Background:Puerarin(Pue)has been reported to be a natural active ingredient with multiple antifibrotic properties.This work aimed at exploring the function of Pue in oral submucousfibrosis(OSF)treatment.Methods:Human or...Background:Puerarin(Pue)has been reported to be a natural active ingredient with multiple antifibrotic properties.This work aimed at exploring the function of Pue in oral submucousfibrosis(OSF)treatment.Methods:Human oral mucosafibroblasts(hOMF)were induced with transforming growth factor beta1(TGF-β1)and intervened with Pue.Expressions offibrosis-related markers were analyzed by Western blot and IF staining.Cell viability was characterized by the CCK-8 assay.Expressions of miR-30 family members were quantified by qRT-PCR.The correlation betweenfibroblast activation protein(FAP)and miR-30 family expression was evaluated by the Pearson correlation coefficient.Bioinformatics prediction and dual-luciferase reporter assay were employed to verify the regulation between FAP and miR-30b-5p.The specific mechanism of Pue on OSF was explored through the promotion or inhibition of miR-30b-5p.Results:After induction by TGF-β1,hOMF showed upregulated Collagen I,Collagen III,and FAP expressions,while miR-30 family expression was downregulated with miR-30b-5p being the most significant.Pue intervention inhibited the excessive proliferation of TGF-β1-induced hOMF,downregulated FAP,collagen type 3(COL3A1),collagen type 1(COL1A1),matrix metalloproteinase 1(MMP1),and matrix metalloproteinase 3(MMP3)expressions,and restored miR-30 family expression.Bioinformatics prediction and dual-luciferase reporter assay revealed that miR-30b-5p selectively inhibited FAP expression.Mechanistically,miR-30b-5p mimic suppressed the excessive proliferation of TGF-β1-induced hOMF and declinedfibrosis levels.Pue intervention significantly reversed the promotion of TGF-β1-induced OSF by miR-30b-5p inhibition.Conclusion:Pue mediated miR-30b-5p targeting FAP against OSF,which provided a theoretical basis for the pathogenesis research and Pue application in OSF.展开更多
目的:探讨葛根素能否通过调控miR-30e-5p的表达减轻高糖所致的足细胞损伤。方法:高糖诱导小鼠肾足细胞MPC5建立足细胞损伤模型,使用不同剂量的葛根素处理细胞;将miR-NC、miR-30e-5p mimics、anti-miR-NC、anti-miR-30e-5p转染至MPC5细...目的:探讨葛根素能否通过调控miR-30e-5p的表达减轻高糖所致的足细胞损伤。方法:高糖诱导小鼠肾足细胞MPC5建立足细胞损伤模型,使用不同剂量的葛根素处理细胞;将miR-NC、miR-30e-5p mimics、anti-miR-NC、anti-miR-30e-5p转染至MPC5细胞后采用高糖诱导建立足细胞损伤模型,并给予不同剂量的葛根素处理细胞。采用流式细胞术检测细胞凋亡率;采用RT-qPCR法检测miR-30e-5p的表达量;Western Blot法检测半胱氨酰天冬氨酸特异性蛋白酶3(cysteinyl aspartate-specific protease-3,caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bcl-2-associated X protein,Bax)、p62、微管相关蛋白轻链3Ⅱ(microtubule-associated protein l light chain 3,LC3-Ⅱ)、LC3-Ⅰ蛋白表达水平并计算LC3-Ⅱ/LC3-Ⅰ的比值。结果:高糖处理小鼠肾足细胞MPC5后,可明显提高MPC5细胞的凋亡率及caspase-3、Bax、p62蛋白表达水平(P<0.05),显著降低Bcl-2蛋白及miR-30e-5p的表达水平和LC3-Ⅱ/LC3-Ⅰ的比值(P<0.05),而葛根素可显著降低细胞凋亡率及caspase-3、Bax、p62的蛋白表达水平(P<0.05),显著升高Bcl-2蛋白、miR-30e-5p的表达水平及LC3-Ⅱ/LC3-Ⅰ的比值(P<0.05);与miR-NC组比较,miR-30e-5p组细胞凋亡率显著降低(P<0.05),caspase-3、Bax、p62蛋白表达水平显著降低(P<0.05),Bcl-2蛋白水平、LC3-Ⅱ/LC3-Ⅰ的比值显著升高(P<0.05);与高剂量葛根素+anti-miR-NC组比较,高剂量葛根素+anti-miR-30e-5p组细胞凋亡率显著升高(P<0.05),caspase-3、Bax、p62蛋白表达水平显著升高(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.05),LC3-Ⅱ/LC3-Ⅰ的比值显著降低(P<0.05)。结论:葛根素可通过上调miR-30e-5p的表达抑制高糖诱导的足细胞凋亡,其作用机制可能与诱导细胞自噬有关。展开更多
基金This work was supported by the National Natural Science Foundation of China(Nos.81874496,82374530)the Clinical Medical Technology Innovation Guide Project of Hunan Province(No.2020SK53206)+3 种基金the Natural Science Foundation of Hunan Province(No.2021JJ70062)the Changsha Natural Science Foundation Project(No.kq2014019)the Health Special Fund Research Project of Hunan Province(No.B2020-07)the Clinical Pharmaceutical Research Fund of Hunan Medical Association(No.B202012).
文摘Background:Puerarin(Pue)has been reported to be a natural active ingredient with multiple antifibrotic properties.This work aimed at exploring the function of Pue in oral submucousfibrosis(OSF)treatment.Methods:Human oral mucosafibroblasts(hOMF)were induced with transforming growth factor beta1(TGF-β1)and intervened with Pue.Expressions offibrosis-related markers were analyzed by Western blot and IF staining.Cell viability was characterized by the CCK-8 assay.Expressions of miR-30 family members were quantified by qRT-PCR.The correlation betweenfibroblast activation protein(FAP)and miR-30 family expression was evaluated by the Pearson correlation coefficient.Bioinformatics prediction and dual-luciferase reporter assay were employed to verify the regulation between FAP and miR-30b-5p.The specific mechanism of Pue on OSF was explored through the promotion or inhibition of miR-30b-5p.Results:After induction by TGF-β1,hOMF showed upregulated Collagen I,Collagen III,and FAP expressions,while miR-30 family expression was downregulated with miR-30b-5p being the most significant.Pue intervention inhibited the excessive proliferation of TGF-β1-induced hOMF,downregulated FAP,collagen type 3(COL3A1),collagen type 1(COL1A1),matrix metalloproteinase 1(MMP1),and matrix metalloproteinase 3(MMP3)expressions,and restored miR-30 family expression.Bioinformatics prediction and dual-luciferase reporter assay revealed that miR-30b-5p selectively inhibited FAP expression.Mechanistically,miR-30b-5p mimic suppressed the excessive proliferation of TGF-β1-induced hOMF and declinedfibrosis levels.Pue intervention significantly reversed the promotion of TGF-β1-induced OSF by miR-30b-5p inhibition.Conclusion:Pue mediated miR-30b-5p targeting FAP against OSF,which provided a theoretical basis for the pathogenesis research and Pue application in OSF.
文摘目的:探讨葛根素能否通过调控miR-30e-5p的表达减轻高糖所致的足细胞损伤。方法:高糖诱导小鼠肾足细胞MPC5建立足细胞损伤模型,使用不同剂量的葛根素处理细胞;将miR-NC、miR-30e-5p mimics、anti-miR-NC、anti-miR-30e-5p转染至MPC5细胞后采用高糖诱导建立足细胞损伤模型,并给予不同剂量的葛根素处理细胞。采用流式细胞术检测细胞凋亡率;采用RT-qPCR法检测miR-30e-5p的表达量;Western Blot法检测半胱氨酰天冬氨酸特异性蛋白酶3(cysteinyl aspartate-specific protease-3,caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bcl-2-associated X protein,Bax)、p62、微管相关蛋白轻链3Ⅱ(microtubule-associated protein l light chain 3,LC3-Ⅱ)、LC3-Ⅰ蛋白表达水平并计算LC3-Ⅱ/LC3-Ⅰ的比值。结果:高糖处理小鼠肾足细胞MPC5后,可明显提高MPC5细胞的凋亡率及caspase-3、Bax、p62蛋白表达水平(P<0.05),显著降低Bcl-2蛋白及miR-30e-5p的表达水平和LC3-Ⅱ/LC3-Ⅰ的比值(P<0.05),而葛根素可显著降低细胞凋亡率及caspase-3、Bax、p62的蛋白表达水平(P<0.05),显著升高Bcl-2蛋白、miR-30e-5p的表达水平及LC3-Ⅱ/LC3-Ⅰ的比值(P<0.05);与miR-NC组比较,miR-30e-5p组细胞凋亡率显著降低(P<0.05),caspase-3、Bax、p62蛋白表达水平显著降低(P<0.05),Bcl-2蛋白水平、LC3-Ⅱ/LC3-Ⅰ的比值显著升高(P<0.05);与高剂量葛根素+anti-miR-NC组比较,高剂量葛根素+anti-miR-30e-5p组细胞凋亡率显著升高(P<0.05),caspase-3、Bax、p62蛋白表达水平显著升高(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.05),LC3-Ⅱ/LC3-Ⅰ的比值显著降低(P<0.05)。结论:葛根素可通过上调miR-30e-5p的表达抑制高糖诱导的足细胞凋亡,其作用机制可能与诱导细胞自噬有关。
