Shrinivas K. Kulkarni

CURCUMIN, THE ACTIVE PRINCIPLE OF TURMERIC (CURCUMA LONGA), AMELIORATES DIABETIC NEPHROPATHY IN RATS

1 Chronic hyperglycaemia in diabetes leads to the overproduction of free radicals and evidence is increasing that these contribute to the development of diabetic nephropathy. Among the spices, turmeric (Curcuma longa) is used as a flavouring and colouring agent in the indian diet every day and is known to possess anti‐oxidant properties. The present study was designed to examine the effect of curcumin, a yellow pigment of turmeric, on renal function and oxidative stress in streptozotocin (STZ)‐induced diabetic rats. 2 Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg) in rats. Four weeks after STZ injection, rats were divided into four groups, namely control rats, diabetic rats and diabetic rats treated with curcumin (15 and 30 mg/kg, p.o.) for 2 weeks. Renal function was assessed by creatinine, blood urea nitrogen, creatinine and urea clearance and urine albumin excretion. Oxidative stress was measured by renal malonaldehyde, reduced glutathione and the anti‐oxidant enzymes superoxide dismutase and catalase. 3 Streptozotocin‐injected rats showed significant increases in blood glucose, polyuria and a decrease in bodyweight compared with age‐matched control rats. After 6 weeks, diabetic rats also exhibited renal dysfunction, as evidenced by reduced creatinine and urea clearance and proteinuria, along with a marked increase in oxidative stress, as determined by lipid peroxidation and activities of key anti‐oxidant enzymes. Chronic treatment with curcumin significantly attenuated both renal dysfunction and oxidative stress in diabetic rats. 4 These results provide confirmatory evidence of oxidative stress in diabetic nephropathy and point towards the possible anti‐oxidative mechanism being responsible for the nephroprotective action of curcumin.

Possible role of nitric oxide in the nootropic and antiamnesic effects of neurosteroids on aging- and dizocilpine-induced learning impairment

The ability of the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), to modulate the attenuating effects of neurosteroids on the aging- and NMDA receptor antagonist dizocilpine-induced learning impairment, was tested in mice using two different behavioral models of long-term memory. The performance of aged mice (16 months old) in step-down type of passive-avoidance and elevated plus-maze paradigms was significantly impaired compared to that of young mice (3 months old). Neurosteroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS), at 1-20 mg/kg, s.c., significantly improved the passive-avoidance and plus-maze performances in aged mice. Neurosteroids PS and DHEAS, at doses 1-20 mg/kg, s.c., significantly attenuated dizocilpine (0.1 mg/kg, i.p.)-induced amnesia, without producing any promnestic effects alone in adult mice. In both cognitive tasks, the effects exhibited by the neurosteroids tested had a bell-shaped curve. Preadministration of L-NAME (10 and 20 mg/kg, i.p.), at doses that did not disrupt cognition alone in either young or aged mice, significantly blocked the beneficial and antiamnesic effects of neurosteroids PS (5 mg/kg) and DHEAS (10 mg/kg). A selective action of L-NAME on the effects of neurosteroids was indicated, since the effects of L-NAME were completely reversed by L-arginine (300 mg/kg, i.p.), a competitive substrate for NO synthase. Neither L-NAME nor L-arginine alone affected the antinociception, locomotor activity or rota-rod performance of young or aged mice. These observations suggest that a NO-dependent mechanism may be involved in the beneficial and antiamnesic effects of neurosteroids PS and DHEAS on the aging- and dizocilpine-induced impairment of learning and memory processes.

Anti-depressant like effect of curcumin and its combination with piperine in unpredictable chronic stress-induced behavioral, biochemical and neurochemical changes

Curcumin, a yellow pigment extracted from rhizomes of the plant Curcuma longa (turmeric), has been widely used as food additive and also as a herbal medicine throughout Asia. The present study was designed to study the pharmacological, biochemical and neurochemical effects of daily administration of curcumin to rats subjected to chronic unpredictable stress. Curcumin treatment (20 and 40 mg/kg, i.p., 21 days) significantly reversed the chronic unpredictable stress-induced behavioral (increase immobility period), biochemical (increase monoamine oxidase activity) and neurochemical (depletion of brain monoamine levels) alterations. The combination of piperine (2.5 mg/kg, i.p., 21 days), a bioavailability enhancer, with curcumin (20 and 40 mg/kg, i.p., 21 days) showed significant potentiation of its anti-immobility, neurotransmitter enhancing (serotonin and dopamine) and monoamine oxidase inhibitory (MAO-A) effects as compared to curcumin effect per se. This study provided a scientific rationale for the use of curcumin and its co-administration with piperine in the treatment of depressive disorders.

Heat and other physiological stress-induced analgesia: Catecholamine mediated and naloxone reversible response

Abstract Acute environmental heat (40±2°C) and other physiological stressful situations increased the pain threshold to radiant heat in rats and mice. Naloxone pretreatment or chronic exposure to stress antagonised this response. After pretreatment with catecholamine depleters, α-methyl-p-tyrosine, reserpine or with adrenoceptor blockers, haloperidol and chlorpromazine, the stress-induced analgesic effect was abolished. Cyproheptadine, a serotonin antagonist, also blocked this response. The results suggest the role of brain monoamines in stress-mediated analgesia.

Sigma (σ1) receptor mediated antidepressant-like effects of neurosteroids in the Porsolt forced swim test

THIS study examined the effects of neurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and progesterone on the Porsolt forced swim test of depression in mice, and investigated the possible involvement o σ receptors. The immobility time in the mouse forced swimming test was significantly reduced by DHEAS (5 and 2 0 mg/kg, s.c.) and PS (5 mg/kg) without accompanying changes in the ambulatory or open-field activity. Pretreatment with DHEAS (&PHgr; mg/kg) or PS (10 and &PHgr; mg/kg), however, failed to modify the immobility. The relief of behavioral despair in the immobility test by DHEAS (5 and 20 mg/kg) was dose-dependently blocked by preadministration of NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl-ethylamine monohydrochloride; 0.5 and 1 mg/kg), a putativeσ1 receptor antagonist, or progesterone (1 0 mg/kg), aσ receptor antagonistic neurosteroid. On the other hand, PS (5 mg/kg)-induced decrease in the immobility was significantly blocked by NE-100 (0. 5 mg/kg), but not by progesterone (1 0 mg/kg). Neither NE-100 nor progesterone influenced the immobility alone. These data suggest a role for centralσ receptor in the antidepressant-like effects of neurosteroids, and reinforced their potential therapeutic use in depression.

Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice

1. The effect of drugs affecting learning and memory was investigated using transfer latency (TL) as parameter for acquisition and retention of memory process on elevated plus-maze both in rats and mice. Further the validity of the procedure was envisaged. 2. The results provide an evidence for utility of shortened TL on 2nd day trial in old rats and mice as a parameter for retention or consolidation of memory, while treatment of drugs 30 min prior to 1st day may also be utilised for acquisition related action of drugs. 3. The drugs producing acquisition deficits namely scopolamine (0.1-0.5 mg/kg) and MK 801 (0.05-0.1 mg/kg) did not affect the shortened TL on elevated plus-maze in rats while nootropics, like piracetam (150 mg/kg) and captopril (30 mg/kg) reduced the shortened TL. The memory enhancing effect of these agent was reversed by scopolamine (0.3 mg/kg) and MK 801 (0.1 mg/kg) both in rats and mice. The results suggested the acquisition affecting drugs, however, did not show any effect on retention parameter (shortened TL) but can reverse the retention facilitatory action of nootropics. The results also provide indirect evidence for participation of cholinergic and NMDA-receptor blockade in the mechanism of these drugs. 4. Scopolamine and MK 801 produced acquisition deficits in mice, as they increased the TL on 1st and 2nd day trial while physostigmine (0.05 mg/kg) decreased the 2nd day TL. Physostigmine (0.1 mg/kg) reversed scopolamine and MK 801 induced acquisition deficits suggested participation of cholinergic and NMDA- receptor in learning process. 5. The results validate the utility of the elevated plus-maze for evaluation of possible nootropic action of drugs.

Minocycline prevents the development of neuropathic pain, but not acute pain: possible anti-inflammatory and antioxidant mechanisms.

Glia, particularly astrocytes and microglia, are known to play an important role in central sensitization and are strongly implicated in the exaggerated pain states. In the present study, we determined the effect of minocycline, an inhibitor of microglial activation, in acute nociception, peritonitis, and the development and maintenance of hypersensitivity following chronic constriction injury of the sciatic nerve in rats. A single dose of minocycline (30 or 100 mg/kg, i.p.) 30 min before acetic acid or zymosan injection did not attenuate the nociceptive behavior in mice. It had no effect on the early events of peritoneal inflammation (vascular permeability, inflammatory cell infiltration, and release of pro-inflammatory cytokines) in acetic acid or zymosan-injected mice. In addition, minocycline (30 or 100 mg/kg, i.p.) did not alter basal nociceptive responses in the tail immersion test. Chronic administration of minocycline (10 or 30 mg/kg, i.p.) for 7 days started before nerve injury significantly prevented the development of neuropathic pain, interestingly, it further delayed the development of hypersensitivity. In contrast, single injection of minocycline failed to reverse hypersensitivity when administered during the development of neuropathic pain. No significant effects were observed on hypersensitivity when treatment was started once neuropathic state was established. Pre-treatment, but not post-treatment, with minocycline markedly attenuated increased pro-inflammatory cytokines release and oxidative and nitrosative stress in mononeuropathic rats. These results suggest that minocycline had no effect on acute peritoneal inflammation, nociception, and chronic administration of minocycline when started early before peripheral nerve injury could attenuate and further delays the development of neuropathic pain. Concluding, this study clearly shows minocycline, an inhibitor of microglial activation, by inhibiting the release of pro-inflammatory mediators and reducing oxidative stress prevented the development of neuropathic pain.

Resveratrol, a polyphenolic phytoalexin, attenuates diabetic nephropathy in rats

Diabetic nephropathy is a serious microvascular complication and one of the main causes of end-stage renal disease. Various studies have revealed that increased oxidative stress is a major pathophysiological mechanism which is involved in the etiology of diabetic nephropathy. Resveratrol, a polyphenolic phytoalexin present in red wine, is known to possess potent antioxidant properties and thus we aimed to examine its effect on renal function and oxidative stress in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg) in rats. After 4 weeks of STZ injection, rats were divided into four groups: the control rats, diabetic rats and diabetic rats treated with resveratrol (5 and 10 mg/kg, orally) respectively from week 4 up till week 6. At the termination of the experiments, urine albumin excretion, urine output, serum creatinine, blood urea nitrogen, creatinine and urea clearance were measured. The levels of the renal oxidative stress markers malonaldehyde and glutathione and the antioxidant enzymes superoxide dismutase and catalase were measured in kidney homogenate. STZ-injected rats showed significant increases in blood glucose, polyuria, proteinuria and a decrease in body weight compared with age-matched control rats. After 6 weeks, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine and urea clearance, and proteinuria along with a marked increase in oxidative stress, as determined by lipid peroxidation and activities of key antioxidant enzymes. Treatment with resveratrol significantly attenuated renal dysfunction and oxidative stress in diabetic rats. The present study reinforces the important role of oxidative stress in diabetic kidney and points towards the possible antioxidative mechanism being responsible for the renoprotective action of resveratrol.

Sildenafil-induced peripheral analgesia and activation of the nitric oxide–cyclic GMP pathway

Although several lines of evidence have shown a role of the nitric oxide/cyclic guanosine monophosphate signaling pathway in the nociceptive mechanism, the exact role of the phosphodiesterase (PDE) 5 enzyme via the NO-cGMP pathway is not fully understood in pain response. The present study was aimed at exploring the role of the NO-cGMP pathway in nociceptive conditions in experimental animals. Peripheral nociception was assessed by acetic acid-induced chemonociception or carrageenan-induced hyperalgesia and central nociception was assessed by tail-flick and hot-plate methods. Sildenafil exhibited dose-dependent (1, 2, 5 and 10 mg/kg, i.p.) antinociception in both male and female mice against acetic acid-induced writhing. However, it did not alter the pain threshold in central nociception (5 and 10 mg/kg, i.p.). Local administration of sildenafil (50-200 microg/paw, i.pl) also attenuated carrageenan-induced hyperalgesia. In the peripheral nociceptive reaction (acetic acid-induced chemonociception), the antinociceptive effect of sildenafil (2 mg/kg, i.p.) was enhanced by co-administration of sodium nitroprusside (0.25 mg/kg), and L-arginine (50 mg/kg). Sildenafil-induced analgesia was significantly blocked by methylene blue (1 mg/kg), a guanylate cyclase inhibitor, but was not reversed by L-NAME (10 mg/kg), a nitric oxide synthase inhibitor. But a higher dose of L-NAME (20 mg/kg) significantly reversed sildenafil analgesia. Both of these agents also reversed the facilitatory effect of L-arginine (50 mg/kg) and sodium nitroprusside (0.25 mg/kg) on sildenafil analgesia. These results suggest that sildenafil-induced analgesia is mediated via the inhibition of PDE5. The results also indicate that the guanylate cyclase system is stimulated in the peripheral nociceptive reaction. In conclusion, sildenafil produces antinociception and its effect can be potentiated by sodium nitroprusside and L-arginine, probably through the activation of the NO-cyclic GMP pathway.

Effect of natural and synthetic antioxidants in a mouse model of chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is an illness characterized by persistent and relapsing fatigue, often accompanied by numerous symptoms involving various body systems. The etiology of CFS remains unclear; however, a number of studies have shown that oxidative stress may be involved in its pathogenesis. In the present study, a mouse model of CFS was used in which mice were forced to swim for one 6-minute session on each day for 15 days and the immobility period was recorded. There was a significant increase in immobility period in saline-treated mice on successive days. Intraperitoneal treatment with the potent antioxidants carvedilol (5 mg/kg) and melatonin (5 mg/kg) produced a significant reduction in immobility period. Similar results were observed with herbal preparations administered orally: Withania somnifera (100 mg/kg), quercetin (50 mg/kg), and St. Johns wort (Hypericum perforatum L., 10 mg/kg). Biochemical analysis revealed that chronic swimming significantly induced lipid peroxidation and decreased glutathione (GSH) levels in the brains of mice. The rats also showed decreased levels of antioxidant defense enzymes, superoxide dismutase (SOD), and catalase. Co-administration of antioxidants carvedilol, melatonin, W. somnifera, quercetin or St. Johns wort significantly reduced lipid peroxidation and restored the GSH levels decreased by chronic swimming in mice. Further, the treatment increased levels of SOD in the forebrain and of catalase. The findings strongly suggest that oxidative stress plays a significant role in the pathophysiology of CFS and that antioxidants could be useful in the treatment of CFS.