Effects Of Delayed Overexpression Of Smad7 Gene On Peritoneal Inflammation In Rat Peritoneal Fibrotic Model

Peritoneal dialysis(PD) as a main style of replacement treatment,is popular in end-stage of renal failure patients .Compared with hemodialysis(HD), PD has many advantages,for example,it is easy and convenient to implement, doesn't need complicated apparatus,has little influence on the patients lives and the patients are stable in hemodynamics,especially in protection of residual renal function. However, long time peritoneal dialysis can lead to peritoneal fibrosis and loss of dialysis effeciency. During PD,contact continuously with peritoneal dialysis soluetion that are non-physiology and have a high glucose content,infection repeatedly and influenced by Lipopolysaccharide(LPS) all could induce pathologic alteration on peritoneal membrane,even lead to peritoneal fibrosis and functional deterioration.For now,there are no effective methods to prevent or treat peritoneal fibrosis induced by long-term PD treatment.Fibrosis is due to excessive tissue repair, proliferation of fibroblast and excessive sediment of ECM are fibrotic characters.Epithelial-to-mesenchymal transition of mesothelial cells could be a source of myofibroblasts in the peritoneum. TGF-βis one of the key growth factors involved in peritoneal fibrosis.TGF-beta is an important growth factor involved in ECM modulation and can increase ECM protein syntheses and decrease their degradation. TGF-βexerts its biologic effects by signaling through the Smads pathway.The recent reseach demonstrated that Smads take part in the copy and regulation of collagen-Ⅰ,Ⅳcaused by TGF-β. TGF-βmakes Smad2 and Smad3 phosphorylation and makes them translocate into the neucleus. Smad6 and Smad7 are inhibitive Smads.they could inhibit the effect of receptor﹣Smads and antagonize signaling conduction of TGF-β.The pathogenesis of peritoneal fibrosis is not clearly understood, but recurrent bacterial peritonitis and glucose-containing dialysate may be the two most important causes . CAPD induces chronic inflammation in the peritoneum either with or without bacterial infections.Peritoneal inflammation is one of the characteristics of peritoneal fibrosis. The cytokine IL-1βhas also been implicated in peritoneal fibrosis. IL-1βcould stimulate the proliferation of fibroblasts and increase sediment of ECM by increase ECM protein syntheses and decrease their degradation. IL-1βand TNF can upregulate VEGF synthesis in peritoneal mesothelial cells.VEGF is implicated in neovascularization in peritoneum.The TGF-βfamily is a group of pleiotropic secreted cytokines, TGF-βfunctions in both autocrine and paracrine manners to regulate cell proliferation ,differentiation,extracellular matrix production,and anti-inflammation. Smad7 is the main negative regulator of the TGF-β/Smads singaling pathway.YuXueQin's study has proved that peritoneal fibrosis induced by intraperitoneal injection of 4.25% peritoneal dialysate combined with LPS had been alleviated by transferation of Smad7 plasmid to peritoneum.The result suggests transferation of Smad7 gene to peritoneum exhibits potent therapeutic effects on peritoneal fibrosis.It has been reported that overexpression of Smad7 on peritoneum can deteriorate inflammation in E.Coli-induced acut rat peritonitis. Smad7 is the inhibitory protein that negatively regulates TGF-β/Smads signaling pathway.Whether or not overexpression of Smad7 could aggravate peritoneal inflammation by inhibit anti-inflammation role of TGF-βwhile it reduce peritoneal fibrosis? So, we designed and completed the following study.1.To over-express Smad7 on peritoneum by transfer Smad7 plasmid .2.To explore the effect of delayed overexpression of Smad7 on peritoneal inflammation in rat peritoneal fibrotic model.1. Transferring Smad7 gene on peritoneum by ultrasound microbubble systemObjective: By ultrasound microbubble system to make Smad7 over-express for 4 weeks in peritoneum.Methods :( 1 ) To study the appropriated Smad7 injection volume: Respectedly injected mixture of Smad7 plasmid (100ug/ml) and Sonovue(v/v=1) into abdominal cavity by 4ml,5ml and 6ml by ultrasound microbubble system.(2) To investigate the appropriated Smad7 injection concentration: Respectively injected 6ml mixture of Smad7 plasmid and Sonovue into abdominal cavity by 100ug/ml,150ug/ml and 200ug/ml by ultrasound microbubble system.(3) Injected 6ml, 200ug/ml mixture of Smad7 plasmid into rat peritoneum by ultrasound microbubble system on day 15 in peritoneal fibrosis rat model,and rats were sacrificed on day3,7,14,21,28. (4) injected 6ml, 200ug/ml mixture of Smad7 plasmid into rat peritoneum by ultrasound microbubble system on day 15 and day 29 in peritoneal fibrosis rat model, rats were sacrificed on day3,7,10,17,21,28.Results: (1) Transfection efficiency was more than 80% with injection 6ml and 200ug/ml Smad7 plasmid by ultrasound microbubble system. (2) Smad7 level decreased significantly at day29 when Smad7 plasmid was transferred at day15 in peritoneal fibrosis rat model induced by intraperitoneal injection of 4.25%peritoneal dialysate and LPS.(3) Smad7 level could highly express in 4 weeks on peritoneum when it was transferred on day15 and day29. Conclusions: When Smad7 plasmid is transferred on day15 and day29 by ultrasound microbubble system in peritoneal fibrosis rat model,it could highexpress on peritoneum in 4 weeks.2.Effects of delayed overexpression of Smad7 gene on peritoneal inflammation in rat peritoneal fibrotic model Objective:To observe the infiltration of inflammation cells in peritoneum after transferring Smad7 gene into peritoneum using ultrasound microbubble systemMethods:Male SD rats were divided into four groups:control group(N group);model group(M group);vector group(V group);treat group(T group ) .M,V,T groups were received daily intraperitoneal injection with 4.25% peritoneal dialysate(100mg/kg BW) for 4 weeks and LPS(0.6mg/kg BW) at day 1,3,5,8,10,12,V and T groups were transferred individually with Tet-on/vector gene and Smad7 gene at day 15 and day 29.To induce Smad7 transgene expression,a dose of Doxycycline(1ml) with a concentration of 200μg/ml was administered into the peritoneal cavity immediately after ultrasound microbubble gene transfer and followed by additional Dox in the daily drinking water(200μg/ml) for 4 weeks.After 2,4,6 weeks,the samples of visceral peritoneum were collected.The expression of OX-1,ED-1, IL-1 and TNF mRNA were examined with immunofluorscent or immunohistochemical analysis and RT-PCR.Results: The infiltration of macrophage and the white cell in visceral peritoneum was decreased after successful transfection(T group).In comparison with M,V groups ,the mRNA expression of IL-1 and TNF-αin peritoneum T group were also decreased significantly.Conclusions: High glucose-containing peritoneal dialysate combined with LPS could make inflammatory cell infiltration in peritoneum and increase the transcription of IL-1 and TNF-α.Delayed overexpression of Smad7 exhibits potent therapeutic effects on retarding the established peritoneal inflammation in rat peritoneal fibrotic model.Summary1. When Smad7 is transferred on day15 and day29 by ultrasound microbubble system ,it could highly express on peritoneum in 4 weeks.2. High glucose-containing peritoneal dialysate combined with LPS could make inflammatory cell infiltration in peritoneum and increase the transcription of IL-1 and TNF-α3.Infiltrated Macrophages could excrete TGF-β.4.Delayed overexpression of Smad7 could inhibit the peritoneal inflammation in rat peritoneal fibrotic model.These results suggest that exogenous Smad7 exhibits potent therapeutic effects on retarding the established peritoneal inflammation in rat peritoneal fibrotic model.