| Research backgroundInflammatory bowel disease (IBD) is a chronic intestinal autoimmune disease that the etiology remains to be elucidated more clearly. Despite that, IBD has been widely considered as an autoimmune that involves the chaos of intestinal immune system with high hereditary susceptibility, as well as the ambalance of intestinal flora. Indeed, the excessive response of T lymphocyte immune, i.e. the adaptive immune, is the key characteristics of IBD, especially the unbalance of the subsets of helper T cell (Th) within the intestinal musoca. Hence, it is of great importance to investigate the pathogenesis mechanism of IBD via regulation of the Th subsets.Pogostemon cablin (Blanco) Benth is a traditional Chinese medicine with long history for treatments of gastrointestinal disease caused by summer and/or wet, and pogostemonis herba is capable of dispelling pathogens, appetizing, preventing vomiting, and relieving summer-heat. Pogostone (PO) is one of the major components of pogostemonis herba. It was found that the essential oil of pogostemonis herba could ameliorate the inflammation of IBD induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) in a previous experiment by our research team. In the present study, the potential effect of PO against IBD was explored in the view of immunomodulation on T cell, for a better elucidation of the immune pathogenesis of IBD, as well as for the purpose to interpret the active principle of pogostemonis herba.Methods1. Inflammation remission by pogostone against IBD induced by TNBS1) Male SD rats were randomly divided into 6 groups, Normal group, Model group, salazosulfapyridine group (SASP,200mg/Kg) and PO group (20,40,80 mg/Kg). IBD was induced by intra-rectal installation of TNBS (25mg/Kg). SASP and PO groups were administrated by intra-rectal installation for 7 consecutive days, while normal group and model group received equal volume of 0.5% CMC-Na.2) Body weight, feces characteristics and activity were recorded every day, and datas of the 7th day were used for disease activity index (DAI) assessement. All rats were sacrificed on the 8th day and colons (from anus to cecum) were obtaind for macroscopic score (MS). According to DAI and MS, samples of the most effective PO group were subject to the following study together with normal, control, and SASP group.3) Part of the leision (about 8cm from anus) was fixed in 4% formaldehyde (pH～7.2) and embedded in paraffin. Some sections were subjected to hematoxylin-Eosin (H&E) staining for histological score (HS), and the other were subjected to immunohistology chemistry (IHC) to analyze the expression of CD4, Foxp3, IFN-γ, and IL-17. Part of the leision was homogenated for Myeloperoxidase (MPO) activity change detection, transforming growth factor-β (TGF-β) determination by enzyme-linked immunosorbent assay (ELISA). And contents of interleukin-17A (IL-17A), IL-10, IL-4, IL-12p70, and interferon-γ (IFN-γ) were assayed by Luminex xMAP.4) Part of the fresh MLN was analyzed by FCM immediately for the expression of IFN-γ, IL-4、IL-17 and Foxp3, to evaluate ratios of Th1, Th2, Th17, and regulatory T cell (Treg) in the MLN. Positive percentage of Ki67 and CD38/MHC Ⅱ were also assessed for characteristics of proliferation and activation.5) Relative mRNA expression of transcript fators (TF) in the leision tissue and MLN, including T-bet, STAT1, GATA3, STAT6, STAT3, ROR γt, Foxp3Part of Total ribonucleic acid (RNA) were assayed by quantitative realtime Polymerase Chain Reaction (quantitative rt-PCR).2. Proliferation blocking by pogostone on the Concanavalin A (ConA)-stimulated T cell1) Impact of PO on the viability of splenocyte from C57BL/6 mice was assayed using MTS kit after the 48h- and 72h-incubation.2) Splenocytes from C57BL/6 mice were treated with PO (20,40,80μM) under the stimulation by ConA (1μg/mL) for 72h. By flowcytometry (FCM), T cell and subsets of CD4+ and CD8+ were figured out by Anti-CD3、Anti-CD4、Anti-CD8, and stained with CFSE for proliferation analysis. Apoptosis was determined by Annexin V staining and cell cycle distribution was assayed by Annexin V/Pyridine iodide (PI). Expressions of CDK2, cyclin E, CDK1, and Cyclin B were analyzed by FCM in T cells.3) Contents of IL-6, IL-10, monocyte chemotactic protein (MCP-1), IFN-γ, Tumor nerosis factor (TNF) in the supertant were determined by Cytometry Beads Array (CBA).Results1. Inflammation remission by pogostone against IBD induced by TNBS1) After the 25mg/Kg TNBS challenge, rats of the model group exihited typical IBD symptoms including weight loss, diarrhea, and bloody stools, accompanying with serious colon leision of ankylenteron, ulcer, necrosis, and edema. Moreover, MPO activity in tissues of model was found significantly higher than that of normal group. HE histological analysis suggested transmural inflammation affecting the entire colonic mucosa of model group, which is characterized by ulceration, degradation of crypt integrity, massive inflammatory cell infiltration supported by elevated colonic MPO activity, muscularis hypertrophy and angiogenesis. By contrast, PO was capable of improving life quality of the suffering rats and eliminating diarrhea and bloody stools, resulting in an evidently lower DAI than that of model group. Moreover, both macroscopic score and HE histological score of PO group were much lower than those of model group, indicating that inflammation was alleviated by PO treatment as manifested by less edema, ulcer and nerosis. HE staining suggested that 40mg/Kg PO made the best effect on mucous healing, crypt restoration, and leukocyte infiltration inhibition proved by the obvious reduction of MPO activity. Cytokine profiling analysis showed that contents of IL-4, IL-12p70, IL-10, IL-17, and TGF-β were evidently higher than those of control group, while concentrations of IFN-y, IL-12p70, IL-17, and IL-10 in PO (40mg/Kg) were decrease. Additionaly, IHC results showed that TNBS challenge remarkably increased the expressions of CD4, IFN-y, IL-17, Foxp3 in the inflammatory colon tissue, but PO would apparently decrease those of CD4, IFN-y, IL-17, implying that PO would inhibited Th cell infiltration in the colon. Quantitative rt-PCR data showed that mRNA expressions of T-bet, GATA3, STAT6, and Foxp3 in model grouop were much higher, though that of RORyt was found to be lowered. In comparision, PO hardly affected the mRNA expressions of these transcriptional factors, among which only that of RORyt was upregulated.2) Th cell characteristics in MLN were further studied. There is no difference among the populations of Ki67+and CD38/MHC Ⅱ Th cells from control group, model group or PO (40mg/Kg) group. Except the increased IL-4 Th cells, no difference was observed between populations of IFN-y, IL-17, Foxp3 Th cells from control group and those from model group. PO (40mg/Kg) did not affect the populations of these Th cells in MLN. Results showed that possitve percentages of Ki67 and CD38/MHC-Ⅱ were not affected by PO, neither those of IFN-γ、IL-4、 IL-17, and CD25+/Foxp3. In terms of mRNA relative expression of transcriptional factors in MLN, it was found that in MLN from model group, expression of GATA3 was apparently decreased accompanying with an increased RORyt expression, while that of T-bet, STAT6, STAT3, or Foxp3 was not affected. By contrast, PO(40mg/Kg) restored the mRNA relative expressions of STAT-1, GATA-3, STAT6, T-bet, ROR-yt, Foxp3 to the level similar to those of normal group. Results from the present study found that proliferation, activation or differentiation of Th cells in MLN was not affected in this IBD model. PO did not affect the abovementioned characteristics of Th cells in MLN, neither.2. Proliferation blocking by pogostone on the Concanavalin A (ConA)-stimulated T cell1) Firstly, it was found that PO (0～80μM) had no cytotoxicity on splenocyte as indicated that the viability of splenocytes under PO treatment was not affected. In the the ConA-stimulated T cell, there existed slight but significant apoptosis attributed to activation induced cell death (AICD), and T cell population as well as the proliferation ratio were evidently increased. By contrast, PO (20,40,80μM) significantly reduced the T cell population and the proliferation ratio without causing apoptosis, suggesting a direct blocking on the T cell proliferation. Moreover, both CD4+T cell and CD8+T cell proliferations induced by ConA were inhibited by PO.2) Cell cycle analysis showed that the direct proliferation blocking by PO was closely related to cell cycle arrest. It was found that populations of S-phase and G2/M-phase T cell increased evidently with a significant increase of CDK2 but reductions of cyclin E, CDK1 and cyclin B. In comparison, PO (20,40,80μM) caused a remarkable S-phase arrest attributed to down-regulation of cyclin E, cyclin B and CDK1.3) Additionally, it was found that secretion of IL-6, IL-10, MCP-1, IFN-y, and TNF soared under ConA stimulation. PO (20,40,80μM) could inhibit both pro-inflammatory IFN-y and anti-inflammatory IL-10.ConclusionsTaken together, PO was proved to be effective in IBD, in which PO could improve the life quality of the suffering animals, and ameliorate the inflammation as indicated by less edema, ulcer, nerosis and leukocyte infiltration, thereby promoting mucosa healing. It is reasonable to infer that the IBD alleviation by PO would be related to its direct immunosuppression activity as showed in the in vitro study. Via induction of S-phase arrest, PO directly blocked the proliferation of Th cells, thereby inhibiting the infiltration of both inflammatory Th cells, such as Thl, Th2, and Th17, and immunosuppressive Th cell (Treg), and restored secretions of several key cytokines in the triggering of immune response, finally dumped and eliminated the destructive autoimmune response in IBD. From data of the present study, it can be found that pogostone would be the active component of pogostemonis herba that is responsible for its clinical implication in treatment of IBD, which is mainly attributed to the direct immunosuppression on Th cells by pogostone. |
PDF Full Text Request