Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress

Figure 1 Cryptotanshinone reduces collagen deposition and induces cell cycle arrest at the G2/M checkpoint. A: Chemical structure of cryptotanshinone; B: CCK8 assay for LX2 cell viability; C: CCK8 assay for cell viability of LO2; D: Protein levels of collagen I and α-SMA were determined using Western blot analysis; E: mRNA levels of α-SMA and collagen I were determined using reverse transcription polymerase chain reaction (RT-PCR); F: The percentage of cell cycle distribution was determined using flow cytometry. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01 versus control.

Figure 2 Cryptotanshinone induces apoptosis of activated hepatic stellate cells. A: TUNEL staining to assess LX2 apoptosis. Red fluorescence indicates apoptotic cells. Scale baes: 100μm; B: Western blot analysis of Bcl-2 and Bax protein expression in LX2 cells; C: Reverse transcription polymerase chain reaction analysis of Bcl-2 and Bax mRNA levels in LX2 cells; D: Flow cytometric analysis of LX2 cell apoptosis using fluorescein isothiocyanate-labeled Annexin-V/propidium iodide staining. Cells located in the right two quadrants of each figure were considered apoptotic cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 versus control.

Figure 3 Cryptotanshinone activates the endoplasmic reticulum stress pathway in activated LX2 cells. A: Western blot analysis of endoplasmic reticulum stress (ERS)-related protein expression in LX2 cells; B: Reverse transcription polymerase chain reaction (RT-PCR) analysis of Grp78 and CHOP mRNA levels in LX2 cells; C: Western blot analysis of ERS-related protein expression in LO2 cells; D: RT-PCR analysis of Grp78 and CHOP mRNA levels in LO2 cells; E and F: Western blot analyses of ATF4, PERK, and IRE1 signaling pathways in hepatic stellate cells; G: Transmission electron microscopy showing that CPT caused ER swelling and destruction of the mitochondrial membrane integrity. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01 versus control. ER: Endoplasmic reticulum.

Figure 4 The endoplasmic reticulum stress signaling pathway leads to LX2 cell apoptosis and reduces collagen deposition induced by cryptotanshinone. A: Protein expression of Grp78 and CHOP was determined using Western blot analysis; B: mRNA levels of Grp78 and CHOP were determined using reverse transcription polymerase chain reaction (RT-PCR); C: Protein expression of collagen type I and α-SMA was determined using Western blot analysis; D: mRNA levels of α-SMA and collagen I were determined using RT-PCR; E: TUNEL staining was used to assess LX2 cell apoptosis. Red fluorescence indicates apoptotic cells. Scale bars: 100 μm; F: Protein expression of Bax and Bcl2 was determined using Western blot analysis; G: mRNA levels of Bax and Bcl2 were determined using RT-PCR; H: Flow cytometry data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 versus control. CPT: Cryptotanshinone; FITC: Fluorescein isothiocyanate.

Figure 5 Cryptotanshinone protects the liver against carbon tetrachloride-induced injury and inflammation. Cryptotanshinone (CPT) alleviates hepatic fibrotic injury in mice. Mice were injected with carbon tetrachloride (CCL₄) for 8 wk to induce liver fibrosis. During weeks 5 through 8, mice in the treatment groups were given CPT (40 mg/kg) or salubrinal (1 mg/kg). A: CPT treatment protocol in the CCL₄-induced hepatic fibrosis mouse model; B: Western blot analysis of α-SMA and collagen I in the liver tissue; C: Determination of serum alanine aminotransferase levels; D: Determination of serum aspartate aminotransferase levels; E: Enzyme-linked immunosorbent assay (ELISA) measurement of IL-6 levels in the serum; F: ELISA measurement of IL-10 levels in the serum; G: Liver sections were stained with hematoxylin and eosin, Masson reagents, and Sirius red. Scale baes: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with the CCL₄ group. CPT: Cryptotanshinone; CCL₄: Carbon tetrachloride.