| Acute pancreatitis is a critical disease in clinic. With the treatment level of p ancreatitis increasingly enhanced, mortalities of severe acute pancreatitis were still up to 10-30%. The elevated level of cytokines(Interleukin-1, Interleukin-6 and I nterleukin-10) and activated phlogistic cells can star Systemic Inflammatory Respo nse Syndrome and apoptosis of pancreas tissues with acute pancreatitis. So contro lling the inflammation and apoptosis can obviously relieve the pancreatic trauma and improved the survival rate in early SAP. Recent studys have found that tradi tional Chinese medicine piperlonguminine of roots extractson long pepper can reg ulate the ROS(reactive oxygen species(ROS), the partial thromboplastin time an d the ERK-1/2 signal pathways. These functions can repair brain injury, anticoagu lant activity and inhibiting tumor cell growth. This paper aims to study on the m echanism of traditional Chinese medicine in the treatment of SAP and its molecu lar basis by using the modern molecular biology technology. Especially illustrate the Chinese medicine Piperlonguminine in the treatment of SAP targets and clear its molecular mechanism and provide a theoretical basis for the future developm ent of new traditional Chinese medicine in the treatment of SAP.Objective:We established the SAP model and study on its mechanism of Chinese medic ine piperlonguminine on SAP rats and supply datas for clinic using of piperlongu minine.Method:1 Grouping and processing1.1 Grouping: Sixty SD rats by digital table method were randomly divided into sham operation(SO) group(n=10) model(SAP) group(n=10), piperlonguminine(P) group(n=30), gabexate mesilate(GM) group(n=10).1.2 Processing: Pancreatitis was induced by intraductal administration of 5% sodi um taurocholate 0.1ml/kg and the SO group used saline instead of sodium tauroc holate. P group were randomly divided into low dose group, mid dose group and high dose group(2.5, 5, 10 mg.kg-1) and immediately injected with piperlongumi nine by intraperitoneal injection after the building-model. GM group were injected with gabexate mesilate by intraperitoneal injection after the building-model. More over, SO group and SAP group were injected with normal saline by intraperitone al. Blood samples and pancreatic samples were collected and then the rats were sacrificed at 12 h after the establishment of SAP model.2 Experimental observation indexes2.1 The first part of experimental observation indexes: The general state of the r ats was observed after 12 h. The pancreatic water contents were determined by th e dry weight method. The pathologic change of pancreas head was measured with HE staining. The amylase and lipase change were measured by UV Spectrophot ometry.2.2 The secondary part of experimental observation indexes: Myeloperoxidase wer e measured by UV Spectrophotometry. ELISA was used to detect the expression change of Interferon-γ, Interferon-1β, tumor necrosis factor-α and Interferon-10 in r at serum. The Quantitative PCR was used to detect the expression change of TL R-4 m RNA and NF-κB m RNA. Western blot was used to detect the expression c hange of TLR-4 protein and NF-κB protein.2.3 The thirdly part of experimental observation indexes: The apoptosis change w as measured with TUNNEL staining. The Quantitative PCR was used to detect th e expression change of peroxiredoxin-4 m RNA、XBP-1 m RNA and p38 MAPK m RNA. Western blot was used to detect the expression change of peroxiredoxin-4 p rotein.Results:1 Experimental results1.1 The first part of experimental results:(1) The rats in SO group were generall y in good condition. SAP group rats were dispirited. The rats in P group and G M group were better than SAP rats.(2) We have successfully established the SAP mordel by intraductal administration of 5% sodium taurocholate 0.1ml/kg(serum amylase and lipase levels 3 times greater than the upper limit of normal value).compared with SAP group,the AMS and LPS levels in P group, GM group we re decreased(P > 0.05).(3) Compared with SO group, the levels of drying wet i n SAP group were much higher(P<0.05). The levels of drying wet in P group and GM group were much lower than SAP group(P<0.05).(4) HE staining r esults show that the rats in SAP group with severe pancreatic injury and the panc reatic injury was mitigated in P group and GM group.1.2 The secondary part of experimental results:(1)Results of serum MPO level in rats: Compared with SO group, MPO in SAP group were much higher(P<0.05).However, though the levels of MPO in P group and GM group were different with SAP group, no significant difference was seen between them(P>0.05).(2)ELISA results: Compared with SO group, IL-1β, TNF-α and IFN-γ in SAP grou p were much higher(P<0.05,0.01). The levels of IL-1β,TNF-α and IFN-γin P group and GM group were much lower than SAP group(P<0.05,0.01). Tho ught the level of IL-10 in SAP group was higher than SO group, there were not significant between the two groups(P>0.05). At the same time, the levels of IL-10 in P group and GM group were much higher than SAP group(P<0.05).(3) Quantitative PCR results:Compared with SO group, TLR-4 m RNA and NF-κB m RNA in SAP group were much higher(P<0.05). The levels of TLR-4 m RNA and NF-κB m RNA in P group and GM group were much lower than SAP group after intraperitoneal injected with piperlonguminine and gabexate mesilate(P<0.05).(4) The expression of TLR-4 protein and p-NF-κB protein expressio n in SAP group was significantly higher than that in SO group. The levels of TLR-4 protein and p-NF-κB protein in P group and GM group were much lower t han SAP group.1.3 The thirdly part of experimental results:(1) TUNNEL staining results show t hat the apoptosis have no significant difference between SAP group and SO grou p. Compared with SAP group, the apoptosis of P group and GM group were inc reased after treated with piperlonguminime and gabexate mesilate.(2) Quantitative PCR results:Compared with SO group, peroxiredoxin-4 m RNA, XBP-1 m RNA,p38 MAPK m RNA and peroxiredoxin-4 protein in SAP group were much higher(P<0.05,0.01). The levels of peroxiredoxin-4 m RNA, peroxiredoxin-4 protein in P group and GM group were much lower than SAP group(P<0.05). At the sa me time, the levels of p38 MAPK m RNA in P group and GM group were much higher than SAP group(P<0.05). However, though the levels of XBP-1 m RNA in P group and GM group were different with SAP group, no significant differe nce was seen between them(P>0.05).(3) The results of Western blotting: the expression of peroxiredoxin-4 protein in SAP group was significantly higher than that in SO group, and the expression of peroxiredoxin-4 protein was significantl y lower in P group and GM group compared with SAP group(P < 0.05).Conclution:Piperlonguminine could alleviate SAP by reducing the expression of AMS a nd LPS and inhibiting the expression of inflammatory factor. Its mechanism ma y be related with adjusting the TLR-4/NF-kappa B pathway, cell apoptosis and i nflammation. But the endoplasmic reticulum stress with XBP-1 and oxidative str ess with MPO have no obvious effect. |
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