Protection of catalpol against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway

Material and methods

Cell cultures and reagents

HepaRG cells, a preferable cell model for drug-induced liver injury (DILI) (Wu et al., 2016), were used for in vitro experiments. The HepaRG cell line was purchased from Beina Chuanglian Biotechnology (Beijing, China). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics, and then incubated in a cell incubator containing 5% CO₂ at 37 °C.

CAT (purity ≥ 98.0%) and TP (purity ≥ 98.0%) were purchased from Yuanye Biotechnology (Shanghai, China). The stock solution of TP (20 mg/mL) was dissolved in dimethyl sulfoxide (DMSO) and diluted with basal medium to various concentrations (0, 5, 10, and 20 µg/L). CAT (20 mg/mL) dissolved in DMSO was diluted to various concentrations (0, 0.4, 4, 40, 400, 4,000, and 40,000 µg/L) with RPMI 1640.

Cell viability assay using cell counting kit 8 (CCK8)

Cell proliferation was assessed using the CCK8 (Beyotime, Shanghai, China) assay. To detect cell toxicity of TP, HepaRG cells (5 × 10⁴ cells/mL) were plated into 96-well plates for 24 h, and then treated with different doses of TP for 12, 24, 36, and 48 h. To detect the protective effect of CAT, the cells were pretreated with CAT (0, 0.008, 0.04, 0.2, and 1 µg/L) for 12 h, and then incubated with TP (20 µg/L) for 24 h. After incubating with 10 µL CCK8 solution for 3 h, the optical density value was determined at an excitation wavelength of 450 nm using a microplate reader (TECAN, Switzerland).

In addition, to evaluate the hepatotoxicity, the supernatant of cells incubated with TP/CAT was used to detect the total alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels using detection kits (Jiancheng Bioengineering, Nanjing, China).

Detection of autophagosomes by transmission electron microscope (TEM)

After incubation, the cells (3 × 10⁵ cells/mL) were treated with electron microscope fixative, fixed for 2 h at 25 °C, cut into small pieces (1 mm³), fixed, dehydrated, penetrated and embedded, sliced (1–2 µm) and axial lead stained. The specimens were observed by TEM.

To demonstrate the relationship between autophagy and apoptosis, the autophagy inhibitor 3-methyladenine (3-MA) (2.5 mM) and agonist rapamycin (RAPA) (50 nM) were used. Furthermore, the ERS inhibitor 4-phenylbutyric acid (4-PBA) (500 nM) and PERK inhibitor GSK2656157 (1 µM) (Bastida-Ruiz et al., 2019; Wang et al., 2020) were also used to study the relationship between PERK and autophagy.

Autophagosome detection by fluorescence microscope

Acidic autophagic vacuoles were measured using monodansylcadaverine (MDC) staining (Veeran et al., 2017) with an autophagy detection kit (KeyGEN, Nanjing, China). The cells (1 × 10⁵ cells/mL) were plated in 24-well plates and incubated with different concentrations of TP/CAT for 24 h at 37 °C. The cells were treated according to the manufacturer’s instructions and observed at an excitation wavelength of 355 nm using a fluorescence microscope (ZEISS, Thuringia, Germany).

Flow cytometry for apoptosis

The apoptotic ratio was detected using an Annexin V-FITC/PI Apoptosis Detection kit (Vazyme, Nanjing, China) and measured by flow cytometry (Beckman Coulter, CA, USA). Early apoptotic cells are single positive for Annexin V-FITC, and late apoptotic cells are double positive for Annexin V-FITC and PI (Chen et al., 2008). As triptolide-induced apoptosis involves early and late apoptosis (You et al., 2018), the apoptosis rate was calculated as the sum of the ratios of early and late apoptosis. Data were analyzed using FlowJo V 10.

Measurement of reactive oxygen species (ROS)

The content of intracellular ROS was measured using the DCFH-DA fluorescent dye (Wang et al., 2017) with the ROS Assay Kit (Beyotime, Shanghai, China). After incubation, the cells were treated with 10 µM DCFH-DA reagent and incubated in the dark at 37 °C for 20 min. Fluorescence was measured by flow cytometry at an excitation and emission wavelengths of 488 nm and 530 nm.

Western blotting

HepaRG cells (4 × 10⁵ cells/mL) were seeded in six-well plates overnight and then treated with different concentrations of TP/CAT for 24 h. The cells were collected and lysed using RIPA buffer (Beyotime, Shanghai, China) on ice for 30 min. The total protein concentration was detected using a BCA protein assay kit (Beyotime, Shanghai, China). The protein lysates (30 µg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Darmstadt, Germany). The membranes were incubated overnight at 4 °C with the following primary antibodies: p-PERK (CST, Danvers, MA, USA), LC3, SQSTM1/P62, Beclin1, GRP78/BIP, PERK, IRE1, ATF6, ATF4, CHOP, BCL2, Cleaved-caspase3, Cleaved-caspase9, and GAPDH (Proteintech, Chicago, IL, USA). After incubating with the secondary antibodies at room temperature for 1 h, the protein membranes were detected using the ECL system (Bio-Rad, Hercules, CA, USA).

Quantitative real-time PCR (qRT-PCR)

The total RNA from cells was extracted, and the mRNA expression levels of LC3, P62, Beclin1, BAX, BCL2, GRP78, and GAPDH was detected using qRT-PCR. The primer pairs are listed in Table 1. Relative expression was calculated as fold changes compared with the control gene GAPDH using the 2^−△△CT method.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) and were analyzed using GraphPad Prism 8.0.2. One-way ANOVA followed by Dunnett post-hoc test and t-test were used to compare the means of multiple or two groups, respectively. Difference was considered significant at p < 0.05.

CAT defends from TP-induced hepatotoxicity

The results of the CCK8 assay showed that, 5–20 µg/L TP decreased the relative activity of cells for 12, 24, 36, and 48 h in a time- and dose-dependent manner (Fig. 1A). Cell toxicity was moderate under 20 µg/L TP treatment for 24 h, and the concentration was used in the subsequent experiments. Simultaneously, the levels of ALT, AST, and LDH in the culture medium increased (Fig. 1B). Using the CCK8 assay, the relative safe dose of CAT was determined to be 0.4–400 µg/L (Fig. 1C). The protective effect of CAT was the most obvious when CAT was used at 40 and 400 µg/L. Based on this, the actual dose of CAT in the subsequent experiments was set to 40 µg/L. CAT reversed the TP-induced decrease in cell viability (Fig. 1D) and the levels of AST, ALT, and LDH (Fig. 1E). The results indicated that CAT could defend from TP-induced hepatotoxicity.

TP-induced overactivation of autophagy is inhibited by CAT

Excessive autophagy is one of the important mechanisms by which TP induces hepatotoxicity (Huo et al., 2019); thus, we focused on investigating autophagy. At 5–20 µg/L TP, the expression of the autophagy proteins, LC3 and Beclin1, was upregulated, whereas that of P62 was downregulated compared with those in the control group (Fig. 2A). By MDC staining, fluorescence microscopy suggested that the production of autophagosomes also increased as the TP dosage increased (Fig. 2B). Simultaneously, the corresponding mRNA levels of LC3, Beclin1, and P62 also showed tendencies with those of the consistent proteins (Fig. 2C).

When RAPA (50 nM), an effective and specific mTOR inhibitor, was used to further activate autophagy, the expression level of LC3 was upregulated and that of P62 was downregulated (Fig. 2D). Meanwhile, the levels of ALT, AST, and LDH further increased (Fig. 2E). When 3-MA (2.5 mM), a pan class III PI3K inhibitor, was used to inhibit autophagy, the expression of LC3 was downregulated and that of P62 was upregulated (Fig. 2D), and the elevated levels of ALT, AST, and LDH levels were reversed (Fig. 2E). In addition, cell viability also changed in line with liver function indexes when 3-MA, RAPA, and TP were used in combination (Fig. 2F). These results indicated that excessive autophagy is one of the main mechanisms of TP-induced liver cell damage.

As expected, CAT downregulated LC3 and Beclin1, and upregulated P62 (Fig. 2G). Simultaneously, elevated levels of ALT, AST, and LDH were reversed by CAT (Fig. 1E), and cell viability was also improved (Fig. 1D)

TP-induced apoptosis is restrained by CAT via hyperautophagy repression

Considering the role of apoptosis in TP-induced liver injury, we also detected the level of apoptosis. The results showed that with the increase in TP exposure, the apoptotic protein levels of Cleaved-caspase3 and Cleaved-caspase9 increased, and the anti-apoptotic protein level of BCL2 decreased (Fig. 3A). Flow cytometry revealed that 5–20 µg/L TP increased the number of apoptotic cells (Fig. 3B). The mRNA level of BAX increased, whereas that of BCL2 decreased (Fig. 3C). CAT reversed the protein expression levels of Cleaved-caspase3 and Cleaved-caspase9 (Fig. 3D).

To investigate the relationship between autophagy and apoptosis in TP-induced hepatotoxicity, we further used RAPA and 3-MA. When RAPA (50 nM) was used to upregulate autophagy, the level of Cleaved-caspase3 and Cleaved-caspase9 was upregulated and that of BCL2 was downregulated; when 3-MA (2.5 mM) was used to downregulate autophagy, the level of Cleaved-caspase3 and Cleaved-caspase9 was downregulated and that of BCL2 was upregulated (Fig. 3E). Thus, excessive autophagy induced apoptosis in TP-induced live cell damage.

In summary, CAT could restrain apoptosis by inhibiting excessive autophagy in TP-induced liver cell damage.

CAT protects against TP-induced extreme autophagy by inhibiting ERS

TP caused an increase in the ROS ratio as determined flow cytometry (Fig. 4A). Meanwhile, western blotting and qRT-PCR analysis indicated that GRP78, IRE1, ATF6, and PERK, the markers of ERS, were upregulated at the protein (Fig. 4B) and mRNA levels (Fig. 4C). To probe the relationship between autophagy and ERS in TP-induced hepatotoxicity, we used 4-PBA, an ERS inhibitor. When 4-PBA (500 nM) was used along with TP to downregulate ERS, the autophagy marker protein LC3 was downregulated and P62 was upregulated (Fig. 4D).

As expected, CAT decreased the ROS generation ratio (Fig. 4A) and downregulated the protein level of GRP78, which was elevated by TP (Fig. 4E); whereas, the protein level of LC3 and Beclin1 was decreased and that of P62 was increased (Fig. 2G).

In summary, ERS is an important factor in regulating autophagy, and CAT can inhibit excessive autophagy caused by TP by restraining ERS.

CAT restrains fulsome activation of autophagy by the PERK-ATF4- CHOP pathway

Western blotting revealed that the expression of p-PERK, ATF4, and CHOP, three key proteins in the PERK pathway, was upregulated by TP in a dose-dependent manner (Fig. 5A) and the autophagy flux also increased (Fig. 2B).

To explore whether the PERK-ATF4-CHOP pathway is a key regulator of TP-induced hype autophagy, we further applied GSK2656157, a selective ATP-competitive PERK inhibitor. When GSK2656157 (1 µM) inhibited the PERK pathway of ERS, LC3 was downregulated and P62 was upregulated (Fig. 5B), and simultaneously, the ALT, AST, and LDH levels also decreased (Fig. 5C). Thus, the PERK-ATF4-CHOP pathway plays an important role in the regulation of fulsome autophagy.

As envisioned, CAT reversed TP-induced upregulation of autophagic flux under TEM (Fig. 5D), and the changes in GRP78 (Fig. 4E), LC3, Beclin1, and P62 (Fig. 2G) by inhibiting the PERK-ATF4-CHOP pathway (Fig. 5E). Simultaneously, elevated ALT, AST, and LDH levels were also reversed (Fig. 1E).

Overall, CAT inhibited ERS induced by TP through suppressing the PERK-ATF4-CHOP pathway to restrain excessive autophagy to protect liver cells.