| ObjectiveTo study the therapeutic effects for an inflammatory response in SAP rats through siRNA silencing of the CARD9 gene.MethodsSixty adult male Sprague-Dawley(SD)rats were divided into six groups randomly,including control group,control siRNA group,CARD9 siRNA group,severe acute pancreatitis(SAP)group,control siRNA+SAP group,CARD9 siRNA+SAP,with 18 rats in severe acute pancreatitis(SAP)group and CARD9 siRNA+SAP and 6 rats in control group,control siRNA group,CARD9 siRNA group and control siRNA+SAP group.Using a perftusion pump,the induction of SAP rats was made by perfusion of 5%sodium taurocholate in a volume-weight ratio of 1.5 ml/kg.Control rats received an intraperitoneal saline solution injection.CARD9 siRNA+SAP were intravenously injected in the tail with CARD9 siRNA before being induced in the same manner as the SAP group,and siRNA-control groups were intravenously injected in the tail with control siRNA.All procedures were conducted according to the guidelines established by the animal ethical committee of Shanghai Jiao Tong University(China).Rats in all groups were sacrificed at 3h,6h and 12h after induction of model.At different predetermined time-points,the rats were killed and ascites were collected from blood obtained directly from the aorta abdominalis.The amylase activity was examined by an automated biochemistry analyzer.Routine HE staining was used to evaluate pancreatic pathological damage and the myeloperoxidase(MPO)in the pancreas was measured.Serum proinflammatory cytokines TNF-a,IL-1? and IL-6 were detected by enzyme linked immunosorbent assay(ELISA).TNF-?,IL-1?,IL-6,CARD9,Dectin-1,TLR-4,p38 MAPK and NF-?B p65 mRNA expression in pancreas were detected by real-time PCR.The levels of CARD9,Dectin-1,TLR-4,p38 MAPK and NF-?B p65 in pancreas was detected by Western blotting.ResultsCARD9 mRNA in the SAP group significantly increased after three hours and reached apeak at 12 hrs.Up-regulation of CARD9 expression was further confirmed by Western blot analysis.Control siRNA rats failed to reveal any differences in CARD9 expression in pancreatic tissue when compared to wild-type rats.CARD9 siRNA-treated SAP rats indicated a reduction of up to 60%of the targeted gene's mRNA expression,and RT-PCR and Western blot analysis showed that there was a decrease in the level of CARD9 expression at 48 hrs after received the CARD9 siRNA.Little ascites were observed in normal SD rats,while ascite volume was dramatically up-regulated at 3 hrs in SAP rats and maintained at a high level at 6 and 12 hrs.After the in vivo siRNA gene knock-down,ascite volume gradually declined at 3 hrs and reached a relatively low level at 6 and 12 hrs(P<0.05).Serum amylase in the SAP group was significantly higher compared to the control group(P<0.05).However,there was no difference in the levels of serum amylase in siRNA-treated animals when compared to the SAP group.MPO activity in the SAP group significantly increased at 3 hrs and reached a peak at 12 hrs,while the CARD9 gene silencing with siRNA led to a significant decrease in pancreatic MPO activity(P<0.05).There were no remarkable pathological changes in control rats.In SAP rats,histological characterization found that interstitial oedema,haemorrhage,inflammatory cell infiltration and focal necrosis had occurred.Treatment of SAP rats with CARD9 siRNA at 6 and 12 hrs resulted in an obvious amelioration of pancreatic injury.The serum levels of TNF-cc,IL-6 and IL-lpincreased significantly at 3 hrs in the SAP group.After treatment with CARD9 siRNA,there was a significant inhibitory effect on the levels of TNF-?,IL-6 and IL-1?expression when compared to the SAP group.Consistent with serum levels,these inflammatory cytokines showed similar changes in the pancreatic tissue.Accompanied by the noticeable and decreased production of TNF-?,IL-6 and IL-1?pin CARD9 siRNA knock-down rats,the TLR4 and Dectinl receptors appeared to show a similar down-regulation in pancreatic tissue.CARD9 mRNA levels gradually increased and reached a peak at 12 hrs,which was consistent with the NF-?B p65 and P38 MAPK signalling activation in SAP rats.After the in vivo CARD9 gene silencing with siRNA,NF-?B p65mRNA and P38MAPK mRNA levels significantly decreased in siRNA-treated rats at 12 hrs and were accompanied by reduced CARD9 mRNA.CARD9,NF-?Bp65 and P38MAPK expression at protein level were markedly increased in the pancreatic tissue of SAP rats.After in vivo CARD9 gene silencing with siRNA,the level of unphosphorylated and phosphorylated NF-?Bp65 and P38MAPK gene regions was also decreased in siRNA-treated group at 3,6 and 12 hrs(P<0.05).ConclusionCARD9 is up-regulated in SAP rats and acts as a potential therapeutic target for the treatment thereof.CARD9 gene silencing with siRNA in rats with SAP demonstrated a significant reduction in pancreatic injury,neutrophil infiltration,myeloperoxidase activity and pro-inflammatory cytokines.Blocking the activation of NF-?B and P38MAPK via siRNA-mediated gene knock-down of CARD9 appears to reduce the inflammatory response in pancreatic tissue. |
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