Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1ß and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage.

Glycyrrhizic acid (GA) inhibits reactive oxygen Species mediated photodamage by blocking ER stress and MAPK pathway in UV-B irradiated human skin fibroblasts.

BACKGROUND: Previously we have reported that generation of reactive oxygen species is the prime event responsible for calcium mediated activation of PERK-eIF2α-CHOP pathway and apoptosis in UV-B irradiated human skin fibroblasts (Hs68). We have also reported that glycyrrhizic acid (GA) mediates potent photoprotective activity against UV-B - irradiation-induced photodamage in human skin fibroblast. OBJECTIVE: In the present study, we aimed to investigate the role of GA in preventing oxidative stress mediated unfolded protein response (UPR) and mitogen activated protein kinases (MAPK) pathway. METHODS: Human skin fibroblast (Hs68) cells were exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analysed using western-blotting, microscopy and flow cytometry. RESULTS: Our results show that GA has strong photoprotective action against UV-B induced cellular damage. It was observed that: (a) Oxidative disturbances and intracellular Ca(2+) imbalance induced by UV-B irradiation was significantly restored by GA treatment; (b) activation of PERK-eIF2α-CHOP and MAPK pathway induced by UV-B was significantly blocked by GA; (c) Loss of mitochondrial membrane potential and apoptosis induced by UV-B were reduced by GA treatment. CONCLUSION: Based on the above findings we conclude GA has a highly significant ROS quenching activity, thereby blocking the cascade of events including release of calcium from ER and subsequent ER stress, MAPK pathway and cellular demise. GA offers highly potent anti photodamage effect and can be exploited for cosmetic or therapeutic purposes.

Oxidative stress mediated Ca(2+) release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells.

BACKGROUND: Exposure of skin to ultraviolet (UV) radiation, an environmental stressor induces number of adverse biological effects (photodamage), including cancer. The damage induced by UV-irradiation in skin cells is initiated by the photochemical generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress and consequent activation of unfolded protein response (UPR). OBJECTIVE: To decipher cellular and molecular events responsible for UV-B mediated ER stress and UPR activation in skin cells. METHODS: The study was performed on human skin fibroblast (Hs68) and keratinocyte (HaCaT) cells exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analyzed using Western-blotting, microscopic studies and flow cytometry. RESULTS: Our results depicted that UV-B induces an immediate ROS generation that resulted in emptying of ER Ca(2+) stores inducing ER stress and activation of PERK-peIF2α-CHOP pathway. Quenching ROS generation by anti-oxidants prevented Ca(2+) release and subsequent induction of ER stress and UPR activation. UV-B irradiation induced PERK dependent G2/M phase cell cycle arrest in Hs68 and G1/S phase cell cycle arrest in HaCaT. Also our study reflects that UV-B exposure leads to loss of mitochondrial membrane potential, activation of apoptotic cascade as evident by AnnexinV/PI staining, decreased expression of Bcl-2 and increased cleavage of PARP-1 protein. CONCLUSION: UV-B induced Ca(2+) deficit within ER lumen was mediated by immediate ROS generation. Insufficient Ca(2+) concentration within ER lumen developed ER stress leading to UPR activation. These changes were reversed by use of anti-oxidants which quench ROS.

Ethanolic extract of Alternanthera sessilis (AS-1) inhibits IgE-mediated allergic response in RBL-2H3 cells.

The present study was designed to investigate the anti-allergic effects of ethanolic extract of Alternanthera sessilis (AS-1) in rat basophilic leukemia (RBL-2H3) cells. It significantly reduced the ß-hexosaminidase release from anti-DNP-IgE sensitized RBL-2H3 cells. AS-1also inhibited the IgE antibody-induced increase in Interleukin-6 (IL-6), TNF-α, IL-13 and IL-4 production in these cells. The inhibitory effect of AS-1 on these cytokine was found to be nuclear factor-KB (NF-kB) dependent, as it attenuated the degradation of IKBa and nuclear translocation of NFkB. In addition, AS-1 significantly attenuated the DNP HAS-induced intracellular Ca(2+) release from these cells, which makes us speculate strongly that the decreased intracellular Ca(2+) is involved in the inhibitory effect of AS-1 on ß-hexoaminidase release. Taken together, anti-allergic effects of AS-1 suggest possible therapeutic application of this extract in allergic diseases.

Effect of N-acetyl cysteine (NAC), an organosulfur compound from Allium plants, on experimentally induced hepatic prefibrogenic events in Wistar rat.

Aim of present study was to investigate the effect of NAC on experimental chronic hepatotoxicity models induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). CCl4 toxicity was induced by administering 200 µl CCl4 (diluted 2:3 in coconut oil)/100 g body weight, p.o., twice weekly for 8 weeks. TAA toxicity was induced by administering 150 mg/kg b. wt. of TAA i.p., twice weekly for 8 weeks. NAC treatment was started along with toxicants (CCl4 and TAA) for 8 weeks and continued for further 4 weeks. Self reversal group was kept without any treatment for 4 weeks after completion of toxicant treatments. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin were measured in serum. Hydroxyproline (HP), lipid peroxidation (LPO), catalase (CAT), Glutathione peroxidase (GPx) and Glutathione (GSH) were determined in liver samples by colorimetric methods. Cytochrome P450 2E1 (CYP 450 2E1), activity was determined as hydroxylation of aniline in liver microsomes. General examination and histological analysis were also performed. Serum markers of liver damage (AST, ALT, ALP and Bilirubin) were increased by CCl4 and TAA intoxication (p<0.001), whereas co-treatment with NAC reversed such changes (p<0.001). HP was enhanced in toxicant groups (p<0.001 in CCl4 and TAA), but inhibited by NAC (p<0.001). LPO was increased while as GSH, CAT and GPx decreased by the administration of CCl4 and TAA (p<0.001); co-administration of NAC restored these liver markers to normal levels (p<0.001). Biochemical determinations were corroborated by general and histological findings. Keeping in view the biochemical and histopathological studies, it was concluded that CCl4 and TAA are strong hepatotoxic agents that produce liver fibrosis with close proximity to human etiology (micronodular cirrhosis) and NAC has a significant protective activity against CCl4 and TAA. NAC has also been validated as a model against oxidative burden in chronic liver pathology.

Glycyrrhizic acid (GA), a triterpenoid saponin glycoside alleviates ultraviolet-B irradiation-induced photoaging in human dermal fibroblasts.

Glycyrrhizic acid (GA), a triterpenoid saponin glycoside from the roots and rhizomes of licorice is used in traditional and modern medicine for the treatment of numerous medical conditions including skin diseases and beauty care product. In the present study, we investigated the effect of GA against ultraviolet B (UVB) irradiation-induced photoaging in human dermal fibroblasts (HDFs) and its possible mechanism of action. HDFs were subjected to photoaging by sub-toxic dose of UVB (10 mj/cm(2)) irradiation. Cell viability, matrix metalloproteinase 1 (MMP1), pro-collagen 1, cellular and nuclear morphology, cell cycle, intracellular reactive oxygen species (ROS), caspase 3 and hyaluronidase inhibition assays were performed. Western blotting was used to evaluate the expression of NF-kappa B (NF-κB) and cytochrome-C proteins. GA treatment significantly inhibited photoaging. It achieved this by reducing ROS, NF-κB, cytochrome c, caspase 3 levels and inhibiting hyaluronidase enzyme. The main mechanism seems to be, most likely by blocking MMP1 activation by modulating NF-κB signaling. These findings may be useful for development of natural and safe photoprotective agents against UVB irradiation.

Effect of Emblica officinalis (fruit) against UVB-induced photo-aging in human skin fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Emblica officinalis fruit (EO), commonly known as Amla is a reputed traditional medicine and functional food used in Indian subcontinent. It has long been used in Indian folk medicine to treat liver diseases, stomach ulcers, inflammatory diseases, metabolic disorders, geriatric complaints, skin disorders and beauty care. AIM OF THE STUDY: Recently, it has been shown to promote pro-collagen content and inhibit matrix metalloproteinase levels in skin fibroblast. The aim of the present study was to investigate the efficacy of EO to inhibit UVB-induced photo-aging in human skin fibroblasts. MATERIALS AND METHODS: Mitochondrial activity of human skin fibroblasts was measured by MTT-assay. Quantifications of pro-collagen 1 and matrix metalloproteinase 1 (MMP-1) release were performed by immunoassay techniques. Hyaluronidase inhibition assay was studied in vitro using bovine testicular hyaluronidase and human umbilical cord hyaluronic acid. Cell cycle analysis was performed by flowcytometry using propidium iodide. RESULTS: EO stimulated, the otherwise UVB inhibited cellular proliferation and protected pro-collagen 1 against UVB-induced depletion via inhibition of UVB-induced MMP-1 in skin fibroblasts (10-40 µg/mL, p>0.001). EO exhibited inhibitory activity of hyaluronidase (10-40 µg/mL, p>0.001). Treatment with EO also prevented UVB disturbed cell cycle to normal phase. CONCLUSION: The results of the present study suggests that EO effectively inhibits UVB-induced photo-aging in human skin fibroblast via its strong ROS scavenging ability and its therapeutic and cosmetic applications remain to be explored.

Negundoside, an irridiod glycoside from leaves of Vitex negundo, protects human liver cells against calcium-mediated toxicity induced by carbon tetrachloride.

AIM: To evaluate the protective effect of 2'-p-hydroxybenzoylmussaenosidic acid [negundoside (NG), against carbon tetrachloride (CCl(4))-induced toxicity in HuH-7 cells. METHODS: CCl(4) is a well characterized hepatotoxin, and inducer of cytochrome P450 2E1 (CYP2E1)-mediated oxidative stress. In addition, lipid peroxidation and accumulation of intracellular calcium are important steps in the pathway involved in CCl(4) toxicity. Liver cells (HuH-7) were treated with CCl(4), and the mechanism of the cytoprotective effect of NG was assessed. Silymarin, a known hepatoprotective drug, was used as control. RESULTS: NG protected HuH-7 cells against CCl(4) toxicity and loss of viability without modulating CYP2E1 activity. Prevention of CCl(4) toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species (ROS), a decrease in lipid peroxidation and accumulation of intracellular Ca(2+) levels and maintenance of intracellular glutathione homeostasis. Decreased mitochondrial membrane potential (MMP), induction of caspases mediated DNA fragmentation and cell cycle arrest, as a result of CCl(4) treatment, were also blocked by NG. The protection afforded by NG seemed to be mediated by activation of cyclic adenosine monophosphate (cAMP) synthesis and inhibition of phospholipases (cPLA2). CONCLUSION: NG exerts a protective effect on CYP2E1-dependent CCl(4) toxicity via inhibition of lipid peroxidation, followed by an improved intracellular calcium homeostasis and inhibition of Ca(2+)-dependent proteases.

Potentiation of isoniazid-induced liver toxicity by rifampicin in a combinational therapy of antitubercular drugs (rifampicin, isoniazid and pyrazinamide) in Wistar rats: A toxicity profile study.

AIM: Biochemical characterization of long-term toxic manifestations of anti-tubercular (anti-TB) drugs - rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) - individually and in two combinations: (i) RIF + INH, and (ii) RIF + INH + PZA in Wistar rats. METHODS: Animals received anti-TB drugs - alone or in combination - once daily p.o. for up to 90 days (doses, in mg/kg: RIF, 250; INH, 50; PZA, 100). Assays for alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin (serum) and lipid peroxidation (LPO), glutathione (GSH), glutathione peroxidase (GPx), catalase, Na+K+-ATPase and CYP 2E1 (liver) were performed to assess liver toxicity. Clinical biochemistry was done by commercial kits. Determinations were made at 0, 15, 30 and 90 days of treatment schedule. RESULTS: Anti-TB drugs-treated animals showed abnormal rises or falls (>1.5-2 fold) in the serum/liver parameters. Mild hyperlipidemia, hypercholesterolemia and hyperuricemia were the other pathologies. Of all the treated groups, INHalone or in combination with other drugs produced a progressive enhancement of toxicity over 15-90 days. The in vivo results were further supported by in vitro results (MTT assay, GSH and LPO) in primary cultures of rat hepatocyte. RESULTS indicated that anti-TB drugs in combination: (i) caused membrane damage resulting in leakage of ALT, ALP and bilirubin; (ii) caused imbalance in endogenous enzymatic oxidant-antioxidant defense via increased lipid peroxidation and in glutathione homeostasis; and (iii) enhanced the CYP 2E1-mediated bioactivation mechanism. CONCLUSION: Toxicity manifestations seemed to be heptocytic injury targeted at hepatocytes, bile ducts or sinusoidal cells related to hepatitis and primary biliary cholestasis.

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme.

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.