The Role Of NF-kB In The Activation Of Pancreatic Stellate Cells And The Mechanism Of Intervention Of Baicalin

PART ?: The mechanism of NF-?? signaling induced by transforming growth factor-?1 stimulation in pancreatic stellate cells.[AIMS]To induce pancreatic stellate cells(PSCs)activation with administration of transforming growth factor-?1(TGF-?1)in vitro,and to observe the mechanism of NF-?? signal pathway during the elevation of PSC activation.[Methods]Isolating the primary pancreatic stellate cells of healthy Kunming mice,cultured by high glucose DMEM with 15% fetal bovine serum.Cells were used after first passage.Culture medium were replaced by serum-free high glucose DMEM when cells are 80% confluenced,starve 24 hours for synchronize.Cells were divided into 7 groups by time manner,which were0 h,15min,30 min,1h,2h,4h,24 h,and 3 repeating wells for each group.Extracting total protein from PSCs,measuring the expression of nuclear factor-?B p65(NF-?B p65),phosphorylated p65(p-p65),TGF-?1receptor(TGF-?1R),inhibitor of ?B-?(I?B-?)and ?-smooth muscle actin(?-SMA)level by Western-Blot.In addition,to observe the expression of NF-?B p65 in PSCs by immunofluorescence after the administration of TGF-?1.[Results]The Western-Blot result shows the expression of TGF-?1R was elevated15 mins after TGF-?1 administration,at the same time,massive degradation of I?B-? and phosphorylation of p65 were detected.Thereafter,p65 level were increased and reached the peak during 1-2 hour time point.?-SMA expression was increased after NF-?? p65 activated.The immunofluorescence result shows that NF-?? p65 was barely observed in control group,and was strongly expressed in the time point 30min-4h,especially in the nucleus.[Conclusion]TGF-?1 stimulates PSCs to express TGF-?R1,then I?B-? were degraded,NF-?? dimers acquired the ability to enter the nuclei and as the result,PSCs were activatied.PART ?: The effect of NF-?? p65 RNA interfering on PSC activation,cytokine secretion and fibrosis associated factors regulation.[AIMS]To inhibit NF-?? p65 expression in PSCs by RNA interfering strategy,and to observe corresponding changes of PSC activation level,secretion of pro-inflammatory cytokines and fibrosis associated factors.[Methods]Isolating the primary pancreatic stellate cells of healthy Kunming mice,cultured by high glucose DMEM with 15% fetal bovine serum.Cells were used after first passage.Culture medium were replaced by serum-free high glucose DMEM when cells are 80% confluenced,starve 24 hours for synchronize.Cells were divided into 3 groups: Negative control group,TGF-?1 stimulating group and TGF-?1+NF-?? p65 RNAi group.RNAi strategy was performed by using NF-?? p65 si RNA combined with Lipofectamine 3000?.After 36 h transfection,medium was replaced by high glucose DMEM with 5ng/ml TGF-?1,as long as TGF-?1 stimulation group.Four time points for each group(0h,6h,12 h,24h),and three repeating wells for each time point.Culture supernatant,total RNA and total protein were harvested according to the time point.IL-6,MCP-1 level in culture supernatant were measured by ELISA,?-SMA was measured by Western-Blot and IF,IL-6,MCP-1,MMP-1,TIMP-1 m RNA level were measured by RT-PCR.[Results]NF-?? p65 in PSC was successfully inhibited in both m RNA level and protein level by RNAi strategy.Under the stimulation of TFG-b1,?-SMA was elevated strongly in all time point comparing to control.?-SMA expression in TGF-?1+RNAi group was effectively inhibited.IL-6 and MCP-1 level were upregulated in the TGF-?1 stimulation group(P<0.01)and were downregulated in the TGF-?1+RNAi group(P<0.01),similar results were seen in RT-PCR experiment.MMP-1 m RNA in TGF-?1 stimulation group was declined in 6h and 12 h compared with control(P< 0.01),and was lifted in 24 h.TIMP-1 m RNA level was elevated in all time points(P<0.01).Opposite tendencies of MMP-1 and TIMP-1 m RNA level were detected in TGF-?1+RNAi group(P<0.01).[Conclusion]After the inhibition of NF-?? signal pathway activation by RNAi strategy in PSC,the PSC activation level was highly reduced,the level of pro-inflammatory cytokine secretion was reduced,and enhanced the ability of degrading ECM.PART ?: The effect of Baicalin inhibits PSC activation through down-regulating NF-?? signal pathway activity.[AIMS]Performing Baicalin treatment on TGF-?1 induced activated PSCs.To observe the changes of NF-?? activity level,PSC activation level and pro-inflammatory cytokine production.To provide theoretical foundation for the Baicalin in treating patients with pancreatic fibrosis in clinical practice.[Methods]Isolating the primary pancreatic stellate cells of healthy Kunming mice,cultured by high glucose DMEM with 15% fetal bovine serum.Cells were used after first passage.Culture medium were replaced by serum-free high glucose DMEM when cells are 80% confluenced,starve 24 hours for synchronize.Cells were divided into 3 groups: Negative control group,TGF-?1 stimulating group and TGF-?1+Baicalin treatment(50?g/ml)group.Five time points for each group(0h,2h,6h,12 h,24h),and three repeating wells for each time point.Culture supernatant,total RNA and total protein were harvested according to the time point.IL-6,MCP-1 level in culture supernatant were measured by ELISA.?-SMA,FN m RNA expression level were measured by RT-PCR.NF-?B p65??-SMA protein level were measured by Western-Blot.[Results]NF-?? p65 and ?-SMA were highly elevated in protein expression level comparing to negative control,and was obviously inhibited after Baicalin treatment.The m RNA expression level of FN and ?-SMA were highly boosted in TGF-?1 group and were successfully reduced by the interfering of Baicalin.The level of IL-6 and MCP-1 in culture supernatant rose significantly comparing to negative control in all time point(P<0.01)and were effectively reduced in TGF-?1+Baicalin treatment group(P<0.01).[Conclusion]Baicalin(50mg/ml)is capable to reduce PSC activation level and down-regulate the pro-inflammatory cytokine secretion by inhibiting NF-?? signal pathway activity.