The IL-6/STAT3 Signaling Pathway Is Involved In The Ductal Metaplasia Of Pancreatic Acinar Cells And The Intervention Mechanism Of Baicalin

Part I: the role of IL6/STAT3 pathway in the formation of ADM during CP1.Objective Through in vivo and in vitro experiments,explore the changes and regulatory mechanisms of STAT3 signaling pathway in the formation of pancreatic acinar cell ductal metaplasia(ADM)in chronic pancreatitis(CP).2.Methods 1.In vivo experiment: C57BL/6 mice were injected intraperitoneally with cerulein(50ug/kg,6 times/day,3 days/week)to induce the CP model,and the STAT3 inhibitor S31-201NSC(100mg/kg,once/week)was given to the STAT3 inhibitor S31-201NSC(100mg/kg,1 time/week).Day,6 days/week)intraperitoneal injection treatment.The animals were anesthetized at 2,4,and 6 weeks after modeling and treatment,and the pancreas tissues of the mice were completely removed.HE staining was used to observe the changes of mouse pancreas histopathology and the degree of fibrosis;immunofluorescence double-labeling method was used to mark the expression of amylase and CK-19;Western blot was used to detect the changes of CK-19 and IL-6R protein expression in pancreatic tissue.2.In vitro experiment: Purchase 266-6 cell line(mouse pancreatic acinar cell line)and culture it in DMEM medium containing 10% fetal bovine serum.The 266-6 cells were stimulated with 5nmol/L,10nmol/L,and 20nmol/L cerulein.The cells were collected at four time points of 1h,8h,24 h,and 48 h,and the expression of Amylase and CK-19 was detected by the double immunofluorescence method.In addition,266-6 cells were stimulated with 20nmol/L cerulein or 10ng/ml IL-6 for 24 h and 48 h,and the cells were collected.Western blot was used to detect the expression changes of CK-19,p-STAT3,STAT3 protein,and 20nmol/L was further used.The cerulein combined with 10ng/ml IL-6 stimulated 266-6 cells,and the expression of Amylase and CK-19 were labeled with immunofluorescence double labeling at 1h,4h,8h and 24 h.3.Results 1.In vivo experiment:(1)Formation of ADM in a mouse CP model induced by cerulein HE staining showed that after 2 weeks of modeling,mice in the model group had pancreatic acinar damage,inflammatory cell infiltration and a small amount of collagen deposition;with the progress of the disease course,the pancreatic acinar atrophy of the mice after 4 weeks of modeling,and the pancreas gradually Duct epithelial cells and fibroblasts are replaced by ADM.The pancreatic structure is severely destroyed,inflammatory cells are infiltrated extensively,and collagen is deposited in large quantities.After 6 weeks of modeling,part of the pancreas is lost,and a large amount of fibrosis is formed,and ADM is reduced accordingly.Immunofluorescence double-labeling staining of amylase and CK-19 showed that the expression of Amylase increased after 2 weeks of modeling,which may be caused by cerulein stimulating the release of large amounts of amylase in acinar cells.At this time,the expression of CK-19 was weak,suggesting that ADM is less formed,and the expression of Amylase decreased significantly after 4 weeks of modeling,and the expression of CK-19 increased,indicating that acinar cells metabolized into ductal cells in large numbers,and the expression of Amylase further decreased after 6 weeks of modeling,suggesting mice The exocrine function of the pancreas is impaired and the expression of CK-19 is also low,which may be due to the increased destruction of pancreatic tissue,the replacement of fibrotic tissue,and the decrease of tubular acinar cells.(2)The effect of inhibiting STAT3 signal pathway on pancreatic ADM The expression of CK-19 protein in the pancreatic tissue of CP mice was further detected by Western blot.It was shown that the pancreatic tissue of CP model mice expressed CK-19 at 2 weeks,and the expression of CK-19 was further increased at 4 weeks after modeling(p<0.05).),but it was reduced by 6 weeks(p<0.05).In addition,we also found that the expression level of IL-6R in pancreatic tissue increased significantly after 2 weeks of modeling(p<0.01),and increased with the prolongation of modeling time,and maintained a high level at 6 weeks after modeling(p<0.01).After using IL-6/STAT3 signaling pathway inhibitor NSC,it was found that not only the expression of IL-6R at each time point was significantly reduced after NSC treatment(p<0.01),the expression of CK-19 in pancreatic tissue was also significantly lower than that at the same time point.Model group(p<0.01).2.In vitro experiment: Double immunofluorescence staining with Amylase and CK-19 showed that: 5nmol/L,10nmol/L,20nmol/L cerulein stimulated pancreatic acinar cells 266-6,and the expression of CK-19 could be increased after 1h.However,the expression level of Amylase is still high at this time.The expression of CK-19 was further increased after stimulation with 20nmol/Lcerulein for 24h(p<0.01).Western blot showed that the expression of CK-19 was slightly decreased after cerulein stimulated 266-6 cells for 48 h but was still higher than that of the blank control group(p<0.01).The cells showed the peak expression of CK-19(p<0.01)after 24 h stimulation with various concentrations of cerulein.At the same time,the expression changes of p-STAT3 in acinar cells were detected,and the expression trend of p-STAT3 was found to be consistent with CK-19(p<0.01),suggesting that the formation of ADM in acinar cells is related to the activation of STAT3 pathway.Then the 266-6 cells were stimulated with 10ng/ml IL-6,and the test found that: IL-6 can also cause 266-6 cells to express CK-19,but it was later than the cerulein stimulation group,and the expression of CK-19 was obvious after 24 hours of stimulation.Increase(p<0.01).Western blot results also showed that the expression of CK-19 increased,and IL-6 could also increase the expression of p-STAT3.It is suggested that the formation of ADM in pancreatic acinar cells is closely related to the p-STAT3 pathway.After stimulating 266-6 cells with 20nmol/L cerulein and 10ng/ml IL-6,the expression of CK-19 was more obvious by immunofluorescence double-labeling method.4.Conclusions 1.ADM is an important early pathological manifestation in the mouse CP model induced by cerulein,and the formation of ADM may be related to the IL6/STAT3 signaling pathway.Inhibiting the STAT3 signaling pathway can reduce the formation of ADM.2.The formation of ADM is derived from acinar cells.High concentration of cerulein can induce ADM in isolated acinar cells,indicating that over-stimulation is an important cause of tubularization of pancreatic acinar cells.3.IL-6 stimulation can lead to the formation of ADM in pancreatic acinar cells,suggesting that the inflammatory environment is an important reason for the formation of ADM,and the IL6/STAT3 signaling pathway plays a regulatory role in the formation of ADM.Part II: Baicalin reduces CP pancreatic fibrosis by inhibiting the formation of ADM in pancreatic acinar cells1.Objective To explore the effect of baicalin on the formation of ADM and pancreatic fibrosis in mouse CP model pancreatic tissue and 266-6 pancreatic acinar cells and related mechanisms.2.Methods 1.In vivo: The CP model was induced by intraperitoneal injection of cerulein in C57BL/6 mice(same as the first part),and baicalin(50mg/kg,1 time/day,6 days/week)was administered intraperitoneally 1 week after the model was established.At 2,4,and 6 weeks after modeling and treatment,the mouse pancreas tissue was collected completely,Masson staining was used to observe the changes of pancreatic fibrosis,and immunohistochemical staining was used to observe the changes of fibrosis index FN and ADM index CK-19.;Western blot detection of CK-19,p-STAT3,STAT3 expression changes.2.In vitro: The 266-6 cells were stimulated with 20nmol/L cerulein,and 75ug/ml and 50ug/ml baicalin were administered.The cells were collected at 24 and 48 hours.Western blot was used to detect CK-19,p-STAT3,and STAT3.Changes in protein expression.3.Results 1.In vivo: Masson staining showed that in the model group,a small amount of pancreatic acini showed tubular structure and a small amount of collagen fiber deposition at the beginning of the model group.By 4 weeks of modeling,the normal structure of the pancreas was significantly reduced,and it was covered by a large number of duct-like structures and fibrotic tissues.Instead,the pancreatic structure was severely damaged at 6 weeks after the model was created,mainly showing large amounts of fibrotic tissue.Compared with the model group at the same time point in the baicalin treatment group,the complete pancreatic structure was significantly increased,and the blue-stained collagen fibers were significantly reduced.The expression of CK-19,a marker of ADM in pancreatic tissue,was detected by immunohistochemical staining.It was found that the expression of CK-19 in the pancreas increased gradually at 2 and 4 weeks of modeling,until a large number of pancreatic acinars were replaced by fibrous tissue at 6 weeks of modeling.CK-19 expression also decreases. Immunohistochemical staining method was used to detect the expression of fibrosis marker FN: the blank control group had complete pancreatic tissue structure and no FN expression.The expression of FN was positive at 2 weeks after modeling,and the expression of FN increased gradually with the prolonging of modeling time.Western blot detection of CK-19 expression in pancreatic tissue also showed the same trend.The expression of CK-19 and FN in the pancreas of the baicalin treatment group was also significantly lower than that of the model group at the same time point(p<0.01).Western blot detection of the expression of STAT3 and p-STAT3 in pancreatic tissue found that after 2 weeks of modeling,the protein expression of p-STAT3 began to increase(p<0.01),and the expression increased further at 4w and 6 weeks(p<0.01);Compared with the model group at the corresponding time point,the protein expression of p-STAT3 in the baicalin treatment group was significantly lower than that in the model group(p<0.01).2.In vitro: 266-6 cells were stimulated with 20nmol/L cerulein for 24 h and 48 h,and Western blot was used to detect the expression changes of CK-19,STAT3 and p-STAT3.It was found that the expression of CK-19,STAT3 and p-STAT3 in pancreatic acinar cells was stimulated for 24 h and 48 h.It was significantly higher than that of the blank control group(p<0.01).After treatment with different concentrations of baicalin,the expression of CK-19,STAT3 and pSTAT3 decreased significantly.4.Conclusions 1.Baicalin can reduce the formation of ADM in the CP process by inhibiting the activation of the STAT3 pathway,thereby reducing the degree of fibrosis in the mouse CP model.2.Baicalin can also inhibit the activation of IL-6/STAT3 pathway and reduce the ADM metaplasia of pancreatic acinar cells induced by cerulein.