The Molecular Mechanism Of Oxymatrine Against Pancreatic Fibrosis Through The TGF-?1/Smad3/Gli1 Signaling Pathway

Objective Rat pancreatic stellate cells(PSCs)were stimulated by transforming growth factor ?1(TGF-?1),and the cell model of pancreatic fibrosis(PF)in vitro was builded.?-smooth muscle actin(?-SMA)is the marker when PSCs were activated.There are fibrotic markers:fibronectin(FN)and type I collagen(Co LI).The effect of oxymatrine on TGF-?1 stimulated PSCs related factor expression.Moreover,sh Smad3 and sh Gli1 plasmids were transfected into PSCs,and the effects of transfection of the plasmid on Smad3 and/or Gli1 and fibrotic markers in vitro PF model induced by TGF-?1 were examined.EGFP-Smad3 and EGFP-Gli1 high expression plasmids were transfected into PSCs,and the effects of oxymatrine on Smad3 and(or)Gli1 and pancreatic fibrosis markers were detected after transfection of the plasmid.The study in mechanism of OM against chronic pancreatitis by inhibiting TGF-?1/Smad3/Gli1 signaling pathway may provides new targets and therapeutic agents for the diagnosis and treatment of chronic pancreatitis.Methods1.Western Blot technique was used to detect the effects of different concentrations of TGF-?1 on the expression of key factors in Smad3/Gli1 signaling pathway related proteins and ?-SMA in PSC cells.2.The use of Western Blot and real-time quantitative PCR technology to detect the expression of key factors in TGF-?1/Smad3/Gli1 pathway and fibrosis effect expression of ?-SMA.The use of ELISA method to detect the expression of markers of fibrosis FN and Co L-I in transfected sh Smad3 RNAi plasmid of TGF-?1-induced LTC-14 cells.3.The use of Western Blot and real-time quantitative PCR technology to detect the expression of key factors in TGF-?1/Smad3/Gli1 pathway and fibrosis effect expression of ? –SMA.The use of ELISA method to detect the expression of markers of fibrosis FN and Co L-I in transfected sh Gli1 RNAi plasmid of TGF-?1-induced LTC-14 cells.4.After joining the OM pretreatment in TGF-?1 induced LTC-14 cells,use Western Blot technology and real-time quantitative PCR technology to detect the protein and m RNA expression of key factors in TGF-?1/Smad3/Gli1 pathway and fibrosis effect expression of ?-SMA.The use of ELISA method to detect the expression of markers of fibrosis FN and Co L-I.5.Western Blot technology was used to detect the effect of OM on the expression of key factors in TGF-?1/Smad3/Gli1 signal pathway and fibrosis marker ?-SMA after transfection of EGFP-Smad3 high expression plasmid into LTC-14 cells.The expression of fibrosis markers FN and Co L-I was detected by ELISA method.6.Western Blot technology was used to detect the effect of OM on the expression of key factors in TGF-?1/Smad3/Gli1 signal pathway and fibrosis marker ?-SMA after transfection of EGFP-Gli1 high expression plasmid into LTC-14 cells.The expression of fibrosis markers FN and Co L-I was detected by ELISA method.Results1.The expression of Smad3,Gli1 and ?-SMA was increased induced by TGF-?1,especially the concentrations up to 10 ng/m L.When concentrations exceed 10 ng/m L,the expression of Smad3,Gli1 and ?-SMA significantly increased compared with control group LTC-14 cells.2.The basal protein and m RNA levels of Smad3,Gli1 and ?-SMA decreased compared with blank transfection group when Smad3 RNA interference plasmids were transfected.The expression of protein and m RNA of the group which was transfected Smad3 RNA interference plasmids and then treatment with TGF-?1 significantly reduce the protein and m RNA levels of Smad3,Gli1 and ?-SMA compared with TGF-?1-induced group.The secretion of FN and Co L-?decreased compared with blank transfected group.The group which was transfected Smad3 RNA interference plasmids and then treatment with TGF-?1significantly reduce the secretion of FN and Co L-I compared with TGF-?1-induced group LTC-14 cells.3.The basal protein and m RNA levels of Gli1 and ?-SMA decreased compared with blank transfection group when Gli1 RNA interference plasmids were transfected.The expression of protein and m RNA of the group which was transfected Gli1 RNA interference plasmids and then treatment with TGF-?1significantly reduce the protein and m RNA levels of Gli1 and ?-SMA compared with TGF-?1-induced group.The secretion of FN and Co L-? decreased compared with blank transfected group.The group which was transfected Gli1 RNA interference plasmids and then treatment with TGF-? 1 significantly reduce the secretion of FN and Co L-I compared with TGF-?1-induced group LTC-14 cells.4.The protein and m RNA levels of Smad3,Gli1 and ?-SMA increased.Pretreatment with OM significantly reduce the protein and m RNA evels of Smad3,Gli1 and ?-SMA compared with TGF-?1-induced group.The secretion of FN and Co L-I in serium was increased by TGF-?1.The secretion of FN and Co L-I in serum was also reduced by OM LTC-14 cells.5.The basal protein levels of Smad3,Gli1 and ?-SMA increased,as so as the secretion of FN and Co L-I in serium,compared with blank transfection group when Smad3 RNA over-expression plasmids were transfected.The group treatment with OM which was transfected Smad3 RNA over-expression plasmids significantly reduce the protein levels of Smad3,Gli1 and ?-SMA,as so as the secretion of FN and Co L-I in serium,compared with transfected group in LTC-14 cells.6.The basal protein levels of Smad3,Gli1 and ?-SMA increased,as so as the secretion of FN and Co L-I in serium,compared with blank transfection group when Gli1 RNA over-expression plasmids were transfected.The group treatment with OM which was transfected Gli1 RNA over-expression plasmids significantly reduce the protein levels of Smad3,Gli1 and ?-SMA,as so as the secretion of FN and Co L-I in serium,compared with transfected group in LTC-14 cells.Conclusion1.TGF-?1/Smad3/Gli1 signaling pathway may involve in pancreatic fibrosis.2.OM can significantly suppress fibrosis by inhibiting TGF-?1/Smad3/Gli1 signaling pathway.