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. Overall design: 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.

1. 白色脂肪组织（White adipose tissues, WAT）不仅可分泌脂肪酸，还能产生一类调控包括肝脏在内的远端器官稳态的分泌蛋白。为筛选参与非酒精性脂肪性肝炎（Non-alcoholic steatohepatitis, NASH）进展的潜在WAT富集分泌因子，本研究将C57BL/6雄性小鼠分为两组，分别饲喂普通维持饲料与高脂高果糖高胆固醇（high fat high fructose high cholesterol, HFHC）饲料，并设置两个饲养周期：12周与28周。随后通过RNA测序（RNA-Sequencing）分析，对比上述非酒精性脂肪肝（Non-alcoholic fatty liver, NAFL）及NASH模型小鼠的附睾白色脂肪组织（epididymal WAT, eWAT）表达谱。
2. 为探究Sparcl1在NASH发病机制中的作用，本研究将雄性C57BL/6J小鼠饲喂HFHC饲料12周后，每隔一日腹腔注射生理盐水或重组Sparcl1蛋白（0.2mg/kg），连续处理3周。为阐明Sparcl1介导NASH进展的分子基础，我们对经慢性重组Sparcl1蛋白或生理盐水处理的小鼠肝脏组织进行RNA测序，以分析差异表达基因。
整体实验设计：
本研究使用的6周龄雄性C57BL/6J小鼠购自上海实验动物有限公司（Shanghai Laboratory Animal Company, SLAC，中国上海）。对于NAFL或NASH模型构建，小鼠饲喂含40%脂肪、22%果糖及2%胆固醇的饲料（D09100310, Research Diets Inc.），饲养时长为12周或28周。后续每隔一日对小鼠进行腹腔注射，给药组为重组Sparcl1蛋白（0.2mg/kg），对照组为生理盐水，持续处理3周。
RNA高通量测序由云序生物（Cloud-Seq Biotech, 中国上海）完成。具体流程简述如下：依照制造商说明书，使用NEBNext rRNA Depletion Kit（New England Biolabs, Inc., 美国马萨诸塞州）对总RNA进行核糖体RNA移除操作。随后按照制造商指导，使用NEBNext® Ultra™ II Directional RNA Library Prep Kit（New England Biolabs, Inc., 美国马萨诸塞州）构建RNA测序文库。采用BioAnalyzer 2100系统（Agilent Technologies, Inc., 美国）对文库进行质量控制与定量分析。随后在Illumina Hiseq测序平台上完成文库测序，采用150bp双端读长模式。测序数据由Illumina HiSeq 4000测序仪获取，并通过Q30标准进行质量管控。使用cutadapt软件（v1.9.3）完成3'接头修剪与低质量reads过滤后，利用hisat2软件（v2.0.4）将高质量clean reads比对至参考基因组（UCSC mm10）。随后以Ensembl gtf基因注释文件为参考，使用cuffdiff软件（cufflinks套件的组成部分）计算基因水平的FPKM值以表征mRNA表达谱，并基于FPKM值计算差异倍数与P值，以此鉴定差异表达mRNA。