| Chronic pancreatitis is a progressive inflammatory disease characterized by irreversible fibrosis of the pancreas,which can lead to impaired of pancreatic exocrine and endocrine function,with clinical manifestations of diabetes mellitus and fatty diarrhea[1,2].CP has variety of etiologies,alcohol is widely considered to be the main cause of acute pancreatitis(AP)and CP[1,2].In the normal pancreas,PSCs are in a quiescent state,storing vitamin A–containing lipid droplets in the cytoplasm[3].However,when PSCs are activated in response to pancreatic inflammation or injury,they are transformed into a myofibroblast-like phenotype characterized by increased expression of alpha–smooth muscle actin(α-SMA)[3].Therefore,the activated PSCs may secreting some cytokines or chemokines(such as transforming growth factor β15),and extracellular matrix components(such as collagen type I and fibronectin),play a central role in the pathogenesis of CP[4,5].There are many studies on the relationship between PSCs and CP fibrosis.However,there is relatively little research on the relationship between IL-18,chemokine CX3CL1,PSCs and the pathological mechanism of CP.In this study,PSCs activation and CX3CL1 expression in CP rats and normal rats were studied,and different concentrations of IL-18 were incubated with PSCs due to understand the effect of IL-18 on PSCs,and investigate the mechanism of IL-18 act on PSCs.Part 1 Expression of IL-18,CX3CL1,and α-SMA in pancreatic stellate cells in rats with chronic pancreatitisObjective: To investigate the activation of pancreatic stellate cells after pancreatic fibrosis in rats with chronic pancreatitis and the expression of IL-18,chemokine CX3CL1 and α-SMA in rats with chronic pancreatitis.Methods: Normal Wistar male rats(body weight 180-200g),Chronic pancreatitis model induced by tail vein injection of DBTC(8mg/Kg).Pancreatic fibrosis was determined by HE staining.The primary pancreatic stellate cells were extracted by digestion and gradient centrifugation.After 4 days,the cells were collected and the total RNA was extracted.The expression of IL-18,CX3CL1,and α-SMA mRNA was detected by RT-PCR method.Results: The mRNA expression of IL-18 in control group was 1.01±0.08,The mRNA expression of IL-18 in CP group was 1.73±0.40.The PSCs IL-18 mRNA expression in the CP group was higher than that in the control group,and there was no significant difference between the control group and the CP group(P>0.05).The mRNA expression of CX3CL1 in control group was 1.07±0.26,The mRNA expression of CX3CL1 in CP group was 2.22±0.13.The PSCs CX3CL1 mRNA expression in the CP group was higher than that in the control group,The difference of PSCs CX3CL1 mRNA expression between the CP group and the control group was statistically significant(t=3.96,P<0.05).The m RNA expression of α-SMA in control group was 1.00±0.05,The mRNA expression of α-SMA in CP group was 9.76±0.28.The PSCs α-SMA mRNA expression in the CP group was higher than that in the control group,The difference of PSCs α-SMA mRNA expression between the CP group and the control group was statistically significant(t=30.78,P<0.01).Conclusion: Pancreatic stellate cell activation occurs after pancreatic fibrosis,The expression of α-SMA was up-regulated.and The expression chemokine CX3CL1 was up-regulated after pancreatic fibrosis.Part 2 The influence of IL-18 on Pancreatic Stellate Cell and CX3CL1 levelObjective: To evaluate the effect of different concentration gradient of IL-18 on pancreatic stellate cell(PSCs)and the expression level of CX3CL1.Methods: The Human pancreatic stellate cells were cultured in the medium of stellate cells and passaged.The logarithmic growth phase were inoculated in 6-well plates,each hole of 5X105 cell.After culture,the cell fusion degree reached 80%,then IL-18 of different concentration gradient(5、25、50、100ng/ml)was used to treat PSCs for 72 hours and the untreated PSCs were used as control.Real time PCR and Western blot were used to detect mRNA and protein expression of E-cadherin,α-SMA and CX3CL1 respectively.To observe whether IL-18 can activate human PSCs;whether IL-18 can up regulate the expression of chemokine CX3CL1 in PSCs.Results:The mRNA expression of E-cadherin in control group and 5,25,50,100ng/ml IL-18 treated group were 1.03±0.17,0.77±0.15,0.89±0.12,0.54±0.11,0.46±0.06.The mRNA expression of α-SMA in control group and 5,25,50,100ng/ml IL-18 t reated group were 1.03±0.19,0.85±0.14,1.33±0.22,1.60±0.14,1.94±0.09.The mRNA e xpression of CX3CL1 in control group and 5,25,50,100ng/ml IL-18 treated group were 1.01±0.08,0.88±0.25,0.86±0.17,1.58±0.26,1.83±0.13.The mRNA expression of E-cadherin in IL-18 treated group were down-regulated and the difference betweencontrol group and 100ng/ml IL-18 treated group were statistically significant(P<0.05),The mRNA expression of α-SMA and CX3CL1 were up-regulated,and the di fference between control and IL-18 100ng/ml treated group was statistically signifi cant(P<0.05).The protein expression of E-cadherin in control group and 5,25,50,100ng/ml IL-18 treated group were 1.00±0.14,1.14±0.04,1.14±0.07,0.85±0.08,0.80±0.06,The protein expression of α-SMA in control group and 5,25,50,100ng/ml I L-18 treated group were 1.00±0.02,0.77±0.07,1.29±0.02,1.59±0.07,1.70±0.02,The protein expression of CX3CL1 in control group and 5,25,50,100ng/ml IL-18 treat ed group were 1.00±0.05,1.03±0.05,1.37±0.06,1.46±0.18,1.45±0.12.The protein e xpression of E-cadherin was down-regulated and without significant differences sta tistically between the groups.The protein expression of α-SMA was up-regulated and the difference between control group and 25ng/ml IL-18 treated groups were statistically significant(P<0.01);the difference between control group and 50ng/ml I L-18 treated groups were statistically significant(P<0.01);the difference between c ontrol group and 100ng/ml IL-18 treated groups were statistically significant(P<0.01).The protein expression of CX3CL1 was up-regulated and the difference betwe en control group and 100ng/ml IL-18 treated group were statistically significant(P<0.05).Conclusions IL-18 can activates PSCs and up-regulate the expression of CX3CL1.Part 3 Whether PDTC can alleviate the IL-18 induced activation of pancreaticstellate cells and the up regulation of CX3CL1 expressionObjective: To investigate whether the NF-κb pathway inhibitor PDTC can alleviate the IL-18 induced activation of pancreatic stellate cell activation and the increased expression of chemokine CX3CL1.Methods: The Human pancreatic stellate cells were cultured in the medium of stellate cells and passaged.The logarithmic growth phase were inoculated in 6-well plates,each hole of 5X105 cell.After culture,the cell fusion degree reached 80%,then give IL-18 and PDTC.The experiment was divided into 4 groups: PDTC and IL-18 treatment group(10μmol/L,30μmol/L),IL-18 treatment group and normal control group.The expression levels of E-cadherin,α-SMA and CX3CL1 mRNA and protein were detected by RT-PCR and Western Blot.Result: The mRNA expression of E-cadherin in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.01±0.08,0.47±0.03,0.51±0.11,0.90±0.10,There was significant difference between groups(F=10.64,P<0.01).The mRNA expression of α-SMA in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.02±0.16,4.22±0.57,4.05±0.44,1.82±0.17,There was significant difference between groups(F=17.88,P<0.01).The mRNA expression of CX3CL1 in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.01±0.12,2.38±0.19,2.51±0.19,1.37±0.13,There was significant difference between groups(F=21.62,P<0.01).Compared PDTC(10 μmol/L,30 μmol/L)+IL-18 treatment group with the IL-18 treatment group,E-cadherin mRNA expression was up-regulated,CX3CL1 and α-SMA mRNA expression were down regulation,the difference between the groups were not statistically significant(P>0.05).The protein expression of E-cadherin in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.03±0.08,0.40±0.02,0.49±0.09,1.09±0.04,There was significant difference between groups(F=33.04,P<0.01).The protein expression of α-SMA in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.07±0.08,1.87±0.21,1.92±0.21,1.34±0.08,There was significant difference between groups(F=6.82,P<0.05).The protein expression of CX3CL1 in the control group,IL-18 treatment group,PDTC(10 μmol/L)+ IL-18 treatment group,PDTC(30 μmol/L)+ IL-18 treatment group were 1.02±0.04,1.38±0.14,1.61±0.08,1.28±0.04,There were significant difference between groups(F=8.01,P<0.01).Compared PDTC(30 μmol/L)+IL-18 treatment group with IL-18 treatment group,the E-cadherin protein expression increased and the difference between the two groups were statistically significant(P<0.01).There was no significant difference between the two groups in the protein expression of CX3CL1 and α-SMA.Conclusion: PDTC could not reduce the pancreatic stellate cells activation which induced by IL-18,and could not down regulate the expression of chemokine CX3CL1 in PSCs... |
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