Mice and NASH model

C5−/− (B10.D2-Hc0H2dH2-T18c/0SnJ) mice and their haplotype (B10.D2-Hc1H2dH2-T18c/nSnJ) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). C5aR1−/− mice with a C57BL/6 background were purchased from GemPharmatech (Jiangsu, China). All mice were maintained in a specific pathogen-free facility at Guangxi Medical University. A Western diet (WD) and chemically induced murine NASH model as described previously [22] were used. Briefly, male mice were fed normal chow diet/corn oil (ND + Oil), WD/corn oil (WD + Oil) or WD combined with low weekly dose of intraperitoneal carbon tetrachloride (CCl4) for 12 weeks. Male C5−/− and their haplotype, C5aR1−/− mice and C57BL/6 wild-type mice, aged 8–12 weeks, were fed a WD/CCl4 (WD + CCl4) for 12 weeks. Corn oil or CCl4 were intraperitoneally injected into male mice once a week. All animal experiments were approved by the Animal Care and Use Committee of Guangxi Medical University, Guangxi, China.

Histopathologic and immunohistochemistry (IHC)

Fresh liver tissues were frozen, and sliced into 8 μm thick sections. The sections were stained with an Oil Red O staining. Liver biopsy specimens were also fixed with 10% neutral formalin and embedded in paraffin, and then tissue sections (5 μm thick) were stained with hematoxylin–eosin (H&E) and Sirius Red. Histopathological changes in the liver biopsies were assessed using a NanoZoomer S60 digital slide scanner (Hamamatsu, Japan).

Paraffin-embedded sections (4 μm thick) were processed for immune-histochemical staining. Antigen retrieval was performed by pressure cooking for 5 min in citrate buffer (pH 6), followed by peroxidase and serum blocking steps. The sections were incubated with goat anti-C3d antibody (R&D Systems, 1:100 dilution), anti-F4/80 (CST, 1:500 dilution) or anti-α-SMA (Abcam, 1:500 dilution) for 2 h at room temperature, followed by antibody detection with an anti-goat ImmPRESS kit (Vector Laboratories). The images were collected using the NanoZoomer S60 digital slide scanner.

Biochemical indicators and serum C5a analyses

Following killing, retro-orbital blood of the experimental mice was collected under isoflurane anesthesia to obtain serum for analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured with an autoanalyzer (ANTECH Diagnostics, Los Angeles, CA, USA). Serum C5a level was measured using commercially available ELISA kits (CUSABIO, Wuhan, China), according to the manufacturer’s instructions.

Measurement of triglyceride (TG), malondialdehyde (MDA), and glutathione (GSH) levels in liver homogenates

The levels of TG, MDA, and GSH in liver tissue homogenates from each group were measured using the corresponding kits (Catalog# A110-1, A003-1 and A006-2, respectively) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols.

Western blot analysis

Proteins of hepatic samples or cells were analyzed using standard western blotting techniques. The antibodies of anti-α-SMA (ab32575, Abcam), anti-TGF-β1 (SAB4502954, Sigma), anti-P65 (10,745–1-AP, Proteintech), anti-p-P65 (AF2006, Affinity), anti-iNOS (13120S, CST), anti-CD86 (19,589, CST), anti-CD163 (16,646–1-AP, Proteintech), anti-CD206 (24,595, CST), anti-TLR4 (19,811–1-AP, Proteintech), anti-NLRP3 (A5652, ABclonal), anti-AKT (10,176–2-AP, Proteintech), anti-p-AKT (4060S, CST), anti-HO-1 (43,966, CST), anti-SOD1 (10,269–1-AP, Proteintecch), anti-MDA (MDA11-S, ADI), or anti-CASP1 (24232S, CST) were used as primary antibodies. Anti-GAPDH (10,494–1-AP, Proteintech) or anti-TUBULIN (#2144, CST) was used to normalize the signals. Bands were quantified by ImageJ software.

RNA isolation and quantitative (q) RT-PCR

Total RNA was extracted from 0.1 g frozen hepatic tissues according to the TRIzol reagent protocol (No. 15596–026, Invitrogen). Next, 2 μg total RNA was reverse transcribed to complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (No.K1622, Thermo Scientific) according to the manufacturer’s protocol. Relative mRNA levels of genes were measured by qRT-PCR, using a SYBR Green PCR master mix (No.1725125, Bio-Rad). All experiments were performed in triplicate. The qRT-PCR primers used in this study were shown in supplementary Table 1. GAPDH was used to normalize the signals of target gene in the same sample.

Transcriptional profiling

Total RNA was extracted from flash-frozen liver tissues of C5aR1−/− and WT NASH mice with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality of the RNA samples was evaluated with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent’s Bioanalyzer. Sequencing libraries were generated by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The PCR products were sequenced on a HiSeq 2500 sequencing system (RIBOBIO, Guangzhou, China). Transcriptional profiling data were deposited at Mendeley Data (https://data.mendeley.com), which can easily be accessed at http://dx.doi.org/10.17632/j8v2c8z7zt.1

Cell experiments

RAW264.7 cells were kindly provided by Kunming Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Sciences, Kunming, China. RAW264.7 cells were cultured in Dulbecco’s modified Eagle medium (C11995500BT, Gibico) supplemented with 10% fetal bovine serum. The cells were grown at 37 °C in an atmosphere of 5% CO2. The RAW264.7 cells were cultured overnight to about 80% confluent and treated by the recombinant protein C5a (HY-P7695, MCE) with 50 ng/mL for 24 h.

Antagonist PMX-53 treatment

The antagonist PMX-53 was purchased from Nanjing Peptide Industry (Nanjing, China) and diluted in saline. PMX-53 (1 mg/kg) was administered i.p. to the experimental group and saline administration served as the control for the last 4 weeks of the experimental protocol. Mice subject to NASH model were treated with PMX-53 every other day from week eight forward.

Data analysis

Data are shown as the mean ± standard deviation. Significant differences between groups were determined by ANOVA, with a Bonferroni correction for continuous variables and multiple groups. Two-tailed Student’s t test was used for the comparison of a normally distributed continuous variable between two groups. Nonparametric statistical analysis was performed using the Mann–Whitney test. Statistical analysis was performed using GraphPad Prism (Version 7.0.4) software. P values less than 0.05 were considered as statistically significant.