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克老素衍生肽7对慢性胰腺炎小鼠模型胰腺纤维化的影响及其机制
DOI: 10.12449/JCH260419
伦理学声明：本研究方案于2023年8月18日经由广州医科大学附属清远医院实验动物伦理委员会审批，批号：LAEC-2023-042，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：李雨欣负责课题设计，起草论文；李雨欣、傅家财负责实验操作，研究过程的实施；陈赛负责数据收集，统计学分析，绘制图表；齐玲、李凤金负责拟定写作思路、指导撰写文章并最后定稿。
Effect of Klotho-derived peptide 7 on pancreatic fibrosis in a mouse model of chronic pancreatitis and its mechanism
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摘要:
目的 通过观察克老素衍生肽7（KL7）对雨蛙素诱导的慢性胰腺炎（CP）小鼠模型的影响，了解该药物抗胰腺纤维化的作用机制，进而为临床用药提供依据。 方法 将40只雄性BALB/c小鼠随机分为对照组、模型组、KL7低剂量组（2 mg/kg）和KL7高剂量组（4 mg/kg），每组10只。除对照组外，其余各组小鼠腹腔注射雨蛙素（50 μg/kg），每天注射6次，每次间隔1 h，每周注射3 d，连续注射4周，进行CP造模。KL7低剂量、KL7高剂量组分别给予不同剂量的KL7治疗，每天给药1次，连续4周。活体成像观察KL7在胰腺中的富集情况；分子对接检测KL7与转化生长因子β Ⅱ型受体（TβRⅡ）的结合情况；称量小鼠体重及胰腺湿重；苏木素-伊红染色观察胰腺组织的病理变化；马松染色观察胰腺组织纤维化程度；免疫组化染色检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原α1链（COL1A1）的表达情况；蛋白质印迹法检测胰腺组织中α-SMA、TβRⅡ、磷酸化的母系抗十五表态蛋白同源物2/3（p-Smad2/3）蛋白的表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t和Dunnett’s-T3检验。 结果 KL7能够显著富集在CP小鼠胰腺组织，与TβRⅡ蛋白存在强结合活性。与对照组相比，模型组小鼠胰腺湿量和相对胰腺质量均显著降低（P<0.000 1），胰腺组织结构紊乱、炎细胞浸润增加，纤维化程度显著增加，α-SMA和COL1A1阳性面积均显著增加（P值均<0.000 1），α-SMA、TβRⅡ和p-Smad2/3蛋白表达水平显著升高（P值均<0.05）。与模型组相比，KL7高剂量组小鼠胰腺湿量和相对胰腺质量均显著升高（P值均<0.01），胰腺组织结构损伤和炎细胞浸润得到缓解，纤维化程度显著降低，α-SMA和COL1A1阳性面积均显著降低（P值均<0.001），α-SMA、TβRⅡ和p-Smad2/3蛋白表达水平均显著降低（P值均<0.01）。 结论 KL7对CP小鼠胰腺纤维化具有显著的靶向治疗效果，其作用机制涉及KL7与TβRⅡ特异性结合，从而抑制TGF-β/Smad信号通路的激活。 Abstract:Objective To investigate the anti‑pancreatic fibrosis mechanism of Klotho‑derived peptide 7 （KL7） by observing its effect on a mouse model of chronic pancreatitis （CP） induced by cerulean， and to provide a basis for clinical medication. Methods A total of 40 male BALB/c mice were randomly divided into control group， model group， low-dose KL7 group （2 mg/kg）， and high-dose KL7 group （4 mg/kg）， with 10 mice in each group. All mice except those in the control group were given intraperitoneal injection of cerulean （50 μg/kg） 6 times a day at an interval of 1 hour， twice a week for 4 consecutive weeks to establish a model of CP. The mice in the low-dose KL7 group and the high-dose KL7 group were treated with different doses of KL7 once a day for 4 consecutive weeks. In vivo imaging was used to observe the accumulation of KL7 in the pancreas； molecular docking was used to detect the binding of KL7 to transforming growth factor-β type Ⅱ receptor （TβRⅡ）； the mice were measured in terms of body weight and pancreatic weight； HE staining was used to observe the pathological changes of pancreatic tissue； Masson staining was used to observe the degree of pancreatic fibrosis； immunohistochemical staining was used to measure the expression of α-smooth muscle actin （α-SMA） and type Ⅰ collagen （COL1A1）； Western blotting was used to measure the protein expression levels of α-SMA， TβRII， and phosphorylated small mothers against decapentaplegic homolog 2/3 （p-Smad2/3） in pancreatic tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test and the Dunnett’s-T3 test were used for further comparison between two groups. Results KL7 was significantly enriched in the pancreatic tissue of CP mice， and there was a strong binding activity between KL7 and TβRⅡ. Compared with the control group， the model group had significant reductions in pancreatic mass and relative pancreatic mass （P<0.000 1）， with disordered structure of pancreatic tissue， an increase in inflammatory cell infiltration， and significant increases in fibrosis degree， the positive areas of α-SMA and COL1A1 （P<0.000 1）， and the protein expression levels of α-SMA， TβRⅡ， and p-Smad2/3 （P<0.05）. Compared with the model group， the high-dose KL7 group had significant increases in pancreatic mass and relative pancreatic mass （P<0.01）， with alleviation of structural damage of pancreatic tissue and inflammatory cell infiltration， a significant reduction in fibrosis degree， and significant reductions in the positive areas of α-SMA and COL1A1 （P<0.001） and the protein expression levels of α-SMA， TβRⅡ， and p-Smad2/3 （P<0.01）. Conclusion KL7 has a significant targeted therapeutic effect on pancreatic fibrosis in CP mice through specific binding of KL7 to TβRⅡ， thereby inhibiting the activation of the TGF-β/Smad signaling pathway. -
Key words:
- Pancreatitis， Chronic /
- Fibrosis /
- Klotho-Derived Peptide
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注： a，脏器明场；b，脏器克老素衍生肽7-花青素7荧光成像。
图 1 克老素衍生肽7在各组小鼠主要脏器中的富集情况
Figure 1. Enrichment of KL7 in the main organs of mice in various group
注： a，各组小鼠体重；b，各组小鼠胰腺湿重；c，各组小鼠相对胰腺质量。KL7，克老素衍生肽7。
图 3 各组小鼠体重及胰腺湿重情况
Figure 3. Body weight and pancreatic weight of mice in various group
注： KL7，克老素衍生肽7。
图 4 各组小鼠胰腺组织大体形态和苏木精-伊红染色结果（×100）
Figure 4. Gross morphology and HE staining results of pancreatic tissue in various group
注： a，马松染色（×100）；b，α-SMA表达情况（免疫组织化学染色，×100）；c，COL1A1表达情况（免疫组织化学染色，×100）；d， 各组小鼠胰腺组织马松染色和免疫组织化学染色面积半定量结果。α-SMA，α平滑肌肌动蛋白；COL1A1，Ⅰ型胶原α1链；KL7，克老素衍生肽7。
图 5 各组小鼠胰腺组织染色情况
Figure 5. Staining status of pancreatic tissues in various groups of mice
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