Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (4): 661-666.DOI: 10.19852/j.cnki.jtcm.2023.04.003
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Synergistic effect of schizandrin A and DNase I knockdown on high glucose induced beta cell apoptosis by decreasing intracellular calcium concentration
ZHU Bin1(
), YU Ning2, WANG Lei3, TIAN Yue1, WU Mingfen1, ZHAO Zhigang1()
- 1 Department of Pharmacy, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China
2 Accreditation Center of Traditional Chinese Medicine Physician of National Administration of Traditional Chinese Medicine, Beijing 100120, China
3 Department of Endocrinology, the Third Affiliated Hospital of Beijing University of Chinese Medicine, Beijing 100050, China
-
Received:2022-03-16Accepted:2022-07-08Online:2023-08-15Published:2023-07-03 -
Contact:ZHAO Zhigang, Department of Pharmacy, Beijing Tiantan Hospital Affiliated to Capital Medical University, Beijing 100070, China. 1022zzg@sina.com. Telephone: +86-10-59975442; +86-010-59976856
ZHU Bin, Department of Pharmacy, Beijing Tiantan Hospital Affiliated to Capital Medical University, Beijing 100070, China. zbtcm@163.com -
Supported by:Natural Science Foundation of China: the Protective Effect of Schizandrin A to β Cell Apoptosis by DNase I in Type 2 Diabetes(81803909)
Cite this article
ZHU Bin, YU Ning, WANG Lei, TIAN Yue, WU Mingfen, ZHAO Zhigang. Synergistic effect of schizandrin A and DNase I knockdown on high glucose induced beta cell apoptosis by decreasing intracellular calcium concentration[J]. Journal of Traditional Chinese Medicine, 2023, 43(4): 661-666.
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Figure 1 Analysis of apoptosis-associated protein level by Western blot A: immunoblot assay for determining apoptosis protein. B-F: quantitative analysis of immunoblotting results of DNase I, BAX, Bcl-2, Caspase-3, and CaBP4 in each group. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48 h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. DNase I: deoxyribonuclease I; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma-2; CaBP4: calcium binding protein 4; Sch A: schizandrin A; HG: high glucose; NC: negative control. Data are expressed as the mean ± standard deviation (n = 3). aP < 0.05, dP < 0.001, eP < 0.01, compared with control group; bP < 0.05, cP < 0.01, compared with HG group.
Figure 1 Analysis of apoptosis-associated protein level by Western blot A: immunoblot assay for determining apoptosis protein. B-F: quantitative analysis of immunoblotting results of DNase I, BAX, Bcl-2, Caspase-3, and CaBP4 in each group. Densitometry was used to compare the expression levels. β-actin was used as an internal loading control. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48 h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. DNase I: deoxyribonuclease I; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma-2; CaBP4: calcium binding protein 4; Sch A: schizandrin A; HG: high glucose; NC: negative control. Data are expressed as the mean ± standard deviation (n = 3). aP < 0.05, dP < 0.001, eP < 0.01, compared with control group; bP < 0.05, cP < 0.01, compared with HG group.
Figure 2 Cell apoptosis rate analyzed by flow cytometry analysis A: the control group; B the HG group; C: the Sch A + HG group; D: the NC + HG group; E: the si-DNase I + HG group; F: the si-DNase I + HG + Sch A group. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48 h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I+HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control.
Figure 2 Cell apoptosis rate analyzed by flow cytometry analysis A: the control group; B the HG group; C: the Sch A + HG group; D: the NC + HG group; E: the si-DNase I + HG group; F: the si-DNase I + HG + Sch A group. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48 h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I+HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control.
Figure 3 Level of cell membrane potential, Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity A: the cell membrane potential; B: the Na+-K+-ATPase activity; C: the Ca2+-Mg2+-ATPase activity. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. ATPase: adenosine triphosphatease; DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control. Data are expressed as the mean ± standard deviation (n = 3). aP < 0.001, compared with control group; bP < 0.01, cP < 0.001, compared with HG group.
Figure 3 Level of cell membrane potential, Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity A: the cell membrane potential; B: the Na+-K+-ATPase activity; C: the Ca2+-Mg2+-ATPase activity. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. ATPase: adenosine triphosphatease; DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control. Data are expressed as the mean ± standard deviation (n = 3). aP < 0.001, compared with control group; bP < 0.01, cP < 0.001, compared with HG group.
Figure 4 Intracellular Ca2+ concentration detection A: the control group; B the HG group; C: the Sch A + HG group; D: the negative control + HG group; E: the si-DNase I + HG group; F: the si-DNase I + HG + Sch A group. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. The Ca2+ concentration was evaluated by the average fluorescence intensity. DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control.
Figure 4 Intracellular Ca2+ concentration detection A: the control group; B the HG group; C: the Sch A + HG group; D: the negative control + HG group; E: the si-DNase I + HG group; F: the si-DNase I + HG + Sch A group. HG: treated with 30 mmol/L high glucose; SchA + HG: treated with 9 μmol/L Sch A and 30 mmol/L high glucose for 48h; NC: treated with negative control siRNA and 30 mmol/L high glucose; si-DNase I + HG: treated with DNase I siRNA and 30 mmol/L high glucose; si-DNase I + HG + SchA: co-treated with 9 μmol/L Sch A and DNase I siRNA under 30 mmol/L high glucose condition for 48 h; Control group: treated with equivalent volume of normal saline. The Ca2+ concentration was evaluated by the average fluorescence intensity. DNase I: deoxyribonuclease I; Sch A: schizandrin A; HG: high glucose; NC: negative control.
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