A novel function of bamboo extract in relieving lipotoxicity

Abstract

Lipotoxicity is closely related to the etiology and complications of type 2 diabetes mellitus. This study investigated the protective effect of an extract from bamboo Phyllostachys edulis against palmitic acid (PA)-induced lipoapoptosis. The lipo-detoxification function of the bamboo extract (BEX) was evaluated using cell culture models. Cell viability was measured by MTT assay and cell apoptosis was monitored by Annexin V staining. Cellular uptake of fluorescent free fatty acid (FFA) analog was measured by flow cytometry. Protein levels of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) were measured by western blotting. The results show that co-incubating BEX with mouse myoblast C2C12 cells had no effect on the cellular uptake of FFA, but dramatically decreased PA-induced cell apoptosis and protected cell viability. A similar antilipotoxicity effect of BEX was observed in other mammalian cells. BEX significantly decreased the protein levels of both Akt and p-Akt in C2C12 cells under normal cell culture conditions but not under lipotoxic conditions, indicating the regulatory effect of BEX on cell signaling pathways and its response to a high FFA environment. This study demonstrated a novel function of bamboo extract in preventing lipotoxicity in mammalian cells, implicating a promising phytotherapeutic approach for lipo-detoxification.

Figure 1

The morphology of C2C12 cells after exposure to 0.4 mM PA for 26 h. (A) PA free medium + 0.3% (v/v) ethanol as control; (B) 0.4 mM PA + 0.3% ethanol; (C) 0.4 mM PA + 0.3% BEX; (D) 0.4 mM PA + 0.5% BEX.

Figure 2

The MTT-based cell viability of C2C12, myotubes differentiated from C2C12 and Vero cells after PA treatment. (A) C2C12 cells exposed for 26 h to 0.4 mM PA in combination with 0–0.5% (v/v) of BEX or ethanol; PA-free medium + 0.3% BEX or ethanol were used as control. Average ± SD, n = 3. Samples treated with BEX were compared with those treated with ethanol at the same concentration. (B) Myotubes differentiated from C2C12 cells and Vero cells were treated for 24 h with PA-free media, media containing 0.4 mM PA, or 0.4 mM PA in combination with 0.5% BEX or DMSO. Average ± SD, n = 3. Samples treated with PA and BEX are compared with those treated with PA and DMSO.

Figure 3

The apoptosis assay on C2C12 cells after treated by 0.4 mM PA for 16 h in the presence of 0.2% to 0.5% of BEX or ethanol (control). The cells were stained by Annexin V labeled with fluorophore and the fluorescence intensity of the cells was measured by flow cytometer. The histogram in Panel A shows that 30.5% of the control cells treated with PA and 0.5% ethanol became apoptotic as indicated by region M1. Replacing ethanol with 0.5% BEX reduced the apoptotic rate to less than 5.6% (Panel B). The results are summarized in Panel C, average ± SD, n = 3. Samples treated with BEX were compared with those treated with ethanol at the same concentration.

Figure 4

Influence of BEX on cellular properties of C2C12 cells. (A) BEX did not alter the cellular uptake of FFA. C2C12 cells were incubated with BLLA in the presence of 0.5% BEX or ethanol for 1 and 4 h. The intracellular level of BLLA is indicated by the mean fluorescent intensity. The background was adjusted with DMSO treated cells. Average ± SD, n = 3. (B, C) BEX significantly decreased the protein level of total Akt and phosphorylated Akt in C2C12 cells under normal cell culture condition. C2C12 cells were incubated with normal medium containing 0.5% BEX or 0.5% ethanol for 4 h. The protein levels of total Akt and p-Akt were normalized against alpha-tubulin. Average ± SD, n = 3. (D, E) BEX tended to increase the protein level of total Akt and phosphorylated Akt in C2C12 cells under lipotoxic condition. C2C12 cells were incubated with medium containing 0.4 mM PA in the presence of 0.5% BEX or 0.5% ethanol for 4 h. The protein levels of total Akt and p-Akt were normalized against alpha-tubulin. Average ± SD, n = 3.