| Part One Effects of Caerulein on Gelsolin and Autophagy in Pancreatic Ductal Epithelial Cells in Acute Pancreatitis in RatsObjective: To investigate the relationship between autophagy and gelsolin(GSN)in a rat model of caerulein(CAE)-induced acute pancreatitis(AP).Methods: Sprague-Dawley healthy rats(n=64)were randomly divided into two groups according the types of treatment: the control group(C,n=32)and the AP group(AP,n=32).The AP group was intraperitoneally injected with CAE,and the control group was injected with the same amount of saline.The rats were sacrificed at 6,12,24,and 48 h.Serum amylase(AMY)levels in rats were detected using an biochemical analyzer,the morphology of pancreas was observed and the pancreatic histopathologic scores were evaluated by hematoxylin-eosin(HE)staining.Changes of ultrastructure and autophagy in main pancreatic duct epithelial cells of rats were observed by transmission electron microscopy(TEM).The protein expressions of GSN,microtubuleassociated protein 1-light chain 3(LC3),P62,and lysosomal membraneassociated protein-2(LAMP2)at different timepoints of AP were analyzed by western blotting or immunohistochemistry.Results:(1)The serum AMY level in rats was the highest in the AP-6h group(P<0.01),and then went down gradually.(2)The morphology of pancreas showed that the pancreas was coated with gelatinous substance in the AP-6h and AP-12 h group,multiple calcifications were found in the peripheral tissue of the pancreas or peritoneum in the AP-24 h group,even worse the pancreas adhered to peripheral tissue in the AP-48 h group.Moreover,Pancreatic histopathologic scores increased with time.(3)Nuclear chromatin condensation,mitochondrial swelling or dissolving into vacuole,and autophagy were observed in main pancreatic duct epithelial cells of rats in the AP-24 h group by TEM.(4)The results from western blot and IHC showed that the protein expression of GSN was increased with time and peaked in the AP-48 h group(P<0.01),the expression of total LC3 was the highest in AP-12 h group(P<0.05),while LC3II/I ratio was the highest in AP-6h group(P<0.01),and the expression of LAMP2 was the highest,but the expression of P62 was the lowest in the AP-6h group(P<0.05).Conclusions:(1)Ultrastructural impairment and autophagy was observed in main pancreatic duct epithelial cells of rats in CAE-induced AP.(2)In the early stage of AP,the expression of GSN was increased with time,autophagosomes form and autophagy pathway was activated initially,but was impaired with time.Part Two The Changes of Glesolin,Actin Filaments and Autophagy in Pancreatic Ductal Epithelial Cells in CAE-induced Acute pancreatitis in VitroObjective: To investigate the changes of glesolin,actin filaments and autophagy at different timepoints in CAE-treated pancreatic ductal epithelial cells HPDE6-C7.Methods: The HPDE6-C7 cells were divided into five groups,including the C,CAE-6h,CAE-12 h,CAE-24 h and CAE-48 h groups according to treatment timepoints of CAE.Changes of ultrastructure and autophagy in HPDE6-C7 cells treated with CAE for 24 h were observed by TEM.The protein expression of total LC3 and LAMP2 in CAE-treated cells was analysed by immunofluorescence assay(IFA).The protein expressions of GSN,LC3,P62,and LAMP2 in CAEtreated cells were analyzed using western blot.Actin filaments were stained and analyzed under a fluorescence microscope.Results :(1)Nuclear chromatin condensation,mitochondrial swelling or dissolving into vacuole,and autophagy were observed in HPDE6-C7 cells treated with CAE for 24 h by TEM.(2)The results from IFA showed that the protein expression of total LC3 was higher in the CAE-24 h group than that in the control group(P<0.05),while the LAMP2 expression was the highest in the CAE-6h group(P<0.05).(3)The results from western blot showed that the GSN expression was increased with time and peaked in the CAE-48 h group(P<0.01),the LC3-II/I ratio was the highest in the CAE-24 h group,the P62 expression was the lowest(P<0.01)and the LAMP2 expression was the highest in the CAE-6h group(P<0.05).(4)The results from actin filaments staining showed that the depolymerization of actin filaments was increased in a time-dependent manner.The arrangement of actin filaments was disordered in the CAE-6h group and it was more pronounced with time,and the actin filaments depolymerized and collapsed in the CAE-48 h group.Conclusions:(1)CAE may damage the ultrastructure of pancreatic duct epithelial cells directly and induced autophagy.(2)Autophagosomes form and autophagy pathway was activated initially,but was impaired with time in CAE treated cells in vitro.(3)CAE may damage the actin filaments by upregulating the GSN expression.Part Three Effects of Cytochalasin B and Caerulein on Autophagy and Gelsolin in Human Pancreatic Duct Epithelial Cells in VitroObjective: To investigate the effects of actin filaments on autophagy and GSN in pancreatic ductal epithelial cells in AP using actin polymerization inhibitor cytochalasin B(CB).Methods: The optimal intervention concentration of CB was analysed by CCK-8 assay and cell morphological observation.The cells were treated with CAE and CB at 24 h of AP and divided into four groups: the C,CAE,CB,CB+CAE groups.The protein expressions of GSN,LC3,P62 and LAMP2 were analysed by western blot and the LAMP2 expression was also analysed by IFA in CB + CAE-treated cells for 24 h in vitro.Actin filaments were stained and analyzed under a fluorescence microscope.Results:(1)Low concentrations of CB(0.125,0.25,and 0.5 μg/m L)had no effect on cell growth and proliferation rate(P>0.05).However,high concentrations of CB(1.0 and 2.0 μg/m L)influenced the shape of cells,inhibit the cell growth and decreased the proliferation rate of cells(P<0.01).(2)Actin filaments in CAE or CB-treated cells depolymerized and collapsed,and the collapse was the most pronounced in the CB+CAE group.(3)The results from western blot or IFA showed that there was no difference in the GSN expression between CB group and control group(P>0.05).The LC3-II/I ratio was higher in the CAE,CB,CB+CAE groups than those in the control group(P<0.01),lower in the CB,CB+CAE groups than that in the CAE group(P<0.05).The P62 expression was lower in the CAE group than that in the control group(P<0.05),higher in the CB,CB+CAE groups than that in the CAE group(P<0.05).The LAMP2 expression was similar between the CAE group and the control group,lower in the CB,CB+CAE groups than that in the control group(P<0.05).Conclusions:(1)CB can make the depolymerization of actin filaments in CAEtreated cells more serious.(2)CB can inhibit autophagosomes autophagosome maturation and fusion with lysosomes by depolymerizing actin filaments,but was no effect on the protein expression of GSN in HPDE6-C7 cells treated with CAE.Part Four The Effect of Gelsolin Knock-down on Autophagy in Human Pancreatic Duct Epithelial Cells in Caerulein-Induced Acute Pancreatitis in VitroObjective: To investigate the effect of GSN on actin filaments and autophagy in GSN knock-down cells treated with CAE for 24 h.Methods: RNA interference(RNAi)was used to knock down GSN in HPDE6-C7 cells treated with CAE for 24 h.The cells were divided into six groups: the control group(C),the CAE group(CAE),the negative control group(NC)(the cells infected with empty lentivirus alone),the CAE-treated NC group(NC+CAE),the GSN knock-down group(KD)and the CAE-treated KD group(KD+CAE).(1)The m RNA and protein expression of GSN was analysed by q RTPCR and western blot in GSN knock-down cells.(2)The protein expressions of GSN,LC3,P62 and LAMP2 were analysed by western blot in GSN knock-down cells treated with CAE for 24 h.Meanwhile,the LAMP2 expression was analysed by IFA.Actin filaments were stained and analyzed under a fluorescence microscope.Results:(1)The mRNA and protein expression of GSN were lower in the KD group than that in the NC group in GSN knock-down cells(P<0.05).(2)The protein expression of GSN was higher in the CAE and NC+CAE groups than those in the control and NC groups in GSN knock-down cells treated with CAE(P<0.05),while there was no differences between the KD+CAE group and the KD group(P>0.05).The LC3-II/I ratio was higher in the CAE,NC+CAE,KD+CAE groups than those in the control,NC,KD groups(P<0.05),and lower in the NC+CAE group than that in the KD+CAE group(P<0.05).The P62 expression was lower in the CAE,NC+CAE,KD+CAE groups than those in the control,NC,KD groups(P<0.05),and there was no differences between the KD+CAE group and the NC+CAE group(P>0.05).The LAMP2 expression was similar in the CAE,NC+CAE groups to the control,NC groups(P>0.05),higher in the KD+CAE group than that in the KD,NC+CAE groups(P<0.05).The changes in the expression of LAMP2 analysed by IFA was similar to that analysed by western blot.(3)the depolymerization and collapse of actin filaments were found in the CAE group and the NC+CAE group,and were significantly reduced in the KD+CAE group.Conclusions: Activated GSN may inhibit autophagosome maturation and fusion with lysosomes by depolymerizing actin filaments in HPDE6-C7 cells treated with CAE for 24 h. |
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