1.Angiotensin (1-7) activates autophagy to regulates apoptosis and angiogenesis of oral submucosal fibroblasts
Lehong Qiu ; Wei Deng ; Chengwen Gan ; Ying Sun
Acta Universitatis Medicinalis Anhui 2023;58(3):457-464
Objective:
To observe the effect of angiotensin ( 1-7) [Ang( 1-7) ]on the apoptosis and angiogenesis of fibroblasts in the oral submucosal fibrosis ( OSF) ,and to explore the effect preliminarily mechanism.
Methods:
Fibroblasts were isolated and cultured from human buccal mucosal tissue,the cell morphology was observed by inverted microscope,and the expression of vimentin was detected by immunofluorescence staining ; areca nut extract (ANE) was used to induce human fibroblasts to simulate the in vitro model of fibroblasts in OSF,the experimental groups included control group ( normally cultured cells) ,ANE group ( 100 μg/ ml ANE cultured cells for 48 hours) ,ANE + low-dose Ang( 1-7) group ( 100 μg/ ml ANE + 10-7 mol /L Ang ( 1-7) cultured cells for 48 h) , ANE + high-dose Ang( 1-7) group ( 100 μg/ ml ANE + 10-5 mol /L Ang( 1-7) cultured cells for 48 h) ,immunofluorescence staining detected the expression of α-smooth muscle actin ( α-SMA) ,ELISA method detected the content of Collagen I and Collagen Ⅲ in the cell culture supernatant,MTT method detected cell proliferation activity, flow cytometry detected cell apoptosis ,the tubule formation experiment detected the vascularization of human umbilical vein endothelial cell(HUVEC) ; After the mRFP-GFP-LC3 virus was transferred to the cells,the level of autophagy was detected by immunofluorescence staining,Western blot detected the expression of autophagy-related protein Beclin-1 and the ratio of LC3-Ⅱ/ LC3-Ⅰ .
Results:
The isolated and cultured cells were in a long spindle shape,and Vimentin was positively expressed,indicating that fibroblasts were successfully isolated ; Compared with the ANE group,the fluorescence expression of α-SMA protein in ANE low dose Ang( 1-7) group and ANE + high dose Ang( 1-7) group significantly decreased,the contents of Collagen I and Collagen Ⅲ in the culture supernatant were reduced (P<0.05) ,cell proliferation activity decreased (P<0.05) ,and cell apoptosis rate increased (P < 0.05) ,the cell culture supernatants of the two groups inhibited the angiogenesis of HUVEC (P<0.05) ,endophagosomes were reduced (P<0.05) ,Beclin-1 protein expression was reduced (P <0.05) ,and the ratio of LC3- Ⅱ / LC3-Ⅰ was down-regulated (P <0.05 ) ; in addition ,the effect of ANE + high-dose Ang ( 1-7 ) group was stronger than that of ANE + low-dose Ang( 1-7) group (P<0.05) .
Conclusion
Ang( 1-7) can inhibit the activation of fibroblasts induced by ANE,promote cell apoptosis,and reduce the angiogenesis of HUVEC,this mechanism may be related to the regulation of cell autophagy.
2.The effects of sonicated extract of Porphyromonas endodontalis on the cell cycle and apoptosis of osteo-blasts
Yaguang TIAN ; Tianan LIAO ; Xiaobin CHEN ; Puwu WANG ; Lehong QIU
Journal of Practical Stomatology 2015;(2):188-191
Objective:To observe the effects of sonicated extract of Porphyromonas endodontalis(P.endodontalis)on the cell cy-cle and apoptosis of human osteoblastic hFOB 1 .1 9 cells.Methods:hFOB 1 .1 9 cells were treated with sonicated extract of P.end-odontalis at 0 (the control),1 ,1 0 and 1 00 μg/ml for 1 2,24 and 48 h respectively.MTT assay was used to examine cell prolifera-tion.The cell cycle distribution and apoptosis were examined by flow cytometry.Results:The sonicated exact of P.endodontalis in-hibited the proliferation of hFOB 1 .1 9 cells in a time-and dose-dependent manner.24 h treatment of 1 0 μg/ml and 1 00 μg/ml ex-act arrested the cell cycle of the cells in G1 phase,48 h treatment induced apoptosis in a dose-dependent manner.Conclusion:Sonicated exact of P.endodontalis may arrest hFOB 1 .1 9 cells in G1 phase and induce cell apoptosis,leading to inhibition of the cell proliferation.


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