Antioxidative Molecular Mechanism Of Traditional Chinese Medicine Qinghuoyihao Against Detrimental Effects Of High Level Glucose On Endothelial Cells

Aims: Our previous study has demonstrated that a prescribed traditional Chinese medicine preparation — Qinghuoyihao(QHYH) can decrease urinary microalbumin excretion in type 2 diabetic patients and improve microvascular endothelial cells growth in high glucose medium with similar effects to a natural antioxidant N-acetylcysteine(NAC). This study was to explore the antioxidative molecular mechanism of QHYH against the noxious effects of high glucose level on endothelial cells.Methods: Electron paramagnetic resonance(EPR) together with spin trap technique was applied to determine the antioxidative capacity of QHYH and its each constitutional material in vitro. And ffects of QHYH on cellular superoxide anion production was observed when bEnd.3 cells were cultured in a high glucose medium. The effects of QHYH on SD rat pulmonary microvascular endothelial cells cultured in high glucose media was investigated with serum pharmacological approaches. Expression of uncoupling proteins(UCPs) mRNA in bEnd.3 cells cultured in media with 5.56mM glucose(NG group), 35mM glucose(HG group), 35mM glucose and 1:100 QHYH (QHYH group), and 35mM glucose plus 10mM NAC(NAC group) were analyzed by real time quantitive RT-PCR, and expression of UCP molecule were studied by Western blot analysis.Results: EPR determination revealed that QHYH could extinguish 82.2% of the radicals generated in a hydroxyl free radical model system and the scavenging capacity of single component medicine ranged from 23.1% to 46.2%. Like NAC, serum from SD rats which were administered with QHYH could improve SD rat pulmonary microvascular endothelial cells' vitality in high glucose milieu. Cellularsuperoxide anion production increased when bEnd.3 cells were cultured in media with 35mM glucose and QHYH could potently scavenge the superoxide anions with a similar effect to superoxide dismutase. No obvious expression of UCP-1 or UCP-3 mRNA was observed in bEnd.3 cells cultured in media with 5.56mM or 35mM glucose while distinct expression of UCP-2 mRNA was detected in bEnd.3 cells of NG, HG, QHYH, and NAC groups. Expression of UCP-2 mRNA in HG group was higher than that in NG group(p=0.009) and UCP-2 mRNA was further upregulated in cells of QHYH and NAC groups(p=0.009, 0.036 respectively). In Western blot analysis, expression of UCP-2 molecule were detected in cells of QHYH group and a blear band of UCP-2 molecule appeared in NAC group while no apparent UCP-2 molecule band occurred in NG or HG groups.Conclusions: The results suggest that the prescribed traditional Chinese medicine preparation — Qinghuoyihao and its each constitutional material can scavenge oxygen free radicals in vitro with Qinghuoyihao compound recipe being more potent than any of its single component remedium. Serum materia medica of Qinghuoyihao recipe, like NAC, can protect endothelial cells against the noxious effects of high level glucose in culture media. Cellular superoxide anion production increases when endothelial cells are exposed to high level glucose and the Qinghuoyihao preparation is effective in extinguishing superoxide anions produced by the endothelial cells. In addition to its direct effects of scavenging oxygen free radicals, the traditional Chinese medicine compound recipe can upregulate expression of UCP-2 molecule in cells exposed to high glucose milieu resulting in decrease of mitochondrial superoxide anion production and this may also play an important role in its antioxidative and protective effects on endothelial cells in high glucose surroundings.Aims: To determine the oxidative stress status and endothelial function in patients with coronary atherosclerotic heart disease (CAD).Methods: The serous scavenging activities for superoxide anion radicals(O₂^-·), malondialdehyde (MDA) level, nitric oxide synthase(NOS) and its subtype inducible nitric oxide synthase(iNOS) activity, and nitric oxide(NO) level were measured by spectrophotometry in 200 healthy individuals(healthy group), 200 patients with CAD diagnosed by coronary angiography(CAD group) and 135 patients with normal coronary angiographic manifestation(CAG negative group). Glucose metabolism status and plasm lipid profile were also analyzed in all the individuals. Results: The average serous scavenging activities for O₂^-·, NOS and iNOS activity, and NO level in CAD group were much lower than that in healthy group(p<0.001) while MDA level was higher than that in healthy group(p<0.001). No obvious difference in serous scavenging activities for O₂^-· was observed between CAD group and CAG negative group(p=0.634) while MDA level in CAD group was higher than that in CAG negative group(p<0.001) and NOS and iNOS activities were lower than that in CAG negative group. Compared with healthy group, CAG negative group had lower average serous scavenging activities for O2^-· (p=0.002), a higher MDA level(p=0.001), similar NOS activities(p=0.967), decreased iNOS activities(p=0.001), and a lower NO level(p<0.001). The prevalence of impaired glucose regulation(IGR) in patients with CAD was 55.5%, which was much higher than that in patients in CAG negative group(p=0.001).Conclusions: Endothelial dysfunction, increased oxidative stress, decreased serous scavenging activities for O2^-· and high prevalence of IGR can be observed in patients with CAD. Antioxidative therapy may benefit patients with CAD.