Chemicals
Saroglitazar [(S)-α-ethoxy-4-{2-[2-methyl-5-(4-methylthio) phenyl)] − 1H-pyrrol− 1-yl]- ethoxy}) benzenepropanoic acid magnesium salt] (supplementatry information Fig. F1), was supplied by Zydus Lifesciences Limited (formerly known as Cadila Healthcare Limited), Ahmedabad, India.
Animal experiment
Four-week-old male C57BL/6 mice bred at the Zydus Research Centre (ZRC) were used in this study. The total number of mice used in this study was 102 with body weight 12.5 to 19.0 g. The animals were housed in individually ventilated cages and maintained on a standard laboratory rodent diet (Teklad 2018C, Harlan Laboratories, USA) and water ad libitum in a temperature (25 ± 3 °C) and humidity (50–70%)-controlled environment with a 12-h/12-h dark-light cycle. The experimental facility was accredited by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International) and all animal experiment protocols were approved by Institutional Animal Ethics Committee (IAEC) of Zydus Lifesciences Limited, Zydus Research Centre under protocol no. ZRC/PH/BP/039/10-2 K17. After acclimatization for 3 days, 92 animals were injected with DEN (25 mg/kg) and 10 animals were injected with saline by intraperitoneal route. During experimental phase, DEN administered animals were maintained on a choline deficient, L-amino acid-defined, high-fat diet (CDAHFD) containing 45 kcal% fat with 0.1% methionine (Product # A06071309, Research Diet, New Brunswick, NJ, USA). The saline administered animals were maintained on the control for CDAHF diet containing 10 kcal% fat and crystalline amino acids (Product # A06071322; Research Diet, New Brunswick, NJ, USA); saline-control diet + Vehicle (Normal control). Eight weeks of CDAHFD feeding was reported to cause liver steatosis, inflammation and stage 1 fibrosis [7]. Therefore, after eight weeks of diet all the animals were bled under isoflurane anesthesia to estimate serum biochemical parameters for liver function ALT and AST levels (day-0 or pretreatment analysis). Animals with increased serum ALT level were selected and randomized into different treatment groups (n = 10–15) based on serum ALT levels and body weights in such a way that mean ALT value and body weight were not significantly different between the groups. DEN with CDAHFD treated animals were divided into three groups (n = 15); DEN-CDAHFD + Vehicle (Disease control), DEN-CDAHFD + Saroglitazar 1 mg/kg,p.o. (D-Saro 1 mg/kg,p.o.) and DEN-CDAHFD + Saroglitazar 3 mg/kg,p.o. (D-Saro 3 mg/kg,p.o.). From next day (Day-1) onwards animals were administered with either vehicle, saroglitazar 1 mg/kg or saroglitazar 3 mg/kg, once daily orally for 27 weeks and maintained on CDAHF diet. After 27 weeks of treatment, 1-h post-dose, blood samples were collected for estimation of non-fasted serum ALT, AST, TG,TC, adiponectin, TNF-α, alpha-fetoprotein (AFP).
Animals were sacrificed and liver was observed for the presence of tumors. Liver was quickly resected, weighed and fixed in 10% formalin for histological analysis or snap-frozen with or without RNAlater solution in liquid nitrogen for RNA sequencing and other biochemical assays like liver lipids (TG and TC) and liver oxidative stress markers like malondialdehyde (MDA).
Biochemical and histological assessment
Various biochemical markers such as serum ALT, AST, TG, TC, adiponectin, Alphafetoprotein (AFP),osteopontin (OPN),TNF-α,liver lipid and liver MDA levels were estimated using standard procedures mentioned in supplementary information: Annexure I.
Serum biochemical analysis
Serum ALT,AST,TC and TG levels were determined using commercial kits (Roche Diagnostics, Germany) on a Cobas c311 auto analyzer (Roche, Germany). The serum levels of AFP, adiponectin and TNF-α were measured using a commercial Quantikine ELISA Kit supplied by R&D Systems, Inc., USA and the osteopontin levels were measured using ELISA kits from KINESISDx, Inc., USA.
Liver biochemistry
Total liver lipids were extracted, and hepatic TG and TC content were quantified using test kits from Agappe Diagnostics, India. Briefly liver tissue was homogenized in thirty volumes of ethanol (Ball Mixer Mill, MM 301, Retsch GmbH, Haan, Germany) for liver lipid (TG and TC) estimations following the method reported by Cool et al. [14]. Samples were vortexed and allowed to settle, and the supernatant was centrifuged at 12,000 rpm for 10 min at room temperature. For the biochemical assays, 10 μl of phosphate-buffered saline (PBS) was added to a flat bottom falcon micro-test 96-well plate followed by 2.5 μl of cleared supernatant. Next, 300 μl of TG reagent or TC reagent (AGAPEE TG/TC kit) was added to the wells, and the plate was incubated at 37 °C for 5 min. The plates were read at 546 nm for TG and 505 nm for TC with a Synergy™ HT Multidetection microplate reader (BioTek Instruments, Inc., Highland Park, Winooski, Vermont 05404–0998 USA). The liver Malondialdehyde (MDA) content levels were measured in tissue homogenates using the QuantiChrom TBARS Assay Kit (BioAssays Systems Inc., USA).
Gene expression analysis
RNA extraction, library construction, and RNA-Seq
Liver tissue samples were collected in RNAlater solution (Sigma-Aldrich Cat# R0901). RNA extraction, library construction and RNA-sequencing were performed as per the procedures described in supplementary information: Annexure-II. Briefly RNA extraction was performed using the RNeasy Mini Kit (QIAGEN, Cat# 74104) and quantified using Qubit RNA Assay HS (Invitrogen, Cat# Q32852). RNA purity and RNA integrity was accessed and the samples were subjected to RNA library preparation using NEB Ultra I RNA-Seq Library Prep kit (NEB, Cat# E7530L). Prepared libraries quantified using Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852). The obtained libraries pooled and diluted to final optimal loading concentration before cluster amplification on the Illumina flow cell. Once the cluster generation was completed, the cluster flow cell was loaded on the Illumina HiSeq 4000 instrument to generate 60 M, 100 bp paired-end reads. The read quality was checked trimmed and the paired-end reads were aligned to the reference mouse genome release downloaded from hisat2 website (GRCm38). The aligned reads are used for estimating the expression of the genes. Differential expression analysis of the raw read counts was performed using DESeq2 (1.16.1). The log2 (foldchange) values were found to be normally distributed. Those genes which were found to have log2(fold change) ≤ − 1 or log2(fold change) ≥ 1 were considered as differentially expressed and those genes which had padj < 0.05 were considered as statistically significant.
Gene ontology (GO) annotation and pathway analysis
Gene Ontology and Reactome Pathway Annotation were done using Amigo2 Gene Ontology.
Quantitative real-time polymerase chain reaction [qPCR]
Tumor containing liver samples around 100 mg (3 from each group) were homogenized in RNA-Xpress reagent (HIMEDIA) with polytron homoginizer. Total RNA was extracted from tissue in accordance with the supplier’s instructions. 1 μg total RNA from each sample was taken for first strand cDNA synthesis using Verso cDNA synthesis kit (Thermo scientific). An equal amount of cDNA from each sample was taken for quantitative real-time PCR using ABIprism-7300 (Applied Biosystems). Differentially expressed genes in microarray were selected (Igf2-F 5′-GTACTTCCGGACGACTTCCC − 3′, Igf2-R 5′- CTTTGAGCTCTTTGGCAAGCA – 3′, Cdc20-F 5′- GATCCTTGATGCCCCCGAAA − 3′,Cdc20-R 5′-TGCAGGATGTCACCAGAACC − 3′, Elovl3-F 5′- AATTCTAGGCCTGGTAGGCG − 3′,Elovl3-R 5′-GCAGCGATCTCTTCTGCAGTT-3′, Acot1-F 5′- TTCAAGGGCTGGGAATGGAG − 3′,Acot1-R 5′-TTTCTCGCAGCTGGATTGAAC − 3′, Acot3-F 5′- TGCCCTTGCTTTTGTAACACG − 3′,Acot3-R 5′-GGGAGTTGGTGTTTTCCAGC-3′, Slc10a1-F 5′- TTACCTACAAGGCTGCTGCAA − 3′, Slc10a1-R 5′- AAGGCCAGGTTGTGTAGGAG − 3′, Fabp1-F 5′- GTGGTCCGCAATGAGTTCAC − 3′, Fabp1-R 5′- CACCTTCCAGCTTGACGACT − 3′, Col5A2-F 5′- TGGAGCAGTTGGCCCATTAG − 3′, Col5A2-R 5′- CCCAGGCAGTCCAGTTATCC -3′, ADAM8-F 5′- AACAAGCAGCGTCTACGAGC -3′, ADAM8-R 5′- TCTCGGAGCCTTTCGGTAGA-3′, Timp1- F 5′- GTGCACAGTGTTTCCCTGTT − 3′, Timp1- R 5′- AGTGACGGCTCTGGTAGTC − 3′, Col1a1-F 5′- TGATGGGGAAGCTGGCAAG − 3′, Col1a1-R 5′- GAAGCCTCGGTGTCCCTTC − 3′, Tgfb1-F 5′- ATTGCTGTCCCGTGCAGAG − 3′, Tgfb1-R 5′- TCAGCAGCCGGTTACCAAG -3′, MMP13 –F 5′- ACGAGCATCCATCCCGAGACCT -3′,
MMP13 –R 5′- GTGAACCGCAGCACTGAGCCT − 3′) and quantified using Kapa SYBR FAST (KAPA, USA, KK4618). Ribosomal acidic protein (F:5′- TACAGCTTCACCACCACAGC − 3′ and R:5′- TCTCCAGGGAGGAAGAGGAT − 3′) was used as an internal control for normalization of the results and fold change was calculated with ∆∆Ct method.
Necropsy examination and histological assessment
At the end of the experiment, the mice were sacrificed with asphyxiation using overdose of isoflurane, and a detailed gross pathological examination was performed. The liver was observed grossly for lesions and the number of tumors was counted. The liver and spleen were dissected, fat was removed and the organ weights were recorded. One portion of liver was collected in RNAlater solution for gene expression and another portion was collected and preserved in 10% neutral buffered formalin for histopathological processing. The formalin-fixed liver (all lobes) were trimmed in a way to cover all the grossly observed tumors, processed and paraffin-embedded. Sagittal sections were taken at 3–4 μm. Light microscopic examination of liver tissue was performed using standard hematoxylin and eosin (H&E) staining. In case there were no abnormalities observed in the liver, it was trimmed by following the standard practice as the guidelines [15]. Hepatic fibrosis was assessed using Sirius Red stain. Specimens were scored using the method described by Kleiner et al. [16]. The staging of hepatic fibrosis was classified as stage 0 to 4 (stage 0: none; stage 1: mild perisinusoidal or periportal; stage 2: moderate perisinusoidal or periportal; stage 3: bridging fibrosis; stage 4: cirrhosis). The diagnosis, classification/histological typing, and nomenclature of foci of cellular alteration (FCA), benign and malignant hepatocellular tumors observed in this study were performed as per International Harmonization of Nomenclature and Diagnostic criteria for Lesions in Rats and Mice [17].
Statistical analysis
For in-vivo studies, data were analysed using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test to evaluate the statistical difference between the various treatment groups. P < 0.05 was considered significant. # indicates significant difference in control diet (normal control) group versus disease control, *p < 0.05; **p < 0.01, ***p < 0.001 indicates significant difference versus disease control in test compound. All data presented as mean ± SEM. All data analysis was performed using GraphPad Prism software (GraphPad La Jolla California USA).