Acid sphingomyelinase as target of Lycium Chinense: promising new action for cell health

Abstract

Background: Sphingomyelin plays very important roles in cell function under physiological and pathological conditions. Physical and chemical stimuli produce reactive oxygen species that stimulate acid sphingomyelinase to induce apoptosis. Antioxidant plants of the traditional Chinese Pharmacopoeia, such as Lycium Barbarum and Lycium Chinense, have become increasingly popular in Western countries. We investigated the effects of Lycium Chinense on acid sphingomyelinase and sphingomyelin species in relation to gene expression.

Methods: We prepared Lycium Chinense berry extracts and evaluated their antioxidant properties. Increasing amount of extracts was used to test cytotoxic and genotoxic effect on HepG2 cells. Gene expression, protein amount and enzyme activity of acid sphingomyelinase were tested by RT-PCR, immunoblotting and enzymatic activity assay, respectively. Sphingomyelin species were analyzed by UFLC MS/MS. A panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used to test the effect of LC on gene expression. GLRX2, RNF7, and PTGS1 proteins were analyzed by immunoblotting.

Results: We showed that Lycium Chinense berries have high antioxidant properties, have an IC50value of 9.55 mg/mL, do not induce genotoxic effect and maintain high level of cell viability. The berry extracts inhibit acid sphingomyelinase activity and increase both very long fatty acid sphingomyelin species and unsaturated fatty acid sphingomyelin species. Among 96 genes, Lycium Chinense berries up-regulate Glutaredoxin 2 and Ring Finger Protein 7 genes and proteins, able to protect cells from apoptosis. Intrigantly, Lycium Chinense berries down-regulates Prostaglandin H synthase 1 gene but the protein is not expressed in HepG2 cells.

Conclusion: The results identify acid sphingomyelinase as a novel target of Lycium Chinense berries to decrease saturated/unsaturated fatty acid sphingomyelin ratio, known to be useful for cell health. Consistent with these data, the berries regulate specifically gene expression to protect cells from apoptosis.

Keywords: Antioxidant; Fatty acids; Lycium chinense; Sphingomyelin; Sphingomyelinase.

Fig. 1

Antioxidant properties of LC berries. Total phenolic content (TPC) and antioxidant capacity (ORAC) were tested as reported in Methods. Bow milk, negative control; LB berry extract, positive control. Data are expressed as mean ± SD of three independent experiments performed in duplicate. Left ordinate, TPC; right ordinate, ORAC. (Significance, *P < 0.001 versus bow milk)

Fig. 2

Effect of LC berry extract on HepG2 cells. a cell viability, measured by MTT assay; b genotoxic effects, valuated by Comet Assay. In a the values are reported as % viability of the control sample, and in b the values are reported as % tail intensity with cells in MEM as negative control and cells in 100 mM 1,2,4-benzentriol (BT) as positive control. Data are expressed as mean ± SD of three independent experiments performed in duplicate

Fig. 3

Effect of LC berry extract on acid sphingomyelinase. a gene expression evaluated by RT-PCR as fold of increase of mRNA expression relative to control sample. b immunoblotting; the position of the 70 kDa protein for aSMase and 48 kDa for beta-tubulin was indicated in relation to the position of molecular size standards. c area density evaluated by densitometry scanning and analyzed with Scion Image. d enzymatic activity; the data are expressed as pmol/mg protein/min. e enzymatic activity/ area density; the data are expressed as pmol/mg protein/min referred to area density of immunoblotting analyzed as reported in c. Data are expressed as area/mg protein. (Significance, *P < 0.001 versus control sample)

Fig. 4

Effect of LC berry extract on sphingomyelin species. a sphingomyelin (SM) species studied by using 12:0 SM, 16:0 SM, and 18:1 SM external calibrators. Data are expressed as nmol/mg protein. Left ordinate, 16:0 SM, and 18:1 SM; right ordinate, 12:0 SM. b SM species studied by evaluating the areas of all the peaks identified on the basis of their molecular weight. Data are expressed as area/mg protein c total saturated and unsaturated fatty acids (FAs) and saturated/unsaturated ratio. Data are expressed as area/mg protein. d total monounsaturated and polyunsaturated FAs. Data are expressed as area/mg protein. Data are expressed as area/mg protein. (Significance, *P < 0.001 versus control sample)

Fig. 5

Effect of LC berry extract on gene and protein expression of HepG2 cells. The study was performed in control (without LC) and experimental cells (with LC) collected after 24 h of culture. a RTqPCR analysis of a panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used. The results are normalized with the levels of the GAPDH and express as mRNA of experimental sample versus control sample. b immunoblotting analysis, the position of the 15 kDa protein for GLRX2, 13 kDa for RNF7, 69 kDa for PTGS1, and 48 kDa for beta-tubulin is indicated in relation to the position of molecular size standards. c area density is evaluated by densitometry scanning and analyzed with Scion Image. The results of GLRX2 and RNF7 are normalized with the levels of beta-tubulin. Data are expressed as the mean ± S.D. of 3 independent experiments performed in threeplicates. (Significance, *P < 0.001 versus control sample)