Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (1): 42-50.DOI: 10.19852/j.cnki.jtcm.20221111.001
• Original articles • Previous Articles Next Articles
JIANG Wen1, ZHANG Wei2, ZHANG Yuxiang3, YANG Hao1, PAN Xiaomei1, CHEN Qiang1(), CHEN Junhui1()
Received:
2021-12-01
Accepted:
2022-02-27
Online:
2023-02-15
Published:
2023-01-10
Contact:
CHEN Qiang,CHEN Junhui
About author:
CHEN Junhui, Department of Pharmacy, the Sixth Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, China. wensmile1987@126.com. Telephone:+86-9912660042; +86-991-2662027Supported by:
JIANG Wen, ZHANG Wei, ZHANG Yuxiang, YANG Hao, PAN Xiaomei, CHEN Qiang, CHEN Junhui. Tilianin extracted from Xiangqinglan (Herba Dracocephali Moldovicae) inhibits apoptosis induced by mitochondrial pathway and endoplasmic reticulum stress in H9c2 cells after oxygen-glucose deprivation/reoxygenation[J]. Journal of Traditional Chinese Medicine, 2023, 43(1): 42-50.
Group | n | Survival rate (100%) | ||||
---|---|---|---|---|---|---|
3 h hypoxia/3 h reoxygenation | 6 h hypoxia/3 h reoxygenation | 12 h hypoxia/3hreoxygenation | ||||
Control | 5 | 100.0±13.0 | 100.0±8.4 | 100.0±2.7 | ||
OGD/R model | 5 | 92.6±4.0 | 84.1±7.4a | 42.4±3.0a |
Table 1 Effects of different hypoxia time on the survival of OGD/R-induced H9C2 cell model ($\bar{x}\pm s$)
Group | n | Survival rate (100%) | ||||
---|---|---|---|---|---|---|
3 h hypoxia/3 h reoxygenation | 6 h hypoxia/3 h reoxygenation | 12 h hypoxia/3hreoxygenation | ||||
Control | 5 | 100.0±13.0 | 100.0±8.4 | 100.0±2.7 | ||
OGD/R model | 5 | 92.6±4.0 | 84.1±7.4a | 42.4±3.0a |
Group | n | LDH activity (U/L) | ||
---|---|---|---|---|
3 h hypoxia/3 h reoxygenation | 6h hypoxia/3 h reoxygenation | 12 h hypoxia/3 hreoxygenation | ||
Control | 3 | 116±12 | 134±3 | 158±5 |
OGD/R model | 3 | 129±5 | 185±7a | 227±15a |
Table 2 Effects of different hypoxia time on LDH activity of OGD/R-induced H9C2 cell model ($\bar{x}\pm s$)
Group | n | LDH activity (U/L) | ||
---|---|---|---|---|
3 h hypoxia/3 h reoxygenation | 6h hypoxia/3 h reoxygenation | 12 h hypoxia/3 hreoxygenation | ||
Control | 3 | 116±12 | 134±3 | 158±5 |
OGD/R model | 3 | 129±5 | 185±7a | 227±15a |
Figure 1 Effect of tilianin on cell morphology and cellular apoptosis A: the cells were intervened with different concentrations of tilianin as indicated. Cell morphology was observed under an inverted microscope (×100). A1: control; A2: model; A3: 1 μM tilianin; A4: 3 μM tilianin; A5: 10 μM tilianin; A6: 20 μM tilianin; A7: 30 μM tilianin. B: the apoptosis rats of the cells of the control, model, and 1 μM tilianin intervention groups, respectively, were detected with flow cytometry. B1-B3: representative flow cytometry results were shown. B1: control group; B2: model group; B3: 1 μM tilianin group; B4: statistical analysis of the apoptosis rates were shown. The H9c2 rat cardiomyocytes were divided into control group: cells were cultured normally without any drug treatment; model group: cells were cultured for 6 h under normal conditions, and cultured for 6 h in serum free and glucose free medium under anoxic conditions; model+tilianin group: cells were cultured under normal culture conditions, pre intervened with tilianin (1, 3, 10, 20 and 30 μM) for 6 h, then cultured with serum free and glucose free hypoxia medium for 6 h (maintaining drug concentration), and then cultured with normal reoxygenation for 3 h (maintaining drug concentration). aP < 0.05, compared with control group; bP < 0.05, compared with model group.
Group | n | Survival rate (%) | LDH activity (U/L) |
---|---|---|---|
Control | 5 | 100±6 | 75±11 |
OGD/R model | 5 | 80±10a | 136±14a |
1 μM tilianin | 5 | 95±12b | 86±10b |
3 μM tilianin | 5 | 96±9b | 106±7abc |
10 μM tilianin | 5 | 83±5ac | 117±5abc |
20 μM tilianin | 5 | 82±6ac | 123±4ac |
30 μM tilianin | 5 | 74±5ac | 143±9ac |
Table 3 Effect of tilianin on the survival and LDH activity in OGD/R-induced H9C2 cell model ($\bar{x}\pm s$)
Group | n | Survival rate (%) | LDH activity (U/L) |
---|---|---|---|
Control | 5 | 100±6 | 75±11 |
OGD/R model | 5 | 80±10a | 136±14a |
1 μM tilianin | 5 | 95±12b | 86±10b |
3 μM tilianin | 5 | 96±9b | 106±7abc |
10 μM tilianin | 5 | 83±5ac | 117±5abc |
20 μM tilianin | 5 | 82±6ac | 123±4ac |
30 μM tilianin | 5 | 74±5ac | 143±9ac |
Figure 2 Effect of tilianin on mitochondrial functions The effects of tilianin on the mitochondrial membrane potential Rh123 fluorescence intensity (A), Mitosox ROS fluorescence intensity (B), and mitochondrial calcium ion FLuo-3 fluorescence intensity (C) were detected with flow cytometry. A1, B1, C1: control group; A2, B2, C2: model group; A3, B3, C3: 1 μM tilianin group; A4, B4, C4: statistical analysis were shown. The H9c2 rat cardiomyocytes were divided into control group: cells were cultured normally without any drug treatment; model group: cells were cultured for 6 h under normal conditions, and cultured for 6 h in serum free and glucose free medium under anoxic conditions; model + tilianin group: cells were cultured under normal culture conditions, pre intervened with tilianin (1, 3, 10, 20 and 30 μM) for 6 h, then cultured with serum free and glucose free hypoxia medium for 6 h (maintaining drug concentration), and then cultured with normal reoxygenation for 3 h (maintaining drug concentration). ROS: reactive oxygen species. aP < 0.05, compared with control group; bP < 0.05, compared with model group.
Figure 3 Effects of tilianin on expression levels ox-CaMKII, p-CaMKII, and VDAC A1: representative Western blot results of the protein expression levels were shown. A2: quantitative Western blot result of the protein expression levels were shown. The H9c2 rat cardiomyocytes were divided into control group: cells were cultured normally without any drug treatment; model group: cells were cultured for 6 h under normal conditions, and cultured for 6 h in serum free and glucose free medium under anoxic conditions; model + tilianin group: cells were cultured under normal culture conditions, pre intervened with tilianin (1, 3, 10, 20 and 30 μM) for 6 h, then cultured with serum free and glucose free hypoxia medium for 6 h (maintaining drug concentration), and then cultured with normal reoxygenation for 3 h (maintaining drug concentration). ox-CaMKII: oxidized Calmodulin-dependent protein kinase II; p-CaMKII: p-Calmodulin-dependent protein kinase II; VDAC: voltage-dependent anion selective channel. aP < 0.05, compared with control group; bP < 0.05, compared with model group.
Group | n | SDH activity (milliunit/μL) | Succinate contents (ng/μL) |
---|---|---|---|
Control | 3 | 0.0067±0.0021 | 0.9743±0.4029 |
OGD/R model | 3 | 0.0113±0.0012a | 2.0843±0.3303a |
1 μM tilianin | 3 | 0.0080±0.0017b | 1.3787±0.2779b |
Table 4 Analysis of mitochondrial SDH activity and succinate contents
Group | n | SDH activity (milliunit/μL) | Succinate contents (ng/μL) |
---|---|---|---|
Control | 3 | 0.0067±0.0021 | 0.9743±0.4029 |
OGD/R model | 3 | 0.0113±0.0012a | 2.0843±0.3303a |
1 μM tilianin | 3 | 0.0080±0.0017b | 1.3787±0.2779b |
Figure 4 Analysis of expression levels of michondrial pathway-related proteins and endoplasmic reticulum stress-related proteins as well as cytokine levels The expression levels of michondrial pathway-related proteins (A) and endoplasmic reticulum stress-related proteins (B) were detected. C: the levels of TNF-α, IL-1β and IL-6 in the culture supernatant were detected with ELISA. A1, B1: representative Western blot results of the protein expression levels were shown. A2, B2: quantitative Western blot result of the protein expression levels were shown. The H9c2 rat cardiomyocytes were divided into control group: cells were cultured normally without any drug treatment; model group: cells were cultured for 6 h under normal conditions, and cultured for 6 h in serum free and glucose free medium under anoxic conditions; model + tilianin group: cells were cultured under normal culture conditions, pre intervened with tilianin (1, 3, 10, 20 and 30 μM) for 6 h, then cultured with serum free and glucose free hypoxia medium for 6 h (maintaining drug concentration), and then cultured with normal reoxygenation for 3 h (maintaining drug concentration). AKT: protein kinase B; p-AKT: phosphorylated protein kinase B; Bax: B-cell lymphoma-2-associated X; Bcl-2: B-cell lymphoma-2; DRP1: dynamin-related protein 1; MFN2: mitochondrial protein 2; ASK1: apoptosis signal-regulated kinase 1; p-ASK1: phosphorylated-apoptosis signal-regulated kinase 1; p38: mitogen-activated protein kinases; p-p38: phosphorylated-mitogen-activated protein kinases; CHOP: C/EBP homologous protein; TNF- α: tumor necrosis factor- α; IL-1β: interleukin-1β; IL-6: interleukin-6; ELISA: enzyme-linked immunosorbent assay. aP < 0.05, compared with control group; bP < 0.05, compared with model group. cP<0.01, eP < 0.001, compared with control group; dP < 0.01, compared with model group.
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