Protective Effects And Mechanism Of Berberine On Vascular Endothelial Dysfunction In Type 2 Diabetic Mellitus

Diabetes mellitus is a serious health threat affecting approxiamtely 5% of the world population. The mortality from cardiovascular complications is almost 3-5 folds higher in the diabetic population than in the general population. Several risk factors in diabetes including hyperglycemia, dyslipidemia, insulin resistance, oxidative stress and hypertension can impair endothelial function and induce serious endothelial dysfunction. Endothelial dysfunction has been known as an initiating, critical factor, and main pathological change during the development of diabetic vascular disease. Accordingly, the prevention of endothelial dysfunction has become the primary target of the treatment of diabetic cardiovascular complications.Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is known as a key factor for maintaining the physiological function of vascular endothelium, especially for regulating the endothelium-dependent relaxations in large conduit arteries. Decreasing of NO production or bioactivity is mainly contributed to endothelial dysfunction of diabetes. eNOS is the key enzyme of NO synthesis in endothelial cell. It has been confirmed that AMPK, PI3K/Akt and PKA are the upstream kinases of eNOS, which can phosphorylate eNOS ser1177 and activate eNOS. Decreasing of eNOS expression and activety and NO production caused by glucose and lipid disorders in diabetes can lead to endothelial dysfunciton. Diabetes not only decreases the production and activity of NO, but also increases the production of O2-. Over-production of superoxide could react with NO to form peroxynitrite (ONOO-), which decreases NO bioavailability, leading to impairment of vasorelaxation. Furthermore, ONOO- can oxidize tetrahydrobiopterin (BH4), an important cofactor for eNOS, and cause uncoupling of eNOS, which produces superoxide instead of NO. Therefore, the over-generation of ROS such as O2- in vessels has been considered as another key factor during the pathogenesis of diabetes-induced endothelial dysfunction. There are a vareity of enzymes involved in ROS generation. NADPH oxidase (NOX) is known as an important enzyme for the production of O2- within the vascular wall. In diabetes, up-regulated NADPH Oxidase activity or expression has been shown to be the main source of O2- production. Therefore, regulating the expression and activity of eNOS and NOX, promoting NO production, reducing NO inactivation and elevating NO bioactivity may become the key target for the prevention of diabetic vascular endothelial dysfunction.Accordingly, therapeutic strategies for vascular complications of diabetes mellitus should be to control blood glucose and simultaneously ameliorate endothelial functions, while the key point of ameliorating endothelial functions is to keep the balance of NO system functions. However, at present, to control blood glucose is still the main method for the prevetion of diabetic cardiovascular diseases, and the lack of available efficient drugs for the treatment of them can still be seen in clinic. Thus, to develop efficient and low-toxic drugs should be considered as the key to prevent diabetes and cardiovascular complications from it.Rhizoma Coptidis (root of Coptis Chinensis from Ranunculaceae) has been used to treat diabetes mellitus for more than 1000 years in China. Berberine, an isoquinoline alkaloid, is one of the main anti-diabetic components of Rhizoma Coptidis. In recent years, numerous animal and clinical studies have demonstrated that berberine could lower blood sugar, and regulate blood lipids, improve insulin resistance, and produce anti-oxidative and antihypertensive effects. More and more reports demonstrated that the underlying mechanism of berberine actions is related to activating AMPK. However, whether berberine attenuates or restores the endothelial dysfunction to prevent the vascular complications in the type 2 diabetes mellitus is still unclear.The present study established experimental type 2 diabetic rat model induced by a high fat diet plus multiple low dosage of streptozotocin (STZ). The blood glucose, triglyceride, total cholesterol and insulin levels in serums from the fasting rats were measured, and IPGTT and ITT were performed. The serum biochemical parameters were tested, and Nitric oxide (NO) levels and SOD activity were tested. The endothelium-dependent aorta vasorelaxation induced by acetylcholine, the endothelium-independent aorta vasorelaxation induced by sodium nitroprusside, and vasocontraction induced by L-phenylephrine were measured for estimating the endothelial function. Hematoxylin and eosin staining was performed for observing the effect of berberine on histopathology in rat aorta. RT-PCR was used to measure the expression of endothelial nitric oxide synthase (eNOS) mRNA. Western blot was used to analyze the protein expressions of eNOS and NADPH oxidase (NOX4). The results showed that the diabetic rats induced by a combination of the high fat diet and the low dose of STZ (30mg/kg, twice) injection exhibited a higher successful rate (70%), and some concomitant clinical characteristics similar to those of type 2 diabetes in humans, such as high blood glucose, high blood lipids, insulin resistance and insufficiency of insulin sectrtion. Berberine significantly decreased fasting blood glucose, total cholesterol, triglyceride levels, increased the insulin sensitivity index and improved the insulin resistance in diabetic rats. Berberine increased serum SOD activity and enhanced anti-oxidant ability of diabetic rats. Berberine also improved endothelium-dependent NO mediated vasorelaxation impaired in diabetic rats'aorta. The expressions of eNOS mRNA and protein were significantly increased, while NOX4 protein expression was decreased in aortas from diabetic rats with berberine treatment. Moreover, serum NO levels were elevated after berberine treatment. Histopathological results showed that berberine attenuated damage of the aortic endothelium in diabetic rats. In conclusion, berberine not only has therapeutic effect on diabetes, but also significantly restores diabetic endothelial dysfunction through enhancing NO bioavailability by up-regulating eNOS expression and down-regulating expression of NADPH oxidase.To further explore the molecular mechanism of berberine on endothelial dysfunction, human umbilical vein endothelial cells (HUVECs) injured model by being exposed to high free fatty acid (palmitate) was developed. HUVECs viability and morphology were measured to observe the protective effect of berberine on HUVEC impaired by palmitate. NO levels in supernatant were tested by using Griss method. To observe whether berberine affects NO levels by regulating eNOS/NO signaling pathway, NG-nitro-L-arginine (L-NA) , an eNOS inhibitor, was used. ROS generations were determined by DCHF-DA fluorescence probe through confocal microcopy. The expression of eNOS mRNA was measured by RT-PCR, and the protein expressions of eNOS, p-eNOS, Akt, p-Akt, AMPK, p-AMPK and NOX4 were analyzed by using western blot. The results demonstrated that berberine slightly elevated the HUVECs viability, restored the cell morphology, and alleviated the HUVECs impairment by being exposed to palmitate. Berberine increased the NO levels in the supernatant and L-NA partially inhibited the action of berberine. Berberine significantly decreased the ROS generation in HUVECs treated with palmitate. We also found that berberine treatment significantly upregulated the expressions of eNOS mRNA and its protein , while markedly downregulated NOX4 protein expression in HUVECs exposed to palmitate. Berberine markedly increased the AMPK and p-AMPK protein expression and had almost no effect on protein expression of Akt and p-Akt. Our data indicate that AMPK activated by berberine contributes to regulating eNOS and NOX4 expression and activity, keeping the balance of NO system function, and protecting HUVECs injured by high FFA.In summary, based on successful development of a type 2 diabetes animal model, the present study demonstrates that berberine not only could improve glucose and lipid metabolism disorders, and insulin resistance in diabetes, but also significantly protect acetylcholine-mediated endothelium-dependent vasorelaxation in the aorta and attenuate the damage of aortic endothelium of diabetic rats. The underlying mechanism can be attributed to increasing NO bioavailability by up-regulating eNOS expression and down-regulating expression of NADPH oxidase. Furthermore, our study shows that berberine alleviated the impairment of HUVECs exposed to palmitate. AMPK activation by berberine might contribute to regulating eNOS and NOX4 expression and activity, keeping the balance of NO system function, and protecting HUVECs injured by high FFA. Therefore, berberine may have beneficial effects on the treatment of type 2 diabetes and its cardiovascular complications. It may provide theoretical basis for the application of berberine to treat type 2 diabetes and cardiovascular complications resulting from it in clinic.