Supplementary Files: Liver transcriptomic profiles of ruminant species fed spent hemp biomass containing cannabinoids

The inclusion of spent hemp biomass (SHB), an extracted byproduct from industrial cannabidiol (CBD) production, in the diets of dairy cows and lambs appear to be safe with minor effects on the metabolism, including a decrease of circulating cholesterol and increase bilirubinemia, both associated with liver metabolism. Those effects could be consequence of the presence of cannabinoids, particularly Δ⁹-tetrahydrocannabinol (THC) and CBD in the SHB. This study aimed to study the transcriptional profile of the liver of dairy cows and lambs fed SHB. Dairy cows received SHB or alfalfa pellet for four weeks of intervention (IP) and four weeks of withdrawal periods (WP). Finishing lambs were fed a control diet (CON), 10% (LH2) or 20% (HH2) SHB for 2 months or 1 month followed by 1-month SHB withdrawal (LH1 and HH1, respectively). RNA sequencing was performed, and the mRNA was annotated using the latest reference genomes. The RNAseq data were filtered, normalized for library size and composition, and statistically analyzed by DESeq2. The bioinformatic analysis was performed by using DAVID, Gene Set Enrichment Analysis (GSEA), and the Dynamic Impact Approach. Using a 0.2 FDR cut-off, we identified only ≤24 differentially expressed genes (DEG) in the liver by feeding SHB in dairy cows and a larger number of DEG in lambs (from 71 in HH1 vs. CON to 552 in LH1 vs. CON). The KEGG analysis demonstrated that feeding SHB in dairy cows and lambs had relatively minor to moderate metabolic alterations in dairy cows and lambs mainly associated with amino acids and lipid metabolism whereas cholesterol synthesis was overall activated in lambs. GSEA identified activation of PPAR signaling pathway only in dairy cows. We found an opposite effect on activation of metabolism of drug and xenobiotics by cytochrome P450 enzymes in dairy cows and lambs receiving less SHB but an inhibition in HH2 lambs. Immune system-related pathways were inhibited by feeding SHB in lambs, but the impact was minor. Cumulatively, inclusion of SHB containing cannabinoids in dairy and lambs demonstrate very little effects on the alteration of transcriptomic profile of the liver.

将工业大麻二酚（cannabidiol, CBD）生产过程中提取得到的副产物废弃大麻生物质（spent hemp biomass, SHB）添加至奶牛与羔羊日粮中，整体安全性良好，仅对代谢产生轻微影响，包括循环胆固醇水平降低与胆红素血症升高，二者均与肝脏代谢相关。上述效应可能源于SHB中存在的大麻素类物质，尤其是Δ⁹-四氢大麻酚（Δ⁹-tetrahydrocannabinol, THC）与CBD。本研究旨在探究饲喂SHB后奶牛与羔羊的肝脏转录组谱。实验将奶牛分为两组，分别饲喂SHB或苜蓿颗粒，干预期（IP）为4周，随后设置4周的撤药期（WP）。育肥羔羊则分为对照组（CON）、10%SHB组（LH2）与20%SHB组（HH2），分别饲喂2个月，或先饲喂1个月再进行1个月的SHB撤药期（对应LH1与HH1组）。本研究采用RNA测序（RNA-seq）技术，并通过最新参考基因组对mRNA进行注释。对RNA-seq数据进行过滤、文库规模与组成标准化，并通过DESeq2进行统计学分析。生物信息学分析采用DAVID、基因集富集分析（Gene Set Enrichment Analysis, GSEA）以及动态影响分析法（Dynamic Impact Approach）。以0.2的错误发现率（false discovery rate, FDR）作为截断值，本研究在饲喂SHB的奶牛肝脏中仅鉴定出≤24个差异表达基因（differentially expressed genes, DEG），而羔羊肝脏中的DEG数量更多，范围为71个（HH1 vs. CON）至552个（LH1 vs. CON）。KEGG富集分析结果显示，饲喂SHB对奶牛与羔羊肝脏的代谢改变相对轻微至中度，主要涉及氨基酸与脂质代谢；而羔羊体内的胆固醇合成整体被激活。基因集富集分析（GSEA）仅在奶牛中检测到过氧化物酶体增殖物激活受体（PPAR）信号通路被激活。本研究发现，在饲喂低剂量SHB的奶牛与羔羊中，细胞色素P450酶介导的药物与外源性物质代谢通路的激活呈现相反趋势，而在HH2组羔羊中该通路则被抑制。饲喂SHB会抑制羔羊的免疫系统相关通路，但影响程度较为轻微。综上，在奶牛与羔羊日粮中添加含大麻素类物质的SHB，对肝脏转录组谱的改变影响极小。