The Effects And Mechanisms Of IL-10 On The Impairment Of Blood-brain Barrier In Severe Acute Pancreatitis Rats

The study is divided into three parts.In the first part,we observed the impairment of blood-brain barrier(BBB)in severe acute pancreatitis(SAP)rats and explored the underlying mechanisms.In the second part,we evaluated the therapeutic effects of interleukin-10(IL-10)on the impairment of BBB in SAP rats and the potential mechanisms were explored.In the third part,we constructed in vitro BBB models and performed relative experiments to verify the results of in vivo parts.The results provide theoretical basis in preventing and treating pancreatic encephalopathy in SAP patients 1 The potential mechanisms of impairment of blood-brain barrier in severe acute pancreatitis rats Objective:To observe the impairment of BBB in severe acute pancreatitis rats and explore the underlying mechanisms.Methods:(1)SAP rat models were established via retrograde injection of 5% sodium taurocholate into biliopancreatic duct.(2)Serum amylase was detected by biochemical technique.(3)Serum TNF-? was measured by enzyme linked immunosorbent assay(ELISA).(4)Pathological changes in the pancreas and brain were evaluated under microscope and with the pathological score system.(5)Brain dry/wet ratio value was calculated to evaluate the extent of encephaledema.(6)Evan's blue assay was performed to assess the permeability of BBB.(7)Expression of Claudin-5 in rat brains were detected by quantitative real-time polymerase chain reaction(qPCR)and immunohistochemistry.(8)Brain microvascular endothelial cell(BMEC)apoptosis in rat brains were detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling(TUNEL).Results:The pancreases of the SAP group exhibited notable pancreatic edema,hemorrhage and necrosis as well as inflammatory cell infiltration.As compared to the control group,the levels of serum amylase,serum TNF-?,Evan's blue value and pancreatic pathological score were increased while brain dry/wet ratio value was decreased in SAP rats.Expression of Claudin-5 in brain decreased remarkedly in the SAP group and BMECs became swelling and apoptotic,resulting in the impairment of BBB with obvious encephaledema and increased BBB permeability in SAP rats.Conclusion:The impairment of BBB with increased permeability in SAP rats was probably associated with the phenomenon of BMECs apoptosis and down-regulation of tight junction protein Claudin-5.2 The effects and mechanisms of IL-10 on the impairment of blood-brain barrier in severe acute pancreatitis rats Objective:To evaluate the effects of interleukin-10(IL-10)on the impairment of BBB in SAP rats and the potential mechanisms were explored.Methods:(1)32 rats were randomly divied into 4 groups with 8 rats in each group: Control,SAP,SAP +IL-10 treatment,SAP+IL-10+specific inhibitor of signal transducer and activator of transcription 3(STAT3)signaling pathway S3I-201(SAP+IL-10 + S3I-201)groups.Exogenous IL-10 or specific STAT3 inhibitor S3I-201 was administered into the rats before the establishment of SAP models.(2)Serum amylase was detected by biochemical technique.(3)Serum TNF-? was measured by ELISA.(4)Pathological changes in the pancreas and brain were evaluated under microscope and with the pathological score system.(5)Brain dry/wet ratio value was calculated to evaluate the extent of encephaledema.(6)Evan's blue assay was performed to assess the permeability of BBB.(7)Expression of Claudin-5 in rat brains were detected by qPCR,Western blot and immunohistochemistry.(8)BMEC apoptosis in rat brains were detected by TUNEL.(9)Expression of p-STAT3,STAT3,Bax and Bcl-2 in rat brains were detected by qPCR,Western blot and immunohistochemistry.Results:(1)IL-10 decreased the levels of serum amylase,serum TNF-? and Evan's blue value and attenuated the impairment of pancreas and brain in SAP rats.(2)IL-10 attenuated the decreased expression of Claudin-5 in the brains of SAP rats.(3)Bax expression was up-regulated and Bcl-2 expression was down-regulated in the SAP group compared to the control group,as was associated with BMEC apoptosis.IL-10 attenuates BMEC apoptosis via the STAT3 pathway by down-regulating the expression of Bax while up-regulating the expression of Bcl-2,while S3I-201 partly inhibited the effects of IL-10.Conclusion:IL-10 alleviated the down-regulation of Claudin-5 expression and BMEC apoptosis via the STAT3 pathway by decreasing Bax expression while increasing Bcl-2 expression,to attenuate brain injury with increased BBB permeability and severity of inflammation in SAP rats.3 The effects and mechanisms of IL-10 on the impairment of in vitro blood-brain barrier models induced by TNF-? Objective:To evaluate the effects of IL-10 on the impairment of in vitro blood-brain barrier models induced by TNF-? and the potential mechanisms were explored.Methods:(1)Primary BMECs were separated,cultured and identified by immunofluorescence stained with von Willebrand factor(vWF)antibody and by flow cytometry with CD31 antibody.(2)In vitro BBB models were constructed and transendothelial electrical resistance(TEER)was detected to evaluate the qualification of constructed BBB models.(3)In vitro experiment groups were randomly divided into four groups as follow: Control,TNF-?,TNF-?+IL-10,TNF-?+IL-10+specific STAT3 inhibitor S3I-201(TNF-?+IL-10+ S3I-201)groups.In vitro BBB models without any treatment were set as Control group,with TNF-? administration to mimic the inflammation situation were set as TNF-? group,with exogenous IL-10 or S3I-201 administration before the exposure of TNF-? were set as TNF-?+IL-10 and TNF-?+IL-10+S3I-201 group,respectively.(4)TEER values were calculated to evaluate the integrity of in vitro BBB models.(5)Expression levels of Claudin-5 in BMECs were measured by qPCR and western blot.(6)Structural changes on peri-cellular tight junction were evaluated by immunofluorescence stained with Claudin-5 antibody and viewed using a ZEISS LSM 780 confocal microscope.(7)BMECs apoptosis was detected by flow cytometry with Annexin-?-FITC/PI staining.(8)Expression levels of p-STAT3,STAT3,Bax and Bcl-2 in BMECs were measured by qPCR and Western blot.Results:(1)Primary BMECs were separated,cultured,purified,identified and monolayer in vitro BBB models were constructed successfully.(2)TNF-? induced breakdown of in vitro BBB models with decreased TEER values.(3)Claudin-5 expression in BMECs was down-regulated and junctional structure was deteriorated with TNF-? treatment.(4)TNF-? increased Bax expression and decreased Bcl-2 expression in BMECs,resulting in BMEC apoptosis.(5)IL-10 alleviated the TNF-?-induced breakdown of the in vitro BBB model by maintaining its integrity.(6)IL-10 attenuated the TNF-?-induced down-regulation of Claudin-5 and impairment of tight junctions in BMECs.(7)IL-10 reduced BMEC apoptosis induced by TNF-? via down-regulating of Bax and up-regulating of Bcl-2 expression,S3I-201 partly reversed the anti-apoptotic effects of IL-10.Conclusions: IL-10 protected the structural continuity of tight junction and alleviated the down-regulation of Claudin-5 expression and BMECs apoptosis by down-regulating the expression of Bax and up-regulating that of Bcl-2 via STAT3 signaling pathway to attenuate the impairment of in vitro BBB models and maintain the integrity.