MgIG exerts therapeutic effects on crizotinib‐induced hepatotoxicity by limiting ROS‐mediated autophagy and pyroptosis

Abstract Crizotinib (CRIZO) has been widely employed to treat non‐small‐cell lung cancer. However, hepatic inflammatory injury is the major toxicity of CRIZO, which limits its clinical application, and the underlying mechanism of CRIZO‐induced hepatotoxicity has not been fully explored. Herein, we used cell counting kit‐8 assay and flow cytometry to detect CRIZO‐induced cytotoxicity on human hepatocytes (HL‐7702). CRIZO significantly reduced the survival rate of hepatocytes in a dose‐dependent manner. Furthermore, the reactive oxygen species (ROS) assay kit showed that CRIZO treatment strongly increased the level of ROS. In addition, CRIZO treatment caused the appearance of balloon‐like bubbles and autophagosomes in HL‐7702 cells. Subsequently, Western blotting, quantitative real‐time PCR and ELISA assays revealed that ROS‐mediated pyroptosis and autophagy contributed to CRIZO‐induced hepatic injury. Based on the role of ROS in CRIZO‐induced hepatotoxicity, magnesium isoglycyrrhizinate (MgIG) was used as an intervention drug. MgIG activated the Nrf2/HO‐1 signalling pathway and reduced ROS level. Additionally, MgIG suppressed hepatic inflammation by inhibiting NF‐κB activity, thereby reducing CRIZO‐induced hepatotoxicity. In conclusion, CRIZO promoted autophagy activation and pyroptosis via the accumulation of ROS in HL‐7702 cells. MgIG exerts therapeutic effects on CRIZO‐induced hepatotoxicity by decreasing the level of ROS.

of advanced ALK-positive NSCLC. It acts by competitively binding to the adenosine triphosphate (ATP) site of ALK and is effective in inhibiting aberrant ALK activation. Since approved by the US Food and Drug Administration (FDA) for NSCLC treatment in 2011, 1 CRIZO has been used worldwide for several years with favourable clinical outcomes. However, in some large clinical trials, as high as 57% of patients receiving a standard dose of CRIZO had elevated aminotransferase (aspartate/alanine) levels, discontinuation of the drug was required in about 2%-4% of patients and mortality was reported in 0.1% of patients. [2][3][4] Currently, effective approaches for preventing and treating CRIZO-induced hepatotoxicity remain poorly understood as the underlying mechanism of CRIZO-induced hepatotoxicity is yet to be fully elucidated.
Drug-induced liver injury (DILI) is often complex and driven by multiple mechanisms, including apoptosis, autophagy, necrosis and oxidative stress. Prior studies evaluating the toxic effects of CRIZO on the liver have primarily focused on oxidative stress and mitochondrial apoptosis. [5][6][7] Besides, although substantial necrosis was identified in CRIZO-treated hepatocytes, the specific necrotic pathway mainly contributing to CRIZO-induced hepatotoxicity remains controversial. Moreover, previous evidence has suggested that ALK inhibitors could induce autophagy in tumour cells by inhibiting mTOR phosphorylation, and this autophagy led to cancer cell death. [8][9][10][11] Thus, the present study also sought to determine whether autophagy is involved in CRIZO-induced hepatotoxicity and explore other mechanisms associated with hepatotoxicity.
The primary principles for DILI treatment in clinical settings are stopping the drug on time and treating DILI with appropriate antiinflammatory and hepatoprotective agents based on the clinical patterns of DILI. Liver transplantation is recommended if DILI damage is severe and irreversible. 12,13 Because DILI pathogenesis is complex, no specific drugs are currently available. Magnesium isoglycyrrhizinate (MgIG) is a novel α-isomer compound synthesized by isomerization and salification from 18β-glycyrrhizic acid, a triterpenoid extracted from Glycyrrhiza glabra. It has been reported to exhibit antioxidant, anti-inflammatory and anti-allergic pharmacological activities and is used for the treatment of viral hepatitis and abnormal liver function. [14][15][16] Herein, we explored the effects and the underlying mechanisms of MgIG on CRIZO-induced hepatotoxicity.

| Drugs and reagents
CRIZO was purchased from Energy Chemical Reagent Co., Ltd.
(Shanghai, China), it dissolved in dimethyl sulfoxide (DMSO) to make a 20 mM stock solution and further diluted to desired concentrations with culture medium. Bafilomycin A1 (Baf A1) and N-acetyl-L-cysteine (NAC) were purchased from MedChemExpress Co., Ltd.
(New Jersey, United States). Baf A1 was dissolved in DMSO to make a 1 mM stock solution, which was diluted with culture media to the 5 and 10 nM concentration during experiments. NAC was straightly dissolved in culture medium to make a 10 mM solution. MgIG (5 mg/ ml) was purchased from Zhengda Tianqing Pharmaceutical Group Co., Ltd. (Jiangsu, China).

| Cell viability assay
Cell viability was evaluated using cell counting kit 8 (CCK8) assay (MedChemExpress, New Jersey, United States). After 24 or 48 h of drug exposure, the supernatants were removed and then 100 μl of CCK8 (10 μl/100 μl culture medium) was added to each well. After 2 h incubation, the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, Massachusetts, United States). Cell viability was calculated based on the absorbance values.

| Cell death analysis
An Annexin V-PE/7-AAD apoptosis assay kit (Lianke Biotech, Hangzhou, China) was used to detect apoptosis and necrosis. After treatment with CRIZO for 48 h, hepatocytes were harvested and washed twice with pre-chilled phosphate-buffered saline (PBS).
Then, the cells were resuspended in a 500 μl binding buffer containing 5 μl Annexin V-PE and 10 μl 7-AAD and incubated at room temperature in the dark for 5 min. After staining, cells were analysed using a flow cytometer.

| Nuclear protein extraction
Nuclear/cytosol protein extraction was performed using a nuclear protein extraction kit (Beyotime, Shanghai, China), according to the manufacturer's instructions. Briefly, cells were lysed with cytoplasmic lysis buffer on ice and vortexed vigorously. The lysates were centrifuged for 5 min at 16,000 g to obtain the supernatant containing cytosolic proteins and the pellet containing the nuclei.
Following the nuclei were resuspended in 50 μl of nuclear extraction buffer, the samples were vortexed vigorously for 30 s with maximum power and then cooled on ice for 1 min, which was repeated 15 times to ensure the nuclei were lysed adequately. Then the samples were centrifuged for 10 min at 16,000 g to obtain nuclear protein.
The proteins were boiled with 5 × loading buffer and then stored at −20°C for further analysis.

| Quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was extracted from samples using an RNA simple Total RNA kit (Tiangen, Beijing, China). RNA was reverse transcribed to synthesize cDNAs using a RevertAid RT Reverse Transcription kit (Thermo Fisher Scientific, Massachusetts, United States).
Subsequently, cDNA was quantified by qPCR using the SYBR-Green qPCR master mix (Toroivd, Shanghai, China). PCR amplification was performed using the following thermocycling protocol: pre- The relative expression of target genes was calculated using 2 −ΔΔCT .
All primer sequences are shown in Table 1.

| Measurement of Caspase1 activity
Caspase-1 activity was determined using a chromogenic substrate Ac-YVAD-pNA (Beyotime, Shanghai, China) according to the manufacturer's instruction. Briefly, treated cells were collected and lysed with a cell lysate to extract cellular proteins. After aspiration of an equal volume of protein supernatant, 2 mM Ac-YVAD-pNA was added and the mixture was incubated at 37°C for 2 h. Finally, caspase-1 activities of the samples were measured using a microplate reader at 405 nm.

| Transmission electron microscopy
Treated and untreated cells (control cells) were harvested from the culture dish using a cell scraper and fixed with 2.5% glutaraldehyde at 4°C overnight. After staining, the cell masses were dehydrated, embedded in epoxy resin and sectioned at a thickness of 1 μm.

| Evaluation of mitochondrial respiratory chain complex I (RCC I) activity
RCC I activity was measured using the mitochondrial respiratory chain complex I activity assay kit (Solarbio, Beijing, China). Cells were collected and cleaved, and an appropriate amount of mitochondria isolation reagent was added (1 ml per 5 × 10 6 cells); then, cell samples were homogenized with a glass homogenizer (30 strokes) under ice bath condition; next, cells homogenate were centrifuged at 600 g for 10 min at 4°C in order to remove cell debris and nuclei; the supernatant was then transferred into a new centrifuge tube before centrifuging at 11,000 g for 15 min at 4°C; then, the supernatant was discarded; the isolated mitochondria (the precipitation) were lysed TA B L E 1 Sequences of the primers used for quantitative real-time PCR
Secondly, cells were washed with PBS and incubated with DCFH-DA working solution at 37°C for 20 min. After washing thrice in a medium without FBS, cells were harvested and analysed using a flow cytometer.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0 (IBM, California, United States). The results were calculated using data from at least three independent experiments. Values are presented as mean ± SD. Comparisons between two groups were performed using a two-tailed Student's t-test, and p < 0.05 was considered statistically significant.

| Effects of CRIZO on hepatocyte cells
As shown in Figure Figure 1C), which implied that CRIZO markedly induced liver cell necrosis. This was further confirmed by propidium iodide (PI) staining ( Figure 1D). Overall, these findings indicated that CRIZO induced cell death by a necrotic mechanism.

| CRIZO promoted hepatocyte pyroptosis and autophagy
To further explore whether CRIZO promotes necroptosis, HL-7702 cells were treated with CRIZO and the key regulator associated with necroptosis or pyroptosis was detected by Western blotting. As shown in Figure  Moreover, CRIZO treatment significantly increased autophagy in hepatocytes.
As shown in Figure 2G,

| MgIG reduced CRIZO-induced ROS via upregulation of respiratory electron transport chain activity and re-activation of Nrf2/HO-1 pathway
The modulatory role of ROS in CRIZO-induced hepatotoxicity prompted us to seek an appropriate antioxidant for treatment.
Acting as a hepatic protectant, MgIG presents significant advantages in the liver targeting distribution, time to effect, curative effect and safety. 34 Figure 4D).

| DISCUSS ION
CRIZO is one of the most potent drugs for ALK-positive NSCLC, whose therapeutic efficacy is limited largely by hepatotoxicity.
Previous developmental studies of CRIZO-induced hepatotoxicity have mainly concentrated on mitochondrial apoptosis. 5,7 However, our study demonstrated that pyroptosis and autophagy are also involved in CRIZO-induced hepatotoxicity.
Pyroptosis is inflammatory necrosis mediated by inflammasome activation. Compared with apoptosis, pyroptosis occurs more rapidly, which is accompanied with the release of inflammatory cytokines. The pyroptosis signalling pathway can be divided into classical Autophagy is a 'self-eating' process negatively regulated by mTOR. 42 DILI is often accompanied with the occurrence of autophagy. 43 Our study found that CRIZO induced autophagic flux in hepatic cells in a dose-dependent manner. However, the phosphorylation level of mTOR showed the opposite trend. Autophagy is known to be a 'double-edged sword' as it is both a self-defence mechanism against harmful stimuli and a programmed cell death mechanism. 42,43 For example, the activation of autophagy was conducive to protect against acetaminophen-induced hepatotoxicity, 44 but the gefitinib-induced liver injury was considered to develop via autophagy 45 ; in addition, some anticancer agents killed cancer cells by inducing autophagy. 8,46,47

ACK N OWLED G EM ENTS
This research was supported by Zhejiang Provincial Natural Science Y20190653.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

O RCI D
Ziye Zhou https://orcid.org/0000-0003-0294-7399 F I G U R E 6 Intervention mechanism of MgIG on CRIZO-induced hepatotoxicity. In hepatocytes, CRIZO activates excessive production of ROS due to RCC I dysfunction and Nrf2 downregulation, which causes the dysregulation of autophagy and activation of inflammasomes. The NLRP3 inflammasome activates caspase-1, in turn cleaves GSDMD into GSDMD-N, which facilitates the formation of membrane pores, ultimately resulting in pyroptosis. MgIG protected mitochondria from CRIZO damage and re-activated Nrf2/HO-1 antioxidant pathway, thus reduced CRIZO-induced ROS accumulation, which in turn inhibits CRIZO-induced autophagy and pyroptosis of hepatocyte.