Dihydroergotamine ameliorates liver fibrosis by targeting transforming growth factor β type II receptor

Figure 1 Screening Food and Drug Administration-approved drugs for binding to transforming growth factor β type II receptor. A: The combination of transforming growth factor β type II receptor (TGFβR2) and transforming growth factor β 1 (TGFβ1); B: The structure of TGFβR2; C: The affinity of each Food and Drug Administration-approved drug and TGFβR2. The affinity of TGFβR2 and darifenacin, cyproheptadine, lifitegrast, difenoxin, phenytoin, dihydroergotamine (DHE), naldemedine, and irinotecan was -7.6, -7.4, -7.4, -7.3, -7.3, -7.3, -7.3, and -7.3 kcal/mol, respectively; D: Cell viability of LX-2 treated with 20 μM of the drugs for 24 h (n = 5); E-H: Cell viability following LX-2 treatment with different concentrations of darifenacin, DHE, lifitegrast, and phenytoin for 24 h (n = 6). All data are presented as mean ± standard error of the mean. One-way ANOVA test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. TGFβ: Transforming growth factor β; TGFβR: Transforming growth factor β receptor; DHE: Dihydroergotamine.

Figure 2 Treatment inhibiting the activation of LX-2 cells. A and B: After LX-2 cell was activated with transforming growth factor β 1 (TGFβ1) (5 ng/mL) for 24 h, different concentrations of lifitegrast and phenytoin were added for 24 h, and the protein levels of collagen III and α-SMA were detected by western blot; C and D: After LX-2 was activated with TGFβ1 (5 ng/mL) for 24 h, different concentrations of darifenacin and dihydroergotamine (DHE) were added for further treatment for 24 h, and the protein levels of collagen III, α-SMA, and p-SMAD3 were detected by western blot; E: Real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of COL1A1, COL1A2, COL3A1, and α-SMA of LX-2 after different concentrations of darifenacin treatment (n = 6); F: RT-PCR was performed to detect the expression of COL1A1, COL1A2, COL3A1, and α-SMA of LX-2 after different concentrations of DHE treatment (n = 5-6). All data are presented as means ± standard error of the mean. One-way ANOVA test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. TGFβ: Transforming growth factor β; TGFβR: Transforming growth factor β receptor; DHE: Dihydroergotamine.

Figure 3 The binding affinity and sites of dihydroergotamine and transforming growth factor β type II receptor. A: Transforming growth factor β type II receptor (TGFβR2) bound to dihydroergotamine (DHE) with a Kd of 17.64 μM; B: Molecular dynamics simulation results of DHE and TGFβR2. Root-mean-square deviation of TGFβR2 skeleton atom; C: Root-mean-square fluctuation of backbone Cα atoms of TGFβR2 skeleton atom; D: The secondary structure components of TGFβR2 and complex simulation systems in 0-200 ns; E and F: The binding sites of DHE and TGFβR2; G: The binding affinity of TGFβR2 mutants with DHE. TGFβ: Transforming growth factor β; TGFβR: Transforming growth factor β receptor; DHE: Dihydroergotamine.

Figure 4 Dihydroergotamine alleviated fibrosis in CCl₄-induced liver fibrosis model mice. A: The width of the portal vein (left panel); portal vein width of mice in each group (right panel, n = 7); B: The velocity of the portal vein (left panel); the velocity of mice in each group (right panel, n = 7); C: Body weight, liver weight, spleen weight, and liver weight/body weight of mice in each group (n = 6-8); D: Gross liver specimens of the mice in each group; E: An automated biochemistry analyzer determined the enzymatic activities of serum levels of alanine aminotransferase and aspartate aminotransferase (n = 6-7); F: Masson’s trichrome staining was performed on liver sections; G: Collagen area in Masson’s trichrome staining (n = 7-8). All data were presented as means ± standard error of the mean. One-way ANOVA test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. TGFβ: Transforming growth factor β; TGFβR: Transforming growth factor β receptor; DHE: Dihydroergotamine.

Figure 5 Dihydroergotamine decreased the inflammatory infiltration of macrophages and extracellular matrix deposition in the liver. A and B: The flow cytometric analysis of CD11b⁺ cells (n = 6-8); C: The effects of dihydroergotamine (DHE) treatment on the mRNA expression levels of inflammation-related genes (n = 6-8); D: The effects of DHE treatment on the mRNA expression levels of extracellular matrix-related genes (n = 6-8); E: The protein levels of p-SMAD3 and α-SMA in liver tissues of mice in each group were detected by western blot (n = 3). All data are presented as means ± standard error of the mean. One-way ANOVA test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. TGFβ: Transforming growth factor β; TGFβR: Transforming growth factor β receptor; DHE: Dihydroergotamine.