Mohammad Rizwan Siddiqui

Reactive oxygen species in inflammation and tissue injury.

Abstract Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. An enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue injury. The vascular endothelium plays an important role in passage of macromolecules and inflammatory cells from the blood to tissue. Under the inflammatory conditions, oxidative stress produced by PMNs leads to the opening of inter-endothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier. The migrated inflammatory cells not only help in the clearance of pathogens and foreign particles but also lead to tissue injury. The current review compiles the past and current research in the area of inflammation with particular emphasis on oxidative stress-mediated signaling mechanisms that are involved in inflammation and tissue injury.

Trigonella foenum graecum seed powder protects against histopathological abnormalities in tissues of diabetic rats

Trigonella foenum graecum is a well-known hypoglycemic agent used in traditional Indian medicines. It was previously reported that oral administration of its seed powder for 3 weeks to alloxan diabetic rats stabilized glucose homeostasis and free radical metabolism in liver and kidney. In the present study, we further investigated the effects of 3 weeks alloxan induced diabetes on the histological structure and function of liver and kidney and the protective effect of T. foenum graecumseed powder (TSP) oral administration to the diabetic rats utilizing enzyme analysis and light and transmission electron microscopy. The activity of the enzyme, glutamate dehydrogenase was significantly higher whereas the activity of d-β-hydroxybutyrate dehydrogenase enzyme was significantly lower in liver and kidney of alloxan-induced diabetic rats. Histopathological studies showed liver degenerative and early nephropathic changes in diabetic rats. Ultrastructure of the diabetic liver revealed a reduction in the rough endoplasmic reticulum and swelling of mitochondria in the hepatocytes. TSP treatment to the diabetic rats effectively prevented the alteration in the activities of the two enzymes and partially prevented the structural abnormalities thus suggesting a protective effect of TSP on the liver and kidney of the diabetic rats. The role of TSP in reversing the diabetic state at the cellular level besides the metabolic normalization further proves its potential as an antidiabetic agent (Mol Cell Biochem 266: 151–159, 2004)

A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis

Apoptosis is a key regulator for the normal turnover of the intestinal mucosa, and abnormalities associated with this function have been linked to inflammatory bowel disease and colorectal cancer. Despite this, little is known about the mechanism(s) mediating intestinal epithelial cell apoptosis. Villin is an actin regulatory protein that is expressed in every cell of the intestinal epithelium as well as in exocrine glands associated with the gastrointestinal tract. In this study we demonstrate for the first time that villin is an epithelial cell-specific anti-apoptotic protein. Absence of villin predisposes mice to dextran sodium sulfate-induced colitis by promoting apoptosis. To better understand the cellular and molecular mechanisms of the anti-apoptotic function of villin, we overexpressed villin in the Madin-Darby canine kidney Tet-Off epithelial cell line to demonstrate that expression of villin protects cells from apoptosis by maintaining mitochondrial integrity thus inhibiting the activation of caspase-9 and caspase-3. Furthermore, we report that the anti-apoptotic response of villin depends on activation of the pro-survival proteins, phosphatidylinositol 3-kinase and phosphorylated Akt. The results of our studies shed new light on the previously unrecognized function of villin in the regulation of apoptosis in the gastrointestinal epithelium.

Amelioration of altered antioxidant status and membrane linked functions by vanadium and Trigonella in alloxan diabetic rat brains.

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on antioxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na+/K+ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P < 0.001), decreased activities of SOD, catalase and Na+/K+ ATPase (P < 0.01,P < 0.001 andP < 0.01), increased levels of GPx and MDA (P < 0.01 andP < 0.001) and decreased membrane fluidity (P < 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose ofTrigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration.

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.

Long-term effect of Trigonella foenum graecum and its combination with sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues

Trigonella foenum graecum seed powder (TSP) and Sodium Orthovanadate (SOV) have been shown to demonstrate antidiabetic effects by stabilizing glucose homeostasis and carbohydrate metabolism in experimental type-1 diabetes. However their efficacy in controlling histopathological and biochemical abnormalities in ocular tissues associated with diabetic retinopathy is not known. The purpose of this study was to investigate the comparative efficacy of individual as well as combination therapy of TSP and SOV in 8 weeks diabetic rat lens and retina. Retinas and lenses were taken from control, alloxan-induced diabetic rats and diabetic rats treated separately with insulin, 5%TSP, SOV (0.6 mg/ml) and a combined dose of SOV (0.2 mg/ml) and 5%TSP for 60 days. Control and each experimental group had six rats. Alterations in the activities of enzymes HK (hexokinase), AR (aldose reductase), SDH (sorbitol dehydrogenase), G-6-PD (glucose-6-phosphate dehydrogenase), GPx (glutathione peroxidase), GR (glutathione reductase) and levels of metabolites like sorbitol, fructose, glucose, MDA (malondialdehyde) and GSH (reduced glutathione) were measured in the cytosolic fraction of lenses besides measuring blood glucose levels and glycosylated haemoglobin. Histopathological abnormalities were studied in the lens using photomicrography and retina using transmission electron microscopy. Blood glucose, glycosylated haemoglobin levels and polyol pathway enzymes AR and SDH increased significantly causing accumulation of sorbitol and fructose in the diabetic lens and treatment with SOV and TSP significantly (p < 0.05) decreased these to control levels. Similarly, SOV and TSP treatments modulated the activities of HK, G-6-PD, GPx and GR in the rat lens to control values. Ultrastructure of the diabetic retina revealed disintegration of the inner nuclear layer cells with reduction in rough endoplasmic reticulum and swelling of mitochondria in the bipolar cells; and these histopathological events were effectively restored to control state by SOV and TSP treatments. In this study SOV and TSP effectively controlled ocular histopathological and biochemical abnormalities associated with experimental type-1 diabetes, and a combination regimen of low dose of SOV with TSP demonstrated the most significant effect. In conclusion, the potential of SOV and TSP alone or in low dose combination may be considered as promising approaches for the prevention of diabetic retinopathy and other ocular disorders.

Improvement of Cardiac Function in Mouse Myocardial Infarction after Transplantation of Epigenetically-Modified Bone Marrow Progenitor Cells

Objective To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice. Methods and Results We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells. Conclusion Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

Low doses of vanadate and Trigonella synergistically regulate Na+/K+-ATPase activity and GLUT4 translocation in alloxan-diabetic rats

Oral administration of vanadate to diabetic animals have been shown to stabilize the glucose homeostasis and restore altered metabolic pathways. However, vanadate exerts these effects at relatively high doses with several toxic effects. Low doses of vanadate are relatively safe but unable to elicit any antidiabetic effects. The present study explored the prospect of using low doses of vanadate with Trigonella foenum graecum, seed powder (TSP), another antidiabetic agent, and to evaluate their antidiabetic effect in diabetic rats. Alloxan diabetic rats were treated with insulin, vanadate, TSP and low doses of vanadate with TSP for three weeks. The effect of these antidiabetic compounds was examined on general physiological parameters, Na+/K+ ATPase activity, membrane lipid peroxidation and membrane fluidity in liver, kidney and heart tissues. Expression of glucose transporter (GLUT4) protein was also examined by immunoblotting method in experimental rat heart after three weeks of diabetes induction. Diabetic rats showed high blood glucose levels. Activity of Na+/K+ ATPase decreased in diabetic liver and heart. However, kidney showed a significant increase in Na+/K+ ATPase activity. Diabetic rats exhibited an increased level of lipid peroxidation and decreased membrane fluidity. GLUT4 distribution was also significantly lowered in heart of alloxan diabetic rats. Treatment of diabetic rats with insulin, TSP, vanadate and a combined therapy of lower dose of vanadate with TSP revived normoglycemia and restored the altered level of Na+/K+ ATPase, lipid peroxidation and membrane fluidity and also induced the redistribution of GLUT4 transporter. TSP treatment alone is partially effective in restoring the above diabetes-induced alterations. Combined therapy of vanadate and TSP was the most effective in normalization of altered membrane linked functions and GLUT4 distribution without any harmful side effect.

Effect of estradiol and progesterone treatment on carbohydrate metabolizing enzymes in tissues of aging female rats

The aim of this study was to determine the effect of administration of estradiol (E2), progesterone (P4), and combination of estradiol and progesterone (EP) in aging female rats. The changes in the activities of hexokinase (HK), glucose-6-phosphatase (G6P′tase) and glucose-6-phosphate dehydrogenase (G6PDH) enzymes, and in protein levels in tissues of rats namely brain (cerebral hemisphere), heart, liver, kidney and uterus have been measured in different age groups. The random blood sugar level was measured in serum and liver. The different age groups of rats were given 0.1 μg/g body weight estradiol, 2.5 μg/g body weight progesterone and a similar concentration of both in a combined treatment for 1 month. This dose was selected after determining estrogen and progesterone levels in 3 month adult female animals so that the aging female animals had circulating hormone levels nearly the same as those of young female animals. The random sugar level was determined in serum and liver cytosolic fractions, and it was increased by combination treatment. The protein content in tissues showed significant changes only with combined hormone administration when compared with age-matched controls. The activity of HK decreased in aged animals and significantly increased by hormone treatments in all the tissues of the aged rats studied. The activity of G6P′tase increased with age up to 1.5 years and decreased in 2 years. Treatment with E2 and EP further decreased the activity significantly in all the tissues. G6PDH showed a similar pattern as was observed in HK in all the age groups. Therefore, the E2 and EP treatments caused an entire series of growth-related responses, including an increased uptake of glucose, increased the protein level in the tissues of aging rats, thereby reducing the risk factors associated with aging by normalizing hormone levels which decreased with aging and resulted in diseases such as Alzheimers diseases and diabetes.

Effect of hormone replacement therapy in normalizing age related neuronal markers in different age groups of naturally menopausal rats.

Aging of the normal brain is accompanied by changes in its structure, function, and metabolism. There are significant gender differences in aging brain. Most of these changes increase during menopausal condition in females when the level of estradiol and progesterone are decreased. The objective of this study was to determine the effect of estradiol and progesterone (separate as well as combined) hormones in neuronal tissues from naturally menopausal rats of different age groups. Results show decreased activity of Acetylcholine esterase (AChE) whereas the level of lipid peroxidation increased with age, and after the hormone treatments both AChE activity and level of lipid peroxidation returned to control values. The deposition of lipofuscin, a pigment that accumulated intraneuronally in brain and other tissues and is considered a marker of aging, was increased with aging and the hormone treatment decreased this deposition. The present study clearly shows reduction in risk factors associated with aging in the murine model system by hormone treatments, namely estrogen and progesterone by increasing the activity of acetylcholine esterase and decreasing the levels of lipid peroxidation and lipofuscin deposition in different parts of aging brain. This study suggests that hormone replacement therapy may either reduce or delay the onset of age related diseases like Alzheimer’s, Parkinson’s and other neurological disorders.