Propofol improves ischemia reperfusion-induced liver fibrosis by regulating lncRNA HOXA11-AS

Abstract

Liver ischemia reperfusion (IR) injury induces hepatic stellate cell (HSC) activation and liver fibrosis. Propofol (PRO) possesses a positive protective effect on liver ischemia reperfusion injury. We aimed to investigate PRO function and mechanism in IR-induced liver fibrosis. A mice model of liver IR was established. Hematoxylin-eosin (HE) staining was utilized to evaluate liver tissue’s pathological changes. Masson staining was applied to evaluate liver fibrosis. The expression level of α-SMA was measured by immunohistochemical (IHC). The expressions of lncRNA HOXA11-AS (HOXA11-AS), PTBP1, HDAC4, α-SMA, COL1A1 and Fibronectin were tested by qRT-PCR or Western blot. The commercial kits detected alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in serum. Enzyme-linked immunosorbent assay (ELISA) measured TNF-α and IL-6 levels. The binding relationship between HOXA11-AS, PTBP1 and HDAC4 was verified by RNA immunoprecipitation (RIP). Our results showed that PRO alleviated liver fibrosis and the inflammation in IR-induced mice. PRO decreased the expression levels of HOXA11-AS, PTBP1 and HDAC4. Furthermore, HOXA11-AS overexpression abolished the protective effect of PRO against liver fibrosis in mice with IR-disposed. HOXA11-AS interacted with PTBP1 to regulate HDAC4 level and prevented its degradation in JS-1 cells. HDAC4 silencing eliminated the regulatory of HOXA11-AS overexpression on fibrosis and inflammation in IR-induced mice PRO inhibited HOXA11-AS expression to regulate HDAC4, thereby influencing liver fibrosis and inflammation induced by IR. It suggesting that PRO plays a protective role in liver fibrosis induced by ischemia-reperfusion in mice by regulating HOXA11-AS/PTBP1/HDAC4 axis.

INTRODUCTION

Liver ischemia reperfusion (IR) is a complication of complex liver surgery, liver transplantation, and liver trauma, resulting in significant morbidity and mortality (Pulitanò and Aldrighetti, 2008). Liver fibrosis is related to liver disease progression and is a key factor in liver disease prognosis and hepatocellular carcinoma (HCC) risk (Roehlen et al., 2020). It is a common pathological change developed via various chronic liver injuries caused by viral infection, drugs, metabolic disorders and autoimmune disorders (Ge et al., 2017). Liver fibrosis is manifested by a large number of hepatocyte apoptosis and necrosis and excessive deposition of extracellular matrix at the tissue level (Jiang et al., 2018). Much evidence showed that hepatic stellate cell (HSC) activation promoted liver fibrosis occurrence and development (Kostallari et al., 2018). Therefore, inhibiting the activation of HSC might be an early preventive and effective method for treating liver fibrosis. In addition, there is increased evidence that acute IR injury triggered pro-fibrotic pathways and interstitial fibrosis, ultimately leading to organ dysfunction (Liu et al., 2019), but the specific molecular mechanisms remain to be determined.

Propofol (PRO) is a widely used clinical anesthetic (Kotani et al., 2008). In recent years, PRO plays positive protective effects on kidney, liver and other organs (Zhang et al., 2019; Hsing et al., 2011). Studies revealed PRO suppressed IR-induced liver injury through repressing the inflammatory response, and renal fibrosis (Liu et al., 2017; Song et al., 2016). However, whether PRO inhibits IR-induced liver fibrosis remains to be further explored. Recent studies clarified the function of lncRNA homeobox A11 antisense (HOXA11-AS) in liver cancer. It was reported that HOXA11-AS was involved in the development of nephritis (Li et al., 2020b). HOXA11-AS promoted Collagen I and α-SMA expressions to exacerbate myocardial fibrosis (Wang et al., 2019). In addition, Song et al. (2020) reported PRO inhibited HOXA11-AS expression and hindered HCC progression. However, whether PRO plays a critical role in IR-induced liver fibrosis by regulating HOXA11-AS expression needs further exploration.

Polypyrimidine tract-binding protein 1 (PTBP1) is associated with different liver diseases. Zhang et al. (2017) reported that lncRNA maternally expressed gene 3 (MEG3) induced cholestatic liver injury by interacting with PTBP1 to facilitate SHP mRNA decay. In CCl₄-induced liver fibrosis, metastasis associated lung adenocarcinoma transcript 1 (MALAT1) promoted ubiquitin-specific protease 8 (USP8) mRNA degradation through PTBP1, thereby promoting macrophage pyroptosis and inflammation (Shu et al., 2021). In addition, lncRNA HOXA transcript at the distal tip (HOTTIP) facilitated cell proliferation, invasion, and migration through interacting with PTBP1 to promote KH-type splicing regulatory protein (KHSRP) expression in osteosarcoma [17]. We predicted that HOXA11-AS could bind to PTBP1 by bioinformatics prediction. And, we speculated PRO might relieve IR-induced liver injury and liver fibrosis through HOXA11-AS/PTBP1. Previous literature reported that overexpression of histone deacetylases 4 (HDAC4) intensified myocardial IR injury (Zhang et al., 2018). Overexpression of HDAC4 promoted HSC activation, upregulated Collagen I and α-SMA expressions, and promoted liver fibrosis process (Li et al., 2017). Through bioinformatics prediction analysis, we found that PTBP1 could bind to HDAC4, but the relationship between HDAC4 and PTBP1 had not been reported.

Based on the above background, we make a reasonable hypothesis that PRO may alleviate IR-induced liver fibrosis by regulating the lncRNA HOXA11-AS/PTBP1/HDAC4 axis. In the present study, we conducted in vivo experiments to explore whether PRO alleviated IR-induced liver fibrosis by regulating the HOXA11-AS/PTBP1/HDAC4 axis. It will help to deepen the understanding of the pathogenesis of IR-induced liver fibrosis and provide novel targets and strategies for treatment of liver fibrosis.

MATERIALS AND METHODS

Establishment of an animal model of liver IR

Sixty C57BL/6 mice (male, 8 weeks, 20–25 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and kept in a 20–22°C environment with a 12-hr light/dark cycle. All mice were randomly divided into Sham (n = 10), IR (n = 10), PRO+IR (n = 10), oe-NC+sh-NC+PRO+IR (n = 10), oe-HOXA11-AS+sh-NC+PRO+IR (n = 10), and oe-HOXA11-AS+sh-HDAC4+PRO+IR groups (n = 10).

The mice received intraperitoneal injection of ketamine (50 mg kg^-1) and xylazine (10 mg kg^-1) for anesthesia. Then midline abdominal surgery was performed on the mice. A non-traumatic clamp was used to clamp the bottom of the left lobe to block the blood supply from the artery and hepatic portal vein for 90 min, and then the clamp was removed to induce reperfusion for 6 hr (Liu et al., 2019). The animal experiments were approved by the Hunan Provincial People’s Hospital (The first-affiliated hospital of Hunan Normal University).

Lentiviral vectors construction and infection

pLV-Puro-HOXA11-AS (oe-HOXA11-AS), pGLVU6-HDAC4 (sh-HDAC4) and their negative control (oe-NC and sh-NC) were provided by GenePharma (Shanghai, China), respectively. Two weeks before IR treatment, the mice in the oe-NC+sh-NC+PRO+IR group, the oe-HOXA11-AS+sh-NC+PRO+IR group, and the oe-HOXA11-AS+sh-HDAC4+PRO+IR group were infected with oe-NC, sh-NC, oe-HOXA11-As or sh-HDAC4 into liver tissue via the tail vein.

PRO treatment

The mice in the IR + PRO group, the oe-NC+sh-NC+PRO+ IR group, the oe-HOXA11-AS+sh-NC+PRO+IR group, and the oe-HOXA11-AS+sh-HDAC4+ PRO+IR group were injected with PRO (40 mg/kg, #2078-54-8, Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally (Ma et al., 2021). After two weeks, all mice were anesthetized through pentobarbital sodium intraperitoneal injection, and blood and liver tissue were collected to the following experiments.

Hematoxylin-eosin (HE) staining

HE staining was performed to detect liver tissue morphological damage. Slices were baked at 60°C for 12 hr, dewaxed, stained with hematoxylin, stained with eosin, and dehydrated with 95–100% gradient alcohol. Then we took it out, and placed it in xylene, 2 times. After sealing with neutral gum, results were monitored with an optical microscope (BA210T, MOTIC, Xiamen, China).

Masson staining

Liver tissue sections were deparaffinized, rehydrated, and stained with Weiger’s iron hematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA). Then, the samples were rinsed with distilled water and stained with Ponceau acid fuchsin solution. Samples were incubated with 1% phosphomolybdic acid aqueous solution and aniline blue solution, respectively. Samples were differentiated using 1% acetic acid solution. After dehydration with 95% alcohol, the sections were mounted with neutral gum, monitored under the microscope (BA210T, MOTIC) and photographed. Masson staining was analyzed for collagen volume fraction, which is the percentage of blue area positive for collagen to the total tissue area.

Immunohistochemistry (IHC)

The liver tissues of mice were taken, and α-SMA positive condition in liver tissues was tested by IHC. Slices were baked at 60°C for 12 hr, deparaffinized to water, and heated antigens. 1% periodic acid was used for 10 min. Then the slices were incubated with α-SMA (14395-1-AP, 1:1500, Proteintech, Chicago, USA) at 4°C overnight, and incubated with secondary antibody at 37°C for 30 min. DAB was used for color rendering, and hematoxylin was redyed. Then the distilled water was used to wash, the tablets were placed in xylene, sealed with neutral gum and monitored with the microscope (BA210T, MOTIC, China) and photographed. Integrated optical density (IOD) analysis was performed using Image-Pro Plus 6.0 image software and expressed as mean optical density (IOD of positive area in the field of view/area of tissue in the field of view).

Cell culture

The mouse hepatic stellate cells JS-1 were provided by American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium with 10% fetal bovine serum (FBS, Gibco), penicillin and streptomycin (100 U/mL). These cells were incubated in an incubator at 37°C and 5% CO₂.

Cell transfection

pcDNA 3.1-HOXA11-AS (oe-HOXA11-AS), pcDNA 3.1-PTBP1 (oe-PTBP1) and their negative pairs (oe-NC) were provided by GenePharma (Shanghai, China). When JS-1 cells were in logarithmic growth phase, the plasmid transfection was performed, and those plasmids were transfected into JS-1 by Lipofectamine 3000 (Invitrogen). After 48 hr, HOXA11-AS and PTBP1 levels were detected by quantitative real-time PCR (qRT-PCR).

Quantitative real-time PCR (qRT-PCR)

RNAiso Plus (Takara, Dalian, China) extracted total RNA, and RNA was reverse transcribed into cDNAs with cDNA Reverse Transcription Kit (#CW2569, ComWin Biotech, Beijing, China). Genes were tested on ABI 7900 system using Ultra SYBR Mixture (#CW2601, ComWin Biotech). The relative expression of mRNA was calculated by the 2^−ΔΔCt method with GAPDH as internal reference gene. Primer sequences are as follows:

HOXA11-AS (F): 5’- AGAAATCTGGACCCGAGACG-3’,

(R): 5’-GTTACACCGGCGATTACGTG-3’;

PTBP1 (F): 5’-CAGAGGACGACCTCAAGAGC-3’,

(R): 5-GGTGGACTTGGAAAAGGACA-3’;

HDAC4 (F): 5’-GGCGAGCACAGAGGTGAAGATG-3’,

(R): 5’-GCTGTGCTGTGTCTTCCCATAC-3’;

α-SMA (F): 5’-TGTGAAGAGGAAGACAGCACAGCT-3’,

(R): 5’-GATGGCTGGAAGAGGGTCTCCGGG-3’’;

COL1A1 (F): 5’-AGATTGAGAACATCCGAGCC-3’,

(R): 5’-TCCAGTACTCTCCGCTCTTCC-3’;

Fibronectin (F): 5’-CCCCAACTGGTTACCCTTCC-3’,

(R): 5’-TGTCCGCCTAAAGCCATGTT-3’;

GAPDH (F): 5’-AGCCCAAGATGCCCTTCAGT-3’,

(R): 5’-CCGTGTTCCTACCCCCAATG-3’.

Western blot

RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China) extracted total protein from tissue, and protein quantification was performed on each group with BCA protein assay kit (#BL521A, Biosharp, Guangzhou, China). 12% SDS-PAGE loading buffer (#MB2479, meilunbio, Dalian, China) was mixed, and protein was adsorbed on PVDF membrane by gel electrophoresis. Then protein was blocked with 5% nonfat milk solution for 1 hr. primary antibodies and were incubated overnight at 4°C. The membrane was washed with 1 × TBST and incubated with secondary antibody conjugate in 1 × TBS for 1 hr at room temperature. Then, the relative levels of each protein were quantified with Quantity One software (Bio-Rad, Hercules, CA, USA). The primary antibodies used in this study were as follows: PTBP1 (#67462-1-Ig, 1:5000, Proteintech), HDAC4 (#66838-1-Ig, 1:1000, Proteintech), α-SMA (14395-1-AP, 1:4000, Proteintech), COL1A1 (67288-1-Ig, 1:5000, Proteintech), Fibronectin (15613-1-AP, 1:2000, Proteintech), β-actin (66009-1-Ig, 1:20000, Proteintech). β-actin was usedas internal reference.

Detection of ALT and AST

Alanine aminotransferase (ALT, #C009-2-1) and aspartate aminotransferase (AST, #C0010-2-1) kits were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The levels of ALT and AST were detected according to the instructions.

Enzyme-linked immunosorbent assay (ELISA)

Tumor necrosis factor-α (TNF-α, #ml077385, Mlbio, Shanghai, China) and interleukin-6 (IL-6, #ml063160, Mlbio) quantitative ELISA kits were utilized to examine TNF-α and IL-6 concentrations in mice serum according to the instructions. Bio-Tek microplate reader (MB-530, Heales, Shenzhen, China) was performed to evaluate TNF-α and IL-6 levels.

RNA pull-down

Pierce Magnetic RNA-Protein Pull-Down Kit (#20164, Invitrogen, Carlsbad, CA, USA) was purchased for RNA pull-down. All steps were followed according to the instructions. A solution of the RNA-binding protein complex was obtained. qRT-PCR was used to detect the RNA-binding protein PTBP1 and HOXA11-AS.

RNA immunoprecipitation (RIP) assay

Pairwise binding between HOXA11-AS, PTBP1 and HDAC4 was verified by EZMagna RIP kit (Millipore, Billerica, MA, USA). In short, JS-1 cells were lysed with RIP lysis buffer for 30 min at 4°C. Anti-PTBP1 (12582-1-AP, 1:50, Proteintech) and anti-IgG (14678-1-AP, Proteintech) were used to immunoprecipitate target RNA. Precipitated RNA was determined by qRT-PCR.

Actinomycin D (Act D) treatment

JS-1 cells were treated with 5 μM Act D (HY-17559, MedChemExpress, New Jersey, USA) for 1, 2, 3, 4, 5, and 6 hr. Then JS-1 cells were collected to extract RNA. The RNA levels were tested by qRT-PCR.

Statistical Analysis

Statistical analysis was performed using Graphpad Prism 8.0 software. Data were expressed as mean ± standard deviation (mean ± SD). Student’s t test or one-way analysis of variance (ANOVA) was performed for two or multiple group comparison. P < 0.05 indicated that the difference was statistically significant.

RESULTS

PRO assuaged IR-induced liver fibrosis

To study the effect of PRO on liver fibrosis, we first established an animal model of liver IR. HE staining results showed that IR-induced liver fibrosis exhibited abnormal histological morphology and diffused nuclei, addition of PRO improved the pathological changes of liver injury (Fig. 1A). Masson staining further revealed that a lot of fibrous septum formation, collagen fiber hyperplasia and deposition in the IR-induced liver tissue, while PRO treatment reduced collagen deposition and decelerated fibrotic degree (Fig. 1B). Moreover, α-SMA level was notably elevated in the IR group; however, α-SMA was reduced after PRO treatment (Fig. 1C). These results suggested that PRO reduced pathological changes of morphological structure and process precipitation of collagen to relieve liver fibrosis in IR-induced mice.

Fig. 1

PRO prevented IR-induced liver fibrosis. A–B. the pathological changes of liver tissue was evaluated by HE and Masson staining. C. α-SMA expression was assessed by IHC. **P < 0.01, ***P < 0.001.

PRO relieved IR-induced inflammation and fibrosis, and reduced HOXA11-AS expression

Next, we further explored the role of PRO in IR-induced liver fibrosis. PRO treatment reversed the increase of the content of AST and ALT in IR mice (Fig. 2A–2B). After IR treatment, the release of inflammatory factors TNF-α and IL-6 was remarkably upregulated, but PRO moderated IR-induced inflammatory response in mice (Fig. 2C–2D). In addition, PRO treatment reversed the up-regulation expressions of α-SMA, COL1A1, and Fibronectin (FN) in the IR group (Fig. 2E–2F). The expressions of HOXA11-AS, PTBP1 and HDAC4 in the IR group were elevated, while the expressions of HOXA11-AS, PTBP1, and HDAC4 were reduced after PRO treatment (Fig. 2G–2J). These results revealed that PRO decreased HOXA11-AS, α-SMA, COL1A1, and Fibronectin expressions in IR-induced liver fibrosis.

Fig. 2

PRO remitted IR-induced liver fibrosis and decreasing HOXA11-AS expression. A–B. ALT and AST levels in serum were tested by commercial kits. C–D. TNF-α and IL-6 levels were tested by ELISA. E–F. α-SMA, COL1A1 and FN expressions were determined by qRT-PCR and Western blot. G–J. HOXA11-AS, PTBP1 and HDAC4 expressions were monitored by qRT-PCR or Western blot. *P < 0.05, **P < 0.01, ***P < 0.001.

HOXA11-AS targeted PTBP1 to regulate HDAC4 expression

Next, we first verified the binding relationship between HOXA11-AS, PTBP1 and HDAC4 in JS-1 cells by RIP. The anti-PTBP1 group enriched more HOXA11-AS and HDAC4 than anti-IgG group, which meant that there was a relationship between PTBP1, HOXA11-AS and HDAC4 (Fig. 3A–3B). The binding relationship between HOXA11-AS and PTBP1 was verified using RNA pull-down (Fig. 3C). The expressions of HOXA11-AS, PTBP1 and HDAC4 were significantly upregulated after overexpression of HOXA11-AS; nevertheless, HOXA11-AS, PTBP1 and HDAC4 were significantly down-regulated by HOXA11-AS knockdown in JS-1 cells (Fig. 3D–3G). Subsequently, HOXA11-AS overexpression inhibited the degradation rate of HDAC4, whereas HOXA11-AS deficiency accelerated the degradation of HDAC4 (Fig. 3H). Additionally, PTBP1 and HDAC4 expression were obviously enhanced after transfection with oe-PTBP1, while PTBP1 deficiency decreased PTBP1 and HDAC4 levels in JS-1 cells (Fig. 3I–3K). Similarly, knockdown of PTBP1 promoted the degradation of HDAC4 (Fig. 3L). These results indicated that HOXA11-AS targeted PTBP1 to regulate HDAC4 expression.

Fig. 3

HOXA11-AS regulated HDAC4 expression via targeting PTBP1. A–B. The binding relationship between HOXA11-AS, PTBP1 and HDAC4 in JS-1 cells was verified by RIP assay. C. The binding relationship between HOXA11-AS and PTBP1 in JS-1 cells was verified by RNA pull-down. D–G. HOXA11-AS, PTBP1, and HDAC4 expressions were detected by qRT-PCR or Western blot. H. HDAC4 stability was determined by qRT-PCR after Act D treatment (1, 2, 3, 4, 5, 6 hr). I–K. PTBP1 and HDAC4 expressions were tested by qRT-PCR and Western blot. L. HDAC4 stability was assessed by qRT-PCR after Act D treatment (1, 2, 3, 4, 5, 6 hr). *P < 0.05, **P < 0.01, ***P < 0.001.

Overexpression of HOXA11-AS reversed PRO protective effect on IR-induced liver fibrosis by regulating HDAC4

HDAC4 was remarkably reduced when knocked down HDAC4 (Fig. 4A–4B). HOXA11-AS sufficiency aggravated pathological changes and degree of fibrosis in PRO treatment liver fibrosis in IR mice, while pathological changes were alleviated by sh-HDAC4 (Fig. 4C–4D). After IR mice were treated with PRO, the α-SMA expression was reduced, while overexpression of HOXA11-AS raised α-SMA level, but silencing of HDAC4 reversed the effect of oe-HOXA11-AS (Fig. 4E). These results suggested that HOXA11-AS overexpression accentuated IR-induced liver fibrosis and counteracted the impacts of PRO by mediating HDAC4 expression.

Fig. 4

HOXA11-AS overexpression offset the role of PRO on liver fibrosis in IR mice. A–B. HDAC4 expression was assessed by qRT-PCR and Western blot. C–D. Liver tissue’s pathological changes was analyzed by HE and Masson staining. E. α-SMA expression in tissue was determined by IHC. *P < 0.05, **P < 0.01, ***P < 0.001.

PRO alleviated liver fibrosis-related molecules expressions by regulating the HOXA11-AS/ HDAC4 axis in IR-induced mice

Finally, we further explored the mechanism of PRO regulated the HOXA11-AS/PTBP1/HDAC4 axis in IR-induced liver fibrosis. HOXA11-AS overexpression promoted HDAC4 expression and abrogated the inhibiting effect of PRO on HDAC4; moreover, the enhancement effect of HOXA11-AS on HDAC4 expression was reversed by sh-HDAC4 in IR-induced mice (Fig. 5A–5B). PRO repressed AST and ALT levels in IR mice; however, AST and ALT levels were promoted after transfection with oe-HOXA11-AS, but co-transfected with HDAC4 silencing had the opposite effect (Fig. 5C–5D). The levels of inflammatory factor (TNF-α and IL-6) release were suppressed in IR mice after PRO treatment; furthermore, the anti-inflammation of PRO was eliminated by HOXA11-AS overexpression, while knockdown of HDAC4 reversed the effect of HOXA11-AS overexpression (Fig. 5E–5F). In addition, overexpression of HOXA11-AS attenuated the inhibitory effect of PRO on the expression of fibrosis-related molecules (α-SMA, COL1A1 and FN), while this phenomenon was abolished by sh-HDAC4 (Fig. 5G–5J). These results displayed that PRO impeded HDAC4 expression via inhibiting HOXA11-AS to alleviate the IR-induced liver fibrosis.

Fig. 5

PRO regulated the HOXA11-AS/HDAC4 axis to impede liver fibrosis-related molecule expressions. A–B. HDAC4 expression was detected by qRT-PCR and Western blot. C–D. ALT and AST levels in serum were evaluated by commercial kits. E–F. TNF-α and IL-6 levels were tested by ELISA. G–J. α-SMA, COL1A1 and FN expressions were determined by qRT-PCR and Western blot. *P < 0.05, **P < 0.01, ***P < 0.001.

DISCUSSION

IR injury induces HSC activation and liver fibrosis (Cheng et al., 2008). Liver fibrosis is the last usual pathway of chronic or recurrent liver damage and is the main cause of morbidity and mortality in chronic liver disease patients (Campana and Iredale, 2017). Prevention or reversal of liver fibrosis became the primary endpoint of clinical trials of novel liver-specific drugs (Schuppan et al., 2018). PRO was widely used in general anesthesia and has been reported to protect various organs including the liver from IR (Kim et al., 2020). In this research, we first explored PRO effect on liver fibrosis by constructing a mice model of liver IR. We found that PRO regulated the HOXA11-AS/PTBP1/HDAC4 axis to mitigate liver fibrosis in IR mice.

PRO was reported to ameliorate IR-induced renal interstitial fibrosis in mice by downregulating TAK1 and inhibiting the apoptosis (AbuHammad et al., 2019). A previous study also indicated that PRO reduced oxidative stress and hepatocyte apoptosis by modulating the inflammatory response in hepatic IR injury [24]. Our study revealed that PRO alleviated IR-induced liver fibrosis. A previous study reported that PRO inhibited HOXA11-AS expression to suppress liver cancer (Song et al., 2020). HOXA11-AS, with a length of 1628 bp, has been overemphasized as an important initiating and promoting factor during the proliferation and metastasis of malignant tumors in recent years (Xue et al., 2018). The expression of HOXA11-AS was upregulated in liver cancer (Zhan et al., 2018). In this study, we found that HOXA11-AS expression was increased in IR-induced liver tissue, and overexpression of HOXA11-AS reversed the inhibitory of PRO treatment liver fibrosis in IR-induced mice.

PTBP1 is one of the most widely studied multiple RNA-binding proteins (RBPs), regulating almost all mRNA metabolic steps and processes (Kang et al., 2019). PTBP1 was a positive regulator of HCC progression (Kang et al., 2019). LncRNA GCAT1 was reported to be associated with premature ovarian insufficiency through regulating p27 translation in granulosa cells through competitive binding with PTBP1 (Li et al., 2020a). In addition, knockdown of lncRNA OIP5-AS1 inhibited hepatoblastoma growth and stemness by binding to PTBP1 and increasing β-catenin (Jiang et al., 2022). In the present study, we discovered that HOXA11-AS recruited PTBP1 to motivate the stability of HDAC4. HDAC4 is a member of the HDACs family, which is closely associated with cell development (Hou et al., 2020). A previous study reported that silencing of HDAC4 significantly inhibited HSC activation in the context of liver fibrosis (Huang et al., 2015). In our research, overexpression of HOXA11-AS promoted HDAC4 expression to reverse the effect of PRO on liver fibrosis in IR-induced mice, while HDAC4 deficiency reduced the release of inflammatory factors (TNF-α and IL-6) and the expression of fibrosis-related factors (α-SMA, COL1A1, and FN) to improve liver fibrosis in mice with IR treatment.

In conclusion, our findings suggested that PRO suppressed IR-induced liver fibrosis via regulating the HOXA11-AS/PTBP1/HDAC4 axis. Our study provided a theoretical basis for liver fibrosis pathogenesis, and new targets and strategies for IR-induced liver fibrosis clinical treatment. However, this study still has some shortcomings. Our study is only a basic study, and PRO protection against liver fibrosis mechanism needs to be verified by more in-depth clinical research in the future.

Conflict of interest

The authors declare that there is no conflict of interest.

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