Effects of Tiaozhi Granule on Regulation of Autophagy Levels in HUVECs

Abstract

Sera from the rats with Tiaozhi granule treatment were collected. Human umbilical vein endothelial cells (HUVECs) were incubated with different dosage of sera with Tiaozhi granule for 48 hours. Rapamycin or angiotensin II was applied to activate autophagy in HUVECs with or without different dosages of sera of Tiaozhi granule. The mRNA expressions of Atg5, Atg7, Beclin-1, and mammal target of rapamycin (mTOR) were detected by real-time PCR. Autophagic flux markers (protein expression of LC3, Beclin-1, and p62) were examined by western blot analyses. The number of autophagosomes was visualized by immunofluorescence analysis with LC3-II labelling. Results showed that Tiaozhi granule sera increase cell autophagic levels by increase of mRNA of Atg5, Atg7, Beclin-1, and mTOR and increase of autophagic flux and also number of autophagosomes. However, in response to rapamycin or Ang II stimulation, activated autophagic levels were alleviated by Tiaozhi granule sera by reduction of mRNA of Atg5, Atg7, Beclin-1, mTOR, autophagic flux, and also number of autophagosomes. Our present data demonstrate that Tiaozhi granule plays a dual role in response to different cell conditions, which is to increase cell autophagy under physiological condition and to suppress cell excessive autophagy under pathological condition.

Figure 1

Effects of Tiaozhi granule on HUVECs viability. HUVECs were incubated with different concentration of Tiaozhi granule sera for 48 hours (a) or were treated with rapamycin (Rapa) for 48 hours (b) or Ang II for 24 hours (c) under different concentration of Tiaozhi granule sera. Cell viability was analyzed by MTT assay. Data from each group is expressed as absorbance of each well at 570 nm and is presented as mean ± SEM (n = 8).

Figure 2

Effects of Tiaozhi granule on autophagy level in HUVECs. (a) Effect of Tiaozhi granule on Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on LC3 protein expression (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on p62 protein expression (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗  P < 0.05 compared with control group.

Figure 3

Immunofluorescent analysis of Tiaozhi granule on the formation of autophagosomes. (a) Representative images of immunofluorescent detection of LC3-II. Nucleus was stained with DAPI in blue color. Autophagosomes were observed with LC3-II protein expression. Green dots in the cell cytosol indicate autophagosomes (marked with white arrows). Magnification of the image is 400X. (b) Numbers of autophagosomes in HUVECs within each treatment group were counted. Data are presented as mean ± SEM (n = 8). ∗p <0.05 versus control.

Figure 4

Effects of Tiaozhi granule on rapamycin activated autophagy in HUVECs. (a) Effect of Tiaozhi granule on rapamycin induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on rapamycin induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on rapamycin induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on rapamycin induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on rapamycin induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on rapamycin induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on rapamycin induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗  P < 0.05 compared with control group. Δ  P < 0.05 compared with rapamycin group.

Figure 5

Immunofluorescent analysis of Tiaozhi granule on rapamycin induced formation of autophagosomes. (a) Representative images of immunofluorescent detection of LC3-II. Nucleus was stained with DAPI in blue color. Autophagosomes were observed with LC3-II protein expression. Green dots in the cell cytosol indicate autophagosomes (marked with white arrows). Magnification of the image is 400X. (b) Numbers of autophagosomes in HUVECs within each treatment group were counted. Data are presented as mean ± SEM (n = 8). ∗p <0.05 versus control. ΔP < 0.05 compared with rapamycin group.

Figure 6

Effects of Tiaozhi granule on Ang II-induced autophagy in HUVECs. (a) Effect of Tiaozhi granule on Ang II-induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Ang II-induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Ang II-induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on Ang II-induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on Ang II-induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Ang II-induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on Ang II-induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗  P < 0.05 compared with control group. Δ  P < 0.05 compared with rapamycin group.

Figure 7

Immunofluorescent analysis of Tiaozhi granule on Ang II-induced formation of autophagosomes. (a) Representative images of immunofluorescent detection of LC3-II. Nucleus was stained with DAPI in blue color. Autophagosomes were observed with LC3-II protein expression. Green dots in the cell cytosol indicate autophagosomes (marked with white arrows). Magnification of the image is 400X. (b) Numbers of autophagosomes in HUVECs within each treatment group were counted. Data are presented as mean ± SEM (n = 8). ∗p <0.05 versus control. ΔP < 0.05 compared with rapamycin group.