Lysophosphatidic acid protects against acetaminophen-induced acute liver injury (2024)

Animal studies

C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). All experiments involving animals were approved by the Institutional Review Committee for Animal Care and Use at Sungkyunkwan University (Suwon, Korea). The mice were fasted for 16 h. APAP (Sigma, St Louis, MO, USA) was dissolved in 55–60 °C warm 1 × PBS, cooled to 37 °C, and administered to the mice by oral gavage (400 mg kg⁻¹). LPA (AVANTI, Alabaster, AL, USA) or NAC was dissolved in PBS with 1% DMSO or in PBS, respectively, and intraperitoneally injected into the mice 30 min prior to or 1 or 3 h after the APAP administration. The LPA receptor antagonists KI16425 (Sigma) and H2L 5765834 (TOCRIS, Minneapolis, MN, USA) were intraperitoneally injected at 30 min prior to the LPA administration.

Histopathology and immunohistochemistry

After isolating the livers from the APAP-induced acute liver injury models, the livers were fixed with 10% neutral buffered formalin. The liver tissues were embedded with paraffin, and 5 μm sections were cut from them. They were stained with hematoxylin and eosin for histopathological analysis. Fixed liver sections were also used for TUNEL assay after they were incubated with terminal deoxynucleotidyl transferase dUTP nick and labeled with enzyme for 1 h at 37 °C in the dark. After the slides were washed three times in PBS for 3 min, they were incubated with POD substrate for 30 min at 37 °C in the dark. TUNEL-positive cells were detected using DAB solution (Roche, Basel, Switzerland). Necrotic cells were evaluated using a Leica ICC50 HD microscope (Leica Microsystems, Wetzlar, Germany) with magnification of × 200.

Measurement of serum alanine transaminase (ALT) and aspartate aminotransferase (AST)

ALT and AST levels were measured using ALT and AST activity assay kits (Sigma) according to the manufacturer’s instructions.

Western blot analysis

After isolating the livers from the APAP-induced acute liver injury model, the livers were homogenized with RIPA buffer (150 mM NaCl, Tris-HCl, pH 7.5, SDS 0.1%, Triton X-100 1%, EDTA 2 mM, 0.5% Na-deoxycholate) containing proteinase inhibitor. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane, which was then probed with primary antibodies against phosphorylated JNK, total JNK, phosphorylated ERK, total ERK, phosphorylated GSK3β or total GSK3β (Cell Signaling Technology, Danvers, MA, USA). These membranes were then incubated with anti-rabbit (Enzo Life Sciences Inc., Farmingdale, NY, USA) or anti-mouse (KPL Inc., Gaithersburg, MD, USA) secondary antibodies.

GSH measurement

Liver tissues (60 mg) from APAP-induced acute liver injury mice were homogenized with 5% metaphosphoric acid (10 mg of liver per 500 μl of 5% metaphosphoric acid). Lysates were centrifuged at 12 000 r.p.m. for 10 min at 4 °C. GSH measurement was performed for supernatant using a glutathione (GSSG/GSH) detection kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions. The total GSH level was calculated based on the net slope of the standard curve.

ELISA

Blood was harvested from the acute liver injury mice at 12 h post-APAP treatment. After removing cells from the biofluids via centrifugation (12 000 r.p.m. for 1 min), the concentrations of the indicated cytokines were determined by ELISA (eBioscience Inc., San Diego, CA, USA) according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

Total RNA was isolated from whole liver lysates or primary hepatocytes using TRIzol according to the manufacturer’s instructions (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). One microgram of RNA was reverse-transcribed using the Maxime RT PreMix Kit (Intron, Seongnam, Korea). The cDNA was subjected to QPCR using the Rotor-Gene SYBRGreen PCR Kit (QIAGEN, Hilden, Germany) with gene-specific primers: Gpx-1: forward 5′-CCAGGAGAATGGCAAGAATGA-3′, reverse 5′-TCTCACCATTCACTTCGCACTT-3′ Sod2: forward 5′-TCCCAGACCTGCCTTACGACTAT-3′, reverse 5′-GGTGGCGTTGAGATTGTTCA-3′ PGC-1α: forward 5′-AGCACACGTTTATTCACGGGT-3′, reverse 5′-GCCCCCAAGTCCTCACATG-3′ FGF21: forward 5′-AGATCAGGGAGGATGGAACA-3′, reverse 5′-CACCCAGGATTTGAATCAGC-3′ LPA1: forward 5′-CCAGGAGGAATCGGGACAC-3′, reverse 5′-CAATAACAAGACCAATCCCGGA-3′ LPA2: forward 5′-GTCAAGACGGTTGTCATCATTCT-3′, reverse 5′-GAAGCATGATCCGCGTGCT-3′ LPA3: forward 5′-CCACTTTCCCTTCTACTACCTGCT-3′, reverse 5′-GACGGTCAACGTTTTCGACACC-3′ LPA4: forward 5′-CAGTGCCTCCCTGTTTGTCTTC-3′, reverse 5′-GAGAGGGCCAGGTTGGTGAT-3′ LPA5: forward 5′-TCCACGCTGGCTGTATATGG-3′, reverse 5′-TCGCGGTCCTGAATACTGTTC-3′ LPA6: forward 5′-GATCACTCTCTGCATCGCTGTTTC-3′, reverse 5′-CCCTGAACTTCAGAGAACCTGGAG-3′.^{13, 18, 19} Relative gene expression levels were normalized to that of the GAPDH expression level.²⁰

Isolation of primary hepatocytes for quantitative real-time polymerase chain reaction

Primary hepatocytes were isolated from C57BL/6N mice following liver perfusion with 50 ml in a buffer containing 66.7 mM NaCl, 6.7 mM KCl, 50 mM HEPES and 4.8 mM CaCl₂ 2H₂O, collagenase type IV after 20 ml of a buffer containing 66.7 mM NaCl, 6.7 mM KCl, 50 mM HEPES and 2.5 mM EGTA. The cells were centrifuged at 500 r.p.m. for 4 min. Primary hepatocytes were separated from dead cells and other cell types by Percoll gradient centrifugation (1250 r.p.m. for 5 min).

Statistical analysis

The results that were collected were evaluated using GraphPad prism software. All of the results are expressed as the mean±standard error of the mean (s.e.m.). The statistical analysis was performed using one-way analysis of variance test followed by Student’s t-test. The survival data were analyzed using the log-rank test and ANOVA. A P-value⩽0.05 was considered statistically significant.

LPA administration blocks APAP overdose-induced acute liver injury

We investigated the effect of LPA administration in APAP-induced acute liver injury. An APAP challenge induced substantial liver damage in a time-dependent manner based on histopathological analysis with hematoxylin and eosin staining of liver sections (Figure 1a). LPA administration markedly blocked liver damage induced by the APAP challenge (Figure 1a). Serum levels of AST and ALT, two well-defined markers of liver damage, were strongly elevated in a time-dependent manner in APAP-challenged mice (Figure 1b). AST and ALT showed maximal activities at 6 h after APAP gavage. However, liver damage-associated enzyme levels were significantly reduced by LPA administration (Figure 1b). LPA administration alone without the APAP challenge did not affect the levels of AST or ALT (Figure 1b). APAP challenge-induced liver damage was also assessed by TUNEL histology. TUNEL-positive cells were strongly decreased after LPA administration in APAP challenged mice (Figure 1c). We also examined the effect of LPA on APAP-induced mortality. LPA administration significantly protected mice against APAP challenge-induced mortality, showing a survival rate of 100 vs 10% for the vehicle-administered mice at 72 h post-APAP challenge (Figure 1d). The only pharmacological option available for APAP overdose patients since the 1970s is N-acetylcysteine (NAC), the precursor of GSH.²¹ Therefore, we compared the effects of LPA on APAP-induced mortality with that of NAC administration. LPA (4 mg kg⁻¹) administration showed the same protective effects as NAC (150 mg kg⁻¹) against APAP-induced mortality (Figure 1e).

LPA enhances the recovery of antioxidant GSH levels and decreases JNK, ERK and GSK3β phosphorylation in the liver following APAP challenge

GSH can detoxify NAPQI to prevent accumulation of APAP-protein adducts and oxidative stress in a liver injury animal model.² GSH depletion is associated with APAP overdose-induced liver damage in mice.²² Liver GSH levels were markedly depleted after the APAP challenge compared to those in the vehicle-challenged mice (at the 0 h time point). However, liver GSH levels were slightly recovered at 6 h post APAP (Figure 2a). The administration of LPA significantly enhanced GSH recovery at 1–6 h in APAP-challenged mice (Figure 2a). We also investigated whether LPA affects the expression of antioxidant genes, including PGC-1α, Sod2, Gpx-1 and FGF21, in the APAP model. LPA administration did not increase the expression of these antioxidant genes (Figure 2b).

Previously, the phosphorylation of JNK and ERK has been reported to be associated with APAP-induced acute liver injury.²³ In this study, we also observed that APAP challenge strongly elicited JNK and ERK phosphorylation in the liver compared to that of vehicle (PBS) controls based on western blot analysis (Figure 2c). However, APAP-induced JNK and ERK phosphorylation levels were strongly decreased in the livers of mice that were administered LPA (Figure 2b). It has been reported that GSK3β is an upstream molecule of JNK and ERK involved in the pathological process of APAP-induced acute liver injury.⁴ The APAP challenge induced GSK3β phosphorylation, which was markedly attenuated by LPA administration (Figure 2c).

LPA administration decreases TNF-α and IL-1β in experimental drug-induced liver injury

It has been reported that some inflammatory cytokines, such as TNF-α and IL-1β, can mediate the pathological process of APAP-induced drug-induced acute liver injury.⁵ Therefore, we examined the effect of LPA administration on the production of these inflammatory cytokines in this study. The APAP overdose challenge strongly increased the production of TNF-α and IL-1β in the APAP acute liver injury model (Figure 3). However, the production of these inflammatory cytokines was significantly decreased by the administration of LPA (Figure 3). The TNF-α level was significantly decreased by LPA administration at 3 h. This inhibitory effect was sustained at 6 h after the APAP overdose administration. The IL-1β level was significantly decreased by LPA administration at 3 h but not at 6 h after APAP administration (Figure 3). Unlike TNF-α or IL-1β, the production of IL-6, another important proinflammatory cytokine, was not affected by LPA administration (Figure 3).

LPA-induced protective effects against acute liver injury are independently mediated by LPA₁, LPA₃ and LPA₅

Previously, LPA was reported to be able to bind to several different cell surface GPCRs such as LPA_1–6.¹¹ Other previous reports have demonstrated that LPA₅ and a low level of LPA₁ are expressed in mouse and human livers.¹² In this study, we performed q-PCR analysis to investigate which members of the LPA receptor were expressed in primary hepatocytes. We found that mouse primary hepatocytes expressed LPA_1,3–6 but not LPA₂ (Figure 4a). As we detected the expression of LPA₁ and LPA₅ in mouse primary hepatocytes, we examined whether LPA could induce a protective effect against APAP overdose-induced acute liver injury via well-known surface GPCRs using pharmacological inhibitors. KI16425, an LPA_1,3-selective inhibitor, and H2L 5765834, an LPA_1,3,5-selective inhibitor, failed to block LPA-induced inhibitory effects against acute liver injury (Figure 4b). The two LPA receptor antagonists (KI16425 and H2L 5765834) failed to affect the LPA-induced decrease of ALT in the APAP overdose-induced acute liver injury model (Figure 4b). Moreover, liver histology showed that these LPA receptor antagonists (KI16425 or H2L 5765834) did not reverse the LPA-induced protective effect on liver damage (Figure 4c). These results suggest that LPA-induced protective effects against drug-induced liver injury (DILI) are not mediated by LPA₁, LPA₃ or LPA₅.

LPA shows therapeutic effects in experimental DILI

We also examined whether LPA shows therapeutic effects against APAP-induced acute liver injury. To this end, we administered LPA 1 or 3 h after the APAP challenge and measured liver damage by histopathological analysis with hematoxylin and eosin staining of liver sections. APAP-induced liver damage was strongly blocked by the therapeutic administration of LPA (Figure 5a). Moreover, the therapeutic administration of LPA protected mice against APAP-induced mortality, showing a survival rate of 75% vs 0% for vehicle-administered mice at 72 h post-APAP challenge (Figure 5b).

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