Effect And Mechanism Of IL-6 On PSC-Mediated Pancreatic Fibrosis Of Alcoholic Chronic Pancreatitis

Background and aims: Morbidity of alcoholic chronic panreatitis(ACP)is getting higher and higher.Although alcohol is considered as an independent risk factor of ACP,only 5–10% of chronic alcoholics suffered from pancreatic fibrosis.Endotoxin lipopolysaccharide(LPS)is one of the most important triggers of ACP.In vivo and in vitro studies have shown that LPS could induce activation of PSC and increase synthesis of Collagen type I(Col1).TGF-?1 as an important pro-fibrotic factor that is produced in an autocrine and paracrine fashion,could increase the expression of ?-SMA and Col1 in PSC.Recent studies have found that interleukin-6(IL-6)plays an important role in the fibrogenesis of many diseases.IL-6 activates the JAK2/STAT3 signaling pathway through IL-6 receptor(IL-6R),which promotes the fibrogenesis of liver,lung and kidney.So far the effect of IL-6/JAK2/STAT3 pathway on the fibrogenesis of ACP has not been fully clarified.The purpose of this study was to elucidate the role and mechanism of IL-6 and its signaling pathway on the fibrogenesis in ACP patients and alcohol-fed rat model as well as in vitro PSC and macrophage(M?)models.Methods: Thirty-three male SD rats(120 ± 20g)were randomly divided into 3 groups: control diet group,Lieber-DeCarli alcohol liquid diet group andcontrol diet plus LPS group.The rats of control diet plus LPS group were injected with LPS(3 mg/kg)weekly via the tail vein at the end of week 8,9,or10.The rats of other two groups were injected with 1 ml normal saline weekly at the same time point.All rats were sacrificed at 24 h after normal saline or LPS challenge.Pancreatic tissues were fixed in 10% neutral buffer formalin for preparation of paraffin sections or snap-frozen in liquid nitrogen.A stable activated human PSC line(designated as HP-1 cell)was used in this study,which was established by RSV promoter/enhancer-driven SV40 T antigen expression.To prepare monocyte-derived M?s,human peripheral blood mononuclear cells(PBMCs)were isolated using human lymphocyte separation medium and then cultured in RPMI 1640 culture medium containing 10 ng/ml GM-CSF for 6 day.Thereafter the M? culture was stimulated with alcohol or LPS for additional 24 h.Immunohistochemistry was used to detect IL-6R expression in human pancreatic tissues.Double immunostains was used to recognize the expression of IL-6R in PSCs.Immunocytochemistry was used to determine the expression of IL-6 or IL-6R in M?s or HP-1 cells.ELISA was used to determine the production of IL-6,TGF-?1,?-SMA and Col1a1 in M?s,HP-1 cells,rat pancreatic tissues or human sera.Real-time quantitative PCR(RT-qPCR)was used to determine the levels of IL-6,TGF-?1,?-SMA and Col1?1 mR NAs in HP-1 cells.Western blot was used to detect the synthesis of p-JAK2,JAK2,p-STAT3,STAT3,p-Smad2,Smad2,p-Smad3 and Smad3proteins in HP-1 cells.Results: Immunohistochemical stain showed enhanced expression of IL-6protein and its receptor in the pancreas of ACP patients as compared with healthy controls(HC),The number of IL-6+M?s and IL-6+PSCs or IL-6R+M?s and IL-6R+PSCs of ACP patients were significantly increased as compared with HC(all P<0.001).Our further study found higher levels of IL-6,TGF-?1 and Col1?1 in the sera of ACP patients as compared with HC(all P<0.001)and positive correlations between IL-6 level and either TGF-?1 or Col1?1 content in ACP without or with endotoxemia patients(r=0.6609,P<0.05;r=0.6293,P<0.05;r=0.6422,P<0.01;r=0.5006,P<0.05).In vivo study showed increased contents of IL-6,TGF-?1 and Col1?1 in the pancreases of ALC-fed rats or rats with LPS injections(all P<0.001)and positive correlations between IL-6content and either TGF-?1 or Col1?1 content in the pancreases of ALC or LPS rats(r=0.7369,P<0.01;r=0.6445,P<0.05;r=0.7380,P<0.01;r=0.6344,P<0.05).In vitro study showed enhanced expression of IL-6R and increased secretion of IL-6 and TGF-?1 in both M?s and HP-1 cells treated by ALC or LPS(all P<0.001).Mutually induced synthesis between exogenous TGF-?1and IL-6 in HP-1 cells was observed.The expression of ?-SMA and Col1?1mR NAs and proteins were partially inhibited by IL-6 neutralizing antibody(IL-6ab)in HP-1 cells treated by ALC or LPS(all P<0.001),suggesting IL-6 as a stimulator of ?-SMA and Col1?1 synthesis in ALC or LPS-treated HP-1 cells.Further study demonstrated that IL-6 induced TGF-?1 synthesis through IL-6R/JAK2/STAT3 pathway.The production of ?-SMA and Col1?1 in IL-6-treated HP-1 cells was significantly antagized by TGF-?1 neutralizing antibody(TGF-?1ab),suggesting TGF-?1 as a mediator of IL-6-induced?-SMA and Col1?1 synthesis in the HP-1 cells.IL-6 could induce a delayed Smad2/3 phosphorylation in HP-1 cells.The expression of ?-SMA and Col1?1mR NA induced by IL-6 was significantly suppressed by small interfere Smad2(si-Smad2)and si-Smad3 respectively(both P<0.001).Conclusion: ACP is a key player in the fibrogenesis of ACP.IL-6 induces TGF-?1 production by IL-6R/JAK2/STAT3 pathway.IL-6 also promotes PSC activation and collagen I production through up-regulation of TGF-?1/Smads pathway.This study demonstrates that IL-6 may be a potential therapeutic target for pancreatic fibrosis.