Supplementation of curcumin and vitamin E enhances oxidative stress,but restores hepatic histoarchitecture in hypothyroid rats

AimsIn the present study, the effects of vitamin E and curcumin on hepatic dysfunction, mitochondrial oxygen consumption as well as hyperlipidemia in hypothyroid rats are reported.Main methodsAdult male rats were rendered hypothyroid by administration of 0.05% 6-n-propyl-2-thiouracil (PTU) in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days.Key findingsHypothyroidism-induced elevation in serum aspartate aminotransferase activity was found to decline in vitamin E and curcumin treated rats. Nevertheless, distorted histoarchitecture revealed in hypothyroid rat liver was alleviated to normal by vitamin E and curcumin treatment. Regulation of hypothyroidism induced decrease in complexes I and II mediated mitochondrial respiration by vitamin E and curcumin was found to be different. Administration of curcumin to hypothyroid rats alleviates the decreased state 4 respiration and increased respiratory control ratio (RCR) level in complex I mediated mitochondrial oxygen consumption, whereas complex II mediated respiration was not influenced by exogenous antioxidants. Although, increase in serum concentration of total cholesterol was not modified by exogenous antioxidants, increased level of non-high-density lipoprotein cholesterol (non-HDL-C) in serum of hypothyroid rats was further enhanced by vitamin E and curcumin. Moreover, a significant elevation in mitochondrial lipid peroxidation and protein carbonylation was noticed in hypothyroid groups treated with vitamin E and curcumin.SignificanceThe present study suggests that supplementation of curcumin and vitamin E enhances oxidative stress parameters and hyperlipidemia; nevertheless, it protects hypothyroid-induced altered rectal temperature, serum transaminase activity and hepatic histoarchitecture.     

Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.     

Garlic and vitamin E provides antioxidant defence in tissues of female rats treated with nicotine

Nicotine is known to induce oxidative stress in rat tissues and the antioxidant properties of garlic have been reported. This study was designed to determine if the peroxidative damage caused by nicotine administration can be effectively prevented with garlic juice, and vitamin E, a known antioxidant.Four groups of six rats each were divided into: Group I: (control) received 0.2ml of 0.9% normal saline, group II (received nicotine 0.6mg/kg b.w subcutaneously), group III (received nicotine 0.6mg/kg b.w + garlic juice 100mg/kg b.w orally), and group IV (received nicotine 0.6mg/kg b.w + Vitamin E 100mg/kg b.w orally). All animals were treated for 21 days. The pituitary gland, ovary, uterus, heart, liver and kidney of the animals were harvested, weighed and homogenized. Malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) were then measured.Concentration of MDA was significantly increased in tissues of nicotine treated rats when compared with the control. In group III and IV, MDA levels were significantly reduced when compared with nicotine group. The activities of SOD and GSH significantly decreased in group II (nicotine only) rat tissues, while it was significantly increased in group III and IV rat tissues. The study showed that garlic juice extract (100mg/kg b.w) and vitamin E (100mg/kg b.w) administration prevented oxidative damage in rat tissues treated with nicotine. The study also showed that vitamin E has a more potent antioxidant activity than garlic juice in preventing nicotine induced oxidative damage in rat. Keywords: Nicotine, Vitamin E, Garlic, antioxidant.     

Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1α,25 dihydroxy-vitamin D₃. Recent in vitro studies suggested that curcumin and polyunsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin and PUFAs would induce expression of known VDR target genes in cells. In this study, we tested whether these compounds regulated two important VDR target genes — human cathelicidin antimicrobial peptide (CAMP) and 1,25-dihydroxyvitamin D₃ 24-hydroxylase (CYP24A1) — in human monocytic cell line U937, colon cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do not function as ligands for the VDR.     

Increased sensitivity to dietary cholesterol in diabetic and hypothyroid rats associated with low levels of hepatic HMG-CoA reductase expression

We recently postulated that hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase functions as a cholesterol buffer to protect against the serum and tissue cholesterol raising action of dietary cholesterol. This postulate predicts that diminished basal expression of hepatic HMG-CoA reductase results in increased sensitivity to dietary cholesterol. Because diabetic and hypothyroid animals are known to have markedly reduced hepatic HMG-CoA reductase, these animals were selected as models to test our postulate. When rats were rendered diabetic with streptozotocin, their hepatic HMG-CoA reductase activity decreased from 314 to 22 pmol. min(-1). mg(-1), and their serum cholesterol levels increased slightly. When the diabetic animals were challenged with a diet containing 1% cholesterol, their serum cholesterol levels doubled, and their hepatic reductase activity decreased further to 0.9 pmol. min(-1). mg(-1). Hepatic low-density lipoprotein (LDL) receptor immunoreactive protein levels were unaffected in the diabetic rats whether fed cholesterol-supplemented diets or not. In rats rendered hypothyroid by thyroparathyroidectomy, serum cholesterol levels rose from 100 to 386 mg/dl in response to the 1% cholesterol challenge, whereas HMG-CoA reductase activity dropped from 33.8 to 3.4 pmol. min(-1). mg(-1). Hepatic LDL receptor immunoreactive protein levels decreased only slightly in the hypothyroid rats fed cholesterol-supplemented diets. Taken together, these results show that rats deficient in either insulin or thyroid hormone are extremely sensitive to dietary cholesterol largely due to low basal expression of hepatic HMG-CoA reductase.     

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.     

Dietary beta- and gamma-cyclodextrins stimulation of hepatic metallothionein gene expression in rats

This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, beta- and gamma-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the gamma-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both beta- and gamma-cyclodextrins-fed rats, but not in alpha-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver.     

IL-1beta and IL-6 modulate apolipoprotein E gene expression in rat hepatocyte primary culture

Incubation of rat hepatocytes in primary culture with IL-1beta at a concentration of 2.5 units/ml resulted in an increase (+80%) in the amount of apoE mRNA without any effect upon apoE synthesis. IL-6 at a low concentration (10 units/ml) induced a decrease (-35%) in the amount of apoE mRNA, but increased apoE synthesis (+28%). No effect was observed with higher concentrations of IL-1beta (10 units/ml) or IL-6 (100 units/ml). These results suggest that inflammatory cytokines IL-1beta and IL-6 modulate the expression of apoE gene in cultured rat hepatocytes, at a concentration that does not induce the acute phase response.     

The extraordinary antioxidant activity of vitamin E phosphate

The antioxidant activities of RRR-vitamin E (VE), all-rac-vitamin E (all-rac-VE), trolox, RRR-vitamin E acetate (VEA), all-rac-vitamin E phosphate (VEP) and RRR-vitamin E succinate (VES) were compared. In this study, the rank order in the inhibition of lipid peroxidation (LPO) of VE and its derivatives was trolox>VE approximately all-rac-VE>VEA>VES. VE and trolox inhibited LPO in non-heated and heated rat liver microsomes. It has generally been accepted that this is due to scavenging of free radicals by these antioxidants, and during this protection the antioxidants are oxidized. VEA and VES have to be converted into VE by esterases to obtain antioxidant activity against LPO. VEP, however, had a potent antioxidant effect of its own without conversion to VE. In contrast to VE, VEP is not consumed during this protection. Of the compounds tested, VEP is the most potent in induction of hemolysis of erythrocytes. EPR experiments using the spin label 16-doxylstearic acid showed that VEP reduces membrane fluidity, in contrast to VE. This indicates that VEP acts as a detergent and forms a barrier that might inhibit the transfer of radicals from one polyunsaturated fatty acid to another. This new mechanism may form the basis for a new class of antioxidants.     

小麦维生素E基因TaHGGT-7AL的克隆与表达分析

从小麦( Triticum aestivum L.)品种‘科农199’籽粒中克隆出维生素E基因 TaHGGT-7AL 及其另外两个拷贝,通过生物信息学分析,对其序列结构特征及蛋白序列的系统发育关系进行了初步研究。结果显示: TaHGGT- 7AL 基因编码区长1227 bp,共编码408个氨基酸;TaHGGT-7AL与另外两个拷贝的序列一致性为95. 45%。TaHGGT-7AL蛋白序列具有9个α-螺旋,该序列与禾本科HGGT蛋白的同源性在58. 7%~ 98. 5%之间。3个 TaHGGT 基因分别位于小麦基因组的7AL、7BL和7DL染色体上,均具有与膜相关的UbiA异戊烯基转移酶家族的保守结构域和一个转运肽。系统进化分析结果表明,TaHGGT-7AL与禾本科植物的亲缘关系较近。qRT-PCR分析结果显示, TaHGGT-7AL 只在小麦颖壳和籽粒中表达,且在花后13 d籽粒的表达量最高。在ABA、4℃低温、干旱及黑暗胁迫处理下, TaHGGT-7AL 表达量上调;NaCl处理24 h后,该基因表达量升高,表明 TaHGGT-7AL 可以对非生物胁迫产生响应。     

小麦维生素E基因TaHGGT-7AL的克隆与表达分析

从小麦（Triticum aestivum L.）品种‘科农199’籽粒中克隆出维生素E基因TaHGGT-7AL及其另外两个拷贝,通过生物信息学分析,对其序列结构特征及蛋白序列的系统发育关系进行了初步研究。结果显示：TaHGGT-7AL基因编码区长1227 bp,共编码408个氨基酸;TaHGGT-7AL与另外两个拷贝的序列一致性为95.45%。TaHGGT-7AL蛋白序列具有9个α-螺旋,该序列与禾本科HGGT蛋白的同源性在58.7%~98.5%之间。3个TaHGGT基因分别位于小麦基因组的7AL、7BL和7DL染色体上,均具有与膜相关的UbiA异戊烯基转移酶家族的保守结构域和一个转运肽。系统进化分析结果表明,TaHGGT-7AL与禾本科植物的亲缘关系较近。qRT-PCR分析结果显示,TaHGGT-7AL只在小麦颖壳和籽粒中表达,且在花后13 d籽粒的表达量最高。在ABA、4℃低温、干旱及黑暗胁迫处理下,TaHGGT-7AL表达量上调;NaCl处理24 h后,该基因表达量升高,表明TaHGGT-7AL可以对非生物胁迫产生响应。     

Dietary supplementation of vitamin A, C and E prevents p-dimethylaminoazobenzene induced hepatic DNA damage in rats

The preventive effect of antioxidant vitamins A, C, E and their analogues against DNA damage induced by a hepatocarcinogen p-dimethylaminoazobenzene (DAB) was assessed by comet assay. For genotoxicity (DNA damage) study, male albino rats were divided into 11 groups, consisting of four rats each. Group I served as control. Group II to VII received 1, 10, 100, 200, 300 and 400 mg per kg body wt of DAB respectively; group VIII to XI received 500 mg/kg body wt of DAB. They were sacrificed by cervical decapitation 3, 6, 12 and 24 h after treatment; livers were excised immediately and subjected to comet assay to measure DNA damage. To study the effect of vitamins, experiments were conducted on a group of 275 rats divided into 3 sets of 25 rats each. First set served as control; second set received 0.06% DAB and third set received 0.06% DAB, along with analogues of vitamins A, C and E. Rats fed with 0.06% DAB were provided water ad libitum for a period of 4 months, followed by a normal (basal) diet for further 2 months. Vitamins A (10,000-50,000 IU), C (75-1000 mg) and E (50-500 mg) and their analogues were given (per kg body wt) to the third set of rats by gavage route once in a week for a period of 6 months. The DAB induced DNA damage only at the highest tested dose of 500 mg/kg body wt. Administration of high doses of vitamin A acid, L-ascorbic acid and vit. E succinate individually prevented the DNA damage. However, administration of a mixture of these vitamins at low doses prevented the DAB-induced DNA damage, which may be due to their synergistic effect. The results indicate that there is a significant advantage in mixed vitamins therapy at low dose over the treatment with individual vitamins.     

Curcumin influences hepatic expression patterns of matrix metalloproteinases in liver toxicity

Hepatic fibrosis is a result of an imbalance between enhanced matrix synthesis and diminished breakdown of connective tissue proteins, the net result of which is increased deposition of Extra Cellular Matrix. In this concept Matrix Metalloproteinases play an important role because their activity is largely responsible for extra cellular matrix breakdown. In the present study we have tested the influence of curcumin, the active principle of turmeric, on matrix metalloproteinase expression during alcohol and thermally oxidised sunflower oil induced liver toxicity. Male albino Wistar rats were used for the study. The matrix metalloproteinase expressions were found to be increased significantly in alcohol as well as thermally oxidised sunflower oil groups and on treatment with curcumin there was a significant decrease. In alcohol + thermally oxidised sunflower oil group, we found a significant decrease in matrix metalloproteinase activities. Administration of curcumin significantly improved their activities. From the results obtained, we could conclude that curcumin influences the hepatic matrix metalloproteinases and effectively protects liver against alcohol and delta PUFA induced toxicity.     

Simvastatin and vitamin E effects on cardiac and hepatic oxidative stress in rats fed on high fat diet

High fat diet (HFD) is a common cause of metabolic syndrome and type 2 diabetes mellitus. Published data showed that HFD and subsequent dyslipidemia are major triggers for oxidative stress. Forty-eight male Sprague–Dawley rats, weighing 170–200 g, were divided into six groups: control, control with vitamin E (100 mg/kg/day, i.p.), control with simvastatin (SIM) (10 mg/kg of body weight/day), HFD, HFD with vitamin E, and HFD with SIM. Standard and high cholesterol diets were given for 15 weeks and SIM and vitamin E were added in the last 4 weeks. In all rats, serum vitamin E, total cholesterol (TC), triglycerides (TG), low (LDL) and high (HDL) density lipoproteins, alanine (ALT) and aspartate (AST) transaminases, alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (GGT) as well as cardiac and hepatic thiobarbituric acid-reactive substances (TBARS) and antioxidants (reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT)) were measured. Also, electrocardiogram (ECG) was recorded. HFD significantly increased QTc interval, heart rate (HR), serum TC, TG, LDL, ALT, AST, ALP, GGT, liver TG, and cardiac and hepatic TBARS but decreased antioxidants and HDL, while SIM decreased HR, liver TG, serum TC, TG, and LDL and increased HDL in HFD rats. Vitamin E had no effect. Moreover, SIM and vitamin E decreased QTc interval, serum ALT, AST, ALP, GGT, and cardiac and hepatic TBARS and increased antioxidants in HFD rats. Histopathological observations confirm the biochemical parameters. SIM and vitamin E slow progression of hypercholesterolemia-induced oxidative stress in liver and heart and improve their functions.