Research On Wip1/NF-?B Pathways Effects On Mechanism Of Liver Regeneration

1 Objectives(1)To investigate the effect of Wip1 inhibitors CCT007093 and NF-?B inhibitors JSH-23 impact on C57BL6/J mouse after resection 70%liver.(2)To investigate the effect of on the Wipl inhibitors CCT007093 and NF-?B inhibitors JSH-23 impact on expression of Wipl and NF-?B signaling pathway in liver regeneration tissue.2 Methods2.1 Animal modelForty-five male on a C57BL/6 background(6-8 weeks old)were randomly divided into three groups:One group was injection CCT007093 a Wipl inhibitor and one group was injected CCT007093-and JSH-23 as well as control group was injected equal amounts of DMSO.All the mice were performed 2/3 partial hepatectomy,At 24h?36h and 48h after PH,mice of each group were sacrificed and all the regenerated liver samples were collected.Liver tissues were immediately frozen in liquid nitrogen and stored at-80? for RNA and protein isolation.2.2 Western Blot analysisTake liver tissue from stored in liquid nitrogen,respectively,followed by the extraction of three treatment groups in the three time points in liver tissue total protein,hepatic tissue protein phosphorylation levels of NF-?B,proliferating cell nuclear antigen(PCNA),wild type p53-induced phosphatase 1 expression(Wip1),mammalian target of rapamycin(m-TOR)and cyclin D and other proteins.2.3Quantitative real-timePCRFrom liver tissue extracted Total RNA,the expression levels of Wipl?NF-?B and PCNA mRNA were analysis by RT-PCR.2.4 Data Analysis and StatisticsStatistical analyses were performed using SPSS statistical software(version 19.0)and GraphPad Prism 5.0 software.The data are presented as mean±S.E.M.The differences between groups were performed withone-way analysis of variance followed by Student-Newman-Keuls post hoc test for pairwisecomparison,in which P<0.05 is considered to be statistically significant.3 Results3.1 Wipl inhibitors and NF-?B inhibitor impact on postoperative the index of liver to body weightIn two-thirds of the mouse liver resection,liver tissue weight statistical analysis showed that the injection of the inhibitor Wipl group mice liver the index of liver to body weight(ILBW)ratio was significantly higher at 48h with compared to control group mice(p<0.001),but in mice injected Wipl inhibitor group ILBW than CCT007093 and JSH-23-injectioned in mice at 24h increased in no statistics significance.(Fig.2)3.2 Liver regeneration was increased in injection Wipl inhibitors and NF-?B inhibitorAt 48h after PH the PCNA protein were up-regulated in CCT007093-injected compared with that in the control group(p<0.05).while at 36h after PH,the expression were similar between the CCT007093-injected group and the injection CCT007093 and JSH-23 group in mice(Fig.3A).we then detected the level of cyclinD using Western Blot.Levels of cyclinD in CCT007093-injected group was higher than the control group at 48 hours after PH in mice(Fig.3B).The mRNA level of PCNA was also observed higher in CCT007093-injected group than that of control in mice at 48h(p<0.01)(Fig.3C).RT-PCR results showed that the level of PCNA mRNA in CCT007093-injected group was higher than that in CCT007093 and JSH-23-injected group(P<0.05).The results also showed that the changes of cp in mice injected with CCT007093 were significantly higher than those in CCH007093 and JSH-23-injected group(P<0.05)at 48h,However,the cp value of CHT007093-injected group mice was not significantly different from that of CCT007093 and JSH-23-injected group mice(P>0.05)at 24h.3.3 The expression of p-NF-KB in liver tissues in injection Wipl inhibitor and NF-?B inhibitorWestern Blot analysis showed that compared to injection Wipl inhibitor the p-NF-?B protein(p<0.01)expression levels increased with the control group at 36 h(Fig.4B).However,RT-PCR results showed that CCT007093-injected group the NF-?B mRNA level(p<0.01)at 36h was lower than the control groupwas statistically significant change.3.4 The expression of Wipl in liver tissues in injection Wipl inhibitor and NF-?B inhibitorImmunoblotting analysis showed that after injected Wipl inhibitor in mice Wipl expression levels were significantly lower than the expression level of control group in mice(P<0.05)at 48 h(Fig.4A).RT-PCR showed that after injected Wipl inhibitor in mice Wipl cp value change is lower than in the control group cp value change(P<0.05)at the same point.3.5The expression of mTOR in liver tissues in injection Wipl inhibitor and NF-?B inhibitorImmunoblotting analysis showed that after injected Wipl inhibitor group mice,the expression of mTOR levels higher than the same observation point(P<0.05)compare to control group mice,and the activated form of the protein p-mTOR increased after in injection Wipl inhibitors in mice.4 Conclusions(1)Inhibition of Wild-type p53-induced phosphatase 1 promotes liver regeneration in mice by direct activation of NF-?B.(2)CCT007093 can effectively inhibit the expression of Wipl promotion after 2/3 partial hepatectomy(PH).(3)JSH-23 can inhibit the expression NF-?B,and its phosphorylated form of p-NF-?B expression was suppressed.(4)CCT007093 is Wipl inhibitors can increase protein expression of mTOR,and its phosphorylated form of p-mTOR expression was increased.