Effects Of Oxymatrine Against Pancreatic Fibrosis Through JAK2/STAT3 Signal Pathway In Chronic Pancreatitis

Objectives This experiment was carried out to explore the effects of Janus kinase 2(Janus kinase 2,JAK2)/signal transduction and transcriptional regulation kinase 3(signal transducer and activator of transcription 3,STAT3)signaling pathway on activation of pancreatic stellate cells(PSC)in rats,and provide the theoretical basis for oxymatrine(OM)against pancreatic fibrosis in chronic pancreatitis(CP).Methods This study includes the following two parts.One experiment was intended to find out whether JAK2 / STAT3 signaling pathway involved in the activation of PSC in cell experiment.Another experiment was aimed at testing the inhibition effect of OM on pancreatic fibrosis via JAK2 / STAT3 signaling pathway in animal experiment.In cell experiment part,LTC-14 cells,immortalized pancreatic stellate cells,were stimulated with TGF-β1 for concentration gradient(0,4,8,12,16,and 20 ng/ml)and time gradient(6,12,18 and 24 h)respectively.The optimal time and the optimal concentration for highest cell viability was detected using the MTT assay.Realtime PCR and Western Blot were used to detect molecule expression in α-SMA and JAK2 / STAT3 signal transduction in PSCs under stimulus of TGF-β1 in optimum concentration and time.Specific JAK2 antagonist,AG490,was used to observe the effect of the JAK2 / STAT3 signaling pathway on LTC-14 cells activation.The effect of AG490 on IL-6 and IL-1β secretion induced by TGF-β1 was detected by using ELISA.Interference plasmids for STAT3 in different sites were synthesized to detect the best interference site of STAT3 RNA.The effects of plasmid transfection were dectected to identify STAT3 expression inhibition on LTC-14 cells activation.In animal experiment part,according to the body weight,all the thirty-two male Wistar rats(weight 250 – 300 g)were randomly divided into four groups: normal control group(NC group),model group(CP Group),OM prevention group(OP group)and OM negative control group(OM group).Each group has 8 rats.Rats of CP group and OP group received intraperitoneal injection of DDC with a dose of 500 mg/kg/2d to establish chronic pancreatitis model.At the same time,rats of NC group and OM group were injected intraperitoneally with a same volume of saline.Rats in OP group received intraperitoneal injection of OM with a dose of 100 mg/kg/d for 2 hour before DDC injection.Rats in OM group were injected with an equal volume of OM as a negative control.Rats in NC group and CP group received an equal volume of saline.Injection experiment last four weeks,Fibrosis level of pancreatic tissue was detected using Masson staining and the expression of JAK2,STAT3 and SOCS3 were detected by Realtime PCR.All experiments were independent repeated three times.Data from two groups were analyzed with t test,and data from multiple groups were analyzed with one-way analysis of variance.The level of statistical significance was set at P < 0.05.Results In cell experiment part,the MTT results showed that the best concentration was 8 ng/ml and the best time was 12 h for TGF-β1 stimulation.After stimulated with 8 ng/ml TGF-β1 for 12h,we found protein expression levels of JAK2,STAT3,SOCS3,p-STAT3 and α-SMA in TGF-β1 stimulation group were significantly higher than that of control group(P﹤0.05).The molecular transcription levels of JAK2,STAT3,SOCS3 and α-SMA in TGF-β1 stimulation group were higher than that of control group(P﹤0.05).After AG490 administration,we found that the activation of STAT3 and the expression of protein and m RNA in SOCS3 and α-SMA induced by TGF-β1 were significantly inhibited by AG490(P﹤0.05).ELISA detection results showed that TGF-β1 could obviously induced secretion of IL-6 and IL-1β,which could be inhibited by AG490(P﹤0.05).By design multiple si RNA for STAT3 different locus,we found si RNA at the point of 1345 in STAT3 gene could significantly suppress the STAT3 m RNA and protein expression(P﹤0.05).After using the STAT3 si RNA,we found the activation of STAT3 and the expression of m RNA and protein in STAT3,SOCS3 and α-SMA induced by TGF-β1 in LTC-14 cells could be significantly reduced(P ﹤ 0.05).In animal experiment part,Pancreatic fibrosis level in CP group was significantly higher than that of the rest three groups presented by Masson staining(P﹤0.05).Compared with CP group,percentage of collagen fiber area was decreased significantly in OP group(P﹤0.05).There was no significant difference among OP group,NC group and OM group(P﹥0.05).Realtime PCR results showed that the m RNA expression levels of JAK2,STAT3 and SOCS3 in pancreatic tissue of CP group were significantly higher than that of NC and OM group(P﹤0.05).Compared with CP group,the levels in JAK2,STAT3 and SOCS3 m RNA were significantly lowered in OP group after injection of OM(P﹤0.05),while there was no significant difference among OP group,NC group and OM group(P﹥0.05).Conclusions 1 TGF-β1 can activate PSCs by promoting activation and generation in JAK2/STAT3 signaling pathway.2 OM may inhibit pancreatic fibrosis by inhibiting JAK2/STAT3 signaling pathway.