Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (3): 457-465.DOI: 10.19852/j.cnki.jtcm.20220707.005
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Salvianolic acid B promotes the invasion and migration of H2O2-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway
ZHAO Zhiqiang, ZHANG Chong, ZHU Yunxia(
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- Department of Gynecology and Obstetrics, Beijing You'an Hospital of Capital Medical University, Beijing 100069, China
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Received:2021-06-27Accepted:2021-09-30Online:2023-06-15Published:2022-07-07 -
Contact:ZHU Yunxia, Beijing You'an Hospital of Capital Medical University, Beijing 100069, China. yunxiadyxh@163.com. Telephone: +86-13522674990
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ZHAO Zhiqiang, ZHANG Chong, ZHU Yunxia. Salvianolic acid B promotes the invasion and migration of H2O2-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway[J]. Journal of Traditional Chinese Medicine, 2023, 43(3): 457-465.
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Figure 1 SalB promoted the proliferation of HTR-8/ Svneo cells A: MTT detected the cell viability after treatment with different concentrations (10, 20, 40 and 80 μM) of SalB for 24 h; B: CCK-8 detected the viability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.001 vs H2O2. SalB: salvianolic acid B; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 1 SalB promoted the proliferation of HTR-8/ Svneo cells A: MTT detected the cell viability after treatment with different concentrations (10, 20, 40 and 80 μM) of SalB for 24 h; B: CCK-8 detected the viability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.001 vs H2O2. SalB: salvianolic acid B; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 2 SalB inhibited oxidative damage and apoptosis in HTR-8/Svneo cells induced by H2O2 A. ELISA assay detected the expression of SOD, GSH-Px and MDA in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. B. TUNEL assay detected the apoptosis in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x200. C. Western blot detected the expression of apoptosis-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs control; bP<0.05, dP<0.01, cP<0.001 vs H2O2. SalB: salvianolic acid B; SOD: superoxide dismutase; GSH-Px: glutathione-Px; MDA: malondialdehyde; Bcl-2: B cell lymphoma-2; Bax: Bcl-2 associated X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme-linked immunosorbent assay; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; DAPI: 4',6-diamidino-2-phenylindole.
Figure 2 SalB inhibited oxidative damage and apoptosis in HTR-8/Svneo cells induced by H2O2 A. ELISA assay detected the expression of SOD, GSH-Px and MDA in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. B. TUNEL assay detected the apoptosis in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x200. C. Western blot detected the expression of apoptosis-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs control; bP<0.05, dP<0.01, cP<0.001 vs H2O2. SalB: salvianolic acid B; SOD: superoxide dismutase; GSH-Px: glutathione-Px; MDA: malondialdehyde; Bcl-2: B cell lymphoma-2; Bax: Bcl-2 associated X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme-linked immunosorbent assay; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; DAPI: 4',6-diamidino-2-phenylindole.
Figure 3 SalB promoted invasion, migration and EMT of HTR-8/ Svneo cell induced by H2O2. A. Wound healing detected the cell migration ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x100. B. Transwell detected the cell invasion ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x100. C. Western blot detected the expression of EMT-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.05, dP<0.01, cP<0.001 vs H2O2. SalB: salvianolic acid B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
Figure 3 SalB promoted invasion, migration and EMT of HTR-8/ Svneo cell induced by H2O2. A. Wound healing detected the cell migration ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x100. B. Transwell detected the cell invasion ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, x100. C. Western blot detected the expression of EMT-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.05, dP<0.01, cP<0.001 vs H2O2. SalB: salvianolic acid B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
Figure 4 SalB upregulated the expression of MMP-9 in H2O2-induced HTR-8/Svneo cells through PI3K/Akt pathway. A. RT-qPCR detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. B. Western blot detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. C. Western blot detected the expression of PI3K/AKT in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. D. RT-qPCR detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and LY294002 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. E. Western blot detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and LY294002 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; dP<0.01, cP<0.001 vs H2O2; fP<0.05, eP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; MMP9, Matrix metallopeptidase 9; p-PI3K: phosphorylated phosphatidylinositol-3-kinase; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; p-Akt: phosphorylated protein kinase B; Akt: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: reverse transcription-quantitative real-time polymerase chain reaction.
Figure 4 SalB upregulated the expression of MMP-9 in H2O2-induced HTR-8/Svneo cells through PI3K/Akt pathway. A. RT-qPCR detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. B. Western blot detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. C. Western blot detected the expression of PI3K/AKT in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by different concentrations (10, 20 and 40 μM) of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. D. RT-qPCR detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and LY294002 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. E. Western blot detected the expression of MMP9 in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and LY294002 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; dP<0.01, cP<0.001 vs H2O2; fP<0.05, eP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; MMP9, Matrix metallopeptidase 9; p-PI3K: phosphorylated phosphatidylinositol-3-kinase; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; p-Akt: phosphorylated protein kinase B; Akt: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: reverse transcription-quantitative real-time polymerase chain reaction.
Figure 5 MMP-9 inhibitor GM6001 reversed the oxidative damage and apoptosis of SalB on HTR-8/ Svneo cells induced by H2O2 A: ELISA assay detected the expression of SOD, GSH-Px and MDA in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h; B:TUNEL assay detected the apoptosis in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×200; C: Western blot detected the expression of apoptosis-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.001 vs H2O2; cP<0.05, dP<0.01, eP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; SOD: superoxide dismutase; GSH-Px: glutathione-Px; MDA: malondialdehyde; Bcl-2: B cell lymphoma-2; Bax: Bcl-2 associated X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme-linked immunosorbent assay; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; Dapi: 4',6-diamidino-2-phenylindole.
Figure 5 MMP-9 inhibitor GM6001 reversed the oxidative damage and apoptosis of SalB on HTR-8/ Svneo cells induced by H2O2 A: ELISA assay detected the expression of SOD, GSH-Px and MDA in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h; B:TUNEL assay detected the apoptosis in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×200; C: Western blot detected the expression of apoptosis-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs Control; bP<0.001 vs H2O2; cP<0.05, dP<0.01, eP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; SOD: superoxide dismutase; GSH-Px: glutathione-Px; MDA: malondialdehyde; Bcl-2: B cell lymphoma-2; Bax: Bcl-2 associated X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme-linked immunosorbent assay; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; Dapi: 4',6-diamidino-2-phenylindole.
Figure 6 MMP-9 inhibitor GM6001 reversed the invasion, migration and EMT of SalB on HTR-8/Svneo cells induced by H2O2 A: wound healing detected the cell migration ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×100; B: transwell detected the cell invasion ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×100; C: Western blot detected the expression of EMT-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs control; bP<0.001 vs H2O2; eP<0.05, dP<0.01, cP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
Figure 6 MMP-9 inhibitor GM6001 reversed the invasion, migration and EMT of SalB on HTR-8/Svneo cells induced by H2O2 A: wound healing detected the cell migration ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×100; B: transwell detected the cell invasion ability in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. magnification, ×100; C: Western blot detected the expression of EMT-related proteins in untreated cells or in cells exposed to 300?μmol/L of H2O2 for 3 h alone or in cells treated by 40 μM of SalB for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h or in cells co-treated by SalB (40 μM) and GM6001 for 24 h after being exposed to 300?μmol/L of H2O2 for 3 h. aP<0.001 vs control; bP<0.001 vs H2O2; eP<0.05, dP<0.01, cP<0.001 vs H2O2 + SalB. SalB: salvianolic acid B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
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