Pabitra Bikash Pal

Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis

Oxidative stress basically defines a condition in which prooxidant–antioxidant balance in the cell is disturbed; cellular biomolecules undergo severe oxidative damage, ultimately compromising cells viability. In recent years, a number of studies have shown that oxidative stress could cause cellular apoptosis via both the mitochondria-dependent and mitochondria-independent pathways. Since these pathways are directly related to the survival or death of various cell types in normal as well as pathophysiological situations, a clear picture of these pathways for various active molecules in their biological functions would help designing novel therapeutic strategy. This review highlights the basic mechanisms of ROS production and their sites of formation; detail mechanism of both mitochondria-dependent and mitochondria-independent pathways of apoptosis as well as their regulation by ROS. Emphasis has been given on the redox-sensitive ASK1 signalosome and its downstream JNK pathway. This review also describes the involvement of oxidative stress under various environmental toxin- and drug-induced organ pathophysiology and diabetes-mediated apoptosis. We believe that this review would provide useful information about the most recent progress in understanding the mechanism of oxidative stress–mediated regulation of apoptotic pathways. It will also help to figure out the complex cross-talks between these pathways and their modulations by oxidative stress. The literature will also shed a light on the blind alleys of this field to be explored. Finally, readers would know about the ROS-regulated and apoptosis-mediated organ pathophysiology which might help to find their probable remedies in future.

Mangiferin Attenuates Diabetic Nephropathy by Inhibiting Oxidative Stress Mediated Signaling Cascade, TNFα Related and Mitochondrial Dependent Apoptotic Pathways in Streptozotocin-Induced Diabetic Rats

Oxidative stress plays a crucial role in the progression of diabetic nephropathy in hyperglycemic conditions. It has already been reported that mangiferin, a natural C-glucosyl xanthone and polyhydroxy polyphenol compound protects kidneys from diabetic nephropathy. However, little is known about the mechanism of its beneficial action in this pathophysiology. The present study, therefore, examines the detailed mechanism of the beneficial action of mangiferin on STZ-induced diabetic nephropathy in Wister rats as the working model. A significant increase in plasma glucose level, kidney to body weight ratio, glomerular hypertrophy and hydropic changes as well as enhanced nephrotoxicity related markers (BUN, plasma creatinine, uric acid and urinary albumin) were observed in the experimental animals. Furthermore, increased oxidative stress related parameters, increased ROS production and decreased the intracellular antioxidant defenses were detected in the kidney. Studies on the oxidative stress mediated signaling cascades in diabetic nephropathy demonstrated that PKC isoforms (PKCα, PKCβ and PKCε), MAPKs (p38, JNK and ERK1/2), transcription factor (NF-κB) and TGF-β1 pathways were involved in this pathophysiology. Besides, TNFα was released in this hyperglycemic condition, which in turn activated caspase 8, cleaved Bid to tBid and finally the mitochorndia-dependent apoptotic pathway. In addition, oxidative stress also disturbed the proapoptotic-antiapoptotic (Bax and Bcl-2) balance and activated mitochorndia-dependent apoptosis via caspase 9, caspase 3 and PARP cleavage. Mangiferin treatment, post to hyperglycemia, successfully inhibited all of these changes and protected the cells from apoptotic death.

Involvement of both intrinsic and extrinsic pathways in hepatoprotection of arjunolic acid against cadmium induced acute damage in vitro.

Cadmium (Cd) is one of the ubiquitous environmental pollutants and is responsible for various organ pathophysiology including hepatic disorders. It is extremely toxic even in low concentrations and bioaccumulate in organisms. The present study has been carried out to investigate the cytoprotective role of arjunolic acid (AA), a tri terpenoid saponin, against Cd induced oxidative impairment and cell death in murine hepatocytes. Administration of cadmium (30 μM), in the form of chloride (CdCl(2)) for 2h, significantly enhanced the ALT, ALP and LDH leakage, increased reactive oxygen species (ROS) production, reduced hepatocytes viability and altered the antioxidant status of hepatocytes by reducing intracellular GSH level, anti-oxidant enzymes activity and increasing intracellular GSSG and lipid peroxidation. Evidence for Cd-induced nature of cell death was sought by flow cytometric analysis. Signal transduction studies revealed that Cd markedly increased the levels of caspase-9, -8, -3, Fas and Bid, decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol, disturbed the Bcl-2 family protein balance, cleaved PARP protein and ultimately led to apoptotic cell death. Results showed that Cd could trigger both intrinsic and extrinsic apoptotic pathways. In addition, Cd markedly increased NF-κB nuclear translocation in association with IKKα/β phosphorylation and IκBα degradation. Simultaneous treatment with AA (200 μM), however, reduced Cd-induced oxidative stress, attenuated the nuclear translocation of NF-κB and protects the hepatocytes from Cd-induced apoptotic death. Combining, data suggest that Cd-induced hepatic dysfunction and apoptosis might be supported by the ROS formation and mediated via the activation of NF-κB. AA treatment, on the other hand, reduced Cd-induced oxidative stress, attenuated the activation of NF-κB and mitochondrion-dependent and independent apoptotic signaling pathways.

Morin protects gastric mucosa from nonsteroidal anti-inflammatory drug, indomethacin induced inflammatory damage and apoptosis by modulating NF-κB pathway.

BACKGROUND Deregulation in prostaglandin (PG) biosynthesis, severe oxidative stress, inflammation and apoptosis contribute to the pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy. Unfortunately, most of the prescribed anti-ulcer drugs generate various side effects. In this scenario, we could consider morin as a safe herbal potential agent against IND-gastropathy and rationalize its action systematically. METHODS Rats were pretreated with morin for 30 min followed by IND (48 mgkg(-1)) administration for 4 h. The anti-ulcerogenic nature of morin was assessed by morphological and histological analysis. Its effects on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COXs, PGE(2)), and signaling parameters (NF-κB and apoptotic signaling) were assessed by biochemical, RP-HPLC, immunoblots, IHC, RT-PCR, and ELISA at the time points of their maximal changes due to IND administration. RESULTS IND induced NF-κB and apoptotic signaling in rats gastric mucosa. These increased proinflammatory responses, but reduced the antioxidant enzymes and other protective factors. Morin reversed all the adverse effects to prevent IND-induced gastric ulceration in a PGE2 independent manner. Also, it did not affect the absorption and/or primary pharmacological activity of IND. CONCLUSIONS The gastroprotective action of morin is primarily attributed to its potent antioxidant nature that also helps in controlling several IND-induced inflammatory responses. GENERAL SIGNIFICANCE For the first time, the study reveals a mechanistic basis of morin mediated protective action against IND-induced gastropathy. As morin is a naturally abundant safe antioxidant, future detailed pharmacokinetic and pharmacodynamic studies are expected to establish it as a gastroprotective agent.

Mangiferin, a natural xanthone, protects murine liver in Pb(II) induced hepatic damage and cell death via MAP kinase, NF-κB and mitochondria dependent pathways.

One of the most well-known naturally occurring environmental heavy metals, lead (Pb) has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II)] in the form of Pb(NO3)2 (at a dose of 5 mg/kg body weight, 6 days, orally) induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally), on the other hand, diminished the formation of reactive oxygen species (ROS) and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT) and alkaline phosphatase (ALP)]. Mangiferin also reduced Pb(II) induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II)-induced activation of mitogen-activated protein kinases (MAPKs) (phospho-ERK 1/2, phosphor-JNK phospho- p38), nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II) induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II). Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II) induced hepatic dysfunction.

Cadmium (Cd2+) exposure differentially elicits both cell proliferation and cell death related responses in SK-RC-45

Cadmium (Cd(2+)) is a major nephrotoxic environmental pollutant, affecting mostly proximal convoluted tubule (PCT) cells of the mammalian kidney, while conditionally Cd(2+) could also elicit protective responses with great variety and variability in different systems. The present study was designed to evaluate the molecular mechanism of Cd(2+) toxicity on human PCT derived Renal Cell Carcinoma (RCC), SK-RC-45 and compare its responses with normal human PCT derived cell line, NKE. Exposure of SK-RC-45 cells with different concentrations of CdCl2 (e.g. 0, 10 and 20μM) in serum free medium for 24h generate considerable amount of ROS, accompanied with decreased cell viability and alternations in the cellular and nuclear morphologies, heat shock responses and GCLC mediated protective responses. Also phosphatidylserine externalization, augmentation in the level of caspase-3, PARP, BAD, Apaf1 and cleaved caspase-9 along with decreased expression of Bcl2 and release of cytochrome c confirmed that, Cd(2+) dose dependently induces solely intrinsic pathway of apoptosis in SK-RC-45, independent of JNK. Furthermore, the non-toxic concentration (10μM) of Cd(2+) induced nuclear translocation of Nrf2 and increased expression in the level of HO-1 enzyme suggesting that at the milder concentration, Cd(2+) induces protective signaling pathways. On the other hand, exposure of NKE to different concentrations of CdCl2 (e.g. 0, 10, 20, 30 and 50μM) under the same conditions elevate stronger heat shock and SOD2 mediated protective responses. In contrary to the RCC PCT, the normal PCT derived cell follows JNK dependent and extrinsic pathways of apoptosis. Cumulatively, these results suggest that Cd(2+) exposure dose dependently elicit both cell proliferative and cell death related responses in SK-RC-45 cells and is differentially regulated with respect to normal kidney epithelia derived NKE cells.

A 35 kD Phyllanthus niruri protein modulates iron mediated oxidative impairment to hepatocytes via the inhibition of ERKs, p38 MAPKs and activation of PI3k/Akt pathway.

It has been reported that the herb, Phyllanthus niruri, possess antioxidant, anti-infection, anti-asthmatic, anti-diuretic, anti-soresis and many more beneficial activities. The goal of our present study was to evaluate the protective role of a 35 kD protein (PNP) isolated from this herb against iron-induced cytotoxicity in murine hepatocytes. Exposure of hepatocytes to iron (FeSO4) caused elevation of reactive oxygen species (ROS) production, enhanced lipid peroxidation and protein carbonylation, depleted glutathione levels, decreased the antioxidant power (FRAP) of the cells and reduced cell viability. Iron mediated cytotoxicity disrupted mitochondrial membrane potential (Δψm) and thereby caused apoptosis mainly by the intrinsic pathway via the down-regulation of IκBα with a concomitant up-regulation of NF-kB as well as the phosphorylation of ERKs and p38 MAP kinases. In addition, iron-induced cytotoxicity disrupted the normal balance of Bcl-2 family proteins in hepatocytes. Incubation of hepatocytes with PNP, however, protected the cells from apoptosis by stabilizing the mitochondria and arresting the release of cytochrome c. It also suppressed caspase activation and cleavage of PARP. Moreover, this protein has strong free radical scavenging activity and thereby scavenged ROS extensively. Combining all, results suggest that simultaneous treatment with PNP might suppress the iron-induced cytotoxicity in hepatocytes.

Traditional extract of Pithecellobium dulce fruits protects mice against CCl4 induced renal oxidative impairments and necrotic cell death

The present study has been carried out to investigate the role of the aqueous extract of the fruits of Pithecellobium dulce (AEPD) against carbon tetrachloride (CCl(4)) induced renal oxidative injury in mice. HPLC analysis shows that AEPD contains phenolics, flavonoids and saponins as the major active components. Creatinine and blood urea nitrogen (BUN) levels were assayed to determine renal protective action of AEPD in CCl(4)-induced renal pathophysiology. Its antioxidant activity was determined by measuring radical scavenging activity, antioxidant enzymes activities, GSH content, protein carbonylation and lipid peroxidation. In addition, FACS analysis, DNA fragmentation and histological studies were carried out to determine its effect in CCl(4) induced renal oxidative injury and cell death. CCl(4) exposure increased the intracellular reactive oxygen species production, decreased intracellular antioxidant defence, reduced mitochondrial membrane potential, attenuated the intracellular ATP content and caused renal cell death mainly via the necrotic pathway as revealed by DNA fragmentation analysis. Treatment with AEPD both prior and post to the toxin exposure protected the organ from CCl(4) induced oxidative insult. Histological studies also support our results. Combining, results suggest that the protective role of AEPD against CCl(4) induced renal oxidative impairments is probably due to the antioxidative properties present in its active constituents.