Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (34): 5458-5466.doi: 10.12307/2024.808
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Jiang Zixian1, Lu Yuchun1, Li Chaomeng1, Zheng Meimei2, Li Xiufang1, Wang Wenjing1
Received:
2023-11-20
Accepted:
2023-12-27
Online:
2024-12-08
Published:
2024-03-14
Contact:
Corresponding author: Wang Wenjing, MD, Professor, Doctoral supervisor, Yunnan University of Chinese Medicine, Kunming 650500, Yunnan Province, China
Li Xiufang, MD, Professor, Doctoral supervisor, Yunnan University of Chinese Medicine, Kunming 650500, Yunnan Province, China
About author:
Jiang Zixian, Master candidate, Yunnan University of Chinese Medicine, Kunming 650500, Yunnan Province, China
Supported by:
CLC Number:
Jiang Zixian, Lu Yuchun, Li Chaomeng, Zheng Meimei, Li Xiufang, Wang Wenjing. Extracts of Sambucus adnata Wall. inhibit abnormal angiogenesis in a rat model of osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(34): 5458-5466.
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2.5 SAW-ME、SAW-DCE对EA.hy926细胞成管作用的影响 2.5.1 细胞成管网眼数和分支数分析 与对照组相比,VEGF组网眼数 (P < 0.05)、分支数 (P < 0.001)明显增加;与VEGF组相比,给予SAW-ME 50 μg/mL组网眼数 (P < 0.001)、分支数 (P < 0.001)均明显减少;给予SAW-ME 25 μg/mL和12.5 μg/mL时,分支数明显减少 (P < 0.001),网眼数具有减少的趋势,但差异无统计学意义;与VEGF组相比,给予SAW-DCE 5 μg/mL组网眼数 (P < 0.01)、分支数 (P < 0.05)明显减少;给予SAW-DCE质量浓度为2.5 μg/mL和1.25 μg/mL时,分支数均明显减少 (P < 0.05),网眼数减少,但差异无统计学意义,见图6A-C。 2.5.2 细胞成管节点数和成管主段长度分析 与对照组相比,VEGF组节点数明显增加(P < 0.001),主段长度增加,给予SAW-ME质量浓度为50,25和12.5 μg/mL时,节点数明显减少 (P < 0.001或P < 0.05),主段长度减少,但各组的差异均无统计学意义;给予SAW-DCE质量浓度为5 μg/mL时,节点数明显减少 (P < 0.001),段长度明显减少 (P < 0.05),给予SAW-DCE质量浓度为2.5 μg/mL和1.25 μg/mL时,节点数减少,主段长度减少,差异无统计学意义,见图6A,D,E。 "
2.7 SAW-ME、SAW-DCE对EA.hy926细胞内Ang1和Tie2蛋白表达的影响 2.7.1 Ang1的蛋白表达 与对照组比较,VEGF组中Ang1的蛋白表达水平明显升高 (P < 0.05);与VEGF组相比,给予SAW-ME的质量浓度为50,12.5 μg/mL时,Ang1蛋白表达水平明显降低 (P < 0.001或P < 0.05);与VEGF组相比,给予SAW-DCE的质量浓度为5,2.5,1.25 μg/mL时,Ang1蛋白表达水平均显著降低 (P < 0.001) ,见图8A,B。提示斯赤列这两种提取物均可以抑制Ang1的蛋白表达水平。 2.7.2 Tie2的蛋白表达 与对照组比较,VEGF组中Tie2的蛋白表达水平有升高的趋势,但无统计学意义。与VEGF组相比,给予SAW-ME质量浓度为50,25,12.5 μg/mL时,Tie2蛋白表达水平明显降低 (P < 0.001或P < 0.01);与VEGF组相比,给予SAW-DCE质量浓度为5,1.25 μg/mL时,Tie2蛋白表达水平均显著降低 (P < 0.001或P < 0.05),见图8C,D。在蛋白水平上说明了斯赤列这两种提取物可以抑制Tie2的表达水平。 "
2.8.3 对差异表达基因进行KEGG富集分析 对照组和VEGF组比较的差异基因,富集到了细胞周期、黏着斑、DNA复制、ECM-receptor相互作用、Fanconi anemia通路、PI3K-Akt信号通路等;VEGF组与SAW-DCE组比较的差异基因,富集到了细胞因子-细胞因子受体相互作用、PI3K/Akt信号通路、MAPK信号通路、风湿性关节炎、Ras信号通路、JAK/STAT信号通路等。综合以上KEGG通路富集结果,对富集到的PI3K/Akt信号通路进行分析,对照组与VEGF组比较,在PI3K/Akt信号通路上富集到了20个差异基因;VEGF组与SAW-DCE组比较,在该信号通路上富集到了17个差异基因,见图11。若将两个基因集放在一起比较,在该通路上主要富集到了:PDGFRA;FGF1;NR4A1;JAK1;SPP1;ITGB7;AREG;CCND1;FLT1等基因,生信分析发现,经过SAW-DCE干预之后,这些基因mRNA表达水平都明显下调,推测SAW-DCE可能通过抑制PI3K/AKT信号通路,抑制血管新生和迁移,见图12。 通过转录组分析,对PI3K/AKT信号通路中富集到的差异基因的表达量采用柱状图进行展示,见图12。"
2.9 Western bolt验证测序结果 为进一步验证RNA-seq筛选出基因的表达水平,实验选择了与骨关节炎相关度较高的PI3K/AKT信号通路采用Western bolt进行验证。与对照组比较,VEGF组中内皮细胞P-PI3K、PI3K的表达明显升高 (P < 0.05);与VEGF组相比,SAW-DCE干预后P-PI3K、PI3K的表达均降低,SAW-DCE在5 μg/mL的质量浓度下,P-PI3K、PI3K降低的趋势较为明显 (P < 0.05);在1.25 μg/mL的质量浓度下,PI3K下降趋势较为明显 (P < 0.05);与对照组比较,VEGF组中内皮细胞内AKT的表达有升高的趋势,在SAW-DCE的干预质量浓度为2.5,1.25 μg/mL时,AKT的蛋白表达水平有降低的趋势,但差异没有统计学意义,见图13。"
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