Autophagy protects against palmitate-induced apoptosis in hepatocytes

Abstract

Background: Non-alcoholic fatty liver disease, one of the most common liver diseases, has obtained increasing attention. Palmitate (PA)-induced liver injury is considered a risk factor for the development of non-alcoholic fatty liver disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism in response to various stresses. In this study, we investigated whether autophagy plays a protective role in the progression of PA-induced hepatocytes injury.

Results: Annexin V-FITC/PI staining by FCM analysis, TUNEL assay and the detection of PARP and cleaved caspase3 expression levels demonstrated that PA treatment prominently induced the apoptosis of hepatocytes. Meanwhile, treatment of PA strongly induced the formation of GFP-LC3 dots, the conversion from LC3I to LC3II, the decrease of p62 protein levels and the increase of autophagosomes. These results indicated that PA also induced autophagy activation. Autophagy inhibition through chloroquine pretreatment or Atg5shRNA infection led to the increase of cell apoptosis after PA treatment. Moreover, induction of autophagy by pretreatment with rapamycin resulted in distinct decrease of PA-induced apoptosis. Therefore, autophagy can prevent hepatocytes from PA-induced apoptosis. In the further study, we explored pathway of autophagy activation in PA-treated hepatocytes. We found that PA activated PKCα in hepatocytes, and had no influence on mammalian target of rapamycin and endoplasmic reticulum stress pathways.

Conclusions: These results demonstrated that autophagy plays a protective role in PA-induced hepatocytes apoptosis. And PA might induce autophagy through activating PKCα pathway in hepatocytes.

Keywords: Apoptosis; Autophagy; Hepatocytes; Palmitate; Protector.

Figure 1

PA stimulates apoptosis in hepatocytes. (A) HL-7702 and HepG2 cells were treated with either control or PA (100 μM, 250 μM, 500 μM) for 24 hours. Cell viability was detected by CCK-8 assay. Respectively, at 0, 6, 12, 24 time points, PA (500 μM) and CCK-8 assay were used (*p < 0.05; **p < 0.01). (B) DNA fragmentation detection kit was used to deal with cells after treatment with control or PA for 24h, then cells were observed under a confocal microscopy (bar: 50 μm). (C) The ratio was calculated by counting the percentage of cells exhibiting positive TUNEL staining. Quantization was measured for the three times from the three times independent TUNEL assay (*p < 0.05; **p < 0.01). (D) Western blot analysis detected PARP and Cleaved-caspase3 proteins levels in cells after treatment with control or PA for 24 hours. (E) Cells were dealt with control or PA for 24 hours, and stained with AnnexinV-FITC and PI, and then apoptotic cells were quantified by flow cytometry (FCM). Numbers within quadrants represent the percentages of cells in early apoptosis ( AnnexinV + PI - ; lower right ) and in late apoptosis and necrosis (AnnexinV + PI + ; upper right ).

Figure 2

PA stimulates autophagy activation in hepatocytes. (A) HL-7702 cells were treated with control or PA for 24 hours, and plasmids of GFP-LC3 were transfected into the cells. Cells were observed under fluorescence microscope (bar: 20μm). Quantization was obtained by calculating the ratio of cells with GFP-LC3 dots in one visual field and experiments were repeated three times (*p < 0.05; **p < 0.01). (B) LC3 and P62 protein levels were detected by western blotting analysis after treatment of control or PA for 8 hours. (C) Cells were treated with control or PA for 8 hours before being processed, then electron microscope was performed at 11,500× and 29,500× magnification. The black arrows show membrane-bound vacuoles characteristic of autophagosomes. The number of autophagosomes per cell was quantitated. Date were presented as the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01).

Figure 3

Decreased autophagy level augments apoptosis of PA-induced in hepatocytes. (A) Cells were treated with either control or PA for 24h. CQ (10 μM) was added to pretreatment for 8 hours. Western blotting was used for detecting LC3 and cleaved-caspase3 levels. (B) The relative LC3-II/GAPDH was calculated by normalizing their respective levels to the control level in cells. (C) The relative cleaved-caspase3/GAPDH was quantified in the same way. Date were presented as the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01). (D) Cells were quantified of the viability using CCK-8 assay after treatment with control or PA for 24h. CQ (10 μM) was also added to pretreatment for 8 hours. Data were repeated in three independent experiments and as the mean ± SEM (*p < 0.05; **p < 0.01). (E) Cells were treated in the same way, and then apoptotic cells were quantified by FCM after staining with AnnexinV-FITC and PI. The data represent the mean ± SEM values from three times separately (*p < 0.05; **p < 0.01). (F) Atg5 was knocked down with Atg5 shRNA infection in HL-7702 and HepG2 cells, then western blotting analysis were performed. (G) After cultured with control or PA for 24h, normal cells and the transfected cells were used to perform the apoptosis analysis by FCM. The data were expressed as the mean ± SEM values for three independent experiments (*p < 0.05; **p < 0.01).

Figure 4

Elevated autophagy level by rapamycin reduces apoptosis of PA-induced in hepatocytes. (A) Cells were treated with either control or PA (500 μM) for 24h, Rapa (250 nM) was added to pretreatment for 8 hours, then western blotting analysis tested LC3 and cleaved caspase3 expressions levels. (B) The quantization of relative LC3-II/GAPDH from the treatment groups were calculated by normalizing to the control groups. (C) Relative cleaved-caspase3/GAPDH was calculated in the same way. Data were presented as mean ± SEM from three independent experiments (*p < 0.05; **p < 0.01). (D) Cells were treated with control, PA, Rapa (250 nM) and PA with the addition of Rapa pretreatment for 24 hours, then cell viability was detected by CCK-8 analysis. Data were mean ± SEM from the three times independent experiments (*p < 0.05; **p < 0.01). (E) Cells were stained with AnnexinV-FITC/PI after treatment as above, and were measured by FCM. The results were presented as mean ± SEM values at least three independent experiments (*p < 0.05; **p < 0.01). (F) HL-7702 cells were treated with various strategies, and then CCK-8 detected cell proliferation. (G) The result of cell proliferation in HepG2 cells was shown. Date were presented as the mean ± SEM values for three independent experiments (*p < 0.05; **p < 0.01).

Figure 5

PA induces PKCα activation, but has no influenced on mTOR and ER stress pathway in hepatocytes. (A) The involvement of PA in mTOR signaling was analyzed by western blotting analysis after the treatment with either control or PA for 8 hours. Cells treated with Rapa (250 nM) for 8 hours were used as a positive control. (B) Western blot analysis was applied to detecting Grp78 and HSP70 proteins levels in cells after treated with Control or PA for 8 hours. (C) P-PKCα was measured by western blotting after with treatment as above.