Study On The Effects Of Species-based Immune Differences Of SAK-HV On Lipid-lowering And Its Molecular Mechanism

Cardiovascular disease is the first disease leading to human death worldwide,and hyperlipidemia is one of the main risk factors for cardiovascular disease.Currently,therapeutic drugs target low density lipoprotein(LDL),lipoprotein(a)(Lp[a])and triglyceride(TG)-rich very low density lipoprotein(VLDL)to reduce the risk of atherosclerotic cardiovascular disease.However,there are still serious dyslipidemias that are difficult to cure with existing drugs and also side effects to existing drugs,therefore,the development of drugs with new lipid-lowering mechanisms is required urgently.SAK-HV is a recombinant protein with thrombolytic and anticoagulant function developed previously in our laboratory.In pharmacodynamic studies,it could reduce the blood lipid level of high-fat Apoe-/-animal models,improve lipid deposition in liver,and has better lipid-lowering effect than statins during treatment.In both high-fat quail model and high-fat rat model,it showed similar lipid-lowering effects,suggesting the great potential of SAK-HV in the treatment of hyperlipidemia and non-alcoholic fatty liver.It was found in pharmacological mechanism study that specific antibodies induced by SAK-HV mediated the complement system activation,which in turn regulated the activation of lipid metabolism pathway STAT3-C/EBP?-PGC-1? in liver,indicating that the immune response induced by SAK-HV is very important in the lipid-lowering effect.Animals of different species have their own characteristics and different responses to the same drug.SAK-HV,as a therapeutic protein,its evaluation on immune response and lipid-lowering effect in mice is insufficient.Rhesus monkeys belong to primates in terms of species classification,and the systems of lipid metabolism and immune system are more refined and closer to the human body,so high-fat rhesus monkey model has close similarity to the state of high blood lipid in the human body.To further evaluate the lipid-lowering efficacy of SAK-HV,we successfully constructed a high-fat rhesus monkey model by feeding high-fat diet.The model group was intravenously injected with saline,while the treatment group was intravenously injected with the same volume of 0.04mg/kg of SAK-HV every other day for sever times,and the serum total cholesterol,triglycerides,and LDL were detected.At this dosage,SAK-HV did not reduce serum lipid levels significantly in rhesus monkeys.Liver function test showed that SAK-HV had no effect on liver function of rhesus monkeys,which is consistent with previous toxicological studies,indicating that SAK-HV had good safety in rhesus monkeys.Liver pathology suggested that SAK-HV improved liver lipid deposition to a certain extent,but no significant differences were shown between groups due to the limited number of animals and differences between individuals,so further investigation is needed.Although SAK-HV-specific antibodies were induced in serum of rhesus monkeys,the complement system was not activated,and no significant activation of STAT3-C/EBP?-PGC-1? pathway was detected in liver.The results suggested that the difference in immune response induced by SAK-HV among different species might be the cause of the difference in lipid-lowering efficacy.Previous experiments confirmed lipid-lowering effect of SAK-HV in high-fat mouse,rat and quail models.Compared with mice and rats,guinea pigs are more sensitive to sensitizing substances and are ideal animals for immunological research,while rhesus monkeys have immune system and metabolic system more similar to the human body from an evolutionary perspective.Therefore,we further studied the relationship between the differences in immune response and lipid-lowering effect induced by SAK-HV in the above four animals.Basing on the dosage and method of administration determined in the previous mice study,this study administered SAK-HV to mice,guinea pigs,rats,and rhesus monkeys at the same dosage,and compared the SAK-HV-specific antibody levels and complement C3 a levels in serum.The results showed that the SAK-HV-specific antibody levels in guinea pigs were significantly higher than those in rhesus monkeys,and also higher than those in mice and rats,while the antibody levels in rhesus monkeys were below the level of mice and rats.In addition,although SAK-HV activated the complement system in guinea pigs,mice,and rats,it could not activate complement in rhesus monkeys.Guinea pigs died of severe allergic reactions after SAK-HV administration.The above results proved that SAK-HV induced the strongest immune response in guinea pigs with immunotoxicity,while the immune response triggered in rhesus monkeys was lower than that in mice and rats.Combining the differences in the lipid-lowering efficacy of SAK-HV in different species of animals,we proposed that the moderate immune response induced by SAK-HV had a key role in lipid lowering,and the lack of lipid-lowering effect might be related to the lower level of immune response in rhesus monkeys,whose immune homeostasis are more stable as primate.Based on the above results and analysis,we hoped to achieve immune enhancement by changing the drug delivery route to improve lipid-lowering efficacy.PLGA-PEG-PLGA triblock copolymer hydrogel has the characteristics of high biocompatibility,good biodegradability and thermosensitivity.At low temperature(such as 4?),it is low-viscous liquid,easy for drug loading and injection,and in response to physiological temperature,the micelles automatically convert into viscous gels as an in-situ reservoir,making it a safe and controllable drug delivery vehicle.Herein,it was used for the delivery of SAK-HV recombinant protein taking advantage of its ability to release antigens in a sustainable manner and achieve immune enhancement by promoting antigen-specific Ig G immune responses.At the same time,the thermosensitive and subcutaneously injectable PLGA-PEG-PLGA hydrogel has the advantages of more security,easy application and good compliance.Pharmacodynamic experiments showed that treatment of the same dose of SAK-HV/Gel in Apoe-/-mice produced a more significant lipid-lowering effect than SAK-HV and induced a higher level of immune activation.It suggested that moderate immune enhancement could improve lipid-lowering,and it further verified the essential role of SAK-HV-induced immune response in lipid-lowering.Moreover,transcriptomics,lnc RNA sequencing and metabolomics were combined to explore the lipid-lowering mechanism of SAK-HV/Gel.The results suggested that there were unreported pathways in its lipid-lowering process.Function analysis of differential genes indicated that SAK-HV/Gel reduced lipid by promoting the conversion of free fatty acids to HDL,promoting lipid oxidation,and inhibiting lipid synthesis.The regulation of immune response was important in the process as well.Basing on lnc RNA sequencing and m RNA transcriptome analysis,we proposed that the lnc RNA Gm20649-Onecut1-Cyp7a1 might be one of the important pathways in lipid-lowering mechanism of SAK-HV/Gel,which will become the focus of our future work revealing the lipid-lowering mechanism from a new perspective of non-coding RNA transcription.Furthermore,metabolomics studies combined with m RNA transcriptome analysis have shown that changes of fatty acid oxidation process might be the key point in lipid-lowering of SAK-HV/Gel.Differential metabolites in liver were significantly enriched in linoleic acid metabolism,ketone body synthesis and degradation pathways after SAK-HV/Gel treatment,providing clues for further elucidating its lipid-lowering mechanism.These still needed further verification and exploration through subsequent experiments.Macrophage is essential in immune response and is an important part of the innate immune system,the migration of which is a prerequisite for the development of immune functions such as phagocytosis,secretion and killing.Under different microenvironments,macrophages are activated into different states to participate in the regulation of immune homeostasis,and play a regulatory role in the progression of metabolic diseases such as hyperlipidemia and atherosclerosis.Through previous studies,we found that macrophages were one of the target cells of SAK-HV,and SAK-HV selectively triggered its proliferation,but the effect of SAK-HV on macrophage migration and polarization is still not fully understood.In this work,we further studied the effects of SAK-HV on macrophage migration and explored the molecular mechanism of SAK-HV-induced migration based on RAW264.7 cell.Through wound healing and transwell experiments,it was confirmed that SAK-HV could significantly promote macrophage migration through its SAK mutation domain.In mechanism study,through screening the activation of migration-related pathways and inhibitor experiments,JNK and NF-?B pathways were found to be play a key role in SAK-HV-induced cell migration.Further,we screened migration-related effector molecules and found that SAK-HV induced the upregulation of MCP-1,which was regulated by JNK and NF-?B pathways.Inhibition of MCP-1 or interference with its specific receptor CCR2 partially inhibited SAK-HV-induced cell migration.In conclusion,we found that SAK-HV upregulated MCP-1 expression by activating JNK and NF-?B pathways,thereby promoting macrophage migration in an autocrine manner.Further,it was also confirmed that SAK-HV could promote macrophage M1 polarization.The effects of SAK-HV on macrophages deepened our understanding in the regulation of immune response by SAK-HV,and suggested that the regulation of macrophage function might be involved in its lipid-lowering mechanism through regulating immune response.