Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Michael J. Wannemuehler

Abstract

Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as inflammatory bowel disease (IBD), are inflammatory diseases of the enteric mucosa whose etiology is currently undefined. While hypotheses for causation include genetic susceptibility, environmental risk factors, inappropriate and chronic immune responses to members of the intestinal microflora as well as failed immune regulatory mechanisms, many questions regarding IBD susceptibility, dysbiosis and immune dysfunction remain. Not surprisingly, current therapeutic targets are expensive, have questionable long term efficacy and are associated with risk of secondary bacterial and viral infections. As chronically ill patients substitute or supplement their treatments with alternative, nutraceutical remedies in ever greater numbers, more research is needed to ascertain the safety and efficacy of these products as therapeutics.

Prunella vulgaris is a common `cure all' which is popular in traditional Chinese medicine. P. vulgaris can be steeped in water and drank as a tea or ground into a salve, and is known for its anti-viral, anti-inflammatory and antioxidant benefits. P. vulgaris has never, until now, been studied in the context of colitic therapy and has yet to gain a foothold as a commonly utilized nutraceutical in western medicine.

Hypericum gentianoides, akin to Hypericum perforatum/St. John's Wort, is a unique and understudied species of Hypericum. H. gentianoides lacks the photoactive and cytotoxic compounds routinely found in other Hypericum species. While the bioactivity of this plant is not well characterized, it is known to have anti-inflammatory, immunomodulatory and antibacterial potential. This potential and the lack of cytotoxic, photoactive compounds make H. gentianoides a good candidate for study in colitis models.

Echinacea angustifolia is well studied, commonly utilized world-wide as a preventative or remedy for the common cold, and has great potential as a therapeutic agent in many disease modalities. The roots of this plant contain several types of compounds known to be bioactive. Like other nutraceutical, E. angustifolia is documented anecdotally to cure or reduce symptoms related to countless maladies and diseases without sound scientific proof. E. angustifolia has never been studied as anti-colitic; however, pain suppressive and immunomodulatory capabilities of this plant lend credence to the potential for this type of study.

In this study, the anti-colitic potential of P. vulgaris, H. gentianoides and E. angustifolia were determined in the mdr1a deficient (-/-) mouse model of spontaneous colitis or the chemically induced dextran sodium sulphate (DSS) mouse model of acute colitis. Extracts of E. angustifolia root or above ground sections of P. vulgaris or H. gentianoides were extracted using the Soxhlet method in 95% ethanol and were solubilized in a final concentration of 5% ethanol for use in studies. P. vulgaris and H. gentianoides were tested at daily doses of 2.4 mg (~ 100 mg/kg body weight) and 4.8 mg (~ 200 mg/kg body weight) respectively, while E. angustifolia was tested at daily doses of 100 mg/kg and 200 mg/kg body weight.

In the DSS model of acute colitis, E. angustifolia was not found to be effective during the period of DSS treatment (in low dose 1.75% to high dose 2.5% DSS in male and female C57BL/6 mice), or during restitution. E. angustifolia was not found to be cytotoxic in vivo. Conversely, P. vulgaris was able to significantly (p < 0.05) decrease weight loss and improve macroscopic indicators of severe colitic wasting. The disparity between E. angustifolia and P. vulgaris efficacies in this acute model of colitis could be due to the difference in the constituent types found in these two extracts. E. angustifolia is composed mostly of phenolics and alkylamides, while P. vulgaris contains phenolics, flavonoids and triterpinoids. Certain types of phenols have been shown to have efficacy in low dose DSS colitis; however, flavonoids have efficacy in models of colitis as well, and in combination with phenols may be synergistic. Since flavonoids have recently proven very effective in ameliorating spontaneous colitis, P. vulgaris was also evaluated in the mdr1a-/- model of spontaneous colitis.

In the mdr1a-/- mouse model of spontaneous colitis, P. vulgaris extract treatment was compared to mdr1a-/- mice treated with 5% ethanol vehicle, metronidazole (an antibiotic and anti-colitic control) and FVBWT mice. It was discovered that P. vulgaris was able to delay onset of spontaneous colitis, significantly (p < 0.05) decrease macroscopic typhlocolitic and microscopic cecal disease scores, prevent cecal neutrophil influx, and downregulate nuclear factor-kappaB (NF-kB) related cytokines/chemokines and gene targets. The downregulation of innate immune signals and function is hypothesized to contribute to the observed decrease in cecal tonsil CD4+ helper and CD8+ cytotoxic T cells as well as germinal center B cells. Adaptive immune function was also altered. Loss of immune tolerance to the microflora is a characteristic of chronic colitis. Unlike mdr1a-/- vehicle treated mice, which displayed antibody production to microflora antigens, P. vulgaris treated mice displayed little to no Ab response to the same microflora antigens.

H. gentianoides contains flavonoids, phenolics and a unique antimicrobial compound called uliginosin A. As the mdr1a-/- mouse model of spontaneous colitis is driven by the host immune response to the microflora and Hypericum species are known to be immunomodulatory, this extract was of interest as a therapeutic anti-colitic in the mdr1a-/- model. Onset of spontaneous colitis was significantly delayed by H. gentianoides, but was not completely abolished. Macroscopic scores, microscopic scores, serum cytokine levels and myeloperoxidase production were all significantly (p < 0.05) reduced by H. gentianoides gavage. While H. gentianoides has immunosuppressant potential, a surprising increase in intestinal plasma cell infiltrate and an increase in serum IL-6 correlated with H. gentianoides gavage in mdr1a-/- mice. To date, anti-microbial functions have not been ruled out as a contributing factor in the efficacy of anti-colitic activity of this extract.

Together, these novel experiments have identified new candidates for anti-colitic therapy or therapeutic supplementation of current IBD treatment strategies. P. vulgaris, H. gentianoides and E. angustifolia merit further research as nutraceutical treatments of chronic inflammatory disorders, and these studies make it clear that synergy between phenolics, flavonoids and natural anti-bacterial compounds also warrant further research.

DOI

https://doi.org/10.31274/etd-180810-3048

Copyright Owner

Kelley Marie Kemper Haarberg

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

211 pages

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