The Study On The Lipid-lowering Mechanism Of Recombinant Fusion Protein SAK-HV

Dyslipidaemia is the pathogenesis of both cerebral-cardio vascular diseases and metabolism syndromes.Especially,the accumulations of excess lipids within liver and serum are defined as non-alcoholic fatty liver disease(NAFLD)and hyperlipemia respectively,causing serious harm to human health.Supported by the major project of national science and technology for new drug discovery(2009ZX09103-623,2012ZX09102301-017),our lab researched and developed a novel anti-atherosclerosis recombinant fusion protein SAK-HV.SAK-HV composed of a staphylokinase variant,tripeptide of arg-gly-asp(RGD),and a segment of 12 amino acid residues at the C-terminus of hirudin,is a fusion protein with the combined functions of thrombolysis,anticoagulation,and inhibition of platelet aggregation.It exerts anti-atherosclerosis effects on various atherosclerosis(AS)animal models.Meanwhile,SAK-HV significantly decreased the serum total cholesterol(TC)and triglyceride(TG)in ApoE-/-mice,and also markedly ameliorated the hepatic steatosis.During the observation period,the lipid-lowering effects of SAK-HV were even significantly better than that of the post-marketing drug atorvastatin.We explored the lipid-lowering mechanism of SAK-HV in current study,and found surprisingly that it promoted the macrophage proliferation during in vitro experiments.Therefore,the current study consists of two independent and consecutive chapters,the study on the lipid-lowering mechanism of SAK-HV and the research on the mechanism of SAK-HV-mediated macrophage proliferation.The prior one as the major part provided new targets and thinking for the treatment against both NAFLD and hyperlipemia,while the later one as the expansion research laid a foundation for the further understanding of the drug mechanism of SAK-HV.The study on the lipid-lowering mechanism of SAK-HVWe explored the lipid-lowering mechanism of SAK-HV by the hepatic transcriptome analysis and a serials of experiments both in vivo and in vitro.Our study indicated that the dosage-response relationship of SAK-HV was in a dose-independent U-shape,and its lipid-lowering process was swift and violent.SAK-HV treatment effectively ameliorated the hepatic steatosis and decreased the inflammatory levels in both liver and serum without any bleeding risks or pathological changes in liver.Meanwhile,the lipid-lowering effects of SAK-HV were significantly better than that of atorvastatin during the observation period of 14 days.It was also more effective in improving liver function than atorvastatin.and exerted an effect of anti-oxidative stress comparable to that of atorvastatin.SAK-HV effectively lowers lipids in both liver and serum,and the liver is critically involved in the lipid metabolism.Therefore,we postulated that the liver might be one of the main target organs of SAK-HV.The liver transcriptome analysis suggested that SAK-HV induced energy and material-consuming hepatocytes proliferation,causing lipid metabolism alterations,such as upregulated PPAR signaling pathway.It also suggested that PGC-1? might be involved in the steroid hormone synthesis in liver,to which the decreased serum TC was closely related.The potential complement system-induced STAT3-C/EBP?-PGC-1? as a novel lipid-lowering pathway was further identified by the weighted gene co-expression network analysis.We verified and expanded the results of liver transcriptome analysis via a serials of experiments both in vivo and in vitro.The results demonstrated that SAK-HV induces the classical complement activation during the second week after administration.It also activated the potential lipid-lowering pathway STAT3-C/EBP?-PGC-1? in liver,and effectively improve the hepatic steatosis,accompanied with moderated liver proliferation.On the other hand,the results also showed that the fatty acid oxidation in liver was upregulated,and proofed that the enhanced female hormone synthesis in liver led to the significantly upregulated hepatic levels of female hormones.Besides,we further identified that SAK-HV enhanced LDLr-mediated cholesterol uptake in liver without any significant effects on both catabolism and secretion of cholesterol in liver.Therefore,we hypothesized that SAK-HV decreased the lipid levels via an energy-consuming hepatocytic proliferation,and the complement system-mediated STAT3-C/EBP?-PGC-1? in liver constituted a novel lipid-lowering pathway,in which PGC-1?-triggered hepatic intracrine estrogen may play important roles.PGC-la knockdown significantly inhibited the upregulation of female hormones synthesis in liver by SAK-HV,and effectively blocked the increase of female hormones in liver.The results provided the first evidence that PGC-1? mediated the hepatic intracrine production of female hormones during liver proliferation.Both PGC-la knockdown and the aromatase inhibitor letrozole effectively blocked the liver proliferation triggered by SAK-HV,and significantly inhibited the lipid-lowering effects of SAK-HV in both liver and serum.Letrozole partially inhibited the PPAR?-mediated fatty acid oxidation upregulated by SAK-HV,and blocked the LDLr-mediated cholesterol uptake in liver.While the hepatic TG content was remarkably elevated in PGC-1? knockdown ApoE-/-mice after SAK-HV treatment.This elevated hepatic TG content may be caused by the transient TG accumulation during early liver proliferation,but it was not observed in SAK-HV-treated ApoE-/-mice after estrogen inhibition,indicating that PGC-la partially lowered the hepatic TG level without the effect of estrogen.Similarly,PGC-la knockdown caused a nearly complete inhibition of the PPAR?-mediated fatty acid oxidation in liver after SAK-HV treatment,but not a partial inhibition of them as that caused by estrogen inhibition.These results indicated that the PGC-1?-triggered TG-lowering effect by inducing PPAR?-mediated fatty acid oxidation was further strengthened by PGC-la-induced estrogen in liver,and that the PGC-la-estrogen axis in liver constitutes a novel energy model of liver proliferation.The Chromatin immunoprecipitation(ChIP)assay verified that both C/EBPP occupancy on PGC-la promoter and STAT3 occupancy on C/EBP? promoter were increased by SAK-HV treatment in vivo.The STATTIC,a STAT3 phosphorylation inhibitor,effectively inhibited the potential lipid-lowering pathway STAT3-C/EBP?-PGC-1?.It also significantly blocked the serum lipid-lowering effect of SAK-HV,and remarkably counteracted its therapeutic efficacy on hepatic steatosis.We for the first time demonstrated that the STAT3-C/EBP?-PGC-la in liver constitutes a novel lipid-lowering pathway.The complement system-induced IL-6-STAT3 pathway was critically involved in regulating liver proliferation,and its activation level was reported to be positive associated with the liver proliferation rate.Both complement inhibitor FUT-175 and C3 knockout effectively weekended the upregulation of serum C5a and IL-6 in mice by SAK-HV,and significantly inhibited the STAT3-C/EBP?-PGC-1? pathway,as well as the liver proliferation triggered by SAK-HV.Complement inhibition also broke the serum lipid-lowering effect of SAK-HV,and remarkably counteracted its therapeutic efficacy on hepatic steatosis.Neither proliferation nor activation of STAT3-C/EBP?-PGC-1? was induced by the SAK-HV stimulus to mouse embryonic liver cells BNL-C12 in vitro without complement activation.These results both in vivo and in vitro demonstrated the important role of complement system in the lipid-lowering effect of SAK-HV.PPARa was reported to transcriptionally upregulated C3.However the inhibition of PPARa by PGC-la knockdown caused no significant changes in the level of SAK-HV-induced complement activation,further demonstrated that the complement activation is a cause but not an effect of lipid-lowering process.In summary,this study provided the first evidence that PGC-la mediated the intracrine production of female hormones during liver proliferation,and proposed the complement system-induced PGC-la-estrogen axis via the novel STAT3-C/EBP?-PGC-1? pathway in liver as a new energy model for liver proliferation.In this model,PGC-1? ignited and fueled hepatocyte activation as an"igniter";PGC-1?-induced estrogen augmented the energy supply of PGC-1? as an"ignition amplifier",then triggered the hepatocyte state transition from activation to proliferation as a "starter",causing triglyceride and cholesterol-lowering effects via PPARa-mediated fatty acid oxidation and LDLr-mediated cholesterol uptake,respectively.The SAK-HV-triggered distinctive lipid-lowering strategy based on the new energy model of liver proliferation has potential as a novel short-period biotherapy against NAFLD and hyperlipemia.Meanwhile,this novel energy model may also provide new perspectives on lipid metabolism during liver proliferation.The study on the mechanism of SAK-HV-mediated macrophage proliferationWe demonstrated that SAK-HV promoted hepatocyte proliferation in vivo.The proliferation of macrophage cells and vascular endothelial cells is critically involved in the development of AS.Therefore,we observed whether anti-AS SAK-HV triggered proliferation of these two cell lines during the in vitro experiments of the prior study on the SAK-HV-mediated hepatocyte proliferation.The results showed that SAK-HV stimulus selectively induce proliferative effects in RAW264.7 macrophages and THP-1 monocytes,as well as the primary peritoneal macrophage cells of C57BL/6J mice.However,this activity was not observed in neither mouse aortic endothelial cells(MAEC)nor mouse embryonic liver cells BNL-CL2,indicating that SAK-HV promoted macrophage proliferation.As a result,after clarifying the lipid-lowering mechanism of SAK-HV,the current study used both RAW264.7 and primary peritoneal macrophage cells to further explore the mechanism of SAK-HV-mediated macrophage proliferation.We first screen the proliferation pathways of SAK-HV.The results indicated that the proliferation-related pathways,including PI3K,MAPK P38,JNK and ERK,were activated by SAK-HV treatment in RAW264.7 cells.By inhibiting these activated pathways separatively,we screened out MAPK ERK and JNK pathways were the critical pathways for macrophage proliferation mediated by SAK-HV,which was also demonstrated to be activated in primary macrophages after SAK-HV treatment.We further study the function domain for promoting macrophage proliferation of SAK-HV.Using of each of the functional domains individually,the results showed that only the SAK-mutant functional domain of SAK-HV effectively induced proliferation and was comparable to the level of proliferation induced by SAK-HV treatment.While the wild-type SAK had no effects on macrophage proliferation.These results demonstrated that SAK-HV promoted macrophage proliferation via its SAK-mutant function domain.Both confocal microscopy detection and Western blot analysis of RAW264.7 cell fractions showed that SAK-HV could enter into the cytoplasm,and even the nucleus in smaller amounts.Through mass spectrometric analysis,we screened potential interacting proteins of SAK-HV and used co-immunoprecipitation to further screen out Ubal to be the potential interaction protein of SAK-HV.Meanwhile,confocal microscopy showed both proteins were co-localized in the cytoplasm and within the nucleus of RAW264.7 cells.Based on our experiments data and lots of studies on Uba1,we hypothesized an attractive model of macrophage proliferation mediated by SAK-HV.That is,the interactions between Ubal and specific E2 enzymes are blocked by its interaction with SAK-HV,inhibiting the ubiquitin modifications of their specific target pathways to promote macrophage proliferation.By Ubal inhibition in both RAW264.7 and primary macrophage cells,we demonstrated that the important role of Ubal in the macrophage proliferation mediated by SAK-HV.This result highly supported this hypothesized model of macrophage proliferation.Based on this model,we screened the known regulations of ubiquitination related to MAPK/ERK and JNK,and detected that SAK-HV treatment significantly inhibited the level of ubiquitinated-MEKK1,although the overall cellular ubiquitination levels were upregulated at the same time.The self-ubiquitination of MEKK1 was reported to inhibit its own kinase activity and activate the JNK and ERK/MAPK pathways by the inhibition of their phosphorylation.The results indicated that SAK-HV decreases the self-ubiquitination of MEKK1 to promote macrophage proliferation via MAPK/ERK and JNK pathways.In summary,the current study illustrated that SAK-HV decreases the self-ubiquitination of MEKK1 to promote macrophage proliferation via MAPK/ERK and JNK pathways by its SAK-mutant functional domain.Meanwhile,we proposed a novel model of macrophage proliferation mediated by SAK-HV.That is,the interactions between Ubal and specific E2 enzymes are blocked by its interaction with SAK-HV,inhibiting the ubiquitin modifications of their specific target pathways to promote macrophage proliferation.Although the role of macrophage proliferation effect induced by SAK-HV in its treatments against hyperlipemia,NAFLD and AS seperatively remains need further research,the critical role of macrophage proliferation involved in physiological and pathological states makes the current study has potential clinical prospects.The novel mechanism of SAK-HV-mediated macrophage proliferation,and the hypothesized model of macrophage proliferation based on the distinctive ubiquitination regulation at the level of Ubal,provides new cues for the study on both macrophage proliferation and ubiquitination.