An old herbal medicine with a potentially new therapeutic application in inflammatory bowel disease

Abstract

Inflammatory Bowel Disease (IBD) is a chronic and frequently disabling inflammatory disorder of the intestine. New developments in IBD therapy are primarily focused on biologic treatments; however, they are both expensive and associated with significant side effects. Here, we provide the first preclinical evidence that YunNan BaiYao (YNBY), a well-known traditional Chinese herbal remedy frequently used for treating hemorrhages and wounds, can effectively alleviate experimental colitis. Oral administration of YNBY in drinking water significantly reduced the disease activities of both DSS- and TNBS-induced experimental colitis. Mechanistic studies revealed that the effectiveness of YNBY was not due to an anti-bacterial function since YNBY had no effect on E. coli growth. Rather, it exhibited an anti-inflammatory or immunosuppressive function: In the DSS colitis model, YNBY treatment decreased the levels of several pro-inflammatory cytokines in colonic mucosa, including TNFα, IL-12p40, and IL-17. Similar cytokine changes were also observed in mouse serum, suggesting that systemic changes in general reflect the changes in the affected colon. Significant down-regulation of IL-12p40 and IL-17, in addition to IFNγ, was also seen in TNBS-colitis model. Another potential mechanism for the anti-inflammatory effects of YNBY involves the selective suppression of pro-inflammatory immune cells: YNBY effectively suppressed the growth of multiple T- and B-lymphocytes, including Molt-4, Jurkat, and EBV-transformed human B-lymphocytes, more potently than 6-mecaptopurine (6-MP) and 5-aminosalicylic acid (5-ASA), two of the most commonly used first-line drugs in IBD therapy. In sharp contrast, YNBY exhibited no cytotoxicity to colonic epithelial cells (Caco-2 cells), even at the concentration 10-fold higher than that used in the lymphocyte model; and instead promoted cell spreading and wound healing. These results strongly suggest that YNBY not only has effective anti-inflammatory properties through suppressing lymphocyte growth and pro-inflammatory cytokine expression, but also can promote intestinal epithelial wound-healing and repair. Therefore, YNBY demonstrates strong potential as an alternative herbal therapy for IBD.

Keywords: Crohn's disease; DSS-colitis; IBD; TNBS-colitis; YunNan BaoYao (YNBY); complementary and alternative medicine (CAM); cytokines; herbal medicine; immunesuppression; wound healing.

Figure 1

YNBY greatly improves the disease activity of DSS- induced UC-like colitis. A. A schematic illustration of experimental design: C57BL/6 mice (6-8 weeks old) were given drinking water containing either DSS alone or DSS and YNBY (1.5 mg/ml) at day 0. Clinical activity was scored daily. Mice were sacrificed at day 8 for tissue collection. Mice that received neither DSS nor YNBY were used as negative controls. B. A representative view of the colon morphology at the end of the YNBY treatment. Without YNBY, DSS induced severe colitis with bloody stools (or no stool due to diarrhea) and colon-shortening. Both symptoms were drastically improved by YNBY treatment, and the colon exhibited some well-formed stool pellets and no visible blood. C. Disease activity score of controls (green), DSS alone (red), and DSS +YNBY (blue) mice. At least 10 mice were used in each group.

Figure 2

YNBY effectively suppress TNBS-induced colitis. A. A schematic diagram to illustrate the timing of each treatment. BALB/c mice (6-8 weeks old) were induced for colitis using TNBS (0.5 mg in 0.1 ml 50% ethanol). Control (Ctrl) mice received 50% ethanol. Herbal formula YNBY (1.5 mg/ml) was given to the treatment group in drinking water 1 day following TNBS treatment. Disease activities were scored on day 8, immediately prior to sacrifice. B. A representative view of the colon morphology at the end of the YNBY treatment. TNBS induced severe colitis with bloody stools, and muco-sal thickening and shortening, all of which were markedly improved by YNBY treatment. No blood was visible in the colon. C. Disease activity assessment at day 8. Statistical analysis was done using Origin software (*P< 0.01). At least 12 mice were analyzed in each group.

Figure 3

YNBY does not have anti-bacterial capabilities against E. coli K12. 50 μL of E. coli K12 culture (OD=0.6) was incubated on plates with various concentrations of YNBY (B-F) overnight, at 37°C on LB agar plates. E. coli was also incubated with 50 μg/ml of antibiotic ampicillin (Amp) as a positive control (A), in which no bacteria grew (indicated by translucence). In contrast, bacterial lawns formed at all doses of YNBY (B-F). The hand-written indication at the top of each plate denotes the concentrations of the original YNBY stocks for our reference during the experiments. This suggests that YNBY is not anti-bacterial. At least five independent experiments were performed.

Figure 4

YNBY exhibits dose-dependent cytotoxic-ity to both T- and B- lymphocytes, more potently than 6-mercaptopurine (6-MP) and 5-aminosalicylic acid (5-ASA), two of the most commonly used IBD medications. Total protein expression in cell lysates was used as an indicator of change in cell numbers, as analyzed bySDS-PAGE. A. As the concentration of YNBY treatment increased, the total proteins reflected on the gel decreased dramatically for both B-cells and T-cells (Molt4 and Jurkat), indicating a drastic decrease in cell numbers. B and C. Cytotoxicity of 6-MP (B) or 5 -ASA (C) to Molt4 cells was shown for comparison purpose with YNBY (A). These studies demonstrate that YNBY exhibited more cytoxicity to lymphocytes than 6-MP and 5-ASA. At least four independent experiments were performed.

Figure 5

YNBY shows no cytotoxicity to Caco-2/BBE cells and promotes cell spreading. A. Caco-2/BBE cells were plated at 50% confluence in 6-well plates and incubated with various concentrations of YNBYfor 72 hrs. Cells were harvested and total proteins were analyzed by SDS-PAGE and visualized by Ponceau S staining. No change in protein profiles suggests no cytotoxicity of the drug to intestinal epithelial cells. B. YNBY promotes formation of lamellipodia and wound healing of Caco-2/BBE cells. Compared with the control (B-a; no YNBY), wounds on the cell layer closed more rapidly in cells incubated with YNBY at 1 Mg/ml (B-b). Cells incubated with YNBY exhibited larger and more extensive lamellipodia (edges marked by arrows) protruding from the wounded edges (marked by red dotted lines) compared to these of untreated cells (B-a). Cells images were taken with an Olympus DP72 Digital Camera on a Zeiss Axio Inverted Microscope. C. The rate of wound healing is almost doubled in Caco-/BBE cells treated with YNBY when compared to those untreated. At least five independent experiments were performed.

Figure 6

YNBY significantly decreases the TNFα and IL-12p40 in mice with DSS-induced colitis. Colonic mucosa was isolated from DSS-induced (A) or TNBS-induced (B) mice. Total mucosal proteins were extracted and analyzed with SDS-PAGE Western-blots using primary antibodies for TNFα, IFNγ, IL-17, IL-12p40, and actin (as a protein loading control). While DSS- and TNBS-colitis both had significantly higher IL-12p40 and IL-17 expression in the colon with additional elevations of TNFα in DSS-colitis and IFNγ in TNBS-colitis, YNBY treatment significantly decreased all four proinflammatory cytokines examined. At least 3 mice were analyzed in each group.