Juei Tang Cheng

Changes of superoxide dismutase gene expression and activity in the brain of streptozotocin-induced diabetic rats

In an attempt to discover the changes of superoxide dismutase (SOD) in the brain of diabetic state, we investigated the level of SOD in streptozotocin-induced diabetic rats (STZ-diabetic rats) using enzyme activity assay, Northern blotting analysis of mRNA levels and Western blotting of enzyme amount. Five discrete brain regions, cerebrocortex, hypothalamus, hippocampus, the remaining non-cortex cerebrum (NCC area) and cerebellum, were examined in STZ-diabetic rats to compare with age-matched normal rats. Higher levels of Mn-SOD including the activity, mRNA expression and immunoblot of enzyme were found in all areas of the brain from STZ-diabetic rats as compared with that in Wistar rats. Except in the cerebellum, similar changes of Cu,Zn-SOD were found in the brain of STZ-diabetic rats. These results indicate an increase of SOD both gene expression and activity in the brain of STZ-diabetic rats. This alteration of SOD may be one of the important factors for the vulnerability of the brain to oxygen free radicals or may be related to the pathophysiology of diabetes.

Mechanism of the antihypertensive effect of stevioside in anesthetized dogs

Stevioside is a sweet-tasting glycoside isolated from the leaves of Stevia rebaudiana. It has been used as a noncaloric sugar substitute in Japan and Brazil for decades. Previous studies have shown that it lowered blood pressure in spontaneously hypertensive rats by intravenous injection. This study was designed to evaluate the hypotensive effect of stevioside in dogs and to define the underlying mechanism. After nasogastric administration of stevioside powder (200 mg/kg), the blood pressure of healthy mongrel dogs began to significantly decrease at 60 min and returned to baseline level at 180 min. The reduction of blood pressure was more rapid (at 5–10 min) and effective after intravenous injection. However, no significant change of blood pressure was noted after injection through left vertebral artery, implicating that the hypotensive effect is not related to the central nervous system. Stevioside also showed significant hypotensive effects in renal hypertensive dogs, in a dose-dependent manner. In cultured rat aortic smooth muscle cells (A7r5 cell line), stevioside can dose-dependently inhibit the stimulatory effects of vasopressin and phenylephrine on intracellular Ca2+ in a calcium-containing medium. However, no intracellular Ca2+ inhibitory effect was observed in calcium-free medium, implicating that stevioside may inhibit the Ca2+ influx from extracellular fluid. Our present data show that stevioside did not influence the calcium ionophore (A23187) induced Ca2+ influx, indicating that the antagonistic effect was through Ca2+ channels. This study confirmed that stevioside is an effective antihypertensive natural product, and its hypotensive mechanism may be probably due to inhibition of the Ca2+ influx.

Involvement of serotonin in the hypoglycemic response to 2 Hz electroacupuncture of zusanli acupoint (ST36) in rats

In our previous studies, an insulin-dependent hypoglycemic effect produced by electroacupuncture (EA) was shown to be mediated by endogenous opioid peptides (EOP). In the present study, we applied 2 Hz EA to both zusanli acupoints (ST36) in the test group for 30 min, and to a nonacupoint area in the control group for 30 min to compare the acupoint specific character in the hypoglycemic effect of EA. Assays of plasma beta-endorphin and insulin levels were performed by ELISA kits. The insulin-dependent mechanism of the hypoglycemic effect was also investigated in streptozotocin (STZ)-induced diabetic rats. The mediation of EOP and the role of mu-opioid receptor were examined by naloxone and mu-opioid receptor knockout mice (MOR-KOM). The serotonin depletion was carried out by injecting (i.p.) p-chlorophenylalanine (PCPA); two low doses of serotonin were also injected (i.v.) to analyze the direct effect on plasma glucose levels. The hypoglycemic effect of EA was much greater in rats stimulated at ST36 than in rats receiving the same stimulation at the nonacupoint area. The plasma levels of insulin and beta-endorphin were also significantly elevated after stimulation of both zusanli acupoints, but remained unchanged following stimulation at the nonacupoint area. There was no sharp hypoglycemic response to 2 Hz EA at zusanli acupoint of STZ-induced diabetic rats. However, the hypoglycemic effect of this EA was not totally blocked by the sufficient dose of naloxone (1 mg/kg, i.v.). Additionally, 2 Hz EA at ST36 also showed a sharp decrease in plasma glucose levels of MOR-KOM. Pretreatment with PCPA did not reproduce hypoglycemic response to 2 Hz EA in naloxone-treated rats and MOR-KOM mice. Furthermore, injection of serotonin decreased the plasma glucose levels significantly. Therefore, we suggest that serotonin also involved in the hypoglycemic action of 2 Hz EA at both zusanli acupoints of normal rats.

Activation of opioid μ-receptor by loperamide to lower plasma glucose in streptozotocin-induced diabetic rats

We investigated the effect of loperamide, a selective agonist of opioid μ-receptor, on the plasma glucose in diabetic rats induced by an intravenous injection of streptozotocin (STZ; 60 mg/kg). Intravenous injection of loperamide induced a dose-dependent decrease of plasma glucose in fasting STZ-diabetic rats at 30 min later, but did not modify the plasma glucose level in Wistar rats. Plasma glucose lowering effect of loperamide was abolished by the pretreatment with naloxone or naloxonazine at the dose sufficient to block opioid μ-receptor. In isolated skeletal muscle, loperamide enhanced the glucose uptake into soleus muscles in a concentration-dependent manner. Blockade of this action by naloxonazine indicated the mediation of opioid μ-receptor. These results suggest that an activation of opioid μ-receptor by loperamide can increase the utilization of glucose in peripheral tissue to lower the plasma glucose in STZ-diabetic rats.

Alterations of M2-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder

Diabetes associated alterations of M2-muscarinic receptors (M2-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M2-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M2-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M2-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M2-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.66; that for mRNA was 1:0.97. Two weeks after induction of diabetes, the M2-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 44.4% (P<0.01, n=8) and 28.6% (P<0.01, n=8), respectively. Correspondently, the bladder M2-mAChR protein levels were significantly increased by 33.3% (P<0.001, n=8) in the urothelium and 25.3% (P<0.01, n=8) in the muscle layer of the diabetic rats. In conclusion, M2-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M2-mAChR in the urothelium as well as the muscle layer.

Effect of trilinolein on the activity and gene expression of superoxide dismutase in cultured rat brain astrocytes

Stroke is one of the major causes of morbidity and mortality in recent. Oxygen free radicals produced during cerebral infarction increases the damage to neurons. Superoxide dismutase (SOD) is the endogenous antioxidant enzyme that can effectively scavenge superoxide radicals. Catechin is a hydrophilic antioxidant usually existed in tea, fruits and vegetables. In the cultured rat brain astrocytes (RBA), the activity of SOD (both Cu, Zn-SOD and Mn-SOD subtypes) was markedly increased by incubation with catechin at low concentration (0.1 microM) for 2 days (short-term) and 7 days (long-term). This stimulatory effect of catechin was not related to the incubating concentration. Similar changes were also observed in the gene expression of SOD in RBA. The increase in quantity of SOD-messenger RNA was remarkable and seemed to be more obvious than the other antioxidants such as vitamin E. This result shows that catechin is an effective antioxidant to increase the activity of SOD in RBA which would be beneficial to neurons subjected to oxygen free radical damage.

Alteration of M(3) subtype muscarinic receptors in the diabetic rat urinary bladder.

The M3 receptor (M3-mAChR) is the major muscarinic subtype in the animal bladder responsible for detrusor contraction. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. 3-month-old male Wistar rats were divided into two groups: (1) 2-week diabetic rats and (2) normoglycemic control rats. Diabetes was induced by a single intravenous injection of 60 mg/kg streptozotocin. The amount of M3 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M3 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M3-mAChR protein in the diabetic bladder was significantly increased by about 70.2 ± 8.5% when compared to the control bladder (p < 0.05, n = 8). Northern blotting demonstrated a 54.7 ± 6.0% increase of M3-mAChR mRNA in the diabetic bladder (p < 0.05, n = 8). The findings of the present study demonstrated an upregulation of M3-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon offers an explanation of the increased contractility after muscarinic stimulation of the detrusor muscle of diabetic animals.

Pumpkin seed oil and phytosterol-F can block testosterone/prazosin-induced prostate growth in rats.

Introduction: This study was undertaken to investigate the effects of pumpkin seed oil alone or combined with Phytosterol-F on testosterone/prazosin-induced (T-P) prostate growth in rats. Materials and Methods: Forty adult Wistar rats were divided into five groups, including: one control group, rats treated with vehicle only, one group treated with T-P, and two groups of T-P-treated rats, one receiving orally pumpkin seed oil alone and one group receiving orally pumpkin seed oil combined with Phytosterol-F. Two weeks later, the prostatic weight-to-body weight ratio was determined after sacrifice. The total protein concentration was measured by using a protein assay. Some ventral prostatic tissues were histologically examined after hematoxylin-eosin staining. Results: Histological sections of the ventral prostate showed that the architecture of the prostate glands became hyperplastic in the T-P rats, but not in the control or vehicle-treated animals. As compared with the control or vehicle group, T-P rats had a significantly higher prostatic weight-to-body weight ratio for the ventral prostate (p = 0.05 and p = 0.007, respectively), but not for the dorsolateral prostate (p = 0.53 and p = 0.73, respectively). The T-P rats had significantly higher protein levels within both lobes (ventral lobe, p = 0.02 and p < 0.0001, respectively; dorsolateral lobe, p = 0.06 and p = 0.005, respectively). As compared with the T-P-alone rats, the TP rats treated with pumpkin seed oil alone or pumpkin seed oil combined with Phytosterol-F had a significantly lower weight ratio for the ventral prostate (p = 0.01 and p = 0.004, respectively) and significantly lower protein levels within both lobes (p = 0.03 and p = 0.003, respectively; p = 0.007 and p = 0.002, respectively). In addition, Phytosterol-F had some additive effect on the total protein synthesis within the ventral prostate (p = 0.02). Conclusion: Pumpkin seed oil alone or combined with Phytosterol-F can block the T-P-induced increases in prostatic weight-to-body weight ratio and protein synthesis.

Mediation of β-endorphin in andrographolide-induced plasma glucose-lowering action in type I diabetes-like animals

In the present study, we investigated the mechanism(s) for glucose-lowering action of andrographolide in streptozotocin-induced diabetic rats (STZ-diabetic rats). Andrographolide lowered plasma glucose concentrations in a dose-dependent manner and increased plasma β-endorphin-like immunoreactivity (BER) dose-dependently in diabetic rats. Both of these responses to andrographolide were abolished by the pretreatment of animals with prazosin or N-(2 -(2-cyclopropylmethoxy) ethyl) 5-choro-α-dimethyl-1H-indole-3-thylamine (RS17053) at doses sufficient to block α1-adrenoceptors (ARs). Also, andrographolide enhanced BER release from isolated rat adrenal medulla in a concentration-related manner that could be abolished by α1-ARs antagonists. Bilateral adrenalectomy in STZ-diabetic rats eliminated the activities of andrographolide, including the plasma glucose-lowering effect and the plasma BER-elevating effect. Andrographolide failed to lower plasma glucose in the presence of opioid μ-receptor antagonists and in the opioid μ-receptor knockout diabetic mice. Treatment of STZ-diabetic rats with andrographolide resulted in the reduced expression of phosphoenolpyruvate carboxykinase (PEPCK) in liver and an increased expression of the glucose transporter subtype 4 (GLUT 4) in soleus muscle. These effects were also blocked by opioid μ-receptor antagonists. In conclusion, our results suggest that andrographolide may activate α1-ARs to enhance the secretion of β-endorphin which can stimulate the opioid μ-receptors to reduce hepatic gluconeogenesis and to enhance the glucose uptake in soleus muscle, resulting in a decrease of plasma glucose in STZ-diabetic rats. However, the roles of other endogenous opioid peptides or the mixture of several opioid peptides in the activation of opioid μ-receptors associated with the plasma glucose-lowering action of andrographolide, should be considered and need more investigation in the future.

Changes of M3-muscarinic receptor protein and mRNA expressions in the bladder urothelium and muscle layer of streptozotocin-induced diabetic rats.

Diabetes induced alterations of M3-muscarinic receptors (M3-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M3-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M3-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M3-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M3-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.87; that for mRNA was 1:0.74. Two weeks after induction of diabetes, the M3-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 45.7% (P<0.01, n=8) and 80.8% (P<0.001, n=8), respectively. Correspondingly, the bladder M3-mAChR protein levels were significantly increased by 50.0% (P<0.01, n=8) in the urothelium and 147.1% (P<0.001, n=8) in the muscle layer of the diabetic rats. In conclusion, M3-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M3-mAChR in the urothelium as well as the muscle layer.