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
目的探讨葛根素调控miR-30b-5p表达对类固醇诱导的兔股骨头坏死(osteonecrosis of the femoral head,ONFH)的作用及机制。方法将84只新西兰大白兔分为对照组、ONFH组、葛根素低剂量组、葛根素高剂量组、阳性对照组、葛根素高剂量+antago...目的探讨葛根素调控miR-30b-5p表达对类固醇诱导的兔股骨头坏死(osteonecrosis of the femoral head,ONFH)的作用及机制。方法将84只新西兰大白兔分为对照组、ONFH组、葛根素低剂量组、葛根素高剂量组、阳性对照组、葛根素高剂量+antagomir NC组、葛根素高剂量+miR-30b-5p antagomir组,每组12只。通过Micro-CT检测大白兔股骨头骨小梁数量、骨小梁厚度及骨小梁分离度;HE染色检测股骨头组织病理变化;ELISA法检测股骨头组织中超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;TUNEL染色检测股骨头组织中骨细胞凋亡;qRT-PCR检测股骨头组织中miR-30b-5p表达;Western Blot检测股骨头组织中Dickkopf-1(DKK-1)蛋白表达。结果与对照组比较,ONFH组骨小梁数量、骨小梁厚度、SOD活性、miR-30b-5p表达降低,骨小梁分离度、空骨陷窝率、MDA含量、骨细胞凋亡率及DKK-1蛋白表达升高(P<0.05);与ONFH组比较,葛根素低、高剂量组、阳性对照组骨小梁数量、骨小梁厚度、SOD活性、miR-30b-5p表达升高,骨小梁分离度、空骨陷窝率、MDA含量、骨细胞凋亡率及DKK-1蛋白表达降低(P<0.05);miR-30b-5p antagomir减弱了高剂量葛根素对ONFH兔氧化应激及细胞凋亡的抑制作用。结论葛根素可抑制氧化应激及细胞凋亡进而发挥对ONFH兔的保护作用,其机制可能与上调miR-30b-5p表达有关。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调控miR-30b-5p表达对类固醇诱导的兔股骨头坏死(osteonecrosis of the femoral head,ONFH)的作用及机制。方法将84只新西兰大白兔分为对照组、ONFH组、葛根素低剂量组、葛根素高剂量组、阳性对照组、葛根素高剂量+antagomir NC组、葛根素高剂量+miR-30b-5p antagomir组,每组12只。通过Micro-CT检测大白兔股骨头骨小梁数量、骨小梁厚度及骨小梁分离度;HE染色检测股骨头组织病理变化;ELISA法检测股骨头组织中超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;TUNEL染色检测股骨头组织中骨细胞凋亡;qRT-PCR检测股骨头组织中miR-30b-5p表达;Western Blot检测股骨头组织中Dickkopf-1(DKK-1)蛋白表达。结果与对照组比较,ONFH组骨小梁数量、骨小梁厚度、SOD活性、miR-30b-5p表达降低,骨小梁分离度、空骨陷窝率、MDA含量、骨细胞凋亡率及DKK-1蛋白表达升高(P<0.05);与ONFH组比较,葛根素低、高剂量组、阳性对照组骨小梁数量、骨小梁厚度、SOD活性、miR-30b-5p表达升高,骨小梁分离度、空骨陷窝率、MDA含量、骨细胞凋亡率及DKK-1蛋白表达降低(P<0.05);miR-30b-5p antagomir减弱了高剂量葛根素对ONFH兔氧化应激及细胞凋亡的抑制作用。结论葛根素可抑制氧化应激及细胞凋亡进而发挥对ONFH兔的保护作用,其机制可能与上调miR-30b-5p表达有关。
