溶质载体家族7成员5抑制剂JPH203对单侧输尿管梗阻诱导小鼠肾纤维化的抑制作用

doi:10.12300/j.issn.1674-5817.2025.092

摘要/Abstract

摘要：

目的探究溶质载体家族7成员5（solute carrier family 7 members 5，SLC7A5）抑制剂JPH203对单侧输尿管梗阻诱导小鼠肾纤维化的作用。方法将16只SPF级雄性C57BL/6小鼠随机分为对照组和实验组，每组8只，使用单侧输尿管结扎手术建立小鼠肾纤维化模型。术后第3天开始，对照组小鼠每天经腹腔注射PBS 200 μL，连续11 d；实验组小鼠每天经腹腔注射JPH203（50 mg/kg），连续11 d。术后第14天，小鼠安乐死后取肾脏组织。采用HE染色法分析小鼠肾脏组织损伤情况，采用Masson染色法分析小鼠肾脏细胞外基质中胶原纤维沉积情况，采用免疫组织化学法检测小鼠肾脏组织中成纤维细胞活化指标α平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和Ⅰ型胶原蛋白（collagen typeⅠ，COL-Ⅰ）含量。进一步采用蛋白质印迹法检测小鼠肾脏组织中SLC7A5和转化生长因子-β1（transforming growth factor-β1，TGF-β1）表达水平，以及哺乳动物雷帕霉素靶蛋白复合物1（mammalian target of rapamycin complex 1，mTORC1）信号通路相关分子的磷酸化水平；采用实时荧光定量PCR法验证肾脏组织中SLC7A5、α-SMA和COL-Ⅰ mRNA水平变化。结果与对照组相比，实验组小鼠肾脏组织结构破坏减轻，病理学损伤评分明显降低（P<0.05），同时细胞外间质中胶原沉积减少，胶原纤维占比明显降低（P<0.001）。与对照组相比，实验组小鼠肾脏组织中成纤维细胞活化指标α-SMA和COL-Ⅰ含量均明显降低（均P<0.001），SLC7A5和TGF-β1表达水平也均明显降低（P<0.001），mTORC1信号通路相关蛋白4E-BP1和mTORC1的磷酸化水平也均明显下降（P<0.001）。实时荧光定量PCR法证实实验组小鼠肾脏组织中SLC7A5、α-SMA和COL-Ⅰ mRNA水平均明显降低（P<0.001）。结论 JPH203可能通过抑制SLC7A5表达，调节mTORC1信号通路，改变成纤维细胞活化状态，进而抑制小鼠肾纤维化进展。

关键词: 溶质载体家族7成员5抑制剂, JPH203, 哺乳动物雷帕霉素靶蛋白复合物1, 肾纤维化, 单侧输尿管梗阻模型, 小鼠

Abstract:

Objective To investigate the effect of solute carrier family 7 member 5 (SLC7A5) inhibitor JPH203 on renal fibrosis induced by unilateral ureteral obstruction in mice. Methods Sixteen SPF male C57BL/6 mice were randomly divided into the control group and the experimental group, with 8 mice in each group. The mouse model of renal fibrosis was established by unilateral ureteral obstruction. From the third day after surgery, the mice in the control group were intraperitoneally injected with phosphate-buffered saline (PBS) for 11 consecutive days, and the injection dose was 200 μL/d. Mice in the experimental group received intraperitoneal injection of JPH203 (50 mg/kg) every day for 11 days. On day 14, the mice were euthanized, then the kidney tissues were obtained. Hematoxylin and eosin (HE) staining was used to assess renal tissue damage, Masson staining was used to evaluate collagen fiber deposition in the extracellular matrix, and immunohistochemistry was used to detect the levels of fibroblast activation markers α-smooth muscle actin (α-SMA) and collagen type Ⅰ (COL-Ⅰ) in kidney tissues. Western blotting was further performed to measure the expression levels of SLC7A5 and transforming growth factor-β1 (TGF-β1), as well as the phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway-related molecules. Real-time quantitative PCR was used to verify changes in the mRNA levels of SLC7A5, α-SMA, and COL-Ⅰ in kidney tissues. Results Compared with the control group, the experimental group showed reduced destruction of renal tissue structure and a significantly lower pathological injury score (P<0.05). Additionally, collagen deposition in the extracellular matrix was decreased, and the percentage of collagen fiber area was significantly reduced (P<0.001) in the experimental group. The levels of fibroblast activation markers α-SMA and COL-Ⅰ were significantly lower in the experimental group (both P<0.001). The expression levels of SLC7A5 and TGF-β1 were also significantly decreased (P<0.001), and the phosphorylation levels of mTORC1 signaling pathway-related proteins 4E-BP1 and mTORC1 were significantly reduced (P<0.001). Real-time quantitative PCR confirmed that the mRNA levels of SLC7A5, α- SMA, and COL-Ⅰ in kidney tissues were significantly lower in the experimental group (P<0.001). Conclusion JPH203 may inhibit the progression of renal fibrosis in mice by suppressing SLC7A5 expression, regulating the mTORC1 signaling pathway, and altering fibroblast activation status.

Key words: Solute carrier family 7 member 5 inhibitor, JPH203, Mammalian target of rapamycin complex 1, Renal fibrosis, Unilateral ureteral obstruction model, Mice

中图分类号:

崔畅婉,路一平,于淼,等. 溶质载体家族7成员5抑制剂JPH203对单侧输尿管梗阻诱导小鼠肾纤维化的抑制作用[J]. 实验动物与比较医学, 2026, 46(2): 205-211. DOI: 10.12300/j.issn.1674-5817.2025.092.

CUI Changwan,LU Yiping,YU Miao,et al. Inhibitory Effect of Solute Carrier Family 7 Member 5 Inhibitor JPH203 on Renal Fibrosis Induced by Unilateral Ureteral Obstruction in Mice[J]. Laboratory Animal and Comparative Medicine, 2026, 46(2): 205-211. DOI: 10.12300/j.issn.1674-5817.2025.092.

图/表 3

图1 JPH203处理后UUO诱导C57BL/6小鼠模型的肾脏组织变化
注：A~B，不同组别小鼠肾脏组织的HE染色结果及其评分比较；C~D，不同组别小鼠肾脏组织的Masson染色结果及其阳性面积占比（反映胶原纤维占比）。Control即对照组，经单侧输尿管梗阻（UUO）手术诱导并腹腔注射PBS；JPH203即实验组，经UUO手术诱导并腹腔注射溶质载体家族7成员5抑制剂JPH203。每组8只小鼠（n=8）。与对照组相比，^*P<0.05，^***P<0.001。图中比例尺为200 μm。蓝色箭头指示肾小管；黑色箭头指示炎性细胞；红色箭头代表胶原染色。

Figure 1 Changes of kidney tissues in UUO-induced C57BL/6 mice after JPH203 treatment
Note： A-B， Hematoxylin and eosin （HE） staining of mouse kidney tissues in different groups， and their pathological scores. C-D， Masson's trichrome staining of mouse kidney tissues in different groups， and the percentage of collagen fiber area （Masson staining positive area）. Control， mice induced by unilateral ureteral obstruction （UUO） and injected with PBS； JPH203 as the experimental group， mice induced by UUO and injected with JPH203 （an inhibitor of solute carrier family 7 member 5）. Each group consisted of 8 mice （n=8）. Compared with the control group， ^*P<0.05， ^***P<0.001. The scale bar in the figure represents 200 μm. The blue arrow indicates the renal tubules； The black arrow indicates inflammatory cells； The red arrow represents collagen staining.

图2 JPH203处理对UUO诱导C57BL/6小鼠肾脏组织中成纤维细胞活化分子表达的影响
注：A~B，不同组别小鼠肾脏组织中Ⅰ型胶原蛋白（COL-Ⅰ）的免疫组织化学染色结果及其评分［光密度值（IOD）/%］比较；C~D，不同组别小鼠肾脏组织中α平滑肌肌动蛋白（α-SMA）的免疫组织化学染色及其评分（IOD/%）比较。图中比例尺为200 μm。Control即对照组，经单侧输尿管梗阻（UUO）手术诱导并腹腔注射PBS；JPH203即实验组，经UUO手术诱导并腹腔注射溶质载体家族7成员5抑制剂JPH203。每组8只小鼠（n=8）。累积光密度值（IOD）=阳性细胞相对面积×染色强度。与对照组相比，^***P<0.001。

Figure 2 Effects of JPH203 treatment on the expressions of fibroblast activation molecules in kidney tissues of UUO-induced C57BL/6 mice
Note： A-B， Immunohistochemical staining for collagen typeⅠ（COL-Ⅰ） in mouse kidney tissues of different groups， and their scores ［integrated optical density （IOD）/%］； C-D， Immunohistochemical staining of α-smooth muscle actin （α-SMA） in mouse kidney tissues of different groups， and their scores （IOD/%）. The scale bar in the figure represents 200 μm. Control， the mice induced by unilateral ureteral obstruction （UUO） and injected with PBS； JPH203 as the experimental group， the mice induced by UUO and injected with JPH203 （an inhibitor of solute carrier family 7 member 5）. Each group consisted of 8 mice （n=8）. Compared with the control group， ^***P<0.001. Integrated optical density （IOD）=relative positive cell area×staining intensity.

图3 JPH203处理后UUO诱导C57BL/6小鼠肾脏组织中相关蛋白和mRNA水平的变化
注：A，蛋白质印迹法检测不同组别小鼠肾脏组织中各蛋白含量；B，不同组别小鼠肾脏组织中各蛋白表达水平的统计分析结果；C~E，实时荧光定量PCR法验证不同组别小鼠肾脏组织中各蛋白的mRNA水平变化。SLC7A5，溶质载体家族7成员5；TGF-β1，转化生长因子-β1；mTORC1，哺乳动物雷帕霉素靶蛋白复合物1；4E-BP1，真核翻译起始因子4E结合蛋白1；β-actin，β-肌动蛋白；COL-Ⅰ，Ⅰ型胶原蛋白；α- SMA，α平滑肌肌动蛋白。Control即对照组，经单侧输尿管梗阻（UUO）手术诱导并腹腔注射PBS；JPH203即实验组，经UUO手术诱导并腹腔注射溶质载体家族7成员5抑制剂JPH203。每组8只小鼠（n=8）。与对照组相比，^***P<0.001。

Figure 3 Changes in protein and mRNA levels in kidney tissues of UUO-induced C57BL/6 mice after JPH203 treatment
Note： A， The content of each protein in kidney tissues of mice in different groups was detected by Western blotting. B， Statistical analysis of Western blotting of mouse kidney tissues in different groups. C-E， The changes of some mRNA levels in kidney tissues of mice in different groups detected by real-time quantitative PCR. SLC7A5， solute carrier family 7 member 5； TGF-β1， transforming growth factor-β1； mTORC1， mammalian target of rapamycin complex 1； 4E-BP1， eukaryotic translation initiation factor 4E-binding protein 1；COL-Ⅰ， collagen type Ⅰ； α-SMA， α-smooth muscle actin. Control， mice induced by unilateral ureteral obstruction （UUO） and injected with PBS； JPH203 as the experimental group， mice induced by UUO and injected with JPH203 （an inhibitor of solute carrier family 7 member 5）. Each group consisted of 8 mice （n=8）. Compared with the control group，^***P<0.001.

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