利用转录组测序数据集探究纤维化进程中关键细胞簇及特异转录调控因子，以解析萝卜硫素抗小鼠肾纤维化的机制。

选择单侧输尿管梗阻(unilateral ureteral obstruction，UUO)肾纤维化模型的单细胞转录组测序数据集(GSE175412)，使用Leiden算法进行细胞聚类，对细胞簇进行人工注释；利用CellPhoneDB数据库进行细胞-细胞互作分析以识别肾纤维化关键细胞簇，并以RegDiffusion算法结合AUCell评分识别各细胞簇关键特异转录调控子。选用萝卜硫素干预人多囊肾细胞批量转录组测序数据集(GSE141740)，并利用DESeq2软件分析萝卜硫素处理组与模型组间的差异表达基因，以寻找萝卜硫素的潜在下游靶点。然后采用6~8周龄雄性C57BL/6J小鼠左侧输尿管上极结扎制备UUO模型，验证萝卜硫素的抗肾纤维化作用及其对潜在下游靶点表达水平的影响。小鼠分为UUO模型组和UUO萝卜硫素干预组[造模次日起腹腔注射萝卜硫素12.5 mg/(kg·d)]，每组均为6只。采用实时荧光定量PCR验证Y染色体性别决定区相关高迁移率族盒转录因子4(sex-determining region Y-related high-mobility-group box transcription factor 4, Sox4)的mRNA表达水平，马松染色评估肾间质胶原沉积情况，免疫组化和Western印迹法检测波形蛋白、Ⅰ型胶原蛋白、α-平滑肌蛋白等纤维化指标。采用单因素方差分析等进行多组间比较。

结果显示，UUO模型小鼠肾脏近端小管上皮细胞占比下降，受损肾小管上皮细胞占比增加；受损肾小管上皮细胞可通过多条促纤维化通路与成纤维细胞和周细胞相互作用。Sox4在受损肾小管上皮细胞簇中呈现高特异活性，其mRNA水平显著增高。萝卜硫素可以显著下调人多囊肾细胞Sox4的mRNA水平。UUO萝卜硫素干预组Sox4的mRNA水平显著下调(P<0.05)，肾间质胶原蛋白沉积显著减少(P<0.05)，Ⅰ型胶原蛋白、α-平滑肌肌动蛋白和波形蛋白表达亦显著下调(P均<0.05)。

受损肾小管上皮细胞是UUO肾纤维化进程的关键细胞簇，Sox4是其特异转录调控因子；萝卜硫素可能通过下调Sox4发挥抗纤维化作用。

To utilize transcriptome sequencing datasets for exploring key cell clusters and specific transcriptional regulators during the fibrotic process, aiming to elucidate the mechanism by which sulforaphane ameliorates renal fibrosis in mice.

A single-cell RNA sequencing dataset (GSE175412) derived from a unilateral ureteral obstruction (UUO) renal fibrosis model was utilized. Cell clustering was performed using the Leiden algorithm, followed by manual annotation of the cell clusters. Cell-cell interaction analysis was performed using CellPhoneDB to identify key cell clusters involved in renal fibrosis. Subsequently, the RegDiffusion algorithm combined with AUCell scoring was employed to identify key cell-type-specific transcriptional regulators within each cluster. The bulk RNA-seq dataset of human polycystic kidney disease cells treated with sulforaphane (GSE141740) was utilized. The DESeq2 package was employed to analyze differentially expressed genes between the sulforaphane-treated group and the model group, aiming to identify potential downstream targets of sulforaphane. Subsequently, a UUO model was established by ligating the upper pole of the left ureter in 6 to 8-week-old male C57BL/6J mice to validate the anti-fibrotic effect of sulforaphane and its impact on the expression levels of potential downstream targets. The mice were divided into two groups (n=6 per group): the UUO model group and the UUO plus sulforaphane intervention group. The latter received intraperitoneal injections of sulforaphane [12.5 mg/(kg·d)] starting from the day after surgery. Real-time quantitative PCR was performed to verify the mRNA expression level of sex-determining region Y-related high-mobility-group box transcription factor 4 (Sox4). Masson′s trichrome staining was utilized to evaluate renal interstitial collagen deposition. Additionally, immunohistochemistry and Western blotting were employed to detect fibrotic markers, including vimentin, collagen I, and α-smooth muscle actin. One-way ANOVA was used for comparisons among multiple groups.

The results demonstrated that the proportion of proximal tubular epithelial cells decreased, while the proportion of injured tubular epithelial cells increased in the kidneys of UUO mice. Furthermore, injured tubular epithelial cells were found to interact with fibroblasts and pericytes via multiple pro-fibrotic pathways. Sox4 exhibited high specific activity and significantly elevated mRNA levels in the cluster of injured tubular epithelial cells. Furthermore, sulforaphane treatment significantly downregulated the mRNA expression of Sox4 in human polycystic kidney disease cells. In the UUO plus sulforaphane intervention group, Sox4 mRNA level was significantly downregulated (P<0.05), and renal interstitial collagen deposition was significantly reduced (P<0.05). Besides, the expression of collagen I, α-smooth muscle actin, and vimentin was also significantly decreased (all P<0.05).

Injured tubular epithelial cells served as a key cell cluster in the progression of UUO-induced renal fibrosis, with Sox4 acting as their specific transcriptional regulator. Sulforaphane may exert its anti-fibrotic effects by downregulating Sox4.