Protective Effects Of Allicin On Diabetic Macroangiopathy

Background Due to the changes of lifestyle, diabetes mellitus has risen markedly in the world and become a global public health crisis. Diabetes mellitus is the disorder of insufficient production of insulin or reduced sensitivity to insulin, with the characteristics of chronic hyperglycemia. As we known, diabetic has a more great risk of vascular disease occurrence compared with non-diabetic, which results in an increase of both cardiovascular and cerebrovascular diseases such as myocardial infarction and cerebral ischemic attacks. According to statistics, over 80% of diabetic patients succumb to cardiovascular diseases. Diabetes-related macroangiopathy has become the major cause of mortality for diabetic individuals. Management of diabetic macroangiopathy complications is becoming one of the critical social problems.But so far, the prevention and cure diabetes-related macroangiopathy mainly focus on the risk factors, such as blood glucose, blood pressure and blood fat, which can limit the diabetic microangiopathy, but has no more benefit to the macroangiopathy. Thus, it is important to reinforce the intervention in the pathophysiology of diabetes-related macroangiopathy.Elevated serum homocysteine concentration, termed hyperhomocysteinemia, has been identified as an important risk factor for atherosclerosis-related cardiovascular disease, including coronary artery disease and apoplexy. Homocysteine is a sulfur-containing amino acid generated by the metabolism of methionine. Hyperhomocysteinemia can promote oxidative stress, inflammation, endothelial dysfunction, thrombosis and vascular smooth muscle cell proliferation. Some previous studies have indicated that insulin levels might affect the activity of homocysteine metabolism-related enzymes and diabetic patients with hyperhomocysteinemia have greater risk of development of vascular disease than the patients with normal homocysteine levels. There is a positive association between hyperhomocysteinemia and pathophysiology of diabetes mellitus. Moreover, research has found that there is a hypoxia microenvironment in diabetes, which is closely associated with oxidative stress induced by hyperglycemia and plays an important role in diabetic complications. In the setting of hypoxia, HIF-la increases in response to ischemia. HIF-1α is a key regulator of oxygen homeostasis, which restores blood flow to ischemic regions. However, sustained and prolonged activation of the HIF-1 pathway induces cell death. There is a close relationship between HIF-la and diabetic macrovascular complications.Studies have shown that excessive production of reactive oxygen species (ROS) and the subsequent increase of oxidative stress play a critical role in diabetic pathology. Oxidative stress is considered the final common pathway, through which hyperglycemia-related pathways (protein kinase C, et al) can trigger diabetic complications. Cumulative oxidant-mediated damage and cellular dysfunction result from an imbalance between ROS generation and antioxidant capacity. Hyperglycemia promotes glucose oxidation and protein glycation, impairs DNA repair with resultant DNA cleavage, and generates reactive oxygen species, thereby leads to increased oxidative stress. Therefore, therapeutic strategies aimed at the removal of free radicals or the prevention of their formation, are necessary.Allicin is one of the main active components in garlic, with plenty of obvious therapeutic effects such as regulating lipid metabolism, decreasing blood glucose level, promoting insulin sensitivity, restraining inflammation and fibrogenesis, reducing homocysteine level, and attenuating superoxide production. Our previous study confirmed that allicin decreased homocysteine level of rats and the LDH level of culture solution as well as inhibited the vascular smooth muscle cell proliferation.Given the role of oxidative stress within the etiology and pathogenesis of diabetic complications, in our study, we used allicin to treat diabetic patients with hyperhomocysteinemia and observed the blood glucose, homocysteine and intracarotid intima-media thickness. Furthermore, we set up an in vitro model of diabetes-associated oxidative stress using aorta endothelial cells cultured under high-glucose/hypoxia and high-glucose/homocysteine conditions to clarify the possible mechanism of allicin on diabetic macrovascular complications.Objective1. To observe the effect of allicin on homocysteine and intracarotid intima-media thickness of diabetic patients with hyperhomocysteinemia.2. To investigate the mechanisms of the cell injury induced by high glucose and homocysteine as well as the anti-injury effects of allicin.3. To observe the reaction of allicin with homocysteine under the condition of temperature and PH similar to human body.4. To explore the protective effects of allicin on aortic endothelial cell’injury induced by high glucose/hypoxia, and the corresponding mechanisms.Methods1. Forty diabetic patients with hyperhomocysteinemia were divided randomly into two groups. Before treatment, the blood glucose, homocysteine, blood fat, liver function, renal function and intracarotid intima-media thickness were measured. Application insulin, atorvastatin and allicin and the control group not using allicin for 3 months, the homocysteine level and intracarotid intima-media thickness were evaluated again.2. Endothelial cells of the third passage were randomly divided into five groups: the normal group (NG), the mannitol group (MG), the high glucose/homocysteine group (HG), the allicin group (AG) and the PKC inhibitor (GF109203x) group (GG). Morphological changes were observed under an inverted phase contrast microscope every day. MTT assays were performed to assess cell viability. Dihydroethidium (DHE) staining was adopted to quantify the intracellular reactive oxygen species (ROS) production. The levels of hydrogen sulfide (H2S), NADPH oxidase 4 (Nox4), 8-hydroxydeoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), hypoxia inducible factor-1α (HIF-1α) and the activity of PKC were measured with the enzyme linked immunosorbent assay (ELISA). The mRNA expressions of Nox4, HIF-1α and NF-kB were evaluated by real-time quantitative-polymerase chain reaction (RT-PCR).3. Allicin was added into homocysteine dissolved in PBS(PH7.4) with oil bath at 37℃. Every 30 minutes to 1 hour, the reactant was detected by thin-layer chromatography and iodine coloration. So do the diallyl trithioether and diallyl dithio.4. The primarily cultured mouse aortic endothelial cells were subcultured to passages 3, then randomly divided into five groups:the normal group (NG), the mannitol group (MG), the high glucose/hypoxia group (HG), the allicin group (AG) and the PKC inhibitor (GF109203x) group (GG). The General morphology was observed under the inverted phase contrast microscope. Cell viability was assessed by MTT. Intracellular reactive oxygen species (ROS) production of the endothelial cells was quantified by Dihydroethidium (DHE) staining. The levels of 8-hydroxydeoxyguanosine(8-OHdG), nuclear factor-κB(NF-κB), NADPH oxidase 4(Nox4), hypoxia inducible factor-1α(HIF-1α), and the activity of PKC were measured with enzyme linked immunosorbent assay(ELISA). Real-time quantitative-polymerase chain reaction(RT-PCR)was adopted to evaluate the mRNA expressions of Nox4, HIF-1α and NF-κB.Results1. Allicin decreased the homocysteine level and intracarotid intima-media thickness of diabetic patients with hyperhomocysteinemia.2. In high glucose/homocysteine condition, the morphology of aortic endothelial cells represented an impaired appearance. Treatment with allicin or GF109203x, the appearance was obviously ameliorated. Among the five groups, the cell viability in high glucose/homocysteine condition was the lowest and the levels of 8-OHdG, NF-κB, Nox4, HIF-1α, PKC were highest. Compared with that of high glucose/homocysteine group, the cell viability of allicin group increased obviously and the levels of 8-OHdG, NF-βB, Nox4, HIF-1α, PKC decreased significantly. The level of H2S in allicin group increased obviously compared with that of high glucose/homocysteine group. In high glucose/homocysteine condition, the ROS production increased. There was a noticeable decrease in ROS production when the cells were treated with allicin.3. Allicin had no reaction with homocysteine under the condition of 37℃ and PH7.4.4. The general morphology of aortic endothelial cells was observed an impaired appearance in the high glucose/hypoxia group. In the allicin group and the PKC inhibitor (GF109203x) group, the appearance was obviously ameliorated compared with the high glucose/hypoxia group. The cell activity in the high glucose/hypoxia group was lowest (P<0.05 or P<0.01) of the five groups, but the levels of 8-OHdG, NF-κB, Nox4, HIF-la, PKC, and the mRNA of NF-κB, Nox4, HIF-la were higher (P<0.05 or P<0.01) than those in the other groups. In the allicin group, the cell activity obviously increased compared with that of the high glucose/hypoxia group (P<0.01), and the levels of 8-OHdG, NF-κB, Nox4, HIF-1a, PKC as well as NF-κB, Nox4, HIF-la mRNA also decreased (P<0.05 or P<0.01). The ROS production increased in cells of the high glucose/hypoxia group, and in the allicin group there was a noticeable decrease.Conclusion1. Allicin can decrease the homocysteine level and intracarotid intima-media thickness of diabetic patients with hyperhomocysteinemia.2. Allicin can decrease the injury of aortic endothelial cells induced by high glucose and homocysteine, probably through antioxidation via H2S pathway and inhibition of PKC pathway.3. Allicin has no reaction with homocysteine under the condition of 37℃ and PH7.4.4. Allicin has a protective effect on aortic endothelial cells against injury induced by high glucose/hypoxia through its antioxidation, and the possible mechanism is that allicin can inhibit over-activity of PKC pathway and regulate the hypoxia-related factor, such as HIF-la.