The Influence Of SAK-HV On Mouse Aortic Endothelial Cells And Its Possible Mechanism

Objective:To investigate the drug mechanism of SAK-HV in mouse aortic endothelial cells in vitro. To evaluate the pharmacodynamics effects and to study the drug mechanism on oral administration of SAK-HV in hyperlipidemia rats.Methods:1. Cell proliferation, cell cycle and apoptosis were detected after SAK-HV treatment at all concentrations to MAEC for 24hrs. The activity of inflammatory relative pathways and the expression of inflammatory factors were detected after SAK-HV treatment at all concentrations and at all time to MAEC. Thrombin was used to imitate the inflammatory environment in vivo. SAK-HV and dodecapeptide of hirudin were added respectively to co-stimulate MAEC with thrombin, then the activity of inflammatory relative pathways and the expression of inflammatory factors were detected. MAEC was treated by the same molarity of SAK-HV, SAK, RGD and dodecapeptide of hirudin and the activity of inflammatory relative pathways and the expression of inflammatory factors were detected. Cell immunofluorescence technique was used to detect that whether SAK-HV and SAK could get into the cell or not. Nanomolecular motor and mass spectrum analysis were used to screen out potential interaction proteins for SAK-HV in MAEC.2. Male Wistar rats were fed high-fat diets to build animal models of hyperlipemia, and were randomly divided into model group, solvent control group (NaHC03), treatment group (SAK-HV) and positive control group (atorvastatin), according to their body weight. After administration for six weeks, the levels of TG and TC in serum were detected, hepatic fat deposition detected by oil red staining, the mRNA and protein expression of NPC1L1 in intestine and ABCG5/G8 in liver detected, the content of ROS in aorta detected, the activity of SOD and the content of MDA in serum detected.Results:SAK-HV treatment at all concentrations to MAEC for 24hrs had no proliferation effects, caused no changes in the cell cycle, and did not induce apoptosis either. Meanwhile, SAK-HV upregulated the expressions of inflammatory cytokines in MAEC in a concentration-dependent manner after its treatment for 6hrs. The variation trend of these inflammatory cytokines was basically consistent with that of phosphorylation level of P65. After the thrombin stimulation, the SAK-HV significantly inhibited the thrombin induced activation of P65 and P38 in MAEC after its treatment for a short time (40min), despite the compromised inhibitory effect compared with that of dodecapeptide of hirudin treatment, In contrast, it upregulated the expression of inflammatory cytokines, including IL-6, MCP-1, ICAM-1 and VCAM-1, in a concentration-dependent manner after its treatment for a long time (6hrs). SAK-HV, modified SAK, RGD peptide, and dodecapeptide of hirudin were used to stimulate MAEC cells respectively at the same molar concentration, and only SAK-HV induced the rise of the P65 phosphorylation level and the inflammatory cytokines expression. The immunofluorescence detection result indicated that SAK-HV can penetrate the cell membrane, but not the nuclear membrane, into the cytoplasm. Moreover, HSP70, a potential interaction protein for SAK-HV in MAEC, was screened out by combining with nanomolecular motor and mass spectrum analysis, although their interaction effect remains to be verified.2. The curative effects of SAK-HV on both hyperlipemia and hepatic steatosis were remarkably better than that of atorvastatin. SAK-HV significantly down-regulated the NPC1L1 expression in intestine, and upregulated the ABCG5/G8 expression in liver, compared to model group. Meanwhile, the anti-oxidative stress effect of SAK-HV was significantly better than that of the atorvastatin, including the reduced ROS in aorta, and the increased SOD activity but reduced MDA content in serum.Conclusion:1. SAK-HV plays both pro-and anti-inflammatory roles in MAEC. The anti-inflammatory activity of SAK-HV induced by its dodecapeptide of hirudin functional domain is predominant in thrombin-induced inflammatory environment during its treatment for short time (40min). With increased treatment time, its pro-inflammatory activity emerges gradually. SAK-HV may play a pro inflammatory role by a potential noval functional domain via NF-κB pathway.2. SAK-HV, administrated by oral way, significantly down-regulated the NPC1L1 expression in intestine, and upregulated the ABCG5/G8 expression in liver, which indicated that it lowers cholesterol by reduced intestine cholesterol absorption and enhanced hepatic cholesterol excretion into bile. Moreover, it could reduce hepatic steatosis and the level of oxidative stress.