Effect of Turmeric, Turmerin and Curcumin on H2O2-Induced Renal Epithelial (LLC-PK1) Cell Injury

doi:10.1016/S0891-5849(97)00140-8

Free Radical Biology and Medicine

Volume 24, Issue 1, 1 January 1998, Pages 49-54

https://doi.org/10.1016/S0891-5849(97)00140-8 Get rights and content

Abstract

Cell injury by oxidative stress is an important mechanism for renal epithelial cell destruction. This study observed the protective effect of turmeric and its constituents on H₂O₂-induced injury. Turmeric consists of a water soluble turmerin and lipid soluble curcumin with potent antioxidant properties. Confluent LLC-PK₁ cells were labelled with ³H-arachidonic acid at 0.1 μCi/ml over 18 h and then further labelled with ⁵¹Cr. Turmeric (100 μg/ml-0.1 μg/ml), turmerin (800 ng/ml-0.8 ng/ml), curcumin (100 μg/ml-0.1 μg/ml), vitamin E (100 μM) and 21-aminosteroid (20 μM) were added and incubated for 3 h at 37°C in 24-well plate. The adherent cells were washed and incubated for 3 h with 1.5 mM H₂O₂ at 37°C. ³H-arachidonic acid release, ⁵¹Cr release and lipid peroxidation by the thiobarbituric acid reaction was determined. Turmeric (100 μg/ml) and curcumin (100 μg/ml, 10 μg/ml) gave as much protection as did vitamin E in both chromium release assay and lipid degradation while Turmeric (100 μg/ ml) and curcumin (100 μg/ml) gave comparable inhibition of lipid peroxidation. Turmerin and 21-aminosteroid showed no protection. These findings provide evidence that turmeric and curcumin provide protection against oxidative stress in a renal cell line.

Introduction

Turmeric powder (T) is a yellow powder, which comes from the plant Curcuma Longa. It has been traditionally used in India for food and medicinal purposes. In pharmacologic studies turmeric exhibits antitumor, anti-inflammatory, and anti-infectious activities with low toxicity.1, 2, 3, 4, 5Antioxidant properties have been associated with turmeric powder by itself and its individual lipid soluble component curcumin (Cu) and the aqueous extract turmerin (Tm).6, 7, 8, 9, 10, 11

Turmeric offers protection by inhibiting lipid peroxidation and scavenging free radicals.12, 13Reactive oxygen species (ROS) such as hydrogen-peroxide play a role in the pathogenesis of renal injury.14, 15, 16, 17, 18Injury may be partially caused by damage to membrane lipids,[19]cytoplasmic macromolecules[20]and DNA.21, 22Complete damage leads to tissue necrosis. Lipid peroxidation of cell membrane results in the production of a number of metabolites including malondialdehyde, conjugated dienes and ethane.23, 24These metabolites are markers for the extent of cell oxidation. Lipid peroxidation occurs due to the presence of polyunsaturated fatty acids (PUFA) and arachidonic acid.[25]Sheridan et al. showed that severity of H₂O₂-induced cell death is directly related to the cell membrane PUFA content and to the degree of lipid peroxidation.[26]Salahudeen[18]demonstrated that lipid peroxidation and degradation precedes H₂O₂-induced cytolysis and that α-tocopherol and 21-Aminosteroid ameliorate cell injury. α-Tocopherol and 21-Aminosteroid react with lipid radicals thereby terminating the chain reaction of lipid peroxidation.30, 31

We studied the efficacy of turmeric powder and its lipid soluble and water soluble components curcumin and turmerin respectively on protection of porcine renal tubular LLC-PK₁ epithelial cell from oxidative cell injury by H₂O₂. Employing turmeric, curcumin and turmerin as test reagents and with vitamin E (vit E ) and 21-aminosteroid (AS) as known antioxidants, we demonstrated that curcumin at 100 μg/ml concentration maximally inhibited lipid degradation, lipid peroxidation and cytolysis in H₂O₂-induced LLC-PK₁ cell injury model. This is the first report to describe the capacity of turmeric and its components to inhibit H₂O₂-induced injury in renal cells grown in culture.

Section snippets

Cell Culture

LLC-PK₁ cells, an analogue of proximal tubular epithelial cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C in 95% air-5% carbon dioxide. The medium was supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (25 μg/ml). Cells were grown in 24-well Costar plates and studied in pyruvate free Eagle’s basal medium (BME).[27]

Chemicals

Turmeric powder was

Results

The degree of cell injury was determined at 3 h which was found to be optimum by Salahudeen.[18]At 3 h, ⁵¹Cr release was 30.97 ± 0.58% in control cells vs. 78.42 ± 0.76% in H₂O₂ cells, ³H-arachidonic release was an average 529 ± 65 cpm in control cells vs. an average 1938 ± 300 cpm in H₂O₂ cells (365.83% of control), and TBARS level was 0.23 ± 0.02 nanomole/500 μl of supernatant in control vs. 0.58 ± 0.005 nanomole/500 μl of supernatant in H₂O₂ cells.

Discussion

Oxidative injury is associated with the generation of reactive oxygen species. In H₂O₂-induced renal epithelial LLC-PK₁ cell injury model, lipid peroxidation precedes H₂O₂-induced cytolysis.[18]By undertaking in vitro studies we were able to document whether turmeric or its constituents when compared to AS and vitamin E,30, 31, 32that are predominantly scavengers of lipid radicals, can be used as a protective agent against kidney epithelial cell injury. Because membranes are the richest source

Acknowledgements

We would like to thank Dr. Richard Miller for his valuable support, Dr. Peter Geisswein for reviewing the manuscript, and Heath Scott for final preparation of the manuscript. We also thank Dr. John McCall (Upjohn, Kalamazoo, MI) for providing the gift of 21-Aminosteroid.

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Original ContributionEffect of Turmeric, Turmerin and Curcumin on H2O2-Induced Renal Epithelial (LLC-PK1) Cell Injury

Abstract

Introduction

Section snippets

Cell Culture

Chemicals

Results

Discussion

Acknowledgements

Cancer Lett.

Cancer Lett.

Free Radic. Biol. Med.

Mutation Res.

J. Ethnopharmacol.

Arch. Biochem. Biophys.

Free Radic.Biol. Med.

J. Cell. Biol.

Kidney Int.

Free Radic. Biol. Med.

J. Ethnopharmacol.

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Mol. Cell. Biochem.

Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous ion

Mol. Cell. Biochem.

Effect of dietary turmeric (Curcuma longa) on iron-induced lipid peroxidation in the rat liver

Fd. Chem. Toxic.

Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

Cell Biophys.

Studies on curcumin and curcuminoids. XXIII: Effect of curcumin on liposomal lipid peroxidation

Int. J. Pharma.

Lipid peroxide induced DNA damage: Protection by turmeric (Curcuma longa)

Mol. Cell. Biochem.

Enhanced in vivo H2O2 generation by rat kidney cortex in glycerol-induced renal failure

Am. J. Physiol.

Original Contribution
Effect of Turmeric, Turmerin and Curcumin on H₂O₂-Induced Renal Epithelial (LLC-PK₁) Cell Injury

Enhanced in vivo H₂O₂ generation by rat kidney cortex in glycerol-induced renal failure