Dachaihu decoction inhibits hypernutrition-induced liver metastasis from colorectal cancer by maintaining the gut vascular barrier

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  Figure 1. A high-fat diet induces hyperlipidemia and liver metastasis in colorectal cancer, but Dachaihu decoction counteracts these effects. (A) Serum total cholesterol, triglycerides, and LDL-cholesterol in NC- and HFD-fed mice orthotopically implanted with MC-38 CRC cells in the cecum. (B) Top, macroscopic appearance of livers with metastasized CRC tumor nodules in NC- and HFD-fed mice. Dotted squares and White Arrows indicate metastatic tumors. Bottom, H&E staining of livers. Yellow arrows indicate tumor regions. Scale bar, 100 μm. (C) Number of metastasized liver nodules in mice. (D) Serum lipids in HFD-fed mice orally administrated vehicle control (Ctrl), DCHD at low (L-DCHD), medium (M-DCHD) or high (H-DCHD) dose or positive control OCA. (E) Top, macroscopic appearance of livers with metastasized CRC tumor nodules following OCA or DCHD treatment. White arrows indicate metastatic tumors. Bottom, H&E staining of livers. Yellow arrows indicate tumor regions. Scale bar, 100 μm. (F) Number of metastasized liver nodules in mice following OCA or DCHD treatment. Mean ± SD (n = 5); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.CRC: Colorectal cancer; DCHD: Dachaihu decoction; H&E: Hematoxylin and…

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  Figure 2. Dachaihu decoction prevents gut vascular barrier leakage in high-fat diet-fed mice. (A) Serum fluorescence intensity of FITC-dextran 4 h after administration in HFD-fed orthotopic CRC-bearing mice. Mean ± SD (n = 5) ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (B) qRT-PCR analysis of relative mRNA expression of GVB permeability biomarkers in mouse colon, including TJ (Claudin, Occludin, and ZO-1) and vascular markers (Pv1 and Vegfa). (C) Immunostaining of ZO-1 (red) and CD34 (green), and DNA staining (blue) in frozen sections of the colon. Top, merged images with a dotted line showing where fluorescence intensity was quantified. Middle, ZO-1 staining. Bottom, relative fluorescence intensity profiles for each protein. (D) Quantification of MFI for ZO-1. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (E) Immunostaining of PV-1 (red) and CD34 (green), and DNA staining (blue) in colon sections. Scale bar, 50 μm. (F) Quantification of MFI for PV-1. Mean ± SD (n = 5); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.CRC: Colorectal cancer; FITC: fluorescein-isothiocyanate-labeled; GVB: Gut vascular barrier; HFD: High-fat diet; MFI: Mean fluorescence intensity; qRT-PCR: Quantitative real-time polymerase chain…

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  Figure 3. Dachaihu decoction modulates bile acid metabolism to alleviate high-fat diet-induced gut vascular barrier disruption. (A) qRT-PCR analysis of expression of genes involved in BA synthesis and transport in livers of HFD-fed orthotopic CRC-bearing mice. (B) qRT-PCR analysis of ileal expression of genes involved in BA absorption and secretion. (C) Fluorescence images of FITC-dextran (green) and DNA staining (blue). (D) Quantitation of FITC intensity in liver 1 h after FITC-dextran administration. Scale bar, 50 μm. (E) Serum fluorescence intensity of FITC-dextran in mice 1 h after intestinal loop instillation. (F) FITC-dextran permeability assay in HUVECs treated with 100 μmol/L primary (C-BAs, TCA, TCDCA, GCA, or GCDCA) or vehicle control. Permeability is given as a percentage FI in the lower well. (G) TEER in HUVECs grown with C-BAs; results from 3 independent experiments. Mean ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.BA: Bile acid; C-BAs: conjugated bile acids; CRC: Colorectal cancer; FI: Fluorescence intensity; FITC: Fluorescein-isothiocyanate-labeled; GCA: Glycocholic acid; GCDCA: Glycochenodeoxycholic acid; GVB: Gut vascular barrier; HFD: High-fat diet; HUVECs: Human umbilical…

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  Figure 4. Dachaihu decoction inhibits glycochenodeoxycholic acid-induced permeabilization in cultured human umbilical vein endothelial cells. (A) FITC-dextran permeability in HUVECs treated with GCDCA and DCHD singly or in combination. (B) TEER analysis in HUVECs; results from three independent experiments. (C) qRT-PCR analysis of expression of TJ and vascular markers in HUVECs. (D) Western blot analysis of TJ proteins. (E) Densitometry of TJ proteins and control β-actin. (F) Invasion of GFP-expressing HCT-116 CRC cells across the HUVEC monolayer. GFP (green) and DNA staining (blue). Scale bar, 250 μm. (G) Quantitation of migrated GFP-HCT-116 cells in the lower Transwell chamber; results from three independent experiments. Mean ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.CRC: Colorectal cancer; DCHD: Dachaihu decoction; FITC: Fluorescein-isothiocyanate-labeled; GCDCA: Glycochenodeoxycholic acid; GFP: Green fluorescent protein; HUVECs: Human umbilical vein endothelial cells; MFI: Mean fluorescence intensity; qRT-PCR: Quantitative real-time polymerase chain reaction; SD: Standard deviation; TEER: Transepithelial electrical resistance; TJ: Tight junction.

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  Figure 5. Dachaihu decoction prevents intracellular Ca2+ elevation and reactive oxygen species accumulation induced by glycochenodeoxycholic acid. (A) Live cell imaging of Fluo-4 AM (green) staining of intracellular Ca2+ in HUVECs after treatment with GCDCA alone or in combination with DCHD. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm. (B) Quantitation of Fluo-4 AM intensity. (C) Live cell staining of ROS using H2DCFDA. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm. (D) Quantitation of MFI for H2DCFDA. (E) qRT-PCR analysis of expression of oxidative stress response genes. Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.DCHD: Dachaihu decoction; GCDCA: Glycochenodeoxycholic acid; H2DCFDA: 2′,7′-dichlorodihydrofluorescein diacetate; HUVECs: Human umbilical vein endothelial cells; qRT-PCR: Quantitative real-time polymerase chain reaction; ROS: Reactive oxygen species; SD: Standard deviation.

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  Figure 6. Dachaihu decoction inhibits high-fat-diet-induced reactive oxygen species accumulation in the colon and ileum of high-fat-diet-fed mice. (A) DHE staining of ROS (red) in colon sections of HFD-fed orthotopic CRC-bearing mice and DAPI staining (blue), Scale bar, 50 μm. (B) Quantitation of DHE positive area (%). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (C) DHE staining (red) and DAPI staining (blue) of ileum sections. Scale bar, 50 μm. (D) Quantitation of DHE positive area (%). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (E) qRT-PCR analysis of expression of oxidative stress response genes in the ileum. Means ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. CRC: Colorectal cancer; DHE: Dihydroethidium; HFD: High-fat diet; ROS: Reactive oxygen species; qRT-PCR: Quantitative real-time polymerase chain reaction; SD: Standard deviation.

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  Supplementary Figure 1. Dachaihu Decoction (DCHD) decreases body weight gain and prolongs overall survival (OS) in high-fat diet (HFD)-fed mice following orthotopic implantation of colorectal cancer (CRC). (A) Weekly body weight gain in normal-chow (NC)- and HFD-fed mice orthotopically implanted with MC-38 CRC cells in the cecum. (B) Weekly body weight gain in HFD-fed mice treated with vehicle control (Ctrl), Dachaihu decoction (DCHD) at low (L-DCHD), medium (M-DCHD), or high (H-DCHD) dose or positive control obeticholic acid (OCA). (C) OS of HFD-mice. NC; Normal chow; HFD: High fat diet; Ctrl; HFD-feeding Control; DCHD, Dachaihu Decoction; L-DCHD: Low dose of DCHD; M-DCHD: Medium-dose of DCHD; H-DCHD: High-dose of DCHD; OCA: Obeticholic acid.

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  Supplementary Figure 2. DCHD improves intestinal morphology and restores the expression of tight junction (TJ) proteins. (A) Hematoxylin and eosin (H&E) staining of colon and ileum from HFD-fed mice. Scale bar, 100 μm. (B) Alcian Blue staining of colon and ileum of mice. Scale bar, 100 μm. (C) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of relative expression of TJ genes Claudin, Occludin, and ZO-1 and vascular markers Pv1 and Vegfa in the ileum. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ctrl; HFD-feeding Control; HFD: High fat diet; DCHD, Dachaihu Decoction; L-DCHD: Low dose of DCHD; M-DCHD: Medium-dose of DCHD; H-DCHD: High-dose of DCHD; OCA: Obeticholic acid.

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  Supplementary Figure 3. DCHD prevents gut vascular barrier (GVB) leakage in the ileum of HFD-fed mice. (A) Immunostaining of ZO-1 (red) and CD34 (green), and DNA staining (blue) in frozen sections of the ileum. Top, merged images with a dotted line showing where fluorescence intensity was quantified. Middle, ZO-1 staining. Bottom, relative fluorescence intensity profiles for each protein. (B) Quantification of mean fluorescence intensity (MFI) for ZO-1. (C) Staining of PV-1 (red) and CD34 (green), and DNA staining (blue) in frozen sections of the ileum. (D) Quantification of MFI for PV-1. (E) CD34 staining of the liver. (F) Quantification of CD-34+ LSEC area per field (%). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar 50 μm. Ctrl; HFD-feeding Control; DCHD, Dachaihu Decoction; L-DCHD: Low dose of DCHD; M-DCHD: Medium-dose of DCHD; H-DCHD: High-dose of DCHD; OCA: Obeticholic acid.; LSECs: Liver sinusoidal endothelial cells.

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  Supplementary Figure 4. DCHD rescues ZO-1 expression in the liver following inhibition by HFD feeding. Immunostaining ZO-1 (red) and DNA staining (blue) and quantification of ZO-1 in liver sections from NC- or HFD-fed (A and B) or HFD-fed CRC-implanted mice treated with DCHD or obeticholic acid (OCA) (C and D) as indicated. ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar, 50 μm. MFI: Mean fluorescence intensity; NC; Normal chow; HFD: High fat diet; Ctrl; HFD-feeding Control; DCHD, Dachaihu Decoction; L-DCHD: Low dose of DCHD; M-DCHD: Medium-dose of DCHD; H-DCHD: High-dose of DCHD; OCA: Obeticholic acid.

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  Supplementary Figure 5. DCHD does not affect invasion by MC-38 tumor cells but increases invasion by HCT-116 cells. Effects of DCHD on the migration of MC-38 (A and B) and HCT-116 (C and D) CRC cells in a wound-healing assay. ∗P < 0.05. Scale bar, 200 μm. DCHD: Dachaihu Decoction; L-DCHD: Low dose of DCHD; M-DCHD: Medium-dose of DCHD; H-DCHD: High-dose of DCHD.