Effects And Mechanisms Of2,3,5,4'-Tetrahydroxystilbene-2-O-beta-D-glucoside On Gastrointestinal Motility Disorders In STZ-induced Diabetic Mice

Part Ⅰ2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside improves gastrointestinal motility disorders in STZ-induced diabetic miceBackgroud:Gastrointestinal (GI) motility disorders are very common in diabetic patients. Most of diabetic patients suffer from associated symptoms such as reflux, early satiety, nausea, abdominal pain, diarrhea or constipation. The etiology of altered GI functions in diabetes is multifactorial and the mechanisms involving oxidative stress, apoptosis, neuronal loss, and advanced glycation end products are elaborated. Diabetes mellitus (DM) manifests a state of high oxidative stress due to hyperglycemia-induced ROS generation. ROS as important second messengers at low concentrations are involved in regulating apoptosis and activation of transcription factors such as nuclear factor kappa B (NF-KB), whereas they can cause significant cellular damage when present in excess. More recently, oxidative stress has been considered to play an important role in GI complications of diabetes. Previous studies indicated that increased oxidative stress may contribute to apoptosis and the neuronal degeneration in diabetes. Therefore, antioxidants have therapeutic potentials for the treatment of diabetic GI motility problems.2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG) is one of the active components extracted from the traditional Chinese herb Polygonum multiflorum, which has a strong anti-oxidant property. Therefore, we investigated the effect of THSG on GI problems in STZ-induced diabetic mice as well as the underlying protective mechanisms. Methods:Diabetes was induced in male Kunming mice by a single intraperitonial injection of150mg/kg streptozotocin (STZ) whereas normal control mice were injected with an equal volume of buffer. Mice with fasting blood glucose levels exceeding16.7mmol/L were considered hyperglycemic. Diabetic mice were randomly divided into four groups:(1) untreated diabetic mice;(2) diabetic mice treated with THSG at three different doses (10,30,60mg/kg). THSG at Different doses was administrated to diabetic mice by oral gavage daily for successive8weeks beginning from day7th after STZ injection. Gastric emptying and intestinal transit were determined by a method using Evans blue. Malondialdehyde (MDA) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels were analysized by biochemical methods. Apoptosis was assessed by TUNEL assay. The protein levels of cleaved caspase-3, ERK1/2(both total and phosphorylated), PPAR-γ and SIRT1were measured by western blot analysis.Results:Blood glucose levels of STZ-induced diabetic mice were significantly higher than that of control mice, which was not affected by THSG treatment. The weight of diabetic mice after8weeks was significantly lower than that of control mice, which was partially ameliorated by THSG. The percentage of gastric emptying in STZ-induced diabetic mouse was less than that of control animal. The rate of intestinal transit was significantly increased in diabetic group compared with controls. These alterations in gastric emptying and intestinal transit manifest the occurrence of GI dysmotility in diabetic mice. THSG restored delayed gastric emptying and increased intestinal transit in diabetic mice. A significant increase in MDA level confirms the occurrence of oxidative damage in diabetic ilea and this was dose-dependently reduced by THSG THSG (60mg/kg) reduced MDA level to1.36±0.11nmol/mg protein, which was no significant difference from that of normal control (1.14±0.04nmol/mg protein). Further, THSG reversed the decrease in the activity of GSH-Px during diabetes in a dose-dependent manner from359.98±25.37to848.70±56.85U/mg protein. Compared with controls, SOD activity of THSG-treated group increased in a dose-dependent manner, but it was lower than lower than that of diabetic group. Apoptosis can contribute to the enteric neuronal loss and disorders in GI motility in diabetes. Compared with control group, the number of TUNEL-positive cells significantly increased in diabetic group (49.6±1.1%). Treatment with60mg/kg THSG could significantly reduce the number of TUNEL-positive cells in diabetic mice with the proportion of23.8±1.4%. Caspase-3has been identified as a key mediator in apoptosis. To examine the molecular mechanism of THSG against apoptosis in diabetic ilea, the protein levels of cleaved caspase-3, ERK1/2(both total and phosphorylated), PPAR-y and SIRT1were measured in ileal tissues. Quality of cleaved caspase-3protein expression significantly increased in the diabetic ilea compared with controls, which was attenuated by THSG Furthermore, THSG elevated the p-ERK/total ERK ratio in diabetic intestine, indicating decreased MAPK pathway in diabetes contributes to GI apoptosis and this can be reversed by antioxidant THSG Finally, diabetes also leads to reductions in PPAR-y and SIRT1protein levels, and that was prevented by THSGConclusion:THSG restored delayed gastric emptying and increased intestinal transit in STZ-induced diabetic mice, suggesting the protective effect of THSG on diabetic GI dysmotilities. In addition, THSG dose-dependently ameliorated weight loss in STZ-induced diabetic mice, indicating THSG may promote GI function. Diabetic mice displayed a significant increase in Malondialdehyde (MDA) level and decrease in the activity of glutathione peroxidase (GSH-Px), which were ameliorated by THSG Inhibition of caspase-3and activation of ERK phosphorylation related MAPK pathway were involved in prevention of enhanced apoptosis in diabetes afforded by THSG Moreover, THSG upregulated a significant decrease in PPAR-y and SIRT1expression in diabetic ilea. Our study indicates that THSG improved diabetic gastrointestinal disorders through activation of MAPK pathway and upregulation of PPAR-y and SIRT1. Part Ⅱ2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside restores nitric oxide synthase expression and function that is lost in diabetic intestineBackgroud:The nitregic neurons have been studied to a large extent in diabetes-related GI dysmotility. The nitric oxide (NO) produced by these neurons, as a major NANC inhibitory neurotransmitter, mediates the smooth muscle relaxation in the GI tract. Hence, it is important in gut motility. Accumulative studies have showed that decreased nNOS neurons and impaired NO-mediated NANC relaxation in diabetic gastroenteropathy. Increased apoptosis appears to be responsible for lack of nNOS neurons. THSG is one of the active components extracted from the traditional Chinese herb Polygonum multiflorum, which has been widely used as a tonic, lubricating intestine and anti-aging agent since ancient times. It has been demonstrated that THSG has strong anti-oxidant, anti-apoptosis and neuron protective properties. Thereby, all of these studies suggest that THSG may have the protective effects on nNOS expression and function that is lost in diabetic intestine. In this study, we investigated the effect of THSG on intestinal nitregic neurons in STZ-induced diabetic mice.Methods:Diabetes was induced in male Kunming mice by a single intraperitonial injection of150mg/kg streptozotocin (STZ) whereas normal control mice were injected with an equal volume of buffer. Mice with fasting blood glucose levels exceeding16.7mmol/L were considered hyperglycemic. Diabetic mice were randomly divided into two groups:(1) untreated diabetic mice;(2) diabetic mice treated with THSG (60mg/kg). THSG was administrated to diabetic mice by oral gavage daily for successive8weeks beginning from day7th after STZ injection. Neuronal nitric oxide synthase (nNOS) expression was estimated by immunohistochemistry. Nonadrenergic, noncholinergic (NANC) relaxation of isolated colon was induced by electrical field stimulation (EFS).Results:Compared with control group, nNOS-positive area in diabetic group was remarkably reduced. However, nNOS-positive area was increased in THSG-treated diabetic mice compared with diabetic mice. In the long-term preventive study, EFS-induced NANC relaxation was assessed in control, diabetic and THSG-treated diabetic mouse. EFS-induced NANC relaxation was significantly reduced in diabetic group compared with controls, which was partially restored by long-term (8-week) THSG treatment. In order to further investigate the effect of THSG on colonic strips isolated from control and diabetic mouse, NANC relaxation induced by EFS were recorded after pre-incubation with different concentrations of THSG (10-7～10-4mol/L) in the presence of atropine, propanolol and indomethacin. THSG enhanced NANC relaxation of diabetic colon in a dose-dependent manner but had no obvious influence on that of control colon.Conlusion:In our study, reduced NANC relaxation and loss of nNOS expression in diabetes are relieved by THSG treatment for8weeks, indicating that restorative effect of THSG is probably associated with nNOS neuronal protection. Furthermore, THSG(10-7～10-4mol/L) could also enhance NANC relaxation of diabetic colon in a concentration-dependent manner, suggesting that THSG may enhance NANC relaxation in diabetic mice.