The Impact Of Wip1 Gene Knock Out On Acute Liver Injury And Mechanisms

Background:Liver constitutes 2.5% of the body weight and is the largest organ in the body. It receives-25% of the cardiac output via dual blood flow, the portal vein and the hepatic artery.The portal vein contributes to-75-80% of the total flow and>50% of the total oxygen supply. Warm ischemia/reperfusion injury is a common acute liver injury in clinical scenario. The Pringle manoeuvre in many liver surgeries is one of the many causes.By now researchers have found out that many mechanisms including the change of pressure in liver sinusoids mediated by endothelium and NO, Kupffer cell regulated innate immunity, ATP-depletion-dependent liver cell necrosis and caspase-dependent apoptosis.Akt is a serine/threonine kinase which plays an important role in regulating apoptosis, autophagy, cell growth, regeneration and protein synthesis. It has been revealed that Akt activates downstream proteins (Bad) which regulate pathogenesis of liver ischemia/reperfusion injury. Wipl (Wild-type p53 induced phosphatase 1) is a serine/threonine phosphatase. Recent researches unvailed that Wipl plays a key role in immunity and inflammation. However, it still remains mystery whether it is involved in the progression of liver ischemia/reperfusion injury. In this study, we used partial(2/3) liver warm ischemia/reperfusion injury model to dig into the function of wipl gene in acute liver injury and the underlying mechanisms.Methods:1.129sv genetic background wipl knock out mice were intercrossed with C57BL/6 genetic background wild type mice, then the 129sv/C57BL/6 wip1+/- springs were intercrossed with C57BL/6 wild type mice for 7 serial generations to get C57BL/6 wip1+/-. We mated these wip1+/- mice to breed wip1-/- and wip1+/+ littermates.2.10-14w wip1-/- and and their wildtype littermates with the same gender were assigned as experimental group and control group respectively. We established a 70% hepatic warm ischemia/reperfusion model. Time of ischemia were set as 60min and 90min. After 6h or 24h of reperfusion, we draw blood and harvested the liver shortly after euthanasia. The severity of liver injury were assessed through serum ALT levels and suzuki scores using H&E staining of the ischemic liver lobes. We used TUNEL staining for evaluation of apoptosis. The expression of wipl, Akt, mTOR, p70S6K, S6 and their phosphorylationlevel were determined through western blot assay. Total RNA were extracted from the liver tissues and RT PCR and Realtime PCR were conducted to detect wip1 mRNA levels.Results:1. After 60min of ischemia and 6h of reperfusion, the serum levels of ALT were 1172.5±237.1U/L in wild type mice and 851.3±270.9U/L in wipl knock out mice (P<0.01); while in sham operated mice, serum ALT were 44.3±11.3U/L in wild type mice and 56.6±12.9U/L in wipl knock out mice (P=0.369). Hence, liver injury was attenuated in wipl knock out mice. 2. After 90min of ischemia and 24h of reperfusion, the Suzuki scores from H&E staining of liver samples were 7.75±0.43 in wild type mice and 5.25±0.43 in wipl knock out mice (P<0.01), which implies that wip1 knock out mice indures less liver injury. 3. After 90min of ischemia and 24h of reperfusion, according to the TUNEL staining of liver samples, the rate of apoptosis is ～62.3%±5.6% in wild type mice and ～30.4%±3.7% in wipl knock out mice (P<0.01). 4. After 90min of ischemia and 6h of reperfusion, the expression of Wip1 was downregulated in wild type mice liver. Compared to their wildtype littermates, wipl knock out mice liver showed no significant difference in the expression of mTOR, p-mTOR(Ser2448), Akt and S6 ribosomal protein, while significant increase in the level of p-Akt(Ser473), p-p70S6K(Thr389) and p-S6(Ser235/236) was observed. However, p-mTOR(Ser2481) was somewhat decreased following I/R. 5. After 90min of ischemia and 6h of reperfusion, the wipl mRNA transcription was downregulated in wild type mice.Conclusion:Liver ischemia/reperfusion injury was attenuated in wip knock out mice. This might be the result of increased activation of PI3K/Akt/mTOR signaling and its downstream regulation of apoptosis.