| Part 1:Effects of parthenolide on the secretion function of Kupffer cellsObjective:To explore the effects of parthenolide on the inflammatory cytokines expression in kupffer cells and to elucidate the probable mechanism of the effects.Methods:Kupffer Cells were isolated and purified by situ collagenase perfusion. The isolated and purified cells were then divided into 3 groups:Group A, control group in which kupffer cells were cultured normally; Group B, LPS group in which 10mg/l LPS was added into the nutrient solution; Group C, PTN group in which 5μmol/l parthenolide was added into the nutrient solution 1h before 10mg/l LPS was added. After 3h stimulating to each group, the morphology of kupffer cells were measured by an inverted microscope, the activity of kupffer cell was measured by trypan blue staining, the purity was measured by anti ED-2 immunohistochemical staining, the concentration of IL-1 and TNF-a in the supernatant were measured by ELISA, the NF-kB DNA binding activity were measured by EMSA. Treatments in each group were repeated for 8 times to obtain the mean value of each index as the final result.Results:The shape of newly isolated kupffer cells were sphere like, suspending in the nutrient solution; Most of them became flat and adhered to the wall, pseudopodium formation occurred in few cells; After about 24h, pseudopodium formation occurred in most of the cells and all cells were extended totally with bigger size in star-shape or polygonal shape; The purity ratio of each group was 90%, and the activity ratio was more than 96%. Concentration of TNF-a and IL-1 of the nutrient solution in group B is much higher than that of group A (p＜0.05), Concentration of TNF-αand IL-1 of the nutrient solution in group C is much lower than that of group A,Group B was higher in DNA binding activity of NF-kB than that of group A (P＜0.05), Group C was lower in DNA binding activity of NF-kB than that of group B (P＜0.05).Conclusions:(1) LPS can activate the kupffer cells and as a result induce its ability of releasing cytokines.(2) Parthenolide can inhibit the activation of NF-kB in kupffer cells and as a result reduce its ability of releasing cytokines.Part 2:Effect of parthenolide on hepatic ischemia-reperfusion injuryObjective:To investigate the effect and related mechanisms of parthenolide on liver function and hepatic tissues in the liver ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 4 groups (n=18):sham-operated group(A), model control group(B), low dosage of parthenolide treatment group(C) and large dosage of parthenolide treatment group(D).10 minutes before Ischemia, treatment group rats were intraperitoneally injected 250 or 500μg/kg parthenolide.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO.After 90 min ischemia,6 cases rats of each group were killed at 2h,6h and 24h after residual liver reperfusion respectively, blood and liver tissue samples were collected from the experimental groups. Serum levels of ALT and AST were measured, part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade hepatic IRI(Suzuki's score), the content of MDA and the activity of MPO in liver tissues were detected by ELISA.Results:Compared with the model control group,serum ALT and AST in both treatment group of the low and large dosage parthenolide were significantly decreased(P<0.05),even lower in the large dosage group than in the low dosage group(P<0.05). Hepatic pathology performance was improved and Suzuki's scores was decreased significantly in both dosage group (P<0.05), the large dosage group is more significantly. The content of MDA and the activity of MPO in liver tissues were significantly decreased in both treatment groups when compared with the I/R model control group(P<0.05), also lower in the large dosage group than in the low dosage group(P<0.05).Conclusion:(1) Treatment with parthenolide can effectively improve the rat liver function and liver pathology damage induced by liver ischemia-reperfusion injury;(2) Intervention with parthenolide can inhibit the effection of oxygen free radicals, and reduce the tissues damage induced by lipid peroxidation;(3) Intervention with parthenolide can reduce the aggregation, infiltration and activation of neutrophil in the rat liver induced by ischemia-reperfusion injury,thereby reducing inflammatory response.Part 3:Effects of parthenolide on IKK/NF-κB signaling pathway in the rat hepatic ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of parthenolide inhibiting the inflammation signaling pathway of IKK/ NF-κB in the rat hepatic ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 3 groups (n=6):sham-operated group(A), model control group(B), parthenolide treatment group(C).10 minutes before Ischemia, treatment group rats were intraperitoneally injected 500μg/kg parthenolide.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO.After 90 min ischemia and 6h reperfusion,6 cases rats of each group were killed respectively, blood and liver tissue samples were collected from the experimental groups. Serum levels of TNF-a, MIP-2 and ICAM-1were measured by ELISA,TNF-a, MIP-2 and ICAM-1mRNA of liver were measured by RT-PCR, the NF-kB DNA binding activity were measured by EMSA, the level of protein of NF-kB p65 in caryon and p-IKKser180/βser181 and IkBαin the liver tissues were measured by Western blot.Results:Compared with the model control group, the levels of TNF-a, MIP-2 and ICAM-1 in serum and mRNA of liver in parthenolide treatment group were significantly decreased(P<0.05);Compared with the model control group, the NF-kB DNA binding activity, the level of protein of NF-kB p65 in caryon, the level of protein of p-IKKser180/βser181 in parthenolide treatment group were significantly decreased(P<0.05), the level of protein of IkBbαwere significantly increased(P<0.05).Conclusion:Parthenolide reduces the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury probablely by suppressing IKK/NF-kB signal pathway activation.Part 4:Effects of JAK2/STAT3 signal pathway on the rat hepatic ischemia-reperfusion injury and effects of parthenolide on JAK2/STAT3 signaling pathwayObjective:To identify the role of the activated JAK2/STAT3 in rat hepatic ischemia-reperfusion injury and investigate the mechanisms of JAK2/STAT3 signal pathway causing hepatic damage,to study the molecular mechanism of the effect of parthenolide on the JAK2/STAT3 signaling pathway in the rat hepatic ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 4 groups (n=6):sham-operated group(A), model control group(B), AG490 treatment group(C) and parthenolide treatment group(D).10 minutes before Ischemia, AG490 treatment group and parthenolide treatment group treatment group rats were intraperitoneally injected lmg/kg AG490 and 500μg/kg parthenolide respectively.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO. After 90 min ischemia,6 cases rats of each group were killed at 6h after residual liver reperfusion respectively, blood and liver tissue samples were collected from the experimental groups. The level of protein of p-JAk2 and p-STAT3 were measured by immunohistochemical method and Western blot, TNF-amRNA of liver were measured by RT-PCR, Serum levels of TNF-αwere measured by ELISA.Results:Compared with the sham-operated group, the levels of protein of pJAk2 and pSTAT3, TNF-amRNA of liver, serum levels of TNF-a in model control group(B)were significantly increased(P<0.05); compared with the model control group(B), the levels of protein of p-JAk2 and p-STAT3, TNF-amRNA of liver, serum levels of TNF-a in AG490 treatment group(C) were significantly increased(p<0.05); compared with the model control group(B), the levels of protein of p-JAk2 and p-STAT3, TNF-amRNA of liver, serum levels of TNF-a in parthenolide treatment group(D) were significantly increased(p<0.05).Conclusion:(1) JAK2/STAT3 signal pathway were activated in hepatic ischemia-reperfusion injury;(2) suppressing JAK2/STAT3 signal pathway activation probablely reduce the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury;(3) Parthenolide reduces the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury probablely by suppressing JAK2/STAT3 signal pathway activation.
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