RESUMEN
Endoplasmic reticulum stress (ERS) plays a key role in alcohol liver injury (ALI). Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is a potential modifier of ERS. It was examined whether the protective effect of Qinggan Huoxue Recipe (QGHXR) against ALI was associated with LPCAT3 by suppressing ERS from in vivo and in vitro experiment. Male C57BL/6 mice were randomly divided into five groups (n = 10, each) and treated for 8 weeks as follows: the control diet-fed group (pair-fed), ethanol diet-fed group (EtOH-fed), QGHXR group (EtOH-fed + QGHXR), Qinggan recipe group (EtOH-fed + QGR), and Huoxue recipe group (EtOH-fed + HXR). QGHXR, QGR, and HXR groups attenuated liver injury mainly manifested in reducing serum ALT, AST, and liver TG and reducing the severity of liver cell necrosis and steatosis in ALI mouse models. QGHXR mainly inhibited the mRNA levels of Lxrα, Perk, Eif2α, and Atf4 and activated the mRNA levels of Lpcat3 and Ire1α, while inhibiting the protein levels of LPCAT3, eIF2α, IRE1α, and XBP1u and activating the protein levels of GRP78 to improve ALI. QGR was more inclined to improve ALI by inhibiting the mRNA levels of Lxrα, Perk, Eif2α, Atif4, and Chop and activating the mRNA levels of Lpcat3 and Ire1α while inhibiting the protein levels of LPCAT3, PERK, eIF2α, IRE1α, and XBP1u. HXR was more inclined to improve ALI by inhibiting the mRNA levels of Perk, Eif2α, Atf4, and Chop mRNA while inhibiting the protein levels of LPCAT3, PERK, eIF2α, IRE1α, and XBP1u and activating the protein levels of GRP78. Ethanol (100 mM) was used to intervene HepG2 and AML12 to establish an ALI cell model and treated by QGHXR-, QGR-, and HXR-medicated serum (100 mg/L). QGHXR, QGR, and HXR groups mainly reduced the serum TG level and the expression of inflammatory factors such as IL-6 and TNF-α in the liver induced by ethanol. In AML12 cells, QGHXR and its disassembly mainly activated Grp78 mRNA expression together with inhibiting Lxrα, Lpcat3, Eif2α, Atf4, and Xbp1 mRNA expression. The protein expression of eIF2α and XBP1u was inhibited, and the expression of PERK and GRP78 was activated to alleviate ALI. In HepG2 cells, QGHXR mainly alleviated ALI by inhibiting the mRNA expression of LPCAT3, CHOP, IRE1α, XBP1, eIF2α, CHOP, and IRE1α protein. QGR was more inclined to inhibit the protein expression of PERK, and HXR was more likely to inhibit the protein expression of ATF4.
RESUMEN
AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF). METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-ß1/Smad signaling pathway, including TGF-ß1, Smad3, snail, occludin, ZO-1 and claudin, was also examined. RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 ± 0.05 vs control 1.00 ± 0.08; protein: ALF 0.09 ± 0.05 vs control 0.70 ± 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 ± 0.39 vs 1.00 ± 0.19 and 9.91 ± 0.34 vs 1.00 ± 0.44, respectively, P < 0.01; protein: 1.13 ± 0.42 vs 0.09 ± 0.03 and 1.16 ± 0.43 vs 0.09 ± 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-ß1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-ß1 and snail levels (mRNA: 1.76 ± 0.12 vs 1.00 ± 0.05 and 6.98 ± 0.41 vs 1.00 ± 0.10, respectively, P < 0.01; protein: 1.43 ± 0.05 vs 0.12 ± 0.03 and 1.07 ± 0.29 vs 0.07 ± 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 ± 0.01 vs 1.00 ± 0.12, P < 0.01; protein: 0.06 ± 0.05 vs 0.89 ± 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 ± 0.09 vs 1.00 ± 0.12, 0.11 ± 0.00 vs 1.00 ± 0.12 and 0.60 ± 0.01 vs 1.00 ± 0.08, respectively, P < 0.01; protein: 0.05 ± 0.01 vs 0.87 ± 0.40, 0.09 ± 0.05 vs 0.89 ± 0.18 and 0.04 ± 0.03 vs 0.95 ± 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 ± 0.04 vs ALF model 0.16 ± 0.05, P < 0.01; protein: QGHXR 0.66 ± 0.21 vs ALF model 0.09 ± 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 ± 1.05 vs 11.43 ± 0.39 and 1.45 ± 1.51 vs 9.91 ± 0.34, respectively, P < 0.01; protein: 0.09 ± 0.03 vs 1.13 ± 0.42 and 0.10 ± 0.01 vs 1.16 ± 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-ß1 and increased the expression of Smad3 (mRNA: 1.03 ± 0.11 vs 1.76 ± 0.12, 0.70 ± 0.10 vs 0.05 ± 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 ± 0.03 vs 1.43 ± 0.05 and 0.88 ± 0.20 vs 0.06 ± 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 ± 0.33 vs 6.98 ± 0.41, P < 0.01; protein: 0.08 ± 0.02 vs 1.07 ± 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 ± 0.05 vs 0.60 ± 0.09, 0.57 ± 0.04 vs 0.11 ± 0.00 and 0.68 ± 0.03 vs 0.60 ± 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 ± 0.50 vs 0.05 ± 0.01, 0.94 ± 0.22 vs 0.09 ± 0.05 and 0.94 ± 0.29 vs 0.04 ± 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-ß1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-ß1 mRNA levels were not significant. CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-ß1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.