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

PART Ⅰ: The mechanism of NF-κΒ signaling induced by transforming growth factor-β1 stimulation in pancreatic stellate cells.[AIMS]To induce pancreatic stellate cells(PSCs)activation with administration of transforming growth factor-β1(TGF-β1)in vitro,and to observe the mechanism of NF-κΒ 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-κB p65(NF-κB p65),phosphorylated p65(p-p65),TGF-β1receptor(TGF-β1R),inhibitor of κB-α(IκB-α)and α-smooth muscle actin(α-SMA)level by Western-Blot.In addition,to observe the expression of NF-κB p65 in PSCs by immunofluorescence after the administration of TGF-β1.[Results]The Western-Blot result shows the expression of TGF-β1R was elevated15 mins after TGF-β1 administration,at the same time,massive degradation of IκB-α and phosphorylation of p65 were detected.Thereafter,p65 level were increased and reached the peak during 1-2 hour time point.α-SMA expression was increased after NF-κΒ p65 activated.The immunofluorescence result shows that NF-κΒ p65 was barely observed in control group,and was strongly expressed in the time point 30min-4h,especially in the nucleus.[Conclusion]TGF-β1 stimulates PSCs to express TGF-βR1,then IκB-α were degraded,NF-κΒ dimers acquired the ability to enter the nuclei and as the result,PSCs were activatied.PART Ⅱ: The effect of NF-κΒ p65 RNA interfering on PSC activation,cytokine secretion and fibrosis associated factors regulation.[AIMS]To inhibit NF-κΒ 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-β1 stimulating group and TGF-β1+NF-κΒ p65 RNAi group.RNAi strategy was performed by using NF-κΒ p65 si RNA combined with Lipofectamine 3000?.After 36 h transfection,medium was replaced by high glucose DMEM with 5ng/ml TGF-β1,as long as TGF-β1 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,α-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-κΒ p65 in PSC was successfully inhibited in both m RNA level and protein level by RNAi strategy.Under the stimulation of TFG-b1,α-SMA was elevated strongly in all time point comparing to control.α-SMA expression in TGF-β1+RNAi group was effectively inhibited.IL-6 and MCP-1 level were upregulated in the TGF-β1 stimulation group(P<0.01)and were downregulated in the TGF-β1+RNAi group(P<0.01),similar results were seen in RT-PCR experiment.MMP-1 m RNA in TGF-β1 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-β1+RNAi group(P<0.01).[Conclusion]After the inhibition of NF-κΒ 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 Ⅲ: The effect of Baicalin inhibits PSC activation through down-regulating NF-κΒ signal pathway activity.[AIMS]Performing Baicalin treatment on TGF-β1 induced activated PSCs.To observe the changes of NF-κΒ 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-β1 stimulating group and TGF-β1+Baicalin treatment(50μg/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.α-SMA,FN m RNA expression level were measured by RT-PCR.NF-κB p65、α-SMA protein level were measured by Western-Blot.[Results]NF-κΒ p65 and α-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 α-SMA were highly boosted in TGF-β1 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-β1+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-κΒ signal pathway activity.