Studies On The Anti-diabetic Mechanisms Of Puerarin

Diabetes mellitus is one of the main threats to human health in the 21st century. Ge-gen (also known as"kudzu") is used in Chinese traditional medicine for the therapy of diabetes for over two thousands years. Puerarin (7– Hydroxy– 3 - ( 4– hydroxyphenyl )– 1– benzopyran– 4– one 8-(β-D-glucopyranoside)) is the main isoflavone glycoside derived from ge-gen. Modern pharmacological researches have demonstrated that puerarin has antihyperglycemic effects and can improve insulin resistance, which has been used to treat diabetes mellitus and its complications. Previous studies about the pharmacological mechanisms of puerarin focused on animal and organ levels. However, the cellular and molecular mechanisms involved in its antidiabetic actions remain unclear.The pancreaticβcell dysfunction is one of the main causes in diabetes etiology, and the oxidative stress under diabetic conditions is the leading cause ofβcell dysfunction. Puerarin has been shown to possess anti-oxidant properties such as scavenging reactive oxygen species, increasing superoxide dismutase (SOD) activity and inhibiting protein nonenzymatic glycation. This study was designed to investigate the protective effect against hydrogen peroxide (H2O2)-induced pancreatic islets damage by puerarin. Exposure of islets to 500μM H2O2 could cause a significant viability loss by MTT assay and an increase in apoptotic rate by flow cytometry and AO/EB double staining. Pre-treatment of islets with puerarin for 48 h resulted in a reduction in viability loss and apoptotic rate. 100μM puerarin significantly inhibited the apoptosis of islets induced by H2O2. In addition, preincubation with puerarin could improve the H2O2-induced decrease in glucose-induced insulin secretion in pancreatic islets by radioimmunoassay. Puerarin was also found to inhibit the free radicals production and the loss of mitochondrial membrane potential induced by H2O2 using DCFH-DA and Rhodamine123 as the fluorescent probes, respectively and to increase catalase (CAT) and superoxide dismutase (SOD) activities by enzyme assays in the isolated pancreatic islets. These results suggest that puerarin can protect islets against oxidative stress probably partially due to its antioxidative activity. Puerarin may be effective in preventing islet cells from the toxic action of reactive oxygen species (ROS) in diabetes.The BK_Ca channel, the large-conductance voltage- and Ca²⁺-activated potassium channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. The dysfunction of BK_Ca channels in diabetes mellitus has been reported to lead to diminished vascular relaxation. Puerarin has positive vasodilation effect and the structure of which is similar to some BK_Ca channel openers. We investigated the direct effects of puerarin on cloned pore forming subunit (α) of BK_Ca channel, with or withoutβ1 subunits and on rat thoracic aortas. BK_Ca channels encoded with eitherα(BK-α) orα/β1 subunits (BK-α+β1) were heterologously expressed in Xenopus oocytes or HEK293 cells. The activities of BK_Ca channels were measured using excised patch-clamp recordings. Puerarin intracellularly activated BK-α+β1 currents with a half-maximal concentration (EC50) of 0.8 nM and a Hill coefficient of 1.11 at 10μM Ca²⁺, and with EC50 of 12.6 nM and a Hill coefficient of 1.08 at 0μM Ca²⁺. Puerarin (1 nM) induced a 16 mV leftward shift in the conductance-voltage curve for BK-α+β1 currents at 10μM Ca²⁺, and at 100 nM induced a 26 mV leftward shift at 0μM Ca²⁺. Puerarin mainly increased the BK-α+β1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of theβ1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BK_Ca channels with weaker potency. In addition, puerarin (0.1 to 1000μM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1μM noradrenaline bitartrate (NA) (EC50 = 1.1μM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BK_Ca channels, indicating that the activation of the BK_Ca channel probably contributes to the puerarin-mediated vasodilation action.Further, we explored the effect of puerarin on mslo currents in the presence of Ca²⁺ and the binding sites of puerarin on BK_Ca channel protein. Puerarin blocked mslo currents with an EC50 of 6.2 nM and a Hill coefficient of 0.86 at 10μM Ca²⁺. Puerarin inhibited mslo currents in a Ca²⁺ and voltage-dependent manners, higher Ca²⁺ concentration, higher inhibition while higher voltage, lower inhibition. The chimera construct with dslo S0-S1 Linker to mslo diminished the effect of puerarin on mslo channels, indicated that the sites for puerarin binding may be located at the region of S0-S1 Linker. In summary, the present study provides evidences that puerarin could protect islet cells from oxidative damage and stimulate BK_Ca channel activity. These beneficial effects of puerarin may contribute to the underlying mechanisms by which it acts as an anti-diabetes compound.