| Background and ObjectiveLong-term peritoneal dialysis treatment can result in changes in peritoneal membrance structure, and lead to peritoneal fibrosis, which ultimately limited the development of PD. Peritoneal fibrosis has become a difficult problem for PD patients and researchers. Therefore, preventing or delaying the progression of fibrosis has gained much interest in the PD society.The renin-angiotensin system (RAS) has been implicated in the pathogenesis of fibrosis in various organs. AngⅡ, the main peptide of RAS, is a true cytokine that regulates cell proliferation, apoptosis, and fibrosis, contributing to the progression of many diseases. Recent studies indicate that Peritoneal mesothelial cells (PMC) are capable of producing Ang II and can affect the expression of E-cadherinα-smooth muscle actin(a-SMA),transforming growth factor-1(TGF-β1),plasminogen activator inhibitor-1(PAI-1,monocyte chemoattractant protein-1 (MCP-1) and so on. In recent years, plenty of observations support that AngⅡcan induce the accumulation of extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) and may also play a critical role in the development of peritoneal fibrosis in long-term PD patients.EMT plays an important role in the progression of peritoneal fibrosis, and EMT has been considered to be one important resourse of myofibroblast, which accumulated in peritoneum when peritoneal fibrosis developed. It has been reported that oxidative stress caused by high concentration of glucose in peritoneal solutions is regarded as one of the main factors which promote the progression of EMT. Excessive production or decreased degradation could induce the accumulation of ECM. The ECM-degrading proteases includes serine protease (PA/PAI) and matrix metalloproteinase (MMPs/TIMP), the PA is the key enzyme in the fibrino lytic system, PA activity is regulated by different PAIs, such as PAI-1, PAI-2 and PAI-3, of which PAI-1 is the main inhibitor.In recent years, as signaling messenger, the role of reactive oxygen species (ROS) has received more and more attention in peritoneal fibrosis. Oxidative stress caused by high concentration of glucose in peritoneal solutions is regarded as one of the main factors which initiate functional and structural alterations of the peritoneal mesothelial cells. Generation of ROS is regulated by cytokines and growth factors, and AngⅡis one of the strongest stimulations. NADPH oxidase is a major source of ROS production in many phagocytic or nonphagocytic cells. The NADPH oxidase consists of five components:membrane components p22phox and gp91phox subunit; and the cytosolic components p47phox, p40phox and p67phox. Moreover, there were two low molecular-weight guanosine triphosphatase(GTP ase):rap1a and rac2. When cells were stimulated, cytosolic p47-phox phosphorylated combined with p67-phox moved to the cell membrance as an activity composition of oxidative complex, and generated reactive oxygen species (ROS). As the regulation of ROS, NADPH oxidase subunits p47phox plays the pivotal role in the oxidative stress of peritoneum.The chromatograph fingerprint atlas confirmed that the astragalus intervention(AGI) has the stable function to eliminated free radical and the chromatograph peak of oxidation resistanc, and the ultrastructure assay confirmed that the AGI can oppress oxidized stress damage which is caused by the higher sugar environment of peritoneal mesothelial cells. However, there is scant data regarding the effect of AGI in peritoneal dibrosis from the perspective of molecular biologyTherefore, the present study attempted to test if AngⅡinduces the production of ROS and the expression of NADPH oxidase p47phox subunit and EMT factors, and to observe the effect of AGI on AngⅡ-induced peritoneal fibrosis in rat peritoneal mesothelial cells(RPMCs).1 The effect of AngⅡon ROS and NADPH oxidase subunits in RPMCs and the effects of treatment with AGI1.1 ObjectivesTo investigate the role of AngⅡon the production of ROS and the expression of NADPH oxidase subunits in RPMCs and the effects of AGI1.2 MethodsPrimary mesothelial cells were incubated with serum-free media for 24 h to arrest and synchronize the cell growth. The cells were randomly assigned to the control group(A), the AngⅡ(10-7mol/L) group(B), the AngⅡ+AGI(2g/ml) Group(C) and the AngⅡ+AGI(1g/ml) group (D). The DCF-sensitive cellular ROS were measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for NADPH oxidase p47phox subunits and p47phox protein expression were examined by Western blot.1.3 Results(1) AngⅡat 10-7mol/L significantly induced the production of DCF-sensitive cellular ROS at 20 minutes after stimulation compared with control(p<0.05),and AGI could effectively inhibit AngⅡ-induced ROS generation in a concentration-dependent manner.(2) AngⅡincreased both NADPH oxidase subunits p47phox mRNA expression and protein expression, and AGI inhibited the upregulation of p47phox mRNA and protein overexpression1.4 ConcludesAngⅡinduced the production of ROS and the overexpression of NADPH oxidase subunits. Moreover, AGI could inhibit AngⅡ-induced NADPH oxidase overexpression and ROS generation.2 The effect of Ang II on EMT in RPMCs and the effects of treatment with AGI2.1 ObjectivesTo investigate the role of AngⅡon the EMT in RPMCs and the effects of AGI.2.2 MethodsPrimary RPMCs were cutired to the second generation in vitro.After synchronization for 24 hours,the cells were randomly assigned to the control group,the AngⅡ(10-7mol/L) group, the AngⅡ+AGI(2g/ml)group, the AngⅡ+AGI(1g/ml)group. AngⅡ(10-7mol/L) group:RPMCs was stimulated in vitro with AngⅡ(10-7mol/L).the AngⅡ+AGI(2g/ml)group:RPMCs was stimulated after pretreatment with AGI(1g/ml),and the AngⅡ+AGI(2g/ml)group:with AGI (1g/ml)for1hour, and RPMCs was stimulated in vitro with AngⅡ(10-7mol/L).The expression ofα-SMA,E-Cadherin mRNA was detected by redl-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method,and the expression of a-SMA,protein was detected by western blot method.to observe change of each group index,gene level at 8h,protein level at 24h. 2.3 Results(1) AngⅡstimulatedα-SMA mRNA and protein synthesis in RPMCs,and which were ameliorated by AGI,both P<0.05, Not observed that the inhibited degree for a-SMA mRNA and protein was positively correlated with AGI concentration.(2) AngⅡdown-regulated E-cadherin mRNA expression synthesis in RPMCs, which were ameliorated by AGI, differences was significant P<0.05, Not observed in the expression for E-Cadherin mRNA was positively correlated with AGI concentration.2.4 ConcludesThe treatment of astragalus could inhibit EMT of RPMCs, to prevent the progression of peritoneal fibrosis.3 The effect of AngⅡon the expression of PAI-1 in RPMCs and the effects of treatment with AGI3.1 ObjectivesTo investigate the role of AngⅡon the expression ofPAI-1 in RPMCs, and to observe the effects of AGI.3.2 MethodsPrimary mesothelial cells were incubated with serum-free media for 24 h to arrest and synchronize the cell growth. The cells were randomly assigned to the control group(A), the AngⅡ(10-7mol/L) group(B), the AngⅡ+AGI(2g/ml) Group(C) and the AngⅡ+AGI(1g/ml) group (D). Group B:RPMCs were stimulated with Ang II(10-7mol/L), RT-PCR was employed to detect the mRNA expression at Oh,2h,4h, 8h,12h,24h; group C and group D cells were pretreated with AGI 2g/ml and 1 g/ml respectively before adding AngⅡ, the mRNA expression and protein expression were detected at 8h and 24h respectively.3.3 Results (1) AngⅡstimulated PAI-1 mRNA expression in a time-dependent manner from 2h up to 12h and peaked at 4h, compared with Oh (P<0.05), at the same time,PAI-1 protein expression were also upregulated by AngⅡin RPMCs and peaked at 24h compared with Oh(P< 0.05).(2) Treatment with AGI effectively downregulated AngⅡ-induced PAI-1 mRNA and protein overexpression. AngⅡ-induced PAI-1 mRNA overexpression were significantly downregulated by 39.43% and 37.28%with 2g/ml and 1g/ml AGI treatment respectively after 4h(P<0.05), while its protein overexpression were downregulated by 29.41%and 24.12%respectively after 24h(P<0.05), and there were no differences of PAI-1 expression between the treatment of different AGI concentration (P>0.05).3.4 ConcludesAGI could inhibit the expression of PAI-1, to reduce the accumulation of extracellular matrix in RPMCs and prevent the progression of peritoneal fibrosis.4 The effect of AngⅡon the expression of TGF-β1 in RPMCs and the effects of treatment with AGI4.1 ObjectivesTo investigate the role of AngⅡon the expression of TGF-β1 in RPMCs, and to observe the effects of AGI.4.2 MethodsPrimary rat peritoneum mesothelial cells were cultured to the second generation in vitro.After synchronization for 24 hours, The cells were randomly assigned to the control group(A), the AngⅡ(10-7mol/L) group(B), the AngⅡ+AGI(2g/ml) Group(C) and the AngⅡ+AGI(1g/ml) group (D). Group B:RPMCs were stimulated with AngⅡ(10-7mol/L), RT-PCR was employed to detect the TGF-β1 mRNA expression and protein expression were examined by Western blot. 4.3 Results(1) AngⅡsignificantly increase the TGF-β1 mRNA and protein expression in RPMCs, TGF-β1 mRNA and protein expression peaked at 24h,48h respectively compared with Oh, and there were significant differences between group A and group B(P<0.05);(2) Treatment with AGI effectively downregulated the TGF-β1 mRNA expression at 24h, the expression of TGF-β1 mRNA was 2.50-fold,2.13-fold and 1.67-fold higher in group B than group A, C and D respectively, and 1.25-fold lower in group C than group D. There were positive correlate between TGF-β1 mRNA expression and AGI concentration (P<0.05).At the same tiem, AGI inhibited the TGF-β1 protein expression significantly at 48h, the expression of TGF-β1 protein was 1.84-fold,1.51-fold and 1.23-fold higher in group B than group A, C andD respectively, and 1.22-fold lower in group C and group D. There were significant difference between group C and group D compared with groupB (P<0.05). the TGF-β1 protein expression were alse correlated with AGI concentration (P<0.05).4.4 ConcludesAGI could inhibit TGF-β1 mRNA and protein expression of RPMCs, to prevent the progression of peritoneal fibrosis.5 The effect of AngⅡon the expression of MCP-1 in RPMCs and the effects of treatment with AGI5.1 ObjectivesTo investigate the role of AngⅡon the expression of MCP-1 in RPMCs, and to observe the effects of AGI.5.2 MethodsPrimary mesothelial cells were incubated with serum-free media for 24 h to arrest and synchronize the cell growth. The cells were randomly assigned to the control group(A), the AngⅡ(10-7mol/L) group(B), the AngⅡ+AGI(2g/ml) Group(C) and the AngⅡ+AGI(1g/ml) group (D). Group B:RPMCs were stimulated with AngⅡ(10-7mol/L), RT-PCR was employed to detect the MCP-1 mRNA expression and protein expression were examined by Western blot.5.3 Results(1)AngⅡinduced the upregulation of MCP-1 mRNA expression at 4 hours after stimulation. AGI inhibited the overpression induced by AngⅡ. AngⅡ-induced MCP-1 mRNA overexpression were significantly downregulated by 15.6%and49.9% with 1g/ml and 2g/ml AGI treatment respectively compared with AngⅡgroup(P<0.05), and there were significant differences between group C and group D(P<0.05).(2)Meanwhile, MCP-1 protein expression was also upregulated significantly by AngⅡat 8 hours, and AGI inhibited the upregulation. MCP-1 mRNA overexpression were significantly downregulated by 17.6% and43.7%with 1g/ml and 2g/ml AGI treatment respectively compared with AngⅡgroup(P<0.05), and there were significant differences between group C and group D also.5.4 ConcludesAGI could inhibit MCP-1 mRNA and protein overexpression of RPMCs, to prevent the progression of peritoneal fibrosis.In Summary(1) AngⅡcan induce the production of ROS and the overexpression of NADPH oxidase subunits, and AGI can inhibit AngⅡ-induced NADPH oxidase overexpression and ROS generation. This study confirmed that AGI can relieve the oxidized stress damage induced by AngⅡ.(2) AngⅡcan stimulate the mRNA and protein synthesis of a-SMA,PAI-1,TGF-β1 and MCP-1 in RPMCs, on the other hand, AngⅡcan down-regulated the mRNA expression of E-cadherin in RPMCs,and which can be ameliorated by AGI, it have been proved that those factors playing a critical role in ECM and EMT. This study confirmed that AGI can inhibit the abnormal expression of those role factors and preventing or delaying the progression of fibrosis.Innovation(1) At present, the chromatograph fingerprint atlas confirmed that the astragalus intervention(AGI) has the stable function to eliminated free radical and the chromatograph peak of oxidation resistanc, but the relationships between AGI and oxidized stress damage and ROS, NADPH oxidase subunits p47phox are not clear, our study demonstrated that AGI can down-regulated the expression of p47phox and ROS, and relieve the oxidized stress damage.(2) Although the relationship between AGI and the mRNA and protein expression of TGF-β1 in RPMCs, but there are span data of relationship between AGI and mRNA and protein synthesis of a-SMA,PAI-1,TGF-β1 and MCP-1 in RPMCs. The present study demonstrated that intervented by astragalus can prevent the EMT and decrease the accumulation of ECM.
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