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Abstract

Objective:

To explore the effects of myocardin-related transcription factor A (MRTF-A) knockout on mice with nonalcoholic steatohepatitis (NASH) induced by high-fat diet (HFD).

Methods:

Normal-fat diet (NFD) or HFD was fed to MRTF-A-knockout (MRTF-A^−/−) and wild-type (WT) mice for 16 weeks. Liver histopathological status was observed using Hematoxylin and Eosin (HE) staining, Oil Red O staining, Sirius Red staining, and Immunohistochemical staining. The mRNA and protein levels in liver tissues were measured through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot.

Results:

Compared with WT + HFD group, mice in MRTF-A^−/− + HFD group were decreased in body weight, blood glucose, plasma insulin, liver TG and NAFLD activity score (NAS), with liver function recovery. Besides, compared with HFD-fed WT mice, HFD-fed MRTF-A^−/− mice were improved in hepatic fibrosis, accompanied by decreased collagen content (%) and down-regulated expressions of α-SMA, COL1A2, TGFβ1, and SMAD3. In mice fed with HFD, the expression of MCP-1, CCR2, F4/80 and CD68 declined in liver tissues of MRTF-A^−/− mice as compared with WT mice. Besides, in hepatic macrophages isolated from HFD-fed mice, the observed increased expression of TNF-α, IL-1β, MCP-1, as well as decreased expression of CCR2. Compared with WT + HFD group, MRTF-A^−/− + HFD group mice were decreased regarding NF-κB p65 in liver tissues.

Conclusion:

MRTF-A knockout reduced macrophage infiltration, down-regulated NF-κB p65 expression, and ameliorated inflammation and fibrosis of liver tissues in mice, thereby becoming a potential therapeutic target for NASH treatment.

Keywords

MRTF-A nonalcoholic steatohepatitis HFD NF-κB histopathological and immunohistochemical parameters

Introduction

Liver, as the largest digestive gland in human body, can not only secrete bile, store hepatic glycogen and detoxify, but also function as a vital immune organ.¹ As one of the most common liver diseases in human, nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal results of liver test, including a series of disease states.² Mechanically, initial simple steatosis (cirrhosis) can develop into pathological nonalcoholic steatohepatitis (NASH) to induce further hepatic fibrosis, ultimately leading to end-stage liver diseases, such as liver cirrhosis, liver failure and even liver cancer.³ The incidence of NASH was shown to be often associated with obesity, insulin resistance, mitochondrial dysfunction, type II diabetes mellitus.⁴ Although the living standards are improved in recent years, the NASH prevalence is increasing still without generally accepted and effective therapy.⁵

Myocardin-related transcription factor A (MRTF-A), also widely recognized as megakaryoblastic leukemia factor-1 (MKL1), is a member of the cardiac myosin family of transcriptional regulators and found distributed in many different organs and tissues.^6,7 MRTF-A was initially discovered in a study on chromosome translocation in acute megakaryocytic leukemia and it was a transcriptional coactivator of serum response factor.⁸ It is worth noting that MRTF-A was also susceptible to inflammatory diseases. For example, MRTF-A transduces the stress of AngII challenge to the pro-inflammatory, ultimately leading to development of aortic dissection.⁹ Besides, MRTF-A silencing inhibited the NF-κB-dependent pro-inflammatory transcription, providing insights into the rationalized development of anti-inflammatory therapeutic strategies.¹⁰ As reported by a previous study, MRTF-A^−/−/ApoE^−/− mice had significantly smaller diet-induced atherosclerotic lesions than MRTF-A^+/+/ApoE^−/− mice.¹¹ Additionally, MRTF-A^−/− mice were effectively improved regarding metabolic profiles and also mitigated in terms of diet-induced obesity and insulin resistance.¹² Furthermore, Tian and his group found MRTF-A deficient mice exhibited resistance to CCl₄-induced liver fibrosis compared to wild-type (WT) littermates,¹³ suggesting its pathogenic role in live diseases. Besides, hepatocyte-specific deletion of Brg1 alleviates choline deficient diet (MCD) induced NASH in mice possibly by regulating the interaction between NF-κB and its co-factor MRTF-A.¹⁴ These mentioned above implicated the MRTF-A deficiency may be a potential therapeutic target for NASH.

Therefore, we conducted this in vivo study to explore the effect of MRTF-A knockout on NASH. Till now the study on NASH pathogenesis and treatment outcome evaluation is mainly conducted on suitable animal models, among which diet induction has been most widely used to establish NASH animal models and high-fat diet (HFD) is often used to induce NASH.¹⁵ Therefore, HFD was fed to WT mice and MRTF-A knockout mice for 16 weeks to establish NASH models.¹⁶ then the following experiments were performed, including Hematoxylin and Eosin (HE) staining, Oil Red O staining, Sirius Red staining, immunohistochemical staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, aiming to analyze the effects of MRTF-A deletion on NASH mice, thus finding novel anti-NASH therapeutics.

Materials and methods

Ethics statement

This study was conducted by strictly following the principles in the Guide for the Care and Use of Laboratory Animals,¹⁷ and all animal experiments were performed under the supervision of Ethics Committee of Medical Laboratory Animals in our hospital (2020021301).

Establishment of mice models of NASH

C57BL/6J Slc mice (WT mice, n = 20) and MRTF-A knockout mice (MRTF-A^−/−, n = 20) were provided by the Jackson Laboratory (Bar Harbor, ME, USA) and kept in an environment of specific pathogen-free (SPF) grade, with 12-h light/dark cycle and free access to food and water. HFD was fed to mice for 16 weeks to establish NASH mice models,¹⁶ while normal-fat diet (NFD) was fed to control mice. WT and MRTF-A mice of 7-week-old were assigned into 4 groups with 10 mice in each, including WT + HFD group, MRTF-A^−/− + HFD group, WT + NFD group, and MRTF-A^−/− + NFD group. The HFD (D12492, Research Diet, New Brunswick, NJ, USA) and NFD provide 60 kcal% fat and 10% kcal% fat respectively. At the end of feeding phase, mice underwent anesthetization through the injection of intraperitoneal ketamine (60 mg/kg body weight) and xylazine (7.5 mg/kg body weight). Body weight was recorded weekly. A longitudinal incision was made on the abdominal wall to obtain the blood sample and then the liver was removed. The abbreviations for all terminologies were listed in the Supplemental Table 1.

Detection of biochemical indexes

The biochemical indexes of mice, such as blood glucose, plasma alanine aminotransferase (ALT), and plasma aspartate aminotransferase (AST), were all examined using the automatic biochemical analyzer 7180 (Hitachi Ltd, Tokyo, Japan). Liver triglycerides (TG) were quantified via a colorimetric assay using a triglyceride assay kit (ab65336, Abcam, USA). The plasma insulin level was measured by radioimmunoassay.

Hematoxylin and Eosin (HE) staining

Partial liver tissues of mice were fixed in 4% paraformaldehyde for 48 h, rinsed many times with tap water, dehydrated with gradient alcohol, soaked in paraffin, and embedded. The paraffin sections (5 µm in thickness) were incubated at 80°C for 20 min, de-paraffinized, and soaked for 15 min in hematoxylin solution. Sections were then rinsed with tap water for re-bluing, stained in eosin, hydrated with gradient alcohol, transparentized with xylene, and mounted with neutral resin before air drying and photographing under a microscope. To evaluate the NAFLD activity score (NAS), we referred to the Guidelines for Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease (revised 2010) (http://doctor-network.com/Public/LittleTools/81.html). Steatosis was graded from 0–3 based on the percentage of hepatocytes involved (0: < 5%; 1: 5%–33%; 2: 33%–66%; 3: > 66%). Lobular inflammation was graded from 0–3 based on inflammatory foci (0: none; 1: < 2; 2: 2–4; 3: > 4). Hepatocyte ballooning was limited to three categories (0 = none; 1 = few; 2 = many). NAFLD activity score (NAS, 0–8) was calculated from the sum of scores for steatosis (0–3), lobular inflammation (0–3) and hepatocyte ballooning (0–2).

Oil Red O staining

Formaldehyde calcium was used for 10 min of fixation of tissue sections, which were washed with distilled water, rinsed with 60% isopropanol, and stained in Oil Red O solution for 10 min, followed by color separation using 60% isopropanol and washing with distilled water. Next, tissues sections were counterstained by Mayer Hematoxylin, washed with tap water (bluing) for 1–3 min, rinsed with distilled water, and mounted using glycerin gelatin. Lipid droplets in cells were dyed orange red and nucleus blue.

Sirius Red staining

Tissues were fixed in 10% formalin fixation solution, dehydrated and embedded routinely, sliced into sections of 6 µm in thickness, and de-paraffinized to water. Sirius Red solution was dropped to sections for 1 h staining, followed by rinse with running tap water, staining nucleus with Mayer Hematoxylin for 8–10 min, dehydrated and transparentized, and mounted with neutral resin.

Immunohistochemistry

Paraffin sections were de-paraffinized to water and incubated in 3% H₂O₂ at room temperature for 5–10 min. Next, sections were washed with distilled water, soaked in phosphate buffer saline (PBS) for 5 min, blocked in 5–10% PBS-diluted normal goat serum, and incubated for 10 min at room temperature. Then, primary antibodies were added for 15 min of incubation at room temperature, including anti-alpha smooth muscle actin (α-SMA, ab7817, 0.034 µg/mL, Abcam, USA), anti-chemokine (C-C motif) receptor 2 (CCR2, ab273050, 1/250, Abcam, USA), anti-EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80, ab100790, 1/100, Abcam, USA) and anti-nuclear factor-kappa B (NF-κB) p65 (ab16502, 1 μg/mL, Abcam, USA). After that, secondary antibodies were added for 10–30 min incubation at 37°C, and rinsed with PBS for 5 min × 3 times. Chromogenic agent 1,2-diacetylbenzene (DAB) was used for development and visualization. Tap water was used to rinse sections before counterstaining and section embedment.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA in liver tissues and hepatic macrophages (isolated from mice as previously described) was extracted using the TRIzol kit,¹⁸ and ultraviolet spectrophotometer was used to determine RNA concentration. RNA was reversely transcribed into cDNA using PrimeScript RT (RR014A, Takara Biomedical Technology (Beijing) Co., Ltd., China). Reverse transcription was conducted with RNA 3.988 μg, Oligo (dT) 18 (10 μM) 2 μL, dNTP (2.5 mM) 4 μL, 5 × Hiscript Buffer 4 μL, Hiscript Reverse Transcriptase 1 μL, Ribonuclease Inhibitor 0.5 μL, ddH₂O (Rnasefree) to 20 μL. The reaction conditions include 25°C 5 min, 50°C 15 min, 85°C 5 min, 4°C 10 min. Approprioate volume of cDNA was used as template for PCR. Primer sequences were designed using Primer 5.0 (Table 1), and synthesized by GenScript. qRT-PCR was conducted according to standard instructions of the PCR detection kit, using 4 µL cDNA (10 × dilution), 0.4 µL forward primer, 0.4 µL reverse primer, 10 µL SYBR Green Master Mix, 0.4 µL 50 × ROX Reference Dye 2, and 4.8 µL H₂O. Reaction conditions were: pre-denaturation at 50°C 2 min and at 95°C 10 min, 1 cycle; denaturation at 95°C 30 s and amplification at 60°C 30 s, 40 cycles. Relative transcription level of target genes’ mRNA was calculated using 2⁻^△△Ct method.

Table 1.

Primer sequences for quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

Gene	Primer sequences
α-SMA	Forward: 5’-TGTGAAGAGGAAGACAGCAC AGCT-3’
α-SMA	Reverse: 5’-GATGGCTGGAAGAGGGTCTCCGGG-3’
COL1A2	Forward: 5’-TAGGCCATTGTGTATGCAGC-3’
COL1A2	Reverse: 5’-ACATGTTCAGCTTTGTGGACC-3’
TGFβ1	Forward: 5’-TGACGTCACTGGAGTTGTA CGG-3’
TGFβ1	Reverse: 5’-GGTTCATGTCATGGATGGTGC-3’
SMAD3	Forward: 5’-GCAGAACGTCAACACCAAGT GCAT-3’
SMAD3	Reverse: 5’-ATTCACGCAGACCTCGTCCTT CTT-3’
MCP-1	Forward: 5’-ATTGGGATCATCTTGCTGGT-3’
MCP-1	Reverse: 5’-CCTGCTGTTCACAGTTGCC-3’
CCR2	Forward: 5’-AGCACATGTGGTGAATCCAA-3’
CCR2	Reverse: 5’-TGCCATCATAAAGGAGCCA-3’
F4/80	Forward: 5’-TGCCATCATAAAGGAGCCA-3’
F4/80	Reverse: 5’-GGATGTACAGATGGGGGATG-3’
CD68	Forward: 5’-GGATGTACAGATGGGGGATG-3’
CD68	Reverse: 5’-GGATGTACAGATGGGGGATG-3’
TNF-α	Forward: 5’-AGGGTCTGGGCCATAGAACT-3’
TNF-α	Reverse: 5’-CCACCACGCTCTTCTGTCTAC-3’
IL-1β	Forward: 5’-CAACCAACAAGTGATATTCTCC ATG-3’
IL-1β	Reverse: 5’-CAACCAACAAGTGATATTCTCCATG-3’
18S	Forward: 5’-AGTCCCTGCCCTTTGTACACA-3’
18S	Reverse: 5’-AGTCCCTGCCCTTTGTACACA-3’

Note: Alpha smooth muscle actin (α-SMA); Type I collagen alpha2 (COL1A2); Transforming growth factor beta 1 (TGFβ1); Small mothers against decapentaplegic homolog 3 (SMAD3); Monocyte chemoattractant protein 1 (MCP-1); Chemokine (C-C motif) receptor 2 (CCR2); EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80); Tumor necrosis factor-alpha (TNF-α); Interleukin-1 beta (IL-1β); Ribosomal protein S18 (18 S).

Western blot

Total proteins were extracted from liver tissues and protein samples were adjusted to the same level regarding total protein concentration and loading volume. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins, which were transferred to polyvinylidene fluoride (PVDF) membrane through a semi-dry transfer system (Bio-Rad, USA). PVDF membrane was blocked in skimmed milk at room temperature and washed with PBST buffer, before 1 h hybridization with primary antibodies at room temperature, including anti-NF-κB p65 (ab16502, 1/1000, Abcam, USA) and anti-TATA binding protein (TBP)-nuclear loading (ab63766, 1 µg/mL, Abcam, USA). Next, PVDF membrane was washed with PBS containing 0.05% (v/v) Tween20 (PBST) buffer for 5 times × 3 min, incubated for 1 h with horseradish peroxidase (HRP) goat anti-rabbit IgG H&L (ab97051, 1/20000, Abcam, USA) at room temperature, washed again with PBST buffer for 5 times × 3 min, followed by target protein development and visualization with HRP substrate (Bio-Rad). Optical densities of all protein bands were analyzed using Image Studio Lite software (Licor Biosciences, Lincoln, NE). Relative expression of target proteins was presented as gray value ratio of NF-κB p65/TBP.

Statistical methods

Statistical analysis was conducted by using SPSS 22.0 (SPSS, Inc, Chicago, IL, USA). Measurement data were expressed as mean ± standard deviation ( $\bar{x}$ ± SD). Difference among multiple groups was compared through One-Way ANOVA or Post-hoc Tukey’s test. P < 0.05 indicated the difference was of statistical significance.

Results

Effects of MRTF-A knockout on general profile and biochemical indexes of NASH mice

As shown in Table 2, HFD feeding could increase the body weight, blood glucose, plasma insulin and liver TG of WT mice and MRTF-A^−/− mice relative to NFD-fed mice (all P < 0.05). Furthermore, MRTF-A^−/− + HFD group mice had lower body weight, blood glucose, plasma insulin and liver TG than WT + HFD group (all P < 0.05). Besides, the liver function of mice in each group was examined. As a result, HFD-fed mice decreased in liver function with increased plasma ALT and plasma AST. Compared with WT + HFD group, mice in the MRTF-A^−/− + HFD group presented a certain degree of liver function recovery (all P < 0.05).

Table 2.

Comparison of biochemical changes among the groups.

	WT + NFD (n = 10)	MRTF-A^−/− + NFD (n = 10)	WT + HFD (n = 10)	MRTF-A^−/− + HFD (n = 10)
Body weight (g)
1 weeks	22.10 ± 0.93	21.66 ± 1.08	24.12 ± 1.81*^#	23.19 ± 1.20
2 weeks	23.40 ± 1.01	23.52 ± 1.02	26.39 ± 1.76*^#	25.30 ± 2.83^*
4 weeks	25.10 ± 0.92	25.23 ± 1.81	31.34 ± 2.13*^#	27.26 ± 2.28*^#&
8 weeks	28.70 ± 1.07	28.26 ± 1.76	36.92 ± 1.54*^#	31.68 ± 1.17*^#&
16 weeks	31.89 ± 0.99	30.67 ± 2.13	41.79 ± 1.51*^#	33.15 ± 1.31*^#&
Blood glucose (mg/dL)	131.05 ± 6.00	130.28 ± 13.00	161.33 ± 8.00*^#	143.28 ± 10.00*^#&
Plasma insulin (ng/mL)	1.34 ± 0.11	1.37 ± 0.12	3.43 ± 0.167*^#	1.70 ± 0.25*^#&
Liver TG (mg/g)	8.11 ± 0.30	8.36 ± 1.08	16.62 ± 1.06*^#	11.13 ± 0.19*^#&
Plasma ALT (IU/L)	23.09 ± 5.40	21.00 ± 3.10	41.23 ± 8.60*^#	33.26 ± 6.70*^#&
Plasma AST (IU/L)	30.41 ± 7.30	31.61 ± 3.10	46.85 ± 4.20*^#	39.21 ± 3.20*^#&

Note: Normal-fat diet (NFD); High-fat diet (HFD); Myocardin-related transcription factor A (MRTF-A); Triglyceride (TG); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST);*, P < 0.05 compared with WT + NFD group; ^#, P < 0.05 compared with MRTF-A^−/− + NFD group; ^&, P < 0.05 compared with WT + HFD group.

Effects of MRTF-A knockout on liver tissue histology of NASH mice

As shown in Figure 1, NFD mice had regularly-arranged hepatocytes, clear hepatic lobule structure, obvious hepatocyte cords, and normal stroma according to HE staining. By contrast, WT + HFD mice presented lipid deposits (green arrow) and inflammatory cell clusters (black arrow) in liver tissues. Compared with HFD group, mice in MRTF-A^−/− + HFD group were alleviated to a certain extent regarding HFD-induced liver lesions. Besides, NFD feeding mice had no lipid droplets in liver tissues, while HFD-fed mice showed more red areas indicating the existence of recruited lipid droplets as shown by Oil Red O staining. Compared with WT + HFD group, mice in the MRTF-A^−/− + HFD group exhibited decreased lipid droplets in the liver tissues. NAS (0–8) was calculated from the sum of scores for steatosis (0: < 5%; 1: 5%–33%; 2: 33%–66%; 3: > 66%), lobular inflammation (0: none; 1: < 2; 2: 2–4; 3: > 4) and hepatocyte ballooning (0 = none; 1 = few; 2 = many). And as seen from Table 3, the steatosis score, lobular inflammation score, ballooning score, and NAS were elevated in the HFD groups, but notably, these scores were lower in the MRTF-A^−/− + HFD group than the WT+HFD group (all P < 0.05).

Figure 1.

Effects of MRTF-A knockout on liver tissue histology. Note: A, Representative photomicrographs of hematoxylin-eosin (HE) stained liver sections of mice in each group (× 200); Photomicrograph of the mice showing normal liver morphology; HFD mice show presence of lipid deposits (green arrow) and inflammatory cell clusters (black arrow), which was less in MRTF-A knockout mice; B, Representative photomicrographs of Oil Red O stained liver sections of mice, which stained lipids bright red (× 200).

Table 3.

Comparison of steatosis score, lobular inflammation score, ballooning score, NAS score and collagen content among the groups.

Histological features	WT + NFD (n = 10)	MRTF-A^−/− + NFD (n = 10)	WT + HFD (n = 10)	MRTF-A^−/− + HFD (n = 10)
HE staining
Steatosis (0–3)	0.40 ± 0.52	0.30 ± 0.48	2.40 ± 0.52*^#	1.20 ± 0.79*^#&
Lobular inflammation (0–3)	0.30 ± 0.48	0.40 ± 0.52	2.50 ± 0.71*^#	1.30 ± 0.82*^#&
Ballooning (0–2)	0.10 ± 0.32	0.20 ± 0.42	1.40 ± 0.52*^#	0.80 ± 0.63*^#&
NAS (0–8)	0.80 ± 0.92	0.90 ± 0.74	6.30 ± 1.25*^#	3.30 ± 1.64*^#&
Sirius Red staining
Collagen content (%)	0.52 ± 0.01	0.46 ± 0.09	4.02 ± 0.11*^#	1.81 ± 0.19*^#&

Note: Normal-fat diet (NFD); High-fat diet (HFD); Myocardin-related transcription factor A (MRTF-A); NAFLD activity score (NAS, 0–8) was calculated from the sum of scores for steatosis (0: < 5%; 1: 5%–33%; 2: 33%–66%; 3: > 66%), lobular inflammation (0: none; 1: < 2; 2: 2–4; 3: > 4) and hepatocyte ballooning (0 = none; 1 = few; 2 = many). Collagen content determined by counting positive areas in six randomly selected fields. *, P < 0.05 compared with WT + NFD group; ^#, P < 0.05 compared with MRTF-A⁻^/− + NFD group; ^&, P < 0.05 compared with WT + HFD group.

Effects of MRTF-A knockout on liver fibrosis of NASH mice

Mice in the WT + HFD group showed apparently elevated collagen content (Sirius Red staining, Figure 2A and Table 3) and α-SMA expression (immunohistochemical staining, Figure 2A and Table 4) in liver as compared with the WT + NFD group, but HFD mice with MRTF-A^−/− knockout had less collagen content and α-SMA expression than the WT + NFD group (P < 0.05). According to the detection of qRT-PCR, HFD feeding led to obvious mRNA up-regulations of α-SMA, COL1A2, TGFβ1 and SMAD3 in liver tissues of mice. But compared with WT + HFD group, mice in the MRTF-A^−/− + HFD group were decreased in the expression of the above genes (all P < 0.05, Figure 2B–E). However, mice in the WT + NFD group and MRTF-A^−/− + NFD group had no significant difference regarding liver fibrosis (P > 0.05).

Figure 2.

Effects of MRTF-A knockout on liver fibrosis of NASH mice. Note: A, Representative photomicrographs of Sirius Red stained liver sections of mice in each group (× 200, the red part represented the deposition of collagen fibers), and representative photomicrographs of α-SMA expression (brown staining) quantified by immunohistochemistry (× 200); B–E, Relative mRNA expression of α-SMA (B), COL1A2 (C), TGFβ1 (D) and SMAD3 (E) measured by qRT-PCR; *, P < 0.05 compared with WT + NFD group and MRTF-A^−/− + NFD group; ^#, P < 0.05 compared with WT + HFD group.

Table 4.

Comparison of immunohistochemistry results among the groups.

	WT + NFD (n = 10)	MRTF-A^−/− + NFD (n = 10)	WT + HFD (n = 10)	MRTF-A^−/− + HFD (n = 10)
α-SMA expression (OD)	0.42 ± 0.06	0.44 ± 0.03	0.78 ± 0.08*^#	0.54 ± 0.01*^#&
F4/80-positive cells	28.26 ± 2.30	30.16 ± 5.40	99.36 ± 5.40*^#	52.37 ± 7.80*^#&
CCR2-positive cells	22.59 ± 8.00	23.36 ± 2.00	43.76 ± 3.20*^#	31.29 ± 2.20*^#&
NF-κB p65 expression (OD)	0.23 ± 0.10	0.30 ± 0.05	0.79 ± 0.10*^#	0.56 ± 0.03*^#&

Note: Normal-fat diet (NFD); High-fat diet (HFD); Myocardin-related transcription factor A (MRTF-A); Alpha smooth muscle actin (α-SMA); EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80); Chemokine (C-C motif) receptor 2 (CCR2); Nuclear factor-kappa B (NF-κB); Optic density (OD); *, P < 0.05 compared with WT + NFD group; ^#, P < 0.05 compared with MRTF-A^−/− + NFD group; ^&, P < 0.05 compared with WT + HFD group.

Impact of MRTF-A knockout on hepatic macrophage recruitment in NASH mice

HFD feeding led to significantly up-regulated expressions of MCP-1, CCR2, F4/80 and CD68 in liver tissues of mice (Figure 3A–D), and caused substantial increase in the number of F4/80-positive cells and CCR2-positive cells (all P < 0.05, Figure 3E and Table 4). At the same time, hepatic macrophages isolated from HFD-fed mice presented obvious expression of inflammatory genes, including TNF-α, IL-1β, MCP-1, and CCR2 (all P < 0.05, Figure 3F–I). However, the above-mentioned macrophage infiltration in liver tissue was significantly improved in MRTF-A^−/− + NFD mice (all P < 0.05).

Figure 3.

Effects of MRTF-A knockout on hepatic macrophage recruitment of NASH mice. Note: A–D, qRT-PCR results showed the relative mRNA expression of MCP-1 (A), CCR2 (B), F4/80 (C) and CD68 (D) in liver tissues of mice; E: Representative photomicrographs of F4/80 and CCR2 expression in liver tissues tested by immunohistochemistry (× 200); F4/80-positive cells (black arrow) were expressed in inflammatory foci and hepatic parenchyma, while CCR2-positive cells (black arrow) were predominantly expressed in inflammatory foci; F–I, qRT-PCR results showed mRNA expression of TNF-α (F), IL-1β (G), MCP-1 (H), and CCR2 (I) in hepatic macrophages; *, P < 0.05 compared with WT + NFD group and MRTF-A^−/− + NFD group; ^#, P < 0.05 compared with WT + HFD group.

Effects of MRTF-A knockout on NF-κB p65 in liver tissues of NASH mice

The result of immunohistochemistry demonstrated that NF-κB p65 expression was greatly up-regulated in liver tissues of HFD mice. But compared with WT + HFD mice, mice in the MRTF-A^−/− + HFD group declined dramatically in NF-κB p65 expression in liver tissues (both P < 0.05, Figure 4A, Table 4). Moreover, Western blot confirmed that MRTF-A knockout can significantly inhibit NF-κB p65 expression in nucleus of liver cells in HFD-fed mice (both P < 0.05, Figure 4B–C).

Figure 4.

Effects of MRTF-A knockout on NF-κB p65 in liver tissues of NASH mice. Note: A, Representative photomicrographs of NF-κB p65 expression (brown staining) in liver tissues of mice examined by immunohistochemistry (× 200); B–C, Densitometric analysis of NF-κB p65 expression in liver cell nucleus measured by Western blot and analyzed by Image Studio Lite software using TBP as a loading control. The relative expression of target protein was presented as gray value ratio of NF-κB p65/TBP. *, P < 0.05 compared with WT + NFD group and MRTF-A^−/− + NFD group; ^#, P < 0.05 compared with WT + HFD group.

Discussion

Obesity is one of the major independent risk factors of NAFLD and closely associated with the metabolic balance of glucose and lipid, and higher incidence rate of NASH in obese patients has been revealed in relevant epidemiological data.¹⁹ In our experimental mice feeding with HFD, knockout MRTF-A did reduce the body weight, which was consistent with the finding of a previous study.^20,21 As we know, free fatty acids can be directly used to synthesize TG.²² Because of insulin resistance, the body would present lipid metabolism disorder with significantly increased lipolysis of fat tissues, thus a lot of free fatty acids would be released into the blood to promote liver intake of free fatty acids from the blood, thereby eventually inducing lipid accumulation in liver cells, especially TG accumulation, which is directly related to the development and progression of NASH.^23,24 From this study, MRTF-A knockout can reduce the blood glucose, plasma insulin and hepatic TG of NASH mice. In the similar vein, another study also reported that MRTF-A deficiency can effectively ameliorate HFD-induced obesity, inflammatory responses, insulin resistance and hepatic steatosis.¹² Also, the decreased glucose and insulin levels was identified in MRTF-A-KO mice after HFD diet for 8 and 20 weeks.²⁵ Thus, MRTF-A deletion may play protective roles in NAFLD by improving lipid deposition and insulin condition of liver, which was also supported by the liver tissue pathology with decreased NAS score (including hepatocyte steatosis, intralobular inflammation and hepatocyte ballooning degeneration).

MRTF-A can regulate the expression of collagen in some tissues, which was closely related to various types of fibrosis, including myocardial infarction-induced fibrosis,²⁶ silica-induced pulmonary fibrosis,²⁷ TGFβ1-induced intestinal fibrosis and pleural fibrosis.^28,29 Of note, monomeric G-actin can regulate nucleus-cytoplasm shuttling of serum response factor (SRF) coregulators, namely, MRTFs, and thereby affect the expression of SRF target genes, including smooth muscle actin (SMA).³⁰ Worth mentioning, MRTF-A deletion was protective in cardiac remodeling and fibrosis accompanied with the decreased expression of a fibrosis-related gene COL1A2, a direct transcriptional target of SRF/MRTF-A.³¹ In addition, MRTF-A can function as a coactivator for Smad3.³² Here, the liver fibrosis in this study was improved in HFD-fed MRTF-A knockout mice with the decreased collagen content and down-regulations of α-SMA, COL1A2, TGFβ1 and SMAD3 when compared to those WT mice, suggesting that MRTF-A deletion can significantly mitigate liver fibrosis of NASH mice.

An important manifestation of NASH is infiltration of inflammatory cells, such as macrophages, neutrophils, natural killer (NK) cells and dendritic cells (DC), among which macrophage is regarded as the first barrier against liver injury.³³ Liver macrophages are an important source of liver cytokines and many factors secreted by macrophages are involved in the progression of NASH.^4,34 For example, CCR2 secreted by macrophages can bind to MCP-1 on the surface of peripheral monocytes to induce MCP-1 recruitment in the liver, while knockout of CCR2 gene can alleviate steatohepatitis by reducing the amount of macrophages in the liver, improving insulin resistance and inhibiting the release of pro-inflammatory factors.^35,36 TNF-α produced by macrophages exerts many functions,³⁷ and the up-regulated TNF-α was observed in the steatohepatitis models and the plasma of NASH patients, which could recruit various pro-inflammatory immune cells into the liver, including IL-1β, and cause insulin resistance.^38,39 Besides, MRTF-A over-expression can also remarkably increase the synthesis of endogenous pro-inflammatory mediators in lipopolysaccharide (LPS)-treated macrophages.³⁰ Another important result of this study was that MRTF-A knockout can reduce the expression levels of MCP-1, CCR2, F4/80 (an antigen marker highly specific for mature mouse macrophages⁴⁰) and CD68 (a marker for activated macrophages⁴¹) in liver tissues of HFD-fed mice. Moreover, hepatic macrophages isolated from HFD-fed mice also presented increased expressions regarding inflammatory genes, including TNF-α, IL-1β, MCP-1, and decreased expression level of CCR2. Thus, MRTF-A deficiency attenuated NASH progression by reducing hepatic macrophage infiltration in HFD-fed mice.

In recent years, accumulating evidence has demonstrated the importance of NF-κB in NASH and the activated NF-κB would promote the progression of NASH.⁴² NF-κB family can be classified into 5 subgroups in mammals, including RelA (p65), c-Rel, RelB, NF-κB1 (p50/p105) and NF-κB2 (p52/p100).⁴³ NF-κB family members usually form complexes with their inhibitor protein IKBS in the form of homodimers or heterodimers, which exist in the cytoplasm in an inactive form. When stimulated, lκB would be released with the activation of NF-κB and the phosphorylation of p65, thus the protein expression of p65 in the nucleus was increased.⁴⁴ There was evidence indicating that MRTF-A over-expression can enhance NF-κB-dependent pro-inflammatory transcription, while silencing MRTF-A can inhibit this process.³⁰ MRTF-A can promote the transcription of adhesion molecules in vascular endothelial cells through indirect interaction with p65 subunit of NF-κB.⁴⁵ MRTF-A relies on p65 to access chromatin and also actively affects the nuclear accumulation and target-binding affinity of p65.³⁰ In our study, compared with WT + HFD group, MRTF-A^−/− + HFD group mice had declined NF-κB p65 expression in liver tissues, which suggested that MRTF-A may act on NF-κB p65 to affect the development of NASH.

To sum up, MRTF-A knockout can significantly improve the pathological changes of liver tissues, reduce hepatic fibrosis, and mitigate macrophage infiltration in liver tissues of HFD-fed mice possibly through the inhibition of NF-κB p65.

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_09603271211002886 - Therapeutic effects of myocardin-related transcription factor A (MRTF-A) knockout on experimental mice with nonalcoholic steatohepatitis induced by high-fat diet

Supplemental Material, sj-pdf-1-het-10.1177_09603271211002886 for Therapeutic effects of myocardin-related transcription factor A (MRTF-A) knockout on experimental mice with nonalcoholic steatohepatitis induced by high-fat diet by Lei Zhang, Hua-Long Li, Ding-Ding Zhang and Xiao-Chun Cui in Human & Experimental Toxicology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Lei Zhang

Supplemental material

Supplemental material for this article is available online.

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