Single nuclei sequencing to understand mechanism of action of the melanocortin 1 receptor agonist Pl8177 in Dextran sodium sulfate (DSS)-induced colitis in rats. Single nuclei sequencing to understand mechanism of action of the melanocortin 1 receptor agonist Pl8177 in Dextran sodium sulfate (DSS)-induced colitis in rats

PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed and tested in dextran sulfate sodium induced rat ulcerative colitis model. Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. We used single nuclei RNA sequencing of colon tissues to characterize the mechanism of action and identify relative cell population and key gene expression changes between treated, healthy and vehicle. Overall design: Nuclei were isolated from 9 flash-frozen rat colon tissue samples (n=3 for each treatment group: PL8177-50ug, Sham and placebo) and were used to construct 3’ single cell gene expression libraries (Next GEM v3.1) using the 10x Genomics Chromium system (10x Genomics, Pleasanton, CA). Libraries were sequenced with ~150 million read pairs (PE150) per sample on an Illumina NovaSeq sequencing system (Illumina, San Diego, CA). After sequencing, gene counts were generated by Cell Ranger v6.0.1 (10x Genomics) using the rat reference genome Rattus norvegicus 6.0.97. Introns were included in the analysis. CellBender was applied to eliminate empty droplets and technical artifacts. Only genes detected in a minimum of 3 cells, cells with at least 200 genes, and cells with <5% of mitochondrial reads were retained

PL8177是黑皮质素1受体（melanocortin 1 receptor, MC1R）的强效选择性激动剂。该化合物在插管大鼠溃疡性结肠炎模型中已展现出逆转肠道炎症的功效。为实现口服递送，研究人员开发了一种新型聚合物包载的PL8177制剂，并在硫酸葡聚糖钠诱导的大鼠溃疡性结肠炎模型中开展了测试。相较于不含活性药物的赋形剂组，接受50 μg口服PL8177治疗的大鼠，其宏观结肠损伤评分显著更低，且结肠重量、粪便性状与粪便隐血均得到改善。
本研究通过结肠组织单细胞核RNA测序（single nuclei RNA sequencing）解析其作用机制，并鉴定给药组、健康组与赋形剂组之间的相对细胞群变化及关键基因表达差异。
整体实验设计：从9份速冻大鼠结肠组织样本中分离细胞核（每个处理组n=3：PL8177-50μg组、假手术组与安慰剂组），采用10x Genomics Chromium系统（10x Genomics，美国加利福尼亚州普莱森顿）结合Next GEM v3.1构建3'端单细胞基因表达文库。每个样本在Illumina NovaSeq测序系统（Illumina，美国加利福尼亚州圣地亚哥）上以约1.5亿读对（PE150）进行测序。测序完成后，使用Cell Ranger v6.0.1（10x Genomics）结合大鼠参考基因组Rattus norvegicus 6.0.97生成基因计数矩阵，分析过程纳入内含子区域。随后通过CellBender去除空液滴与技术伪影。最终仅保留在至少3个细胞中被检测到的基因、包含至少200个基因的细胞，以及线粒体reads占比低于5%的细胞。