induced cellular proliferation and the generation of ROS, and improved the antioxidant capacities of MCs. In animal models, BPS attenuated glomerular hyperfiltration by modulating vasomotion of glomerular afferent arterioles, inhibited macrophages infiltration and AGEs formation in diabetic glomeruli, and ameliorated renal function by reduing creatinine clearance and urinary excretion of albumin. In the type2diabetic patients with incipient nephropathy, urinary albumin excretion was significantly decreased after18months of BPS administration, and the level of creatinine clearance was significantly reduced in BPS group after24months. Therefore, BPS may have promising prospects of its applications in the prevention of diabetic nephropathy. To study the potential renoprotective mechamism of BPS may have great clinical significance.In this study, we explored the effects of beraprost sodium on ECM metabolism and MCP-1expression in a rat mesangial cell line under high glucose conditions, and its potential mechanisms, serving as rationale for beraprost sodium in the prevention of diabetic nephropathy.Chapter One Effects of Beraprost Sodium on ECM synthesis induced by high glucose in rat mesangial cells[Objectives]1. To investigate into the effect of Beraprost Sodium (BPS) on proliferative activity of HBZY-1cells induced by high glucose (HG).2. To investigate into the effects of BPS on TGFβ1, FN and MMP-2protein expressions induced by HG in HBZY-1cells.3. To investigate into effects of BPS on mRNA expressions of TGFβ1, FN and MMP-2induced by HG in HBZY-1cells.4. To explore whether BPS can regulate p38and ERK MAPK signalling pathway in HBZY-1cells cultured under high glucose conditions.5. To explore whether BPS can regulate TGFβ1/Smad3signalling pathway in HBZY-1cells cultured under high glucose conditions.[Method]1. Cell culture:HBZY-1cells were cultured at37℃,5%C02with DMEM containing10%FCS, penicillin(100units/mL), and streptomycin(100xg/mL).2. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, the glucose concentration was5.5mmol/L (mM), High glucose (HG) group, the glucose concentration was30mM, HG combined with different concentrations of BPS groups, the BPS concentration were0.1umol/L (uM),0.5uM,1uM,2uM,5uM, and10uM, respectively.3. MTT assay:to investigate into the proliferative activity of HBZY-1cells4. Elisa assaay:to investigate into protein expressions of TGFβ1, FN and MMP-2in the cell culture supernatants.5. Relative mRNA expressions of TGFβ1, FN and MMP-2were detected by real-time PCR.6. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.7. Expression of Smad3phosphorylated protein were analysed by Western blots.8. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett’s T3. P≤0.05was considered as significant.[Results]1. Proliferative activity of HBZY-1cells by MTT assay:compared with NG group, OD values of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, except HG+O.luMBPS group, OD values of HBZY-1cells in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, HG+5uMBPS and HG+lOuMBPS group were significantly decrease after24h (P=0.044) and48h (P<0.001) culture, respectively.According to the results of this experiment, we selected the concentration of0.5,1.2,5uM as BPS concentration processing in the follow-up experiments.2. Protein expression of TGFβ1:compared with NG group, TGFβ1protein expression of HBZY-1cells was significantly increased in HG group (P<0.001) after24h and48h culture, respectively. After24h culture, TGFβ1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.001). After48h culture, TGFβ1protein expressions in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.011).3. Protein expression of FN:compared with NG group, FN protein expression of HBZY-1cells were significantly increased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, FN protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly decreased compared with HG group (P=0.018,0.007). After48h culture, FN protein expressions in HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.005).4. Protein expression of MMP-2:compared with NG group, MMP-2protein expression of HBZY-1cells were significantly decreased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.017,0.001). After48h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.019,0.007).5. Relative mRNA expression of TGFβ1:After24h cultures, compared with NG group, the relative mRNA expression of TGFβ1in HG group was significantly increased (2.45±0.22vs.1.00±0.00,95%CI for Mean [1.92,2.99]). Compared with HG group, the relative mRNA expressions of TGFβ1in HG+0.5uMBPS, HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased (P=0.008).6. Relative mRNA expression of FN:After24h cultures, compared with NG group, the relative mRNA expression of FN in HG group was significantly increased (1.93±0.23vs.1.00±0.00,95%CI for Mean [1.36,2.51]). Compared with HG group, the relative mRNA expressions of FN in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.006,<0.001).7. Relative mRNA expression of MMP-2:After24h cultures, compared with NG group, the relative mRNA expression of MMP-2in HG group was significantly decreased (0.528±0.132vs.1.000±0.000,95%CI for Mean [0.199,0.857]). Compared with HG group, there were no difference among the HG group and HG combined with different concentration BPS groups (F=0.906, P=0.497). 8. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly higher compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated p38MAPK protein in all BPS groups were significantly decreased (P=0.006).9. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated ERK protein in all BPS groups were significantly decreased (P<0.001).10. Smad3protein phosphorylation:after60min stimulation, expression of phosphorylated Smad3protein in HG group was significantly higher compared with NG group (P=0.007). Compared with HG group, expressions of phosphorylated Smad3protein in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.012,0.004).[Conclusions]1. BPS inhibited cell proliferation induced by high glucose in HBZY-1cells.2. BPS inhibited TGFβ1and FN protein and mRNA expressions induced by high glucose in HBZY-1cells, and incresed MMP-2protein synthesis in HBZY-1cells cultured under high glucose conditions.3. BPS inhibited p38and ERK MAPK protein phosphorylation, and Smad3protein phosphorylation, which indicated that BPS could regulate ECM synthesis and metabolic processes in HBZY-1cells cultured under high glucose conditions through different signal pathway interaction. Chapter Two Effects of Beraprost Sodium on MCP-1expression induced by high glucose in rat mesangial cells[Objectives]1. To investigate into the effects of BPS on MCP-1protein expression induced by HG in HBZY-1cells.2. To investigate into the effects of BPS on MCP-1mRNA expression induced by HG in HBZY-1cells.3. To explore whether BPS can regulate NFκB signalling pathway in HBZY-1cells cultured under high glucose conditions.[Method]1. HBZY-1cells were grouped according to different intervention:NG group, HG group, HG combined with different concentrations of BPS groups, the BPS concentration were0.5uM,1uM,2uM, and5uM, respectively, and HG combined with PDTC (1uM) group.2. Elisa assaay:to investigate into MCP-1protein expression in cell culture supernatants.3. Relative mRNA expression of MCP-1was detected by real-time PCR.4. Nuclear translocation of NFκB p65protein was analysed by Western blots and immunofluoscence technique.5. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett’s T3. P=≤0.05was considered as significant.[Results]1. Protein expression of MCP-1:compared with NG group, MCP-1protein expression of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, MCP-1protein expression in HG+PDTC group was significantly decreased (P<0.001) after24h and48h culture, respectively. After24h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.006). After48h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.003).2. Relative mRNA expression of MCP-1:After24h cultures, compared with NG group, the relative mRNA expressions of MCP-1in HG group were significantly increased (4.62±0.12vs.1.00±0.00,95%CI for Mean [4.32,4.93]). Compared with HG group, the relative mRNA expression of MCP-1in HG+PDTC group was significantly decreased (P<0.001), and the relative mRNA expressions of MCP-1in all BPS groups were significantly decreased (P=0.011).3. Nuclear translocation of NFκB p65:after90min stimulation, compared with NG group, NFκB p65protein expression in nucleus in HG group was was significantly increased (P<0.001). Compared with HG group, NFκB p65protein expressions in nucleus in HG+luMBPS, HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.001). In the mean time, NFκB p65protein expression in cytoplasm in HG group was significantly decreased after90min stimulation compared with NG group (P=0.001). NFκB p65protein expressions in cytoplasm in HG+2uMBPS and HG+5uMBPS group were significantly increased (P=0.049,0.034) compared with HG group.4. Immunofluoscence images of NFκB p65nuclear translocation:in NG group, the red fluorescence of NFκB p65was concentrated in the cytoplasm of HBZY-1cells, while the red fluorescence of NFκB p65was concentrated in the nucleus of HBZY-1cells in HG group. The images of red fluorescence of NFκB p65were similar between HG+2uMBPS group and HG+PDTC group. Compared with HG group, the red fluorescence of NFκB p65was reduced in the nucleus in HG+2uMBPS and HG+PDTC groups, which was mainly displayed in the cytoplasm of HBZY-1cells.[Conclusion]1. BPS inhibited MCP-1protein and mRNA expressions induced by high glucose in HBZY-1cells.2. BPS could inhibit NFκB p65subunit nuclear translocation in HBZY-1cells induced by high glucose, thereby inhibiting the activation of NFκB induced by high glucose, which was the potential mechmism of BPS in the regulation of MCP-1synthesis in HBZY-1cells culture in high glucose environment.Chapter ThreeEffects of PPARβ/δ on Beraprost Sodium pretection rat mesangial cells in high glucose enviroment[Objectives]1. To investigate into the effect of BPS on PPARβ/δ protein expression in HBZY-1cells cultured in HG environment.2. To investigate into the effect of BPS on PPARβ/δ mRNA expression in HBZY-1 cells cultured in HG environment.3. To explore whether PPARβ/δ was involved in BPS inhibition of TGFβ1, FN and MMP-2expressions induced by HG in HBZY-1cells.4. To explore whether PPARβ/δ was involved in BPS inhibition of p38and ERK MAPK activation induced by HG in HBZY-1cells.5. To explore whether PPARβ/δ was involved in BPS inhibition of MCP-1expression induced by HG in HBZY-1cells.6. To explore whether PPARβ/δ was involved in BPS inhibition of NFκB-DNA binding activity induced by HG in HBZY-1cells.[Method]1. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, High glucose (HG) group, HG+2uMBPS groups, HG+2uMBPS+GSK0660(5uM) groups, and HG+GSK0660group.2. Protein expression of PPARβ/δ was detected by Western blots.3. The relative mRNA expression of PPARβ/δ was detected by real-time PCR.4. Elisa assaay:to investigate into the protein expressions of TGFβ1, FN, MMP-2and MCP-1in the cell culture supernatants.5. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.6. EMSA:to detect the NFκB-DNA binding activtity.7. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett’s T3. P≤0.05was considered as significant.[Results]1. Protein expression of PPARβ/δ:after24h culture, compared with NG group, PPARβ/δ protein expression of HBZY-1cells in HG group was significantly decreased (P=0.028). Compared with HG group, PPARβ/δ protein expression in all BPS groups were significantly increased (P=0.009). PPARβ/δ protein expression in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and PPARβ/δ protein expression in HG+5uMBPS group was lower than that in HG+2uMBPS group (P=0.017vs. HG+2uMBPS).2. Relative mRNA expression of PPARβ/δ:After12h cultures, compared with NG group, the relative mRNA expressions of PPARβ/δ in HG group were significantly decreased (0.69±0.02vs.1.00±0.00,95%CI for Mean [0.64,0.73]). Compared with HG group, the relative mRNA expressions of PPARβ/δ in all BPS groups were significantly increased (P<0.001). The relative mRNA expression of PPARβ/δ in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and the relative mRNA expression of PPARβ/δ in HG+5uMBPS group was lower than that in HG+2uMBPS group (P<0.001vs. HG+2uMBPS).3. Protein expression of TGFβ1:after24h culture, TGFβ1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. TGFβ1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). TGFβ1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.049). There was no difference between HG+GSK0660group and HG group (P=0.621).4. Protein expression of FN:after24h culture, FN protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. FN protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). FN protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.047). There was no difference between HG+GSK0660group and HG group (P=0.592).5. Protein expression of MMP-2:after24h culture, MMP-2protein expression of HBZY-1cells in HG group was significantly decreased (P<0.001) compared with NG group. MMP-2protein expression in HG+2uMBPS group was significantly increased compared with HG group (P=0.017). There was no difference between HG+2uMBPS+GSK0660group and HG+2uMBPS group (P=0.956), and no difference between HG+GSK0660group and HG group (P=0.958).6. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated p38MAPK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated p38MAPK protein in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.061).7. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated ERK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated ERK in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.093).8. Protein expression of MCP-1:after24h culture, MCP-1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. MCP-1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.004). MCP-1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.042). There was no difference between HG+GSK0660group and HG group (P=0.847).9. NFκB-DNA binding activity:There was no NFκB-DNA binding band detected in NG group. Strong band was detected in HG group, which indicated that HG could induce NFκB bind to DNA by NFκB activation. The grey value of NFκB-DNA binding band was reduced in HG+PDTC group compared with HG group, because PDTC could inhibit the activation of NFκB. The grey value of NFkB-DNA binding band in HG+2uMBPS group was also decreased compared with HG group, which indicated that2uMBPS could reduce the NFκB-DNA binding activity. In HG+2uMBPS+GSK0660group, the grey value of NFκB-DNA binding band was increased compared with HG+2uMBPS group.[Conclusion]1. BPS could increase PPARβ/δ protein and mRNA expression in HBZY-1cells cultured under high glucose conditions.2. GSK0660reversed the inhibiton of BPS on TGFβ1and FN protein expression in HBZY-1cells induced by high glucose, but no changes in the MMP-2protein expression, suggesting that BPS could regluate TGFβ1and FN expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.3. GSK0660reversed the inhibiton of BPS on p38and ERK MAPK activation induced by high glucose in HBZY-1cells, suggesting that BPS could regluate p38and ERK MAPK pathway through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.4. GSK0660reversed the inhibiton of BPS on MCP-1protien expression in HBZY-1cells induced by high glucose, suggesting that BPS could regluate MCP-1expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.5. BPS could inhibit the NFκB-DNA binding activity induced by high glucose in HBZY-1cells, while GSK0660could reverse this effect. It was indicated that BPS could regluate NFκB-DNA binding activity through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions. |
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