Inhibition of TGFβ1 accelerates regeneration of fibrotic rat liver elicited by a novel two-staged hepatectomy

Aims: Emerging evidence is demonstrating that rapid regeneration of remnant liver elicited by associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) may be attenuated in fibrotic livers. However, the molecular mechanisms responsible for this process are largely unknown. It is widely acknowledged that the TGFβ1 signaling axis plays a major role in liver fibrosis. Therefore, the aims of this study were to elucidate the underlying mechanism of liver regeneration during ALPPS with or without fibrosis, specifically focusing on TGFβ1 signaling. Approach: ALPPS was performed in rat models with N-diethylnitrosamine-induced liver fibrosis and no fibrosis. Functional liver remnant regeneration and expression of TGFβ1 were analyzed during the ALPPS procedures. Adeno-associated virus-shTGFβ1 and the small molecule inhibitor LY2157299 (galunisertib) were used separately or in combination to inhibit TGFβ1 signaling in fibrotic rats. Results: Liver regeneration following ALPPS was lower in fibrotic rats than non-fibrotic rats. TGFβ1 was a key mediator of postoperative regeneration in fibrotic liver. Interestingly, AAV-shTGFβ1 accelerated the regeneration of fibrotic functional liver remnant and improved fibrosis, while LY2157299 only enhanced liver regeneration. Moreover, combination treatment elicited a stronger effect. Conclusions: Inhibition of TGFβ1 accelerated regeneration of fibrotic liver, ameliorated liver fibrosis, and improved liver function following ALPPS. Therefore, TGFβ1 is a promising therapeutic target in ALPPS to improve fibrotic liver reserve function and prognosis.


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Forward： TGTCAAGACGCAGTAGCCGTTTAC Note：CST is the abbreviation of "Cell Signaling Technology". the Experimental Animal Center of HMU. After arrival, the animals were given ad libitum access to food and water in a vivarium that was maintained at 23 ± 1 °C with a 12/12 h day/night cycle. Animals were randomly divided into groups, with 3-5 animals sacrificed for each data point.

N-diethylnitrosamine (DEN) and carbon tetrachloride (CCL4) administration
DEN and CCL4 were used to induce liver fibrosis in rats separately. DEN (Sigma Aldrich, 73861, USA) was diluted in sterile phosphate-buffered saline (PBS) and intraperitoneally injected into rats twice a week at a dose of 25 mg/kg from the age of 6 weeks. Sterile PBS was injected into the control group. CCl4-treated rats were intraperitoneally injected with 40 % CCl4 olive oil solution (v/v) at 1ml/kg body weight twice a week. The DEN treatment and CCL4 treatment lasted up to 14 weeks.
METAVIR scoring was used to evaluate the degree of liver fibrosis (15).

Adeno-associated virus (AAV) vectors
AAV9 GFAP vectors designed to specifically transfect HSCs in vivo were generated by Genechem Co. LTD (Shanghai, China). A short hairpin RNA sequence targeting the TGFβ1 gene was cloned and packaged into the AAV vector (CV258, GFAP-EGFP-MIR155). The vector also encoded a green fluorescent protein (GFP) reporter, allowing for cellular visualization. The TGFβ1 RNA interference sequences used were 5'ACCGCTAGCTAACTGGAGGCTTGCTGAAGGCTGTATGCTG and 3'CAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCCCAAGCTTGGT.
TTCTCCGAACGTGTCACGT was used as control.

AAV delivery into rat livers
Rats were anesthetized by intraperitoneal injection of 2% pentobarbital. A longitudinal incision was then made in the abdomen to expose the portal vein. 8 × 10 11 viral particles of AAV9-GFAP-shTGFβ1 or AAV9-GFAP-shNC in a final volume of 2 mL were injected into the portal vein with a 25-gauge needle, as described previously (18). Four weeks after AAV infection, ALPPS was performed.

Development of the ALPPS rat model
After 4-5 days of DEN/PBS treatment (week 12), rats were weighed and anesthetized by intraperitoneal injection of 2% pentobarbital. A state of deep anesthesia was confirmed by the toe-pinch reflex.
Step I: To simulate human ALPPS in rats, portal vein branches were ligated and liver parenchyma was split between the right and left middle lobes. The left lateral lobe (LLL) was resected first, as is often done clinically to remove small colonic liver metastases. Portal branches of the right lobe (RL) and right middle lobes (RML) were individually ligated with 5-0 silk. The portal vein branches and the hepatic artery of the left middle lobe (LML) as well as the caudate lobe (CL), which served as the FLR and represented 26-30% of the total liver volume, were conserved.
The liver parenchyma was split between the deportalized RML and the normally perfused LML with bipolar forceps.
Step II: Rats underwent a relaparotomy 48 h after Step I to remove the deportalized RL and RML.

Liver sample acquisition
Blood was obtained from the intrahepatic vena cava before organ harvesting and centrifuged at 3000 ×g for 30 min. Serum aliquots were collected and stored at -80 ℃.
Serum alanine aminotransferase (ALT), albumin (ALB), and total bilirubin (TBIL) levels were determined using an automated chemical analyzer (Johnson Vitros 5600 automatic biochemical analyzer, USA). Liver tissues were washed repeatedly with precooled PBS, weighed, and aliquoted. Part of the liver tissue was snap frozen directly and preserved at -80 ℃, and part of the tissue later used to extract RNA was placed in a protectant tissue reagent (Qiagen, 76104, Germany) for 24 h at room temperature and then stored at -80 ℃. The remaining liver tissue was fixed in 10% formalin.

Liver regeneration
Liver regeneration was assessed by calculating the ratio of total FLR, including LML and CL, to body weight (FLR/BW). 3-5 animals were used for each time point.

Isolation and culture of primary hepatocytes (PHCs) and HSCs
A two-step collagenase perfusion method was used to isolate and purify rat hepatocytes

Co-culture
PHCs/BRL-3A cells were plated on 10 cm dishes (2 × 10 5 cells/mL) and incubated at 37 °C in 5% CO2 for 48-72 h. When the cultures reached ∼50% confluence, the medium was replaced with medium used to culture HSCs for 48 h. 10 ng/mL cytokine rat TGFβ1 (rTGFβ1) (Novoprotein, Shanghai, China) and 10 μM galunisertib dissolved in DMSO were added to the medium and the cells were incubated for 24 h.

5-Ethynyl-20-deoxyuridine (EdU) assay
Cells were incubated with EdU (Ribobio, Shanghai, China) for 2 h and processed according to the manufacturer's instructions. After washing with PBS, the cells were treated with 300 μL of Apollo reaction cocktail for 30 min. Then, the DNA contents of the cells in each well were stained with Hoechst. In vivo, 5 mg/kg EdU was injected intraperitoneally 24 h before the animals were sacrificed. The tissues were fixed in 10% neutral formalin, dehydrated, embedded in paraffin, and sectioned at 4 μm. The sections were incubated with Apollo reagent for 60 min and the nuclei were stained with Hoechst.
The cells and tissue sections were visualized under a fluorescence microscope.
Quantification of EdU-positive hepatocytes was performed by blinded manual counting of five random visual fields (200×).

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from 50 mg of tissue using GeneJET RNA Purification Kit (Thermo Scientific, k0732, USA). RNA quality and quantity were assessed using a spectrophotometer. After generation of the complementary DNA sequence (Takara reverse transcription system, rr036, China), qRT-PCR amplification and data analysis were performed on an ABI StepOne Plus Biosystem System using TB Green Premix Ex Taq™ II (Takara, rr820, China). The primers used for qRT-PCR are listed in Supplementary Table 1.

Protein analysis
Total protein was extracted from 50 mg of liver tissue using Tissue Extraction Reagent II (Invitrogen, fnn0081, USA) and an EDTA-free protease inhibitor cocktail kit (Roche, 04693132001). Protein concentration was determined using a BCA protein assay kit (Sangon biotech, c503021, Shanghai, China). Total protein was separated by acrylamide gel and transferred to nitrocellulose. After blocking with 5% bovine serum albumin and incubation with primary and secondary antibodies, blots were displayed on the film and quantified using ImageJ v1.53a (Wayne Rasband, National Institutes of Health, USA). The concentration of TGFβ1 in each sample was measured using an ELISA kit (Sangon biotech, d751002, Shanghai, China).

Immunohistochemistry and picric-sirius red (PSR) staining
Ninety rat liver samples were embedded into a tissue microarray by Outdo Biotech

Immunofluorescence assay
Liver tissue sections or cells were permeabilized with 0.1% Triton X-100 and then incubated with TGFβ1, α smooth muscle actin (αSMA), hepatocyte nuclear factor α (HNF4α), and GFP primary antibodies. After washing with PBS, the samples were incubated with goat anti-mouse IgG and goat anti-rabbit IgG secondary antibodies.
Antifade reagent with the nuclear stain Hoechst (Invitrogen, P36985) was added before imaging. Images were captured on a fluorescence microscope.

Statistical analyses
All statistical analyses were blindly performed. Data are presented as mean ± standard deviation. Differences between groups were assessed by Mann-Whitney U Test.
Shapiro-Wilk method was used to test if the data were Gaussian distributed. Two-tailed ANOVA was used for multiple testing and Šidák method was used to justify the data.