| 引用本文: |
李武平,李婕,易南,刘宗义,黄思琪,朱付平.桃红四物汤对大鼠后肢缺血-再灌注损伤骨骼肌CaMKⅡ、PKC表达的影响[J].湖南中医药大学学报,2021,41(5):678-684[点击复制] |
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| 桃红四物汤对大鼠后肢缺血-再灌注损伤骨骼肌CaMKⅡ、PKC表达的影响 |
| 李武平,李婕,易南,刘宗义,黄思琪,朱付平 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 观察桃红四物汤对大鼠后肢缺血-再灌注损伤骨骼肌钙调蛋白依赖性蛋白激酶II（CaMKⅡ）、蛋白激酶C（PKC）表达的影响，从Wnt/Ca2+信号通路角度探讨其作用机制。方法 取2月龄雄性SD大鼠80只，随机分成正常组、模型组、桃红四物汤组、阻滞剂组，每组20只。除正常组外，其他各组均阻断血流复制右后肢缺血-再灌注损伤模型，桃红四物汤组、阻滞剂组（FZR5、Wnt5a阻滞剂）在造模前分别予以桃红四物汤灌胃、阻滞剂腹腔注射预处理。DAPI法观察复灌后各组腓肠肌细胞凋亡变化；RT-qPCR检测复灌后（0、2、4、8 h）腓肠肌CaMKⅡ、PKC mRNA的表达；免疫组化（IHC）检测复灌后8 h腓肠肌中CaMKⅡ、PKC蛋白的阳性表达。结果 DAPI荧光染色结果显示，与正常组比较，模型组、桃红四物汤组、阻滞剂组大鼠腓肠肌细胞凋亡率明显升高（P<0.05）；与模型组比较，桃红四物汤组和阻滞剂组大鼠腓肠肌细胞凋亡率明显降低（P<0.05）。RT-qPCR和IHC结果显示，与正常组比较，模型组、桃红四物汤组、阻滞剂组CaMKⅡ、PKC蛋白和mRNA表达一致性上调（P<0.05）；与模型组相比，桃红四物汤组、阻滞剂组CaMKⅡ蛋白和mRNA表达一致性下调（P<0.05），PKC mRNA表达下调（P<0.05），但PKC蛋白表达下调不明显（P>0.05），不同时间点显示相同倾向性。结论 CaMKⅡ、PKC为大鼠肢体缺血-再灌注骨骼肌损伤中的关键蛋白，桃红四物汤可通过调控Wnt5a/Ca2+信号通路下调CaMKⅡ表达来减轻肢体缺血-再灌注损伤，PKC不是Wnt5a/Ca2+信号通路中桃红四物汤的主要作用靶点。 |
| 关键词: 肢体缺血-再灌注损伤 Wnt5a/Ca2+信号通路 钙调蛋白依赖性蛋白激酶II 蛋白激酶C 桃红四物汤 |
| DOI：10.3969/j.issn.1674-070X.2021.05.005 |
| 投稿时间：2021-01-25 |
| 基金项目:国家自然科学基金项目（81674008）；湖南省自然科学基金项目（2020JJ4070）；湖南省教育厅科学研究基金项目（20C1426）。 |
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| Effect of Taohong Siwu Decoction on Expression of CaMKⅡ and PKC in Skeletal Muscle of Rats with Hindlimb Ischemia-reperfusion Injury |
| LI Wuping,LI Jie,YI Nan,LIU Zongyi,HUANG Siqi,ZHU Fuping |
| (The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To observe the effect of Taohong Siwu Decoction on the expression of calmodulin dependent protein kinase II (CaMK II) and protein kinase C (PKC) in skeletal muscle of rats with hindlimb ischemia-reperfusion injury, and to explore its mechanism from the perspective of Wnt/Ca2+ signaling pathway. Methods 80 2-month-old male SD rats were randomly divided into normal group, model group, Taohong Siwu Decoction group and blocker group, with 20 rats in each group. In addition to the normal group, the right hindlimb ischemia-reperfusion injury model was established by blocking the blood flow in other groups. The Taohong Siwu Decoction group and the blocker group (FZR5, Wnt5a blocker) were treated by intragastric administration of Taohong Siwu Decoction and intraperitoneal injection of blocker respectively. DAPI method was used to observe the apoptosis of gastrocnemius muscle cells in each group after reperfusion. RT-qPCR was used to detect the mRNA expression of CaMKII and PKC in gastrocnemius muscle at 0, 2, 4 and 8 hours after reperfusion. Immunohistochemistry (IHC) was used to detect the protein expression of CaMKII and PKC in gastrocnemius muscle at 8 hours after reperfusion. Results DAPI fluorescence staining results showed that, compared with the normal group, the apoptosis rate of gastrocnemius muscle cells in the model group, Taohong Siwu Decoction group and blocker group was significantly increased (P<0.05); compared with the model group, the apoptosis rate of gastrocnemius muscle cells in the Taohong Siwu Decoction group and the blocker group was significantly decreased (P<0.05). The results of RT-qPCR and IHC showed that, compared with the normal group, the protein and mRNA expression levels of CaMKII and PKC in model group, Taohong Siwu Decoction group and blocker group were up-regulated (P<0.05); compared with the model group, the protein and mRNA expression levels of CaMKII in Taohong Siwu Decoction group and blocker group were down-regulated (P<0.05), the expression of PKC mRNA was down-regulated (P<0.05), but the expression of PKC protein was not significantly down-regulated (P>0.05), and in different time points showed the same tendency. Conclusion CaMKII and PKC are key proteins in limb ischemia-reperfusion injury in rats, Taohong Siwu Decoction can reduce limb ischemia-reperfusion injury by regulating Wnt5a/Ca2+ signaling pathway and down regulating CaMKII expression, but PKC is not the main target of Taohong Siwu Decoction in Wnt5a/Ca2+ signaling pathway. |
| Key words: limb ischemia reperfusion injury Wnt5a/Ca2+ signaling pathway calmodulin dependent protein kinase II protein kinase C Taohong Siwu Decoction |
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