| Background & Objective:So far there is no good way of unified and extensive application for surgical clinical prevention of peritoneal adhesion, researches on optimization and mechanism of drugs have been the focus and hot spot in clinical and basic research. Traditional Chinese medicine has the advantage of multiple links, multiple target in the prevention and treatment of peritoneal adhesions, less toxic and side effect and lower price, among which tetramethylpyrazine shows its talent, being paid more and more attention. The research advisor and team has long been committed to the Chinese traditional medicine for the prevention and treatment of peritoneal adhesions, and TMP’s effect in this aspect as well. We completed the preparation of the TMP nanoparticles spray and establish the quality control system. The study confirmed that TMP nanoparticles spray has the functing that relevant inhibits the increase of models’adhesion related factor TGF-β1, FN, CTGF, and regulated of the balance of t-PA and PAI in fibrinolysis system. Combination of peritoneal mesothelial cells and TGF-β key targets of these adhesion formation mechanism, we open the exhibitionaims to clear efficacy the mechanism of TMP nanoparticles spray affects peritoneal mesothelial cell structure and function and during the process of the TGF-β inducing tissue adhesion, how TMP nanoparticles spray controls the TGF-β/Smad signal pathway and its downstream target gene at the same time, so as to do further study on the mechanism of TMP nanoparticles spray.The study on effect and mechanism of TMP nanoparticles spray anti rat experimenta peritoneal adhesion:Methods:1.Establishing rat model of postoperative abdominal adhesion.120 SD rats were randomly divided into 4 groups,30 rats in each group. Group A, blank group:Did not use any medication; Group B, TMP raw material group:Before suturing, wound coated with TMP raw material,1mg/ml,5ml/kg; Group C, TMP nanoparticles spray group:before suture abdominal spray TMP nanoparticles spray, lmg/ml,5 ml/kg; Group D, hyaluronic acid sodium group: before suture abdominal coated with sodium hyaluronate,1% sodium hyaluronate gel,0.5 ml/kg. 2.Respectively after 1,3,5,7,14d,6 rats of each group were randomly selected, executed and open, according to the Phillips’fivestandard classification, adhesions were, respectively, observed and recorded. The the adhesion tissue were taken to be detected pathologic and transmission electron microscope examination, the pathological morphology and ultrastructure of peritoneal adhesion formation process and different stages of intervention of TMP nanoparticles spray or adhesion after tissue specimens were observed.Results:1.From the overall level, it can decrease the incidence of adhesion, alleviate adhesion grading; 2. from the pathological morphological level, it can obviously inhibit fibroblastcells and connective tissue hyperplasia; 3.from the ultrastructurallevel, it can improve the structure change after damaged mesothelial cells, ihibite transform of fibroblasts.Conclusion:TMP nanoparticles spray has exact effect in preventing and treating experimental peritoneal adhesion in rats.The experimental study on protection mechanism of TMP nanoparticles spray on rat peritoneal mesothelial cells(RPMCs):Methods:1. Enzyme digestion method in vitro RPMCs, immunohistochemical identification; 2. MTT method study on the determination of the effects of TMP nanoparticles spray on activity of normal RPMCs and RPMCs induced by Tnf-a;Results:1. The cultured cells were identified as RPMCs; 2. TMP nanoparticles spray has no influence on the normal RPMCs, the activity of RPMCs induced by Tnf-a was significantly decreased, the decreased activity of RPMCs induced by Tnf-a, which the TMP nanoparticles spray has certain protection for RPMCs functions;Conclusion:TMP nanoparticles spray play the protection of RPMCs by inhibiting RPMCs activity decrease when damaged.The experimental study on anti experimental peritoneal adhesion mechanism of TMP nanoparticles spray through control TGF-β/Smad signal pathway of RPMCs:methods:1. Culture and identification of RPMCs in vitro; 2. RPMCs induced by Tnf-a, activation of TGF-β/Smad signal pathway. Reverse transcription polymerase chain reaction (RT-PCR) detects expression of the TGF-β1mRNA, Smad7mRNA after TMP nanoparticles spray intervention; 3. Dyeing and immunohistochemical analysis of p-Smad2/3’s location and protein expression in RPMCs with TMP nanoparticles spray intervention; 4. Western blotprotein analysis of TGF-β1, Smad7, FN, CTGF, PAH, and COL1 in RPMCs under the intervention of TMP nanoparticles spray;Results:1. The cells were identified as RPMCs; 2. TGF-β1 in Tnf-α induced RPMCs expressed significantly, Smad7 expression was inhibited significantly, while the TMP nanoparticles spray can inhibit expression of TGF-β1mRNA obviously, and increase the expression of Smad7mRNA; 3. P-Smad2/3 in RPMCs induced by tnf-a was activated, and accumulated to the nucleus and the level of its expression was obviously increased. But TMP nanopartieles spray preparation can inhibit accumulation to the nucleus and the level of p-Smad2/3; 4. Protein TGF-β1, FN, CTGF, COL1 and PAH in RPMCs induced by Tnf-a have an increased, whlie protein Smad7 has a decrease; TMP nanoparticles spray can reduce TGF-β1, FN, CTGF, COL1 and PAI1 protein content, and increase the content of protein Smad7;Conclusion:TMP nanoparticles spray can inhibit the activation of TGF-β/Smad signal pathway, and further inhibit the expression of its downstream target genes, there by inhibiting peritoneal fibrosis, and contributing to the prevention and treatment of peritoneal adhesions in rats.Summary:The research defined preventive and the rapeutic effects of TMP nanoparticles spray on peritoneal adhesion in rats from three levels, the whole to the local to the micro; confirmed the protective effect of TMP nanoparticles spray on rat peritoneal mesothelial cell’s damaged structure and activity, and makes it clear that the TMP nanoparticles spray, inhibites of Smad2/3 nuclear cohesion and phosphorylation by inhibiting the expression of TGF-β1, and up regulates Smad7 expression, thus inhibiting the function of TGF-β/Smad singnal pathway so that inhibits the expression of downstream target genes, FN, CTGF, COL 1 and PAI-1, eventually suppressing generation and accumulation of extracellular matrix of the system, preventing peritoneal fibrosis and peritoneal adhesion. This research obtained function evidence of TMP nanoparticles spray inducing postoperative fibrosis change. Combined with the result of downstream target gene being suppressed excited, we analyzed its possible mechanism of the connection and influence between Smad signal pathway and effects, which provides a new target for mechanism of TMP nanoparticles spray anti peritoneal adhesion by controling TGF-β/Smad signal pathway in peritoneal mesothelial cells. |
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