Journal: Journal of Pharmaceutical Analysis

Article Title: Tanshinone IIA ameliorates energy metabolism dysfunction of pulmonary fibrosis using 13 C metabolic flux analysis

doi: 10.1016/j.jpha.2023.09.008

Figure Lengend Snippet: Tanshinone IIA (Tan IIA) inhibits the expression of key enzymes and reduces collagen synthesis. (A) Tan IIA inhibits the expression of hexokinase 2 (HK2) by regulating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor 1α (HIF-1α) pathway. (B) Schematic representation of the possible fate of 2-carbon citrate derived from [U- 13 C] glucose. (C) Fractional m+2 enrichment of tricarboxylic acid (TCA)-cycle intermediates in NIH-3T3 cells induced by transforming growth factor-β1 (TGF-β1) cultured with [U- 13 C] glucose for 24 h. (D) Fractional enrichment of glucose-derived malate M+2 relative to citrate M+2 (mal+2/cit+2) in NIH-3T3 cells induced by TGF-β1 after incubation with Tan IIA (10 μM) or SB204990 (15 μM) for 24 h. (E) ATP-citrate lyase inhibitors dose-dependently inhibit α-smooth muscle actin (α-SMA) and collagen synthesis. (F) SLC25A1 inhibition reduced citrate cytoplasmic transport, and reduced α-SMA and collagen synthesis. (G) Blocking mitochondrial citric acid efflux and decomposition can reduce α-SMA and collagen synthesis. BTA (2 mM) and SB204990 (15 μM) can effectively inhibit α-SMA and collagen synthesis. (H) Tan IIA inhibited the expression of p -Akt and ACLY in a concentration dependent manner. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The membranes were blocked with 5% ( m/ V ) skim milk and incubated with the following primary antibodies: rabbit anti-collagen type I (14695-1-AP, Proteintech, Chicago, IL, USA), rabbit anti-collagen type III (22734-1-AP, Proteintech), rabbit anti-α-smooth muscle actin (#19245, Cell Signaling Technology, Boston, MA, USA), rabbit anti-phospho-Akt (Ser473) (66444-1, Proteintech), rabbit anti-Akt (10176-2-AP, Proteintech), mouse anti-HIF-1α (66730-1-lg, Proteintech), rabbit anti-PI3K (WL02240, Wanleibio, Shenyang, China), rabbit anti-mTOR (WL02477, Wanleibio), rabbit anti-phosphor-mTOR (WL03694, Wanleibio), rabbit anti-hexokinase 2 (WL02454, Wanleibio), rabbit anti-ACLY (R23558, Zenbio, Chengdu, China), rabbit anti-β-actin (81115-1-RR, Proteintech), and rabbit anti-GAPDH (10494-1-AP, Proteintech).

Techniques: Expressing, Derivative Assay, Cell Culture, Incubation, Inhibition, Blocking Assay, Concentration Assay

Journal: Journal of Pharmaceutical Analysis

Article Title: Tanshinone IIA ameliorates energy metabolism dysfunction of pulmonary fibrosis using 13 C metabolic flux analysis

doi: 10.1016/j.jpha.2023.09.008

Figure Lengend Snippet: Tanshinone IIA (Tan IIA) alleviates pulmonary fibrosis (PF) in bleomycin (BLM)-induced mouse model. (A) Schematic workflow for BLM-induced PF mouse model. After intratracheal instillation of BLM, C57BL/6 mice were given 0.5% CMC-Na, Tan IIA (10 mg/kg, 20 mg/kg) by gavage the next day for 21 days. (B) Histopathological changes of lung tissue of mice in different groups. (C) Western blot analysis of the expression levels of alpha-smooth muscle actin (α-SMA), collagen I (Col-I), and collagen III (Col-III) in mouse lungs of different groups. Quantitative results are presented as mean ± standard deviation (SD) of the index's relative protein levels. (D) Western blot analysis of the proteins expression levels of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor 1α (HIF-1α)/hexokinase 2 (HK2) pathway in mouse lungs of different groups. (E) Western blot analysis of the proteins expression levels of ACLY in mouse lungs of different groups. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The membranes were blocked with 5% ( m/ V ) skim milk and incubated with the following primary antibodies: rabbit anti-collagen type I (14695-1-AP, Proteintech, Chicago, IL, USA), rabbit anti-collagen type III (22734-1-AP, Proteintech), rabbit anti-α-smooth muscle actin (#19245, Cell Signaling Technology, Boston, MA, USA), rabbit anti-phospho-Akt (Ser473) (66444-1, Proteintech), rabbit anti-Akt (10176-2-AP, Proteintech), mouse anti-HIF-1α (66730-1-lg, Proteintech), rabbit anti-PI3K (WL02240, Wanleibio, Shenyang, China), rabbit anti-mTOR (WL02477, Wanleibio), rabbit anti-phosphor-mTOR (WL03694, Wanleibio), rabbit anti-hexokinase 2 (WL02454, Wanleibio), rabbit anti-ACLY (R23558, Zenbio, Chengdu, China), rabbit anti-β-actin (81115-1-RR, Proteintech), and rabbit anti-GAPDH (10494-1-AP, Proteintech).

Techniques: Western Blot, Expressing, Standard Deviation