Deficiency of Group IVA Phospholipase A₂ in Collagen-Producing Cells Alleviates the Aggravated Hepatic Fibrosis in High-Fat Diet-Fed Mice after Returning to a Normal Diet

Abstract

Hepatic fibrosis, a primary feature of non-alcoholic steatohepatitis (NASH), develops with inflammation and subsequent activation of hepatic stellate cells (HSCs), the main extracellular matrix-producing cells. Currently, no approved pharmacotherapy is available to treat hepatic fibrosis, even under dietary intervention. The activation of cultured HSCs has been shown to be attenuated by pharmacological inhibition of group IVA phospholipase A₂ (IVA-PLA₂), an enzyme initiating the generation of lipid proinflammatory mediators. We examined the potential utility of IVA-PLA₂ of HSCs as a therapeutic target for hepatic fibrosis in NASH under dietary modification using collagen-producing cell-specific IVA-PLA₂-conditional knockout mice fed a high-fat diet and then returned to a normal one. Apparent hepatic fibrosis and the accumulation of hepatic lipid droplets developed in the IVA-PLA₂-conditional knockout mice on a high-fat diet for nine weeks to a similar degree as in control mice. Most of the lipid droplets disappeared five weeks after switching the diet back to a normal one in both genetic mice. In contrast, the hepatic fibrosis in control mice still progressed even after changing back to a normal diet. However, deficiency of IVA-PLA₂ in collagen-producing cells alleviated the aggravated hepatic fibrosis under dietary modification. Our results revealed that the protective effects of an HSC-specific IVA-PLA₂ deficiency on fibrogenesis appear after switching the diet from a high-fat one back to a normal one, supporting the promising beneficial effects of the inhibition of IVA-PLA₂ on progressive hepatic fibrosis under dietary intervention in NASH treatment.

INTRODUCTION

Non-alcoholic steatohepatitis (NASH) is a chronic and progressive liver disease characterized by histopathological features of steatosis, inflammation, and fibrosis.^1,2) In particular, hepatic fibrosis develops and progresses with the excessive accumulation of collagen fiber, a primary component of the extracellular matrix, mainly produced by proinflammatory and profibrogenic mediator-activated hepatic stellate cells (HSCs), leading to cirrhosis and subsequent hepatocellular carcinoma.^1,3) With no effective pharmacotherapy for hepatic fibrosis available,⁴⁾ the first line of treatment for NASH with fibrosis is weight loss, which is achieved through lifestyle intervention.^5,6) However, long-term adherence to dietary and physical activity modifications is difficult to achieve, so lifestyle modifications alone are not always successful in every individual. In addition, unfortunately, no optimal dietary strategy for preventing and treating NASH with fibrosis exists, despite the improvement in hepatic steatosis under dietary intervention.^7,8) Therefore, effective pharmacotherapy in combination with dietary modifications to prevent or treat hepatic fibrosis in NASH is needed.

Activated HSCs play a pivotal role in fibrogenesis in the liver with inflammation.^3,9) The activation of HSCs is mediated by several factors, including transforming growth factor-beta (TGF-β).^9,10) A previous report showed that TGF-β1-induced activation of rat HSCs is suppressed by inhibitor of group IVA phospholipase A₂ (IVA-PLA₂),¹¹⁾ an enzyme that catalyzes the initial step of the generation of lipid proinflammatory mediators, such as eicosanoids and lysophospholipids.¹²⁾ Furthermore, prostaglandin E₂ and I₂ exhibit an ability to stimulate the expression of TGF-β1 in the LX-2 human HSC line,¹³⁾ although the roles of prostaglandins in HSC activation and hepatic fibrosis remain controversial.^14,15) These findings suggest the involvement of IVA-PLA₂ in HSC-mediated fibrogenesis, supporting the potential utility of IVA-PLA₂ of HSCs as a therapeutic target for hepatic fibrosis.

We previously demonstrated that hepatic fibrosis is suppressed in IVA-PLA₂-conventional knockout mice fed a high-fat, high-cholesterol diet for 16 weeks.¹⁶⁾ Furthermore, our recent study showed that an endothelial cell-specific deficiency of IVA-PLA₂ alleviates hepatic fibrosis in the early stage of NASH in IVA-PLA₂-conditional knockout (IVA-PLA₂-cKO) mice on a choline-deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine (CDAHFD) for 3 weeks, but a collagen-producing cell-specific deficiency of IVA-PLA₂ does not.¹⁷⁾ Our findings suggest that even if IVA-PLA₂ in HSCs is involved in HSC-mediated fibrogenesis, the inhibition of the enzyme is not effective against hepatic fibrosis in mice under high-fat-feeding conditions.

In this context, we suspected that inhibition of IVA-PLA₂ in HSCs would exert a suppressive effect on hepatic fibrosis in a mouse model of NASH under dietary intervention following high-fat feeding. To clarify this possibility, the present study examined the effects of a collagen-producing cell-specific IVA-PLA₂ deficiency on hepatic fibrosis in mice fed a CDAHFD as a high-fat diet for three and nine weeks (to induce early- and advanced-stage fibrosis, respectively), followed by returning to a normal diet as a dietary intervention.

MATERIALS AND METHODS

Animals and Cells

C57BL/6NCrSlc mice (wild-type; WT mice) were purchased from Japan SLC (Shizuoka, Japan). HSC-specific IVA-PLA₂-cKO mice were generated by breeding the IVA-PLA₂-floxed mice¹⁷⁾ with transgenic mice expressing Cre recombinase selectively in collagen-producing cells: Col1a1-cre^KI/+ mice (B6.Cg-Tg(Col1a1-cre)1Haak, RBRC05524, RIKEN BRC). Six-week-old male mice were fed a CDAHFD (A06071302; Research Diets, Inc., New Brunswick, NJ, U.S.A.) for 3 and 9 weeks to induce a high-fat diet-induced NASH pathology, and their chow was switched to an MF diet as a normal diet (ND) (Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were not fasted before sampling. All mice were housed <5/cage with paper chips, with a 12-h light-dark cycle, with ad libitum access to food and water. All experiments were approved by the institutional animal care and use committee of Kyoto Pharmaceutical University (Permit No. A22-019), and were performed in accordance with the institutional guidelines.

TWNT1 (immortalized human hepatic stellate cell line; the Japanese Collection of Research Bioresources Cell Bank, Tokyo, Japan), a model of activated HSCs, was maintained in low glucose-Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum and antibiotics, and treated with pyrrophenone (Cayman Chemical Co., Ann Arbor, MI, U.S.A.), a specific IVA-PLA₂ inhibitor,¹⁸⁾ for 24 h.

Serum Biomarker Measurements

Blood samples collected from the inferior vena cava were left to stand for 1 h. The serum was prepared by centrifugation at 10000 × g for 10 min at room temperature. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Transaminase C II-Test kits (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Histological Analysis

The mice were perfused with saline and 10% buffered formaldehyde, and the livers were post-fixed in 10% buffered formaldehyde for 48 h. The paraffin-embedded 5-µm-thick liver sections were mounted onto MS-coated glass slides, deparaffinized, and stained with picric acid-Sirius red and hematoxylin–eosin (H&E; FUJIFILM Wako Pure Chemical Corporation). Stained sections were photographed using a microscope (model IX71; Olympus, Tokyo, Japan) with a digital camera. Images were taken at full resolution with a single image dimension set at 1360 × 1024 pixels. Collagen fibers stained with Sirius red were quantified by measuring the red areas using the ImageJ software. HE-stained specimens were scored for the severity of inflammation according to the criteria described previously.¹⁹⁾

Total RNA Isolation and RT-Quantitative PCR (RT-qPCR)

Total RNA was extracted from the livers and cells with an RNAiso Plus (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions, and reverse transcribed using deoxyribonucleoside triphosphates (dNTPs), random primers and ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). Amplification reactions were performed using SYBR-Green I (TaKaRa Bio) and LightCycler 96 or Nano System (Roche, Basel, Switzerland). PCR was performed for 55 cycles with 15 s of denaturation at 95 °C and annealing and extension at 63 °C for 34 s. The primer sequences are shown in Supplementary Table 1. The expression levels of all target genes were calculated using the 2^−ΔΔCT method.

Statistical Analyses

Data are presented as the mean ± standard error of the mean (S.E.M.). Differences among means were analyzed using a one-way ANOVA followed by an LSD post-hoc test, or the Kruskal–Wallis followed by Dunn’s t-test. p < 0.05 was taken as the lowest level of significance.

RESULTS

To assess the effect of dietary intervention on NASH pathology, WT mice were fed a CDAHFD for three and nine weeks and then switched to feeding with an ND. After the dietary intervention, the accumulation of lipid droplets in the liver was markedly reduced, but the level of hepatic collagen was increased at both the early and advanced stages of fibrosis (Fig. 1).

Fig. 1. Increase in Hepatic Collagen Deposition after Dietary Intervention for CDAHFD-Induced Hepatic Fibrosis

(A) Representative images of picrosirius red staining are shown. The scale bar corresponds to 100 µm. (B, C) The sirius-red stained areas were measured using the NIH ImageJ software program (B: CDAHFD 3 weeks + ND; C: CDAHFD 9 weeks + ND). The bar graphs represent the mean ± S.E. * p < 0.05, ** p < 0.01. The number of mice in each group was n = 3.

To clarify whether or not IVA-PLA₂ in HSCs mediates the increase in hepatic collagen after dietary intervention, a CDAHFD was fed to collagen-producing cell-specific IVA-PLA₂-cKO mice for nine weeks to induce NASH pathology, and then the mice were switched to an ND for a further five weeks. The amount of food consumed by the IVA-PLA₂-cKO mice was comparable to that of control mice (data not shown). There was no marked difference in collagen deposition between the IVA-PLA₂-cKO and control mice fed a CDAHFD for nine weeks (Fig. 2). However, while the amount of collagen in the liver remarkably increased within five weeks after switching to an ND in the control mice, the increase in collagen fibers was significantly suppressed by the deficiency of IVA-PLA₂.

Fig. 2. Protective Effects of an HSC-Specific IVA-PLA₂ Deficiency on the Increase in Hepatic Collagen Deposition after Dietary Intervention

(A) Representative images of picrosirius red staining are shown. The scale bar corresponds to 100 µm. (B) The sirius-red stained areas were measured using the NIH ImageJ software program. The bar graphs represent the mean ± S.E. ns: non-significant, * p < 0.05, ** p < 0.01. The numbers of the cKO mice and its control mice on a CDAHFD for 9 weeks (on a CDAHFD for 9 weeks and an ND) were n = 4 (n = 8) and n = 5 (n = 10), respectively.

We also confirmed the increase in collagen deposition after the dietary intervention even in the early stage of fibrosis as well as the suppressive effect of the collagen-producing cell-specific deficiency of IVA-PLA₂ on the increased collagen deposition (Supplementary Fig. 1). Notably, the elevated serum levels of AST and ALT and the accumulation of hepatic lipid droplets were dramatically reduced after the dietary intervention in both the IVA-PLA₂-cKO mice and control mice to the same extent (Supplementary Table 2, Figs. 2A, 3A).

Fig. 3. Faster Alleviation of Hepatic Inflammation in HSC-Specific IVA-PLA₂-cKO Mice than in Control Mice under Dietary Intervention

(A) Representative images of H&E staining are shown. The scale bar corresponds to 100 µm. Arrowheads indicate foci. (B) The hepatic inflammation score was evaluated as described in the Materials and Methods. ns: non-significant, ** p < 0.01. The numbers of the cKO mice and its control mice on a CDAHFD for 9 weeks (on a CDAHFD for 9 weeks and an ND) were n = 4 (n = 8) and n = 5 (n = 10), respectively.

In addition, H&E staining of liver sections was performed to elucidate the involvement of IVA-PLA₂ of HSCs in hepatic inflammation. The inflammation scores showed no marked difference in the severity of leukocyte infiltration between the IVA-PLA₂-cKO and control mice fed a CDAHFD for nine weeks (Fig. 3). However, the hepatic inflammation was significantly suppressed in the IVA-PLA₂-cKO mice within five weeks after the dietary intervention, but not in the control mice.

Finally, we analyzed the mRNA levels of monocyte chemoattractant protein-1 (Mcp-1), α-smooth muscle actin (α-Sma) and collagen type I alpha 2 chain (Col1a2) in livers by RT-qPCR (Fig. 4A). The mRNA expression of Mcp-1 in the IVA-PLA₂-cKO mice was lower than that in the control mice even when fed a CDAHFD for nine weeks, and the mRNA levels were markedly reduced in both genetic types of mice after switching to an ND. The mRNA level of α-Sma after dietary intervention tended to be lower in the IVA-PLA₂-cKO mice than in the control mice, while there was no difference in Col1a2 mRNA levels between them. We also measured the mRNA levels of MCP-1, α-SMA, and COL1A2 in TWNT1, a model of activated HSCs, after treatment with pyrrophenone, a specific inhibitor of IVA-PLA₂ (Fig. 4B). Consistent with the results of the in vivo analysis, pyrrophenone (5 or 10 µM) markedly decreased the mRNA level of MCP-1 in TWNT1, and the mRNA expression of α-SMA tended to be reduced by treatment with pyrrophenone.

Fig. 4. Effects of an HSC-Specific IVA-PLA₂ Deficiency and Pyrrophenone on the mRNA Expression of Fibrosis-Related Genes

The mRNA levels of Mcp-1 (MCP-1), α-Sma (α-SMA), and Col1a2 (COL1A2) in livers of HSC-specific IVA-PLA₂-cKO and control mice (A) and in TWNT1 treated with pyrrophenone (5 or 10 µM) or its vehicle for 24 h (B) were determined by qPCR. The expression was normalized to that of ribosomal protein lateral stalk subunit P0 (Rplp0, RPLP0) mRNA (Arbitrary units: A.U.). H: CDAHFD 9 weeks; H + N: CDAHFD 9 weeks + ND 5 weeks. ** p < 0.01. The numbers of the cKO mice and its control mice on a CDAHFD for 9 weeks (on a CDAHFD for 9 weeks and an ND) were n = 4 (n = 8) and n = 5 (n = 10), respectively (A). The numbers of samples were stated as below: vehicle: n = 8, 5 µM pyrrophenone: n = 4, and 10 µM pyrrophenone: n = 3 (B).

Discussion

The present study was conducted to examine whether or not suppression of IVA-PLA₂ in HSCs exerts protective effects on hepatic fibrosis in a mouse model of NASH under dietary intervention following high-fat feeding. The present results with collagen-producing cell-specific IVA-PLA₂-cKO mice showed that deficiency of IVA-PLA₂ in HSCs did not affect collagen deposition as an index of hepatic fibrosis in the mice fed a high-fat diet for nine weeks (Fig. 2). This observation is consistent with the finding of no effect on leukocyte infiltration as an index of hepatic inflammation (Fig. 3) and mRNA expression of α-Sma and Col1a2 (Fig. 4A) as indexes of HSC activation and fibrogenesis, respectively, under high-fat-diet conditions. Our recent study showed similar ineffectiveness on hepatic fibrosis and α-Sma expression, even in an early-stage NASH model, with IVA-PLA₂-cKO mice fed the same high-fat diet (CDAHFD) for three weeks,¹⁷⁾ as confirmed here (Supplementary Fig. 1).

In WT mice and/or the control mice, changing the high-fat diet back to an ND diminished the Col1a2 mRNA expression (Fig. 4A), but rather aggravated hepatic fibrosis (Figs. 1, 2). Similar irreversible fibrosis was also observed in WT mice after changing from a methionine- and choline-deficient diet back to a normal one.²⁰⁾ The present results indicate that even when activated HSCs have lost their ability to produce collagen fibers during dietary intervention, hepatic fibrosis is not ameliorated under our experimental conditions. However, we found in the present study that deficiency of IVA-PLA₂ in HSCs alleviated the progression of hepatic fibrosis observed in control mice after switching the diet from a high-fat one back to a normal one (Fig. 2), and the deficiency and dietary intervention cooperatively suppressed high-fat-diet-induced hepatic inflammation (Fig. 3).

Our findings revealed that the protective effects of the HSC-specific IVA-PLA₂ deficiency on hepatic fibrosis with inflammation are masked during high-fat feeding but appear after returning to a normal diet as dietary intervention. In contrast, deficiency of IVA-PLA₂ in HSCs did not affect the high-fat-diet-induced accumulation of hepatic lipid droplets and their disappearance by returning to an ND (Fig. 2A), implying that dietary intervention alone is sufficient for the treatment of hepatic steatosis. These findings suggest that inhibition of IVA-PLA₂ in HSCs is needed for the preventative treatment of hepatic fibrosis but not hepatic steatosis in NASH during dietary intervention.

Regarding the possible mechanisms underlying the protective effects of the HSC-specific IVA-PLA₂ deficiency on hepatic fibrosis during dietary intervention, we further demonstrated that the deficiency of IVA-PLA₂ reduced the expression of Mcp-1 mRNA, even during high-fat feeding, despite showing no effect on the mRNA expression of α-Sma or Col1a2, hepatic inflammation, or hepatic fibrosis (Figs. 2, 3, 4A). In addition, switching from a high-fat diet to a normal one robustly diminished the expression of Mcp-1 mRNA observed during high-fat feeding to the same level in both the HSC-specific IVA-PLA₂-cKO and control mice (Fig. 4A), although hepatic fibrosis was aggravated under dietary intervention in the control mice only. MCP-1 is a proinflammatory and profibrogenic mediator generated in infiltrated macrophages/Kupffer cells and activated HSCs and contributes to the progression of hepatic fibrosis through amplification of the migration and infiltration of monocytes/macrophages and the activation of HSCs.^21,22) Considering the facilitative roles of MCP-1 in hepatic fibrosis, the suppressed expression of MCP-1 in the IVA-PLA₂-cKO mice on a high-fat diet is likely to explain the more efficiently protective effects of the IVA-PLA₂ deficiency on hepatic fibrosis with inflammation during dietary intervention. Our findings also suggest the possible involvement of IVA-PLA₂ in the expression of MCP-1 in activated HSCs. This notion is supported by the present results showing that pyrrophenone, a specific IVA-PLA₂ inhibitor, suppressed the expression of MCP-1 mRNA in TWNT1, a model of activated human HSCs (Fig. 4B). Given that the tumor necrosis factor α- and interleukin-1α-induced MCP-1 gene expression is suppressed by a selective inhibitor of cyclooxygenase-2 in human HSCs, the contribution of IVA-PLA₂ to the expression of MCP-1 mRNA is probably mediated by prostaglandins, lipid mediators generated by IVA-PLA₂-initiated arachidonic acid cascade.²³⁾

A previous report showed that arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of IVA-PLA₂, suppresses the expression of α-SMA in rat HSCs stimulated with TGF-β1, a profibrogenic mediator,¹¹⁾ suggesting the possible involvement of IVA-PLA₂ in the activation of HSCs. Consistent with this notion, we also observed that a genetic deficiency of IVA-PLA₂ or pyrrophenone, a specific IVA-PLA₂ inhibitor, tended to reduce the expression of α-Sma/α-SMA mRNA in the HSC-specific IVA-PLA₂-cKO mice under dietary intervention and in TWNT1, respectively (Fig. 4). Among the many isozymes of PLA₂, IVA-PLA₂ is the main enzyme catalyzing the initial step of the generation of lipid proinflammatory mediators, including eicosanoids.¹²⁾ Prostaglandin E₂ and I₂ have been shown to stimulate the expression of TGF-β1 in the LX-2 human HSC line.¹³⁾ Furthermore, a recent prospective cohort study suggested that daily aspirin use was associated with a reduced risk of progression to advanced hepatic fibrosis in patients with nonalcoholic fatty liver disease.²⁴⁾ However, the roles of prostaglandins in HSC activation and hepatic fibrosis are complex and controversial (fibrosis-protective and/or fibrosis-promoting).^14,15) As the inhibition of IVA-PLA₂ resulted in the suppression of eicosanoids with protective, promoting, or dual roles in fibrogenesis, cell-type-specific inhibition of IVA-PLA₂ may be more beneficial and less unfavorable for the preventative treatment of hepatic fibrosis than the inhibition in all kinds of cells.

In conclusion, the present study revealed for the first time that genetic inhibition of IVA-PLA₂ in HSCs ameliorates the less beneficial effects of dietary intervention on high-fat diet-induced hepatic fibrosis with inflammation, providing novel insight into pharmacotherapeutic strategies for the preventative treatment of progressive hepatic fibrosis in NASH. In combination with our previous report that IVA-PLA₂ in liver sinusoidal endothelial cells mediates hepatic fibrosis in NASH,¹⁷⁾ our findings also support the potential utility of a dual cell-targeting IVA-PLA₂ inhibitor to HSCs and liver sinusoidal endothelial cells for NASH therapy.

Acknowledgments

This work was supported in part by JSPS KAKENHI Grant No. 20K07077 (to S.A.). The authors would like to thank Ms. Monami Imada, Mr. Daiki Tanekusa, Ms. Ayana Nakomoto, Ms. Saya Tanaka, Ms. Namiki Tanimoto, Ms. Shiori Yasuda, Mr. Tetsuya Kimura, Ms. Ai Kotani, Ms. Mayumi Fukuda, Ms. Mireina Yamamoto, Ms. Ayumi Mizota, and Ms. Saki Kishinaka from Kyoto Pharmaceutical University for the technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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