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中华肾病研究电子杂志

中华肾病研究电子杂志 ›› 2022, Vol. 11 ›› Issue (06) : 318 -326. doi: 10.3877/cma.j.issn.2095-3216.2022.06.004

论著

虾青素通过CCN1调节肾间质纤维化的潜在分子作用机制
尹丽丽1,†(), 管陈2, 赵龙2, 蒋伟2, 秦振志3, 李宸羽4, 徐岩2,†()   
  1. 1. 266003　青岛大学附属医院肾内科；261041　潍坊市人民医院血液净化中心
    2. 266003　青岛大学附属医院肾内科
    3. 999077　香港大学李嘉诚医学院
    4. 266003　青岛大学附属医院肾内科；80336　慕尼黑大学附属医院
  • 收稿日期:2021-08-05 出版日期:2022-12-28
  • 通信作者: 尹丽丽, 徐岩
  • 基金资助:
    国家自然科学基金面上项目(81470973，81770679，81970582); 青岛市科技惠民示范引导专项(20-3-4-36-nsh); 潍坊市卫生计生委科研项目计划(wfwsjs-2018-0045)

Potential molecular mechanism of astaxanthin regulating renal interstitial fibrosis through CCN1

Lili Yin1,†(), Chen Guan2, Long Zhao2, Wei Jiang2, Zhenzhi Qin3, Chenyu Li4, Yan Xu2,†()   

  1. 1. Department of Nephrology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Provine, China; Blood Purification Center, Weifang People′s Hospital, Weifang 261041, Shandong Provine, China
    2. Department of Nephrology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Provine, China
    3. Li Ka Shing School of Medicine, The University of Hong Kong, Hong Kong 999077, China
    4. Department of Nephrology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Provine, China; LMU University Hospital, Munich 80336, Germany
  • Received:2021-08-05 Published:2022-12-28
  • Corresponding author: Lili Yin, Yan Xu
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引用本文：

尹丽丽, 管陈, 赵龙, 蒋伟, 秦振志, 李宸羽, 徐岩. 虾青素通过CCN1调节肾间质纤维化的潜在分子作用机制[J]. 中华肾病研究电子杂志, 2022, 11(06): 318-326.

Lili Yin, Chen Guan, Long Zhao, Wei Jiang, Zhenzhi Qin, Chenyu Li, Yan Xu. Potential molecular mechanism of astaxanthin regulating renal interstitial fibrosis through CCN1[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2022, 11(06): 318-326.

目的

探讨虾青素(AST)对肾间质纤维化的潜在分子作用机制，为临床防治肾脏纤维化提供数据。

方法

动物实验采用单侧输尿管结扎(UUO)法建立肾间质纤维化模型。60只雄性C57BL/6小鼠随机分为6组(10只/组)：假手术组、假手术+AST(200 mg/kg)组、UUO模型组、UUO+AST(50 mg/kg)组、UUO+AST(100 mg/kg)组、UUO+AST(200 mg/kg)组。细胞实验采用TGF-β1诱导HK-2细胞株上皮间充质转化(EMT)模型。质粒转染实验将HK-2细胞分为7组：对照组、空白组、CCN1过表达组、CCN1敲低组、TGF-β1诱导组、TGF-β1+CCN1过表达组、TGF-β1+CCN1敲低组。AST处理实验将HK-2细胞分为5组：对照组、TGF-β1诱导组、TGF-β1+AST组、TGF-β1+CCN1敲低组、TGF-β1+CCN1敲低+AST组。实时定量荧光PCR法检测肾组织肾损伤生物标志物中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、肾损伤分子-1(KIM-1)、血清胱抑素-C(Cys-C)和白介素-18(IL-18)。Western印迹检测肾组织和细胞的纤维化标志物Ⅰ型胶原蛋白(Col-Ⅰ)、纤连蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白和E-钙粘蛋白(E-cad)，以及TGF-β1/Smad和Notch信号通路分子：pSmad3、Notch1、Notch3、Jagged-1的表达。

结果

与假手术组相比，UUO组小鼠肾损伤生物标志物NGAL、KIM-1、Cys-C和IL-18的水平显著升高；肾脏纤维化标志物蛋白表达水平显著升高，E-cad表达水平显著下降；pSmad3、Notch1、Notch3、Jagged-1的蛋白表达水平显著增加，Smad7表达显著减少。AST处理使肾损伤生物标志物(NGAL、KIM-1、Cys-C和IL-18)、纤维化标志物蛋白表达水平显著下降，E-cad蛋白表达显著上升且呈剂量依赖性。细胞实验结果显示：CCN1过表达可以抑制TGF-β1/Smad和Notch信号通路的激活，而敲低CCN1不仅增强了TGF-β1/Smad和Notch信号通路的激活，还减弱了AST在TGF-β1诱导的EMT模型中的保护作用。

结论

AST可能通过CCN1调节TGF-β1/Smad和Notch信号通路，从而可能具有减轻肾间质纤维化、保护肾脏的作用。

关键词: 虾青素, CCN1, 肾间质纤维化, Notch信号通路
Objective

To explore the potential molecular mechanism of astaxanthin (AST) in renal interstitial fibrosis and provide data for clinical prevention and treatment of renal fibrosis.

Methods

In animal experiment, unilateral ureteral ligation (UUO) was used to establish renal interstitial fibrosis model. 60 male C57BL/6 mice were randomly divided into six groups with 10 mice each, including sham operation group, sham operation+ AST 200mg/kg group, UUO group, UUO+ AST 50, 100, 200 mg/kg groups. In cell experiments, the HK-2 cell line epithelial-mesenchymal transition (EMT) model was induced by TGF-β1. Plasmid transfection experiments divided HK-2 cells into 7 groups: control group, blank group, CCN1 overexpression group, CCN1 knockdown group, TGF-β1 group, TGF-β1+ CCN1 overexpression group, and TGF-β1+ CCN1 knockdown group. The AST treatment experiments divided the HK-2 cells into 5 groups: control group, TGF-β1 group, TGF-β1+ AST group, TGF-β1+ CCN1 knockdown group, and TGF-β1+ CCN1 knockdown+ AST group. Real time-PCR was used to detect the biomarkers of kidney injury in kidney tissue: neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), serum cystatin-C (Cys-C), and interleukin-18 (IL-18). Western blot was used to determine expression of fibrosis markers: type I collagen (Col-Ⅰ), fibronectin (FN), α-smooth muscle actin (α-SMA), vimentin, and E-cadherin (E-cad) in the cells supernatant and kidney tissues, as well as expression of TGF-β1/Smad and Notch signaling pathway molecules including pSmad3, Notch1, Notch3, and Jagged-1.

Results

Compared with the sham operation group, the UUO group showed significant higher levels of kidney injury biomarkers as NGAL, KIM-1, Cys-C, and IL-18. The UUO group also displayed significant higher expression level of renal fibrosis marker proteins as well as lower expression level of E-cad. Besides, in the UUO group, the protein expression levels of pSmad3, Notch1, Notch3, and Jagged-1 also increased significantly, and the expression of Smad7 decreased significantly. AST treatment significantly decreased the expression of biomarkers of renal injury (NGAL, KIM-1, Cys-C and IL-18) and protein expression of fibrosis markers, but increased the expression of E-cad protein in a dose-dependent manner. The cell experiments showed that overexpression of CCN1 inhibited the activation of TGF-β1/Smad and Notch signaling pathways, while knocking down CCN1 not only intensified the activation of TGF-β1/Smad and Notch signaling pathways, but also weakened the protective effect of AST in the EMT model induced by TGF-β1.

Conclusion

AST may regulate TGF-β1/Smad and Notch signaling pathways through CCN1, so as to reduce renal interstitial fibrosis and play a role in renal protection.

Key words: Astaxanthin, CCN1, Renal interstitial fibrosis, Notch signaling pathway
图1 各组小鼠肾损伤生物标志物水平注：A：中性粒细胞明胶酶相关脂质运载蛋白(NGAL)mRNA水平比较；B：肾损伤分子-1(KIM-1)mRNA水平比较；C：血清胱抑素-C(Cys-C)mRNA水平比较；D：白介素-18(IL-18)mRNA水平比较；实时定量荧光PCR法检测；与假手术组(Control)相比，aP<0.05；与UUO组相比，bP<0.05
图2 各组小鼠肾纤维化指标比较注：A：Western印迹检测结果；B：Ⅰ型胶原蛋白(Col-Ⅰ)蛋白表达量比较；C：纤连蛋白(FN)蛋白表达量比较；D：α-平滑肌肌动蛋白(α-SMA)蛋白表达量比较；E：波形蛋白(vimentin)蛋白表达量比较；F：E-钙粘蛋白(E-cad)蛋白表达量比较；每组实验均独立重复5次；与假手术组(Control)相比，aP<0.05；与UUO组相比，bP<0.05
图3 各组小鼠TGF-β1/Smad和Notch信号通路蛋白水平比较注：A：Western印迹检测结果；B：转化生长因子-β1(TGF-β1)蛋白表达量比较；C：pSmad3/Smad3蛋白表达量比较；D：Smad7蛋白表达量比较；E：Notch1蛋白表达量比较；F：Notch3蛋白表达量比较；G：Jagged1蛋白表达量比较；每组实验均独立重复5次；与假手术组(Control)相比，aP<0.05；与UUO组相比，bP<0.05
图4 过表达和敲低细胞通信网络因子1对上皮间充质转化模型纤维化指标的影响注：A：Western印迹检测结果；B：Ⅰ型胶原蛋白(Col-Ⅰ)蛋白表达量比较；C：纤连蛋白(FN)蛋白表达量比较；D：α-平滑肌肌动蛋白(α-SMA)蛋白表达量比较；E：波形蛋白(vimentin)蛋白表达量比较；F：E-钙粘蛋白(E-cad)蛋白表达量比较；Control：对照组，Vehicle：空白组，CCN1 OE：CCN1过表达组，CCN1 shRNA：CCN1敲低组，TGF-β1：TGF-β1诱导组，TGF-β1+CCN1 OE：TGF-β1+CCN1过表达组，TGF-β1+CCN1shRNA：TGF-β1+CCN1敲低组；每组实验均独立重复5次；与对照组(Control)相比，aP<0.05；与TGF-β1诱导组相比，bP<0.05
图5 过表达和敲低细胞通信网络因子1对上皮间充质转化模型TGF-β1/Smad和Notch信号通路的影响注：A：Western印迹检测结果；B：转化生长因子-β1(TGF-β1)蛋白表达量比较；C：pSmad3/Smad3蛋白表达量比较；D：Smad7蛋白表达量比较；E：Notch1蛋白表达量比较；F：Notch3蛋白表达量比较；G：Jagged1蛋白表达量比较；Control：对照组，Vehicle：空白组，CCN1 OE：CCN1过表达组，CCN1 shRNA：CCN1敲低组，TGF-β1：TGF-β1诱导组，TGF-β1+CCN1 OE：TGF-β1+CCN1过表达组，TGF-β1+CCN1shRNA：TGF-β1+CCN1敲低组；每组实验均独立重复5次；与对照组(Control)相比，aP<0.05；与TGF-β1诱导组相比，bP<0.05
图6 虾青素对敲低细胞通信网络因子1的上皮间充质转化模型纤维化指标的影响注：A：Western印迹检测结果；B：Ⅰ型胶原蛋白(Col-Ⅰ)蛋白表达量比较；C：纤连蛋白(FN)蛋白表达量比较；D：α-平滑肌肌动蛋白(α-SMA)蛋白表达量比较；E：波形蛋白(vimentin)蛋白表达量比较；F：E-钙粘蛋白(E-cad)蛋白表达量比较；Control：对照组；TGF-β1：TGF-β1诱导组；TGF-β1+AST：TGF-β1+虾青素组；TGF-β1+CCN1 shRNA：TGF-β1+CCN1敲低组；TGF-β1+CCN1 shRNA+AST：TGF-β1+CCN1敲低+虾青素组；每组实验均独立重复5次；与对照组(Control)相比，aP<0.05；与TGF-β1诱导组相比，bP<0.05
图7 虾青素对敲低细胞通信网络因子1的上皮间充质转化模型TGF-β1/Smad和Notch信号通路的影响注：A：Western印迹检测结果；B：转化生长因子-β1(TGF-β1)蛋白表达量比较；C：pSmad3/Smad3蛋白表达量比较；D：Smad7蛋白表达量比较；E：Notch1蛋白表达量比较；F：Notch3蛋白表达量比较；G：Jagged1蛋白表达量比较；Control：对照组；TGF-β1：TGF-β1诱导组；TGF-β1+AST：TGF-β1+虾青素组；TGF-β1+CCN1 shRNA：TGF-β1+CCN1敲低组；TGF-β1+CCN1 shRNA+AST：TGF-β1+CCN1敲低+虾青素组；每组实验均独立重复5次；与对照组(Control)相比，aP<0.05；与TGF-β1诱导组相比，bP<0.05
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