Sparcl1 promotes nonalcoholic steatohepatitis progression through upregulation of CCL2

1. White adipose tissues (WAT) are capable of secreting not only fatty acids but also a class of secretory proteins that modulate the homeostasis of distant organs, including the liver. To identify potential WAT-enriched secretory factors involved in NASH progression, C57BL/6 male mice were fed with a normal chow diet or HFHC diet for two different periods: 12 weeks and 28 weeks. Then, we compared the expression profile of epididymal WAT (eWAT) from mouse models of NAFL and NASH above mentioned by RNA-Sequencing analysis. 2. To investigate the contribution of Sparcl1 in the pathogenesis of NASH, male C57BL/6J mice were fed a HFHC diet for 12 weeks, and then intraperitoneal injected with saline or recombinant Sparcl1 protein (0.2mg/kg) every other day for 3 weeks. To elucidate the molecular basis of Sparcl1-mediated NASH progression, we conducted RNA-Sequencing analysis for differentially expressed genes using the livers of chronic recombinant Sparcl1 protein or saline- treated mice. Male C57BL/6J mice aged 6 weeks were purchased from the Shanghai Laboratory Animal Company (SLAC, Shanghai, China). For NAFL or NASH diet feeding, C57BL/6J mice were fed a diet containing 40% fat, 22% fructose, and 2% cholesterol (D09100310, Research Diets Inc.) for 12 or 28 weeks. Mice were intraperitoneal injected with saline or recombinant Sparcl1 protein (0.2mg/kg) every other day for 3 weeks. RNA high throughput sequencing was performed by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instructions. RNA libraries were constructed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts,USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired end reads. Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3), the high quality clean reads were aligned to the reference genome (UCSC mm10) with hisat2 software (v2.0.4). Then, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and P value were calculated based on FPKM, differentially expressed mRNA were identified.