| Part Ⅰ Critical role of TLR9 in acute pancreatitis and the variation after pretreatment of CpG-ODNObjective:To investigate the critical role of Toll-like receptor 9 in acute pancreatitis and the variation after pretreatment of CpG-ODN.Method:SD rats were randomly divided into eight groups:control group(con group),AP3h group(Acute pancreatitis 3h group),AP6h,AP12h,ligand pretreatment control group(CpG group), Acute pancreatitis 3h after ligand retreatment(CpG+AP3h group), CpG+AP6h, CpG+AP12h.The model of AP rats was induced by retrograde pancreaticobiliary duct injection of 5% sodium taurocholate.The model of CpG rats was intraperitoneal injection of CpG-ODN 1 hour before the AP method. Rats were sacrificed in corresponding time point of different group.The pathological changes in the pancreas were observed by HE staining and electron microscope, TLR9 mRNA and protein expression was detected by qPCR and Western Blot respectively, changes of IL-6 and TNF-a protein in serum were detected by ELISA.Also,serum and ascites pancreatic amylase activity were detected by amylase activity kit.Results:The pancreatic pathological score of AP rats was significantly increased time-dependently(P<0.05), in contrast scores of CpG pretreatment groups was higher(P <0.05). The relative expression of mRNA of AP rats was significantly elevated time-dependently(P<0.05), in contrast expression of CpG pretreatment groups was higher(P<0.05).The TLR9 mRNA expression of pancreatic tissue in AP rats had a positive correlation with pancreatic pathological scores. The TLR9 protein expression was significantly increased in AP rats compared with control rats (P<0.05), TLR9 protein reached a peak at 6h, which was significantly higher than those at 3 h (P<0.05) and 12 h (P<0.05).TLR9 protein of CpG groups were up-regulated compared with AP groups(P<0.05). Levels of serum TNF-α was significantly higher in AP rats compared with control rats (P<0.05),and it reached the peak at AP 12h(P<0.05). In contrast expression of CpG pretreatment groups was higher(P<0.05). Levels of serum IL-6 was significantly higher in AP rats compared with control rats (P<0.05),and it reached the peak at AP 6h(P<0.05). Levels of IL-6 in CpG 3h and 6h was higher than those in AP3h and AP6h(P<0.05).Ascites and serum pancreatic amylase activity were higher in AP rats compared with control group(P<0.05).However, there was not statistically significant between AP groups and CpG groups (P> 0.05).Conclusion:1.Toll-like receptor 9 participates in pathogenesis of acute pancreatitis;2.The expression of toll-like receptor 9 is up-regulated at the early phase of the acute pancreatitis, which has a positive correlation with pancreatic pathological scores;3.This study firstly confirmed that the pretreatment of CpG-ODN could up-regulate the expression of TLR9 mRNA, and aggravate pancreas damage in AP rats.4.TLR9 may play an important role in the conversion of acute edematous to necrotizing acute pancreatitis.PART Ⅱ The variation of acute pancreatitis after TLR9 RNAiObjective:To investigate the variation of the acute pancreatitis after pretreatment of siRNA targeting TLR9mRNA.Method:SD rats were randomly divided into eight groups:control group(con group),AP3h group(Acute pancreatitis 3h group),AP6h,AP12h,siRNA pretreatment control group(siRNA group), Acute pancreatitis 3h after siRNA retreatment (siRNA+AP3h group),siRNA+AP6h, siRNA +AP12h.The model of AP rats was induced by retrograde pancreaticobiliary duct injection of 5% sodium taurocholate.The model of siRNA rats was intraperitoneal and intravenous injection of TLR9 siRNA 48 hours before the AP method. Rats were sacrificed in corresponding time point of different group. The TLR9 mRNA expression was detected by qPCR, pathological changes in the pancreas were observed by HE staining, changes of IL-6 and TNF-α protein in serum were detected by ELISA. Also,serum and ascites pancreatic amylase activity were detected by amylase activity kit.Results:The survival time after TLR9 RNAi was prolonged. The relative expression of TLR9 mRNA was down-regulated after TLR9 RNAi(P<0.05). The pancreatic pathological score was reduced in siRNA+AP6h and siRNA+AP12h groups(P<0.05). Levels of serum IL-6 and TNF-α was significantly lower in siRNA+AP rats compared with AP rats (P<0.05).Conclusion:1.It was the first time that to suppress the TLR9 mRNA expression of pancreatic tissue effectively by the method of intraperitoneal and intravenous injection of TLR9 siRNA in rats.2.This study confirmed that levels of serum IL-6 and TNF-α was significantly lower,the pancreatic pathological score was reduced and the survival time was prolonged after TLR9 RNA interference.3.There are many factors participate in the occurrence and progress of AP. TLR9 may play an important role in the early phase of AP, especially in the conversion of acute edematous to necrotizing acute pancreatitis.4.This study first reveal that the progress of early AP could be significant delayed by the suppression of TLR9 mRNA, which could provide a novel therapeutic target in AP。... |
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