Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: CVB-D exerts a protective effect on LPS-treated H9C2 cells by inhibiting ferroptosis. (A) CCK-8 assay was used to determine the dose of CVB-D and LPS for further study. (B) Effect of CVB-D on LPS-treated H9C2 cell viability. (C) Effect of CVB-D on CK-MB levels, LDH activity and cTnI levels of LPS-treated H9C2 cells. (D) Effect of MDA generation, GSH levels and SOD activity in LPS-treated H9C2 cells. (E) Effect of CVB-D on the expression of ptgs2 mRNA levels (n=3). (F) Representative fluorescent microscopy images of ROS production by H9C2 cells (n=3). (G) SLC7A11 and GPX4 protein expression levels of LPS- and CVB-D-treated H9C2 cells. Western blot images were taken from different gels but a single replicate. * P<0.05 vs. control group, ** P<0.01 vs. control group, ## P<0.01 vs. LPS-treated group. Data are presented as the mean ± SD (n=6). CVB-D, cyclovirobuxine D; LPS, lipopolysaccharide; CK-MB, creatine kinase isoenzyme; LDH, lactate dehydrogenase; cTnI, cardiac troponin I; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde; ptgs2, prostaglandin-endoperoxide synthase 2; ROS, reactive oxygen species; DCFH-DA, 2',7'-dichlorofluorescein diacetate; SLC7A11, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4; CCK-8, Cell Counting Kit-8.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: CCK-8 Assay, Activity Assay, Expressing, Microscopy, Western Blot, Cell Counting

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: CVB-D inhibits cellular iron-overload induced by LPS. (A) Calcein-AM imaging of intracellular iron levels in H9C2 cells. (B) Western blot images and protein expression levels of DMT1, TfR1, FPN1 and FtH in LPS- and CVB-D-treated H9C2 cells. (C) hamp mRNA expression levels in LPS- and CVB-D-treated H9C2 cells. (D) Protein expression levels of pro-IL-1β, TNF-α, IL-6, p-STAT3 and STAT3 in LPS- and CVB-D-treated H9C2 cells. Data are presented as the mean ± SD (n=6). ** P<0.01 vs. control group, # P<0.05, ## P<0.01 vs. LPS-treated group. Western blot images were from different gels, but the same replicate. CVB-D, cyclovirobuxine D; LPS, lipopolysaccharide; hamp, hepcidin; DMT1, divalent metal transporter 1; TfR1, transferrin receptor 1; FPN 1, ferroportin1; IL, interleukin; TNF-α, TNF tumor necrosis factor-α; STAT3, signal transducer and activator of transcription 3; p, phosphorylated.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Imaging, Western Blot, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: CVB-D protects against LPS-induced ferroptosis in H9C2 cells by upregulating Nrf2 expression. (A) Western blot images of protein levels of H9C2 cells treated with LPS and CVB-D. (B) Nrf2 protein expression levels in H9C2 cells treated with LPS and CVB-D. (C) SLC7A11 protein expression levels in H9C2 cells treated with LPS and CVB-D. (D) GPX4 protein expression levels in H9C2 cells treated with LPS and CVB-D. ** P<0.01 vs. control group, ## P<0.01 vs. LPS-treated group, $$ P<0.01 vs. LPS + CVB-D-treated group. Data are presented as the mean ± SD (n=6). Western blot images are from different gels but the same replicate. CVB-D, cyclovirobuxine D; LPS, lipopolysaccharide; SLC7A11, solute carrier family 7 member 11; Nrf2, nuclear factor erythroid 2-related factor 2; GPX4, glutathione peroxidase 4.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: Protective effect of CVB-D on cardiac injury after CLP. (A) Representative histopathological images indicating myocardial tissue damage. Arrows indicate swollen cardiomyocytes and inflammatory infiltrating cells. (B) Serum levels of CK, LDH and cTnI in rats treated with CLP and CVB-D. (C) Representative echocardiographic images of rats treated with CLP and CVB-D. (D) Left ventricular ejection fraction, shortening fraction, left ventricular end-systolic volume and left ventricular end-diastolic volume of rats treated with CLP and CVB-D. Data are presented as the mean ± SD (n=6). ** P<0.01 vs. control group, ## P<0.01 vs. CLP group. CVB-D, cyclovirobuxine D; CLP, cecal ligation and puncture; CK-MB, creatine kinase isoenzyme; LDH, lactate dehydrogenase; cTnI, cardiac troponin I; LVESV, left ventricular end-systolic volume; LVDEV, left ventricular end-diastolic volume.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Ligation

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: CVB-D reduces myocardial ferroptosis induced by CLP surgery in rats. (A) Representative TEM images showing myocardial mitochondria ultrastructure. (B) mRNA expression levels of ptgs2 in hearts of rats treated with CLP and CVB-D. (C) MDA levels in hearts of rats treated with CLP and CVB-D. (D) GSH levels in hearts of rats treated with CLP and CVB-D. (E) Levels of cardiac non-heme iron content in hearts of rats treated with CLP and CVB-D. (F) mRNA expression levels of hamp in livers of rats treated with CLP and CVB-D. (G) Representative echocardiographic images, and left ventricular ejection fraction and shortening fraction of CLP- and Ferr-1-treated rats. Data are presented as the mean ± SD (n=6). ** P<0.01 vs. control group, # P<0.05, ## P<0.01 vs. CLP-treated group. CVB-D, cyclovirobuxine D; CLP, cecal ligation and puncture; PTGS2, prostaglandin-endoperoxide synthase 2; hamp , hepcidin; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Expressing, Ligation

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: CVB-D attenuates calcium overload caused by LPS. (A) Fluorescence microscopy image of different treatment groups of H9C2 cells: CON, LPS, LPS + CVB-D and CVB-D. (B) Exemplary trace and (C) summary data of ICa-L recordings of VER. (D) Typical traces of normal cells. (E) Time course of I Ca-L in normal cells. (F) Pooled data recorded normal cells under CON, 10 µM CVB-D and washout conditions. (G) Typical traces of the CLP group cells. (H) Time course of I Ca-L in the CLP group cells. (I) Pooled data recorded the CLP group cells under CON, 10 µM CVB-D and washout conditions. (J) Exemplary traces and (K) time course of I Ca-L exposure to 0.1, 0.3, 1, 3 and 10 µM CVB-D and 1 µM VER were recorded. (L) Dose-response curves show that CVB inhibited I Ca-L in ventricular myocytes. ** P<0.01 vs. control group. Data are presented as the mean ± SD (n=6). CON, control; CVB-D, cyclovirobuxine D; LPS, lipopolysaccharide; VER, verapamil; ICa-L, L-type Ca 2+ current.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Fluorescence, Microscopy

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: Impact of CVB-D on the current-voltage relationship of I Ca-L . The test voltage is on the X-axis and the current density is on the Y-axis (pA/pF). (A) Representative traces and (B) pooled data demonstrate the impact of CVB-D at different concentrations on the I-V relationship. Effects of CVB-D on (C) steady-state activation and (D) inactivation of L-type Ca 2+ currents. (E) Recordings of the process of cardiomyocytes shortening. (F) A single typical trace recorded in CON, 10 µM CVB-D and without CVB-D. (G) Summarized data were counted under CON, 1 µM CVB-D, 3 µM CVB-D and 10 µM CVB-D. Data are expressed as mean ± SD (n=6). ## P<0.01 vs. control group CVB-D, cyclovirobuxine D; I-V, current-voltage; CON, control; VER, verapamil; I Ca-L , L-type Ca 2+ current.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: Activation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis

doi: 10.3892/etm.2023.12106

Figure Lengend Snippet: Proposed signaling pathways involved in the protective effect of CVB-D in septic cardiomyopathy. Cellular injury, ferroptosis and oxidative stress induced by LPS are attenuated by CVB-D. CVB-D downregulated cellular iron accumulation by alleviating cellular inflammatory responses. Furthermore, CVB-D also upregulated the Nrf2/SLC7A11/GPX4 pathway. CVB-D may reduce the influx of calcium ions by inhibiting LTCCs, thus reducing myocardial injury. CVB-D, cyclovirobuxine D; LPS, lipopolysaccharide; LTCCs, L-type calcium channels; SLC7A11, solute carrier family 7 member 11; Nrf2, nuclear factor erythroid 2-related factor 2; GPX4, glutathione peroxidase 4; ROS, reactive oxygen species; p, phosphorylated; IL, interleukin; R, receptor; SLC3A2, solute carrier family 3 member 2; STAT3, signal transducer and activator of transcription 3; FPN1, ferroportin 1; TfR1, transferrin receptor 1; DMT1, divalent metal transporter 1; GSH, glutathione; GSSH, glutathione disulfide, SOD, superoxide dismutase.

Article Snippet: A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol.

Techniques: