Evidence of Necroptosis in Various Liver Disease

Abstract

Abstract Evidence of Necroptosis in Various Liver Diseases Waqar Khalid Saeed Department of Internal Medicine, Hanyang University School of Medicine Background: For decades, scientists have been struggling to halt the crucial cell death process and thus to prevent parenchymal loss once the disease process has commenced. Apoptosis, necrosis and autophagy are well-known cell death pathways. However, recently, the regulated necrosis, in contrast to accidental necrosis, has been recognized to harbor several diverse regulated cell death types including necroptosis, pyroptosis, mitochondrial permeability transition (MPT)-related regulated necrosis and ferroptosis. Necroptosis axis typically involves receptor interacting serine-threonine kinase-1 (RIP1), RIP3, and mixed lineage kinase domain-like (MLKL). The aim of study was to identify necroptosis relevance in various liver diseases and to identify suitable therapeutic target molecule within necroptosis axis. Methods: Various liver disease models including ischemic reperfusion (IR) injury, high fat (HF) diet induced NAFLD, common bile duct (CBD) ligation and thioacetamide (TAA) induced fibrosis models were used to evaluate necroptosis significance. C57BL/6 wild-type (WT), RIP3-KO and MLKL-KO animals were used for experiments. For IR injury, WT and RIP3-KO mice underwent 70% segmental IR injury by clamping hepatic artery and portal vein for 1hr followed by reperfusion for 4 hr. For HF diet induced NAFLD, normal chow (NC) and HF diet was fed for 12 weeks to WT, RIP3-KO, and MLKL-KO mice. For CBD ligation, WT, RIP3-KO, and MLKL-KO underwent a midline laparotomy to identify and ligate CBD at two sites without cutting. For TAA-induced fibrosis, WT and RIP3-KO animals were divided into control and TAA groups. TAA was injected intraperitoneally for six weeks. After assigned durations (for IR injury; 4hr after reperfusion, for NAFLD; 12 weeks, for CBD ligation; 21 days, and for TAA-induced fibrosis; six weeks) the animals were sacrificed, and serum and liver samples were obtained for further analysis. The serum AST, ALT, TG, LDH, and bilirubin were analyzed. The liver tissue H&E staining, Sirius red, Masson’s Trichrome and immunohistochemistry analysis were performed. qRT-PCR and western blot expression for specified markers was performed. In vitro analysis utilized primary hepatocytes, HepG2 cells, LX-2 cells, RAW 264.7 cells and U937 macrophages. TNF-α/LPS+zVAD, palmitic acid, oleic acid, TGF-β, necrosulfonamide (NSA) and, GSK'843 treated cells were used to elucidate the vitro signaling. Results: Although, necroptosis significance has been evaluated using different disease models; however, our results demonstrate that blocking necroptosis does not always have a beneficial role in all liver disease models. Nec1s pretreatment and RIP3-knockout did not reduce hepatic IR injury. Although, IR injury increased serum hepatic injury markers and Suzuki’s score; however, RIP1, RIP3, MLKL and PGAM5 expressions did not change significantly. Both the Nec1s pretreatment and RIP3-KO groups did not had improved histology or biochemical markers of injury. CyA pretreatment and/or RIP3-KO mice decreased Bax/Bcl2 expression; however, it did not lead to an overall change in serum AST, ALT, LDH and necrotic injury. In IR injury model, necroptosis does not seem to have a role in necrotic injury. In CBD ligated fibrosis model, MLKL-KO mice had reduced serum AST, ALT and fibrosis compared to WT mice. CBD ligated MLKL-KO mice also had decreased adhesion molecules expressions compared to corresponding WT mice. MLKL immunostaining was also increased in NASH patients with fibrosis. LX-2 cells treated zVAD+TNF-α showed increased p-MLKL and RIP3 expressions compared to control. NSA treatment reduced wound healing, α-SMA, collagen1α, vimentin and p-Smad2/3 expressions. Moreover, NSA treated LX-2 cells had quiescent-like morphology and reduced CXCL1, CXCL2, CTSS expressions. CBD ligated RIP3KO mice had reduced Sirius Red and MT staining compared to WT CBD ligated mice. Moreover, α-SMA, collagen1α, and TIMP-1 expressions were also reduced in CBD ligated RIP3-KO mice compared to CBD ligated WT mice. TAA treated RIP3-KO mice also had reduced fibrosis compared to corresponding WT mice. Both the MLKL-KO and RIP3-KO mice were protected against fibrosis. The human NAFLD liver tissue had increased MLKL IHC score compared to healthy control. The overall body weight of HF diet fed WT and MLKL-KO animals was the same. MLKL-KO-HF group had decreased liver weight, serum AST, ALT, and TG. MLKL-KO-HF group also had decreased NAS score, fatty change, lobular inflammation and ballooning degeneration. HF diet fed MLKL-KO mice had reduced SREBP1c, FAS, and SCD-1 expressions. NSA treatment reduced Nile red staining and SREBP1c and SCD-1 expressions in HepG2 cells. The white adipose tissue F4/80 IHC staining was reduced in HF diet fed MLKL-KO mice compared to WT mice. NSA treatment also reduced CXCL1, and CXCL2 expressions in HepG2 cells. MLKL ablation reduces hepatic steatosis and inflammation. After HF diet feeding, RIP3-KO mice had increased gain in body weight, and increased liver weight, liver/body weight ratio, serum AST and ALT. The total NAS score was the same between WT and RIP3-KO HF diet fed groups; however, hepatic TG and fatty change increased while lobular inflammation decreased in RIP3-KO mice. Following HF diet, RIP3 expression increased, while RIP3-KO-HF group had decreased MTTP, PDI, and ApoB expressions compared to WT-HF group. RIP3-KO primary hepatocytes treated with OA showed increased Nile Red staining compared to WT hepatocytes. Necroptotic condition associated increased TNF-α and MCP-1 expressions were exacerbated, while CXCL1 and CXCL2 expressions were decreased with GSK'843 treatment. HF diet fed RIP3-KO mice had increased steatosis while decreased inflammation compared to WT mice. Conclusion: The necroptosis inhibition leads to diverse outcomes in various hepatic disease models. Moreover, blocking the individual necroptosis axis molecules could result in dissimilar outcomes. Our results suggest that MLKL could be a more promising target within necroptosis signaling with significant therapeutic implications.