Mohamed Shafiullah

The protective effect of Tribulus terrestris in diabetes

Abstract:  Tribulus terrestris L (TT) is used in the Arabic folk medicine to treat various diseases. The aim of this article was to investigate the protective effects of TT in diabetes mellitus (DM). Diabetes is known to increase reactive oxygen species (ROS) level that subsequently contributes to the pathogenesis of diabetes. Rats were divided into six groups and treated with either saline, glibenclamide (Glib), or TT for 30 days. Rats in group 1 were given saline after the onset of streptozotocin (STZ)‐induced diabetes; the second diabetic group was administered Glib (10 mg/kg body weight). The third diabetic group was treated with the TT extract (2 g/kg body weight), while the first, second, and third nondiabetic groups were treated with saline solution, Glib, and TT extract, respectively. At the end of the experiment, serum and liver samples were collected for biochemical and morphological analysis. Levels of serum alanine aminotransferase (ALT) and creatinine were estimated. In addition, levels of malondialdehyde (MDA) and reduced glutathione (GSH) were assayed in the liver. The tested TT extract significantly decreased the levels of ALT and creatinine in the serum (P < 0.05) in diabetic groups and lowered the MDA level in liver (P < 0.05) in diabetic and (P < 0.01) nondiabetic groups. On the other hand, levels of reduced GSH in liver were significantly increased (P < 0.01) in diabetic rats treated with TT. Histopathological examination revealed significant recovery of liver in herb‐treated rats. This investigation suggests that the protective effect of TT for STZ‐induced diabetic rats may be mediated by inhibiting oxidative stress.

Amelioration of sodium valproate-induced neural tube defects in mouse fetuses by maternal folic acid supplementation during gestation.

ABSTRACT  Infants of epileptic women treated with valproic acid (VPA) during pregnancy have a higher risk of developing spina bifid a than those of the general population. VPA induces exencephaly in experimental animal embryos. But the pathogenetic mechanism remains rather elusive. Antiepileptic drugs (AED) in general accentuate pregnancy‐imposed fall in maternal folate levels. Periconceptional folic acid supplementation is reported to protect embryos from developing neural tube defects (NTD). Conflicting results have been reported by experimental studies that attempted to alleviate VPA‐induced NTD by folic acid. Our objectives were to determine the critical developmental stages and an effective dose of folic acid for the prevention of VPA‐induced exencephaly in mouse fetuses. A single teratogenic dose of 400 mg/kg of VPA was administered to TO mice on gestation day (GD) 7 or 8. It was followed by (1) a single dose of 12 mg/kg of FA (folinic acid) or (2) 3 doses of FA 4 mg/kg each. In experiment (3), FA (4 mg/kg) was administered thrice daily starting on GD 5 and continued through GD 10. These animals received VPA on GD 7 or 8. VPA and B12 concentrations were determined by radioimmunoassay. The single heavy dose of FA had no rescue effect on NTD. Three divided doses of FA on GD 7 and continuous dosing of FA from GD 5 through GD 10 substantially reduced the VPA‐induced exencephaly in the fetuses. In the later experiments, the neural folds elevated faster than the non‐supplemented group. VPA considerably reduced maternal plasma folate and B12 concentrations. The heavy dose of FA only moderately improved vitamin levels. Three divided doses of FA elevated the vitamin levels slightly better but it was the prolonged dosing of FA that was associated with sustained elevation of plasma levels higher than the control levels and acceleration of neural tube closure thus accounting for the pronounced protection against VPA‐induced NTD development. These data suggest that plasma levels of FA and B12 have to be kept substantially elevated and maintained high throughout organogenesis period to protect embryos against VPA‐induced NTD in this mouse model.

Long‐term effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki rat

In vivo biotelemetry studies have demonstrated a variety of heart rhythm disturbances in type 1 diabetes mellitus. In the streptozotocin (STZ)‐induced diabetic rat, these disturbances have included reductions in heart rate (HR) and heart rate variability (HRV) and an electrocardiogram that displays prolonged QRS duration and Q–T interval. The aim of this study was to investigate the chronic effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki (GK) rat. Transmitter devices were surgically implanted in the peritoneal cavity of young male GK and age‐matched Wistar control rats. Electrodes from the transmitter were arranged in Einthoven bipolar lead II configuration. Electrocardiogram, physical activity and body temperature data were recorded in rats from age 2 to 15 months. Data were acquired for 2 weeks each month. Non‐fasting blood glucose, glucose tolerance and body weight were measured periodically. In GK rats, growth rate and maximal attained body weight were significantly reduced and non‐fasting blood glucose was progressively increased compared with age‐matched Wistar control animals. Heart rate was significantly lower in GK compared with control rats at 2, 7 and 15 months of age. At 2 months of age, HR was 316 ± 6 beats min−1 in GK rats compared with 370 ± 7 beats min−1 in Wistar control animals. There was a progressive age‐dependent decline in HRV in Wistar control rats; however, HRV in GK rats did not alter significantly with age. Heart rate variability was significantly reduced in GK compared with Wistar control rats at 2 and 7 months. At 2 months of age, HRV was 28 ± 2 beats min−1 in GK rats compared with 38 ± 3 beats min−1 in Wistar control rats. Reduced HR in GK rats may be an inherited characteristic. The absence of age‐dependent reductions in HRV in GK rats may be a consequence of an underlying impairment of autonomic control which manifests at early age.

Chronic effects of type 2 diabetes mellitus on cardiac muscle contraction in the Goto‐Kakizaki rat

Type 2 diabetes mellitus accounts for more than 90% of all cases of diabetes mellitus, and cardiovascular complications are the major cause of mortality and death in diabetic patients. The chronic effects of type 2 diabetes mellitus on heart function have been investigated in the Goto‐Kakizaki (GK) rat. Experiments were performed in GK rats and age‐matched Wistar control rats at 18 months of age. The progressive effects of diabetes on glucose metabolism were monitored periodically by application of the glucose tolerance test. Ventricular action potentials were measured in isolated, perfused heart. Shortening and intracellular Ca2+ were measured in electrically stimulated ventricular myocytes. The GK rats displayed mild fasting hyperglycaemia and progressively worsening glucose tolerance. At 18 months of age and 180 min after intraperitoneal injection of glucose (2 g (kg body weight)−1), blood glucose was 436 ± 47 mg dl−1 in GK rats compared with 153 ± 18 mg dl−1 in control animals. Heart weight to body weight ratio was significantly increased in GK rats (4.10 ± 0.09 mg g−1, n= 5) compared with control animals (3.36 ± 0.22 mg g−1, n= 4). Spontaneous heart rate was slightly reduced in GK rats compared with control rats. Although the amplitude of shortening was not altered, the amplitude of the Ca2+ transient was significantly increased in myocytes from GK rats (0.78 ± 0.11 ratio units) compared with control rats (0.50 ± 0.06 ratio units). Despite progressively worsening glucose metabolism, at 18 months of age the contractile function of the heart appears to be well preserved.

Ultra-structural morphological abnormalities of the urinary bladder in streptozotocin-induced diabetic female rats

The objective of this study was to evaluate the ultra-structural changes in the urinary bladder of diabetic rats in relation to disease duration since the morphological bases of diabetes-induced bladder dysfunction are poorly understood. Urinary bladders were examined chronologically by electron microscopy in a female Wistar-rat model of streptozotocin-induced diabetes mellitus and compared to control samples. Numerous dark mitochondria with swollen cristae and electron lucent, large, calcified and degenerated mitochondria were observed first in the urothelium. Intraepithelial capillaries surrounded by thick collagen were also present. Gap junctions between myocytes were interrupted or extensively widened with reduced mitochondria and caveolae. Collagen accumulation, degenerated nerve fibres and myelin bodies were seen between myocytes with increased collagen content and frequent mast cells, phagocytes and lymphocyte aggregates in the stroma. All ultra-structural lesions became augmented with longer duration of diabetes. Diabetes induces time-dependent pathologic changes in the urinary bladder of rats that might account for bladder dysfunction.

Effects of ovariectomy and hormone replacement on collagen and blood vessels of the urethral submucosa of rats

Collagen and blood vessels of the urethral submucosa of ovariectomized rats were studied following 28 daily subcutaneous injections of 17-ß estradiol (n=6, group 1), medroxy-progesterone acetate (n=6, group 2), both drugs (n=6, group 3) or vehicle (n=6, control) and after sham surgery without castration or injection (n=6). Investigations included the immunohistochemistry of estrogen and progesterone receptors and collagen fibres, Western blot analysis of collagen types I and III and counting periurethral vessels by light microscopy. Our results showed positive immunostaining with estrogen, progesterone and collagen types I and III in all samples. Collagen type I and III levels were lower in the controls than in the sham group. The other groups showed increases (2>3>1) over the controls with a relatively higher increase in type III. The type I/III collagen ratio progressively decreased (control>1>2>3) below sham levels. The mean vessel count was significantly lower in control than in sham animals (P<0.00001). However, only estrogen treatment significantly increased the vessel number compared to controls (P=0.04). Our results indicate that estrogen and progesterone, alone or in combination, have an effect on collagen types I and III, and that estrogen has an effect on blood vessels of the urethral submucosa in female rats.

Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol

Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. Therefore, the previously described and structurally strongly related imidazole-based derivatives belonging to carbamate class with high H3R in vitro affinity, in-vivo antagonist potency, and H3R selectivity profile were investigated on their anticonvulsant activity in maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in Wistar rats. The effects of systemic injection of H3R ligands 1-13 on MES-induced and PTZ-kindled seizures were screened and evaluated against the reference antiepileptic drug (AED) Phenytoin (PHT) and the standard histamine H3R inverse agonist/antagonist Thioperamide (THP) to determine their potential as new antiepileptic drugs. Following administration of the H3R ligands 1-13 (5, 10 and 15 mg/kg, ip) there was a significant dose dependent reduction in MES-induced seizure duration. The protective action observed for the pentenyl carbamate derivative 4, the most protective H3R ligand among 1-13, was significantly higher (P <0.05) than that of standard H3R antagonist THP, and was reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine (RAMH) (10mg/kg), or with the CNS penetrant H1R antagonist Pyrilamine (PYR) (10mg/kg). In addition, subeffective dose of H3R ligand 4 (5mg/kg, ip) significantly potentiated the protective action in rats pretreated with PHT (5mg/kg, ip), a dose without appreciable protective effect when given alone. In contrast, pretreatment with H3R ligand 4 (10mg/kg ip) failed to modify PTZ-kindled convulsion, whereas the reference drug PHT was found to fully protect PTZ-induced seizure. These results indicate that some of the investigated imidazole-based H3R ligands 1-13 may be of future therapeutic value in epilepsy.

Pretreatment for acute exposure to diisopropylfluorophosphate: in vivo efficacy of various acetylcholinesterase inhibitors

Prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors before exposure to organophosphorus compounds (OPCs) can reduce OPC‐induced mortality. Pyridostigmine is the only FDA‐approved substance for such use. The AChE‐inhibitory activity of known AChE inhibitors was quantified in vitro and their in vivo mortality‐reducing efficacy was compared, when given prophylactically before the exposure to the OPC diisopropylfluorophosphate (DFP). The IC50 was measured in vitro for the known AChE inhibitors pyridostigmine, physostigmine, ranitidine, tiapride, tacrine, 7‐methoxytacrine, amiloride, metoclopramide, methylene blue and the experimental oxime K‐27. Their in vivo efficacy, when given as pretreatment, to protect rats from DFP‐induced mortality was quantified by determining the relative risk of death (RR) by Cox analysis, with RR = 1 for animals given only DFP, but no pretreatment. Physostigmine was the strongest in vitro AChE‐inhibitor (IC50 = 0.012 µ m), followed by 7‐methoxytacrine, tacrine, pyridostigmine and methylene blue. Ranitidine (IC50 = 2.5 µ m), metoclopramide and amiloride were in the mid‐range. Tiapride (IC50 = 256 µ m) and K‐27 (IC50 = 414 µ m) only weakly inhibited RBC AChE activity. Best in vivo protection from DFP‐induced mortality was achieved when physostigmine (RR = 0.02) or tacrine (RR = 0.05) was given before DFP exposure, which was significantly superior to the pretreatment with all other tested compounds, except K‐27 (RR = 0.18). The mortality‐reducing effect of pyridostigmine, ranitidine and 7‐methoxytacrine was inferior, but still significant. Tiapride, methylene blue, metoclopramide and amiloride did not significantly improve DFP‐induced mortality. K‐27 may be a more efficacious alternative to pyridostigmine, when passage into the brain precludes administration of physostigmine or tacrine. Copyright

Usefulness of administration of non‐organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using paraoxon

Reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of irreversible organophosphorus AChE inhibitors (OPCs), when administered before OPC exposure. We have assessed in vivo the mortality‐reducing efficacy of a group of known AChE inhibitors, when given in equitoxic dosage before exposure to the OPC paraoxon. Protection was quantified in rats by determining the relative risk (RR) of death.

Acetylcholinesterase inhibitors as pretreatment before acute exposure to organophosphates: assessment using methyl-paraoxon.

Prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of organophosphorus compounds (OPCs). The usefulness of pyridostigmine, the only compound approved by the Food and Drug Administration (FDA) for such pretreatment, has been questioned. In search for more efficacious alternatives, we have examined in vivo the efficacy of a group of ten compounds with known anti-AChE activity (pyridostigmine, metoclopramide, tiapride, ranitidine, physostigmine, tacrine, amiloride, methylene blue, 7- methoxytacrine and K-27) to reduce mortality induced by the OPC methyl-paraoxon. AChE inhibitors were given intraperitoneally in equitoxic dosage (25% of LD₀₁) 30 min before OPC exposure. Protection was quantified in rats by determining the relative risk of death (RR) by Cox analysis, with RR=1 for animals given only methyl-paraoxon, but no pretreatment. Only physostigmine (RR=0.39), K-27 (RR=0.40) and tacrine (RR=0.48) significantly (p≤ 0.05) reduced methylparaoxon- induced mortality, when given prophylactically. Pretreatment with pyridostigmine, ranitidine, tiapride, amiloride, metoclopramide and methylene blue did not significantly protect against the lethal effects of methyl-paraoxon. 7-methoxytacrine (7-MEOTA) significantly (p≤ 0.05) increased the relative risk of methyl-paraoxon-induced death (RR=1.31). These results indicate that pretreatment with pyridostigmine cannot be considered a broad-spectrum approach against OPC exposure. K-27 may be a suitable alternative if passage into the brain is contraindicated.