Triptolide Ameliorates Intestinal Inflammation In Inflammatory Bowel Disease (Crohn’s Disease) By Mechanisms Involving Suppression Of IL-6/STAT3 Signaling Pathway

Inflammatory bowel disease (IBD) is comprised of ulcerative colitis (UC) and Crohn’s disease (CD). Based on studies in humans and animal models, it was reported the immune response in CD to be dominated by Thl cells. But this concept has been challenged since a novel T-cell subtype, that is Th17 cells, which play dominant role in inflammatory diseases, has been gradually recognized. Data showed that, both Thl and Th17 cells may contribute to pathogenesis of CD.Accumulated data indicated that, a defect of lamina propria T-cell apoptosis resulting in uncontrolled cell activation represents a crucial mechanism involved in an exaggerated immune response and release of pro-inflammatory cytokines. This hypothesis is supported by the fact that compared to uninflamed mucosal T cells, mucosal T cells from patients with CD are consequently more resistant to apoptosis induced by a variety of factors. Aditionally, the induction of mucosal T-cell apoptosis by drugs has been shown to be beneficial in CD, supporting the concept that depletion of activated T cells in the intestinal mucosa is a highly effective therapeutic strategy in the treatment of CD. Among these regulatory mechanisms, downregulation of activated T lymphocytes via apoptosis is a very potent and effective strategy, which has been considered as a key controlling mechanism. Based on these results, it was considered:a defect in the intrinsic downregulatory apparatus which controls apoptosis of activated mucosal LPMC may result in disease perpetuation and chronic inflammation.IL-6 first binds to a specific receptor, a soluble form of the IL-6R (sIL-6R) which has been found in various cells. IL-6 together with sIL-6R stimulates cells which express gp130, a process which has been called trans-signalling. IL-6 trans-signaling further induces STAT3, a downstream instrument of IL-6 pathway signaling. Recently, the important role played by IL-6/STAT3 was supported by a larger scale study of human CD. It was reported that IL-6, which is abundantly secreted in CD, upregulates expression of antiapoptotic Bcl-2 and Bcl-xl via the IL-6 trans-STAT3 signalling pathway. It was showed the mitochondrial apoptosis pathway is controlled and regulated by the Bcl superfamily. A shift in the Bcl rheostat towards an antiapoptotic constellation results in abrogation of a proapoptotic stimulus. In CD patients, there was a shift in the mitochondrial Bcl-rheostat with upregulation of antiapoptotic Bcl-2 and Bcl-xl molecules, which protected LPMC including T cells from apoptosis. These data strongly indicated that activation of the IL-6/STAT3 pathway has a key role in the development of CD.Recently, it was reported that the retinoid orphan receptor y-T (RORyt), which is critical for Th17 differentiation, could be induced by IL-6 signal in a Stat3-dependent manner. It was indicated that the STAT3-dependent expression of RORyt provides the molecular basis for the critical role played by IL-6-gp130-STAT3 signaling in promoting the development of Th17 from CD4+T cells. Thereby, the classical IL-6/STAT-3 pathway that is well characterized in CD plays an important role in Thl7 response, providing a new function of this signaling pathway. In summary, the IL-6-gp130-STAT3 signaling pathway plays predominant role in autoimmune diseases at least mainly through the protection of LPMC from apoptosis and induction of Th17 response.In addition to spontaneously developing a Thl-mediated chronic enterocolitis, interleukin-10 deficient mice (IL-10-/- mice) which have many similarities to human CD, also showed high expression of IL-17. IL-10-/- mice develop spontaneous inflammation of the large intestine, a process that is T cell dependent. Additionally, elevated IL-6 and activated STAT3 has been observed in the colon both in CD and in IL-107-/- mice colitis. Recently, it has been discovered that Th17 cells are the major effectors cells mediating disease progression in animal IBD, and the author suggested that the early disease in mice colitis is likely mediated by the Thl subset but disease progression is due to Th17 cells. It was reported that blockade of IL-6 trans-signaling reduces STAT3 activation, and approves IL-10-/- mice colitis in a Thl7-independent manner. These accumulated data indicated that similar with the human CD, Thl and Th17 response may contribute together to pathogenesis of animal colitis.Triptolide, a diterpene triepoxide, is an active component of extracts derived from the medicinal vine Tripterygium Wilfordii Hook. F. (TWHF) whose extracts have been used widely to treat autoimmune diseases in China for many years. Triptolide shows therapeutic effects on autoimmune diseases. The immunosuppressive effects by triptolide can be partly attributed to its potent inhibitory effects on monocytes activation. Recently, studies carried out in our laboratory showed that treatment with triptolide improves the spontaneous colitis of IL-10-/- mice in a Thl cytokines dependent way, indicating that triptolide has therapeutic activity in this model, but the potential mechanism of action of treatment with triptolide in CD still needs further studies.Prompted by these important findings, we began an evaluation of the effects of triptolide on IL-6/STAT3 pathway, the IL-17 expression and the apoptosis of LPMC in CD. Our objective was twofold:first, to elucidate whether the intestinal anti-inflammatory effects of triptolide are related to suppression of IL-6/STAT3 signaling pathway in vivo and in vitro; second, to further explore the mechanism of action of this agent in CD. Our results provide evidence that triptolide repress the IL-17 expression and promote apoptois of LPMC in CD by mechanisms involving inhibiting of IL-6/STAT3 signaling pathway.PART 1 Triptolide ameliorates IL-10-deficient mice colitis by mechanisms involving suppression of IL-6/STAT3 signaling pathway in vivoObjective:We hypothesized that triptolide could attenuate the experimental colitis by repressing IL-17, induction of T cells apoptosis and that this would involve down-regulation of IL-6/STAT3 signaling pathway.Methods:C3H/HeJBir.IL-10 gene deficient (C3H.IL-10-/-) mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) and maintained in a SPF animal facility at the Model Animal Research Center of Nanjing University (Nanjing, China). Age- and sex-matched mice between 4 and 5 weeks of age were used for the experiments with chronic triptolide administration. One member of each pair was assigned to the control group and the other was assigned to the triptolide-treated group. In the treatment group, triptolide was intraperitoneally administered every other day for 8 weeks. Control mice were similarly given an equivalent quantity of normal saline. The disease activity index (DAI) was evaluated every week. The colons samples were paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Tissues were reviewed in a blinded fashion and assessed, with the histologic scores ranged from 0 to 10. LP CD4+T cells were then subsequently isolated by positive selection using microbeads and MACS techniques as the manufacture’s instrument. MPO concentration, IL-6 and sIL-6R level were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. The levels of gp130, RORyt, Bcl-2, Bcl-xl and IL-17 of purified CD4+T cells were detectd by quantitative real-time PCR. STAT3 and phospho-STAT3 (p-STAT3) were determined in mouse colon mucosal tissue by western blot. Apoptosis of T cells was detected by using FACS.Result:Since sixth week after administration of triptolide, the DAI were decreased significantly in mice of treatmet group. Histological examination demonstrated that triptolide significantly reduced the severity of colitis in IL-10-/- mice. Triptolide suppressed the IL-6/STAT3 signaling pathway, as well as repressed gene expression of IL-17 in vivo. In addition, triptolide treatment induced the apoptosis of LP T cells.Conclusion:Our results suggested that triptolide attenuates the IBD intestinal inflammation by the mechanism involving downregulation of IL-6/STAT3 signaling pathway, further more, suppressing pathogenic Th17 immune responses and indution of LP T cell apoptosis.PART 2 Triptolide down-regulates IL-6/STAT3 signaling pathway in cultured colonic explants from CD patients in vitroObjective:We hypothesized that triptolide would down-regulat IL-6/STAT3 signaling pathway in cultured inflammed colonic explants from CD patients in vitro.Methods:Colonic specimens were obtained from patients who diagnosed as colonic CD. The diagnosis CD was based on conventional clinical, endoscopic, and histopathological criteria. Surgical specimens derived from involved areas of the colon from CD patients (n=6) were obtained with informed consent.The pieces of tissue were washed with cold PBS and trimmed into explants measuring 2 mm2. Explants were then cultured in serum-free CMRL-1066 tissue culture medium on collagen I-coated plates at 37℃ in 5%CO2. Part of the culture medium was supplemented with 20 ng/ml triptolide to observe the direct effects of triptolide on colonic explants from CD patients. Others were cultured in normal tissue culture medium. After 24h-culture, the tissue were collected and stored at-80℃ until assay. Total RNA of explants preparation and first-strand cDNA synthesis was performed. The levels of IL-17 and IL-6 were detectd by quantitative real-time PCR. STAT3 and phospho-STAT3 (p-STAT3) were determined in cultured explants mucosal tissue by western blot. LPMC were isolated from cultured explants. The levels of Bcl-2, Bcl-xl and Bax in LPMC were detectd by quantitative real-time PCR. Apoptosis of LPMC was detected by using FACS.Result:Triptolide (20 ng/ml) in vitro was able to down-regulate the IL-6/STAT3 pathway, and furthermore reduce IL-17 expression. The expression of Bcl-2 and Bcl-xl were significantly decreased in LPMC from explants cultured with triptolide when compared with those without triptolide. In addition, triptolide significantly premoted LPMC apoptosis in cultured colonic explants from patients with CD. Immunofluorescence assay also indicated that triptolide in vitro also improved the T lymphocytic infiltration in inflammed cultured explants from patients who diagnosed as colonicConclusion:The results of triptolide of down-regulation the IL-6/STAT3 pathway in vitro were in accordance with the results obtained in vivo. Patients with CD may benefit from treatment with triptolide.