Ligustrazine Improve Endothelial Cell Function Damage Induced By High Glucose Mechanism Research

AimsTetramethypyrazine (TMP), crocin, ferulic acid and chlorogenic acid are the potential antioxidant monomers that we isolated from the Traditional Chinese medicin Qing Huo Yi Hao (QHYH), the principles of which are based on’clearing heat and detoxifying’. In this study we aim to determine the endothelial-dependent and independent relaxant effects of these monomers on rat aortic rings and find the most potential one.MethodsThe thoracic aortas were isolated from SD rat and cut into3-4mm ring segments. The rat aortic rings were then divided into enthelium intact group and endothelium stripped group. The endothelium in the internal surface was removed a polyethylene tube in endothelim stripped group. Then the rings of these two groups were suspended between two stainless steel stirrups in an organ bath.10-5mol/L acetylcholine was used to assess the endothelial integrity. After that, the accumulatively relaxant effects of TMP, crocin, ferulic acid and chlorogenic acid on phenylephrine-precontracted rat aortic rings were determined.ResultsThe maximum relaxant effects of TMP on endothelium intact rings was more potential than that on endothelium stripped rings in a concentration dependent manner from10-6mol/L to10-3mol/L (90.36%vs.55.38%, p<0.001); Crocin also shows similar characteristics with concentration from10-7mol/L to10-4mol/L (41.3%vs.9.6%. p<0.001). However, these effects of crocin were not in a concentration dependent manner when the concentration exceeded10-4mol/L, Ferulic acid showed relaxant effects on both endothelium intact ring and endothelium stripped rings in a concentration dependent manner from10-7mol/L-10-3mol/L, but there is no statistical difference between these two effects (59.4%vs.55.4%, P>0.05). Chlorogenic acid showed relaxant effects in a concentration (10-7mol/L-10-4.5mol/L) and endothelium dependent manner. Howerver, with the concentration higher than105mol/L, the relaxant effects of chlorogenic acid reduced.ConclusionsTMP presents potential endothelilum dependent and independent effects on vascular. Crocin and chlorogenic acid show endothelium dependent effects on vascular at the concentration from10"7mol/L to10-4mol/L and from10"7mol/L to10-4.5mol/L, respectively. However, the endothelium independent effects of crocin and chlorogenic acid are not obvious. Ferulic acid only has endothelium independent effects on vascular. Therefore, TMP show more potential endothelial-dependent effects compared to other monomers such as crocin, ferulic acid and chlorogenic acid in QHYH at the organ level, indicating TMP could increase the generation of nitric oxide remarkably. AimsThe results in the first part show that at the organ level TMP present the most potential endothelium dependent effects on rat aortic ring compared to other monomers such as crocin, ferulic acid and chlorogenic acid in QHYH, suggesting TMP could augment the generation of nitric oxide significantly. The aims of this part are to establish the model in which TMP could ameliorate high glucose-induced endothelial dysfunction and determine the antioxidant effects of TMP in this model.MethodsHuman umbilical vein endothelial cells were isolated from the fresh human umbilical vein. Immunofluorescence assay was used to detect the CD31antigen on the cytomembrane of HUVEC. HUVEC an bEnd.3cells were used in4experimental groups:control group, high glucose (30mmol/L) group, TMP (30μmol/L)+high glucose (30mmol/L) group, and treated in corresponding culture condition for48h. DAF-FM indicator was used to determine the nitric oxide concentration and CM-H2DCFDA indicator was applied to assess the change of ROS level in these cell groups.ResultsThe results of Immunofluorescence assay showed CD31antigen was on the cytomembrane of the cells isolated from human umbilical vein, suggesting these cells are HUVEC. Compared to high glucose group, the nitric oxide generation in TMP-pretreated bEnd.3and HUCEC cells was augmentd and ROS production in these cells was decreased.ConclusionsThe cells isolated from human umbilical vein are HUVEC. TMP increase nitric oxide generation and reduce ROS production when cells pretreated with TMP for24h, which are consistent with our previous work, indicating it is successful to establish the cell model in which TMP ameliorate high glucose-induced endothelial dysfunction and the mechanisms underlying this activities of TMP are related to the antioxidant effect of TMP. AimsIn part Ⅱ, HUVEC and bEnd.3cells were used to establish the model in which TMP ameliorate endothelial dysfunction. The aims of this part are to investigate whether TMP performs its antioxidant effects by improving mitochondrial function and to further explore the molecular mechanisms underlying its effects on mitochondrial function involving PGC-la mediated mitochondrial biogenesis by targeting on SIRT1.MethodsSimilar to part Ⅱ, HUVEC and bEnd.3cells were used in following groups:control group, high glucose (30mmol/L) group and TMP (30μmol/L)+high glucose (30mmol/L)group, and treated in corresponding culture condition for48h. Then MitoSOXTm Red mitochondrial indicator was used to detect the effects of TMP on superoxide generation. The activities of TMP on mitochondrial function were assessed by mitochondrial membrane potential and the expression of respiratory chain complex Ⅲ. At last the change of the expression of SIRT1and PGC-1α were evaluated by real-time PCR and immunoblotting assay before and after the SIRT1activity inhibitor EK-527was added.ResultsTMP reduced superoxide generation in mitochonrium, reverse mitochondrial membrane potential and up-regulate the expression of complex Ⅲ when30μmol/L TMP pretreated cells for24h, compared to high glucose group. Moreover, TMP up-regulate the expression of SIRT1, PGC-1α and its downstream genes in both mRNA and protein level. However, after the activity inhibitor of SIRT1was added, TMP-induced effects on up-regulating PGC-1α expression in mRNA level was significantly reduced.ConclusionsTMP improve high glucose-induced mitochondrial dysfunction and relief stress status in mitochondrium. The molecular mechanisms underlying these effects involve the upregulation of SIRT1by TMP and then SIRT1regulates the expression of PGC-la and PGC-la downstream pathway, resulting increased mitochondrial biogenesis. In summary our study indicates that TMP performs protective effects on high glucose-induced endothelial dysfunction by targeting SIRT1.