| BackgroundHMLE is a patented lipid extract from hard-shelled mussel (Mytilus coruscus) with promising anti-inflammation activity, which is produced following supercritical CO2 fluid extraction technology and stabilizing treatment. Research so far on HMLE is limited to studies of its activity and efficacy evaluation in relation to arthritis in animal models of this disease. No studies have been conducted for exploring its activity in other disease models, nor have there been studies evaluating its efficacy in clinical patients. Therefore, this research used cell study, animal study and clinical human study to evaluate whether there could be additional evidence for expanding the use of HMLE.Objective and MethodsThe first objective was to explore the benefits of HMLE on ulcerative colitis improvement and investigate the underlying mechanism. Firstly, classical in-vitro inflammation model induced by lipopolysaccharide (LPS) in RAW264.7 murine macrophages cell line was used, and the anti-inflammatory activity focused on studying the effects on the TLR4/NF-kB signaling pathway, essential in the pathogenesis of inflammatory bowel diseases. Thereafter, an intestinal damage model, caused by LPS challenge in mice, was used to investigate the protective effects of HMLE on intestinal barrier function. Lastly, a chemically-induced mouse model of ulcerative colitis was used to evaluate the effects of HMLE on alleviating disease activity index (DAI), focusing on macroscopic and microscopic colonic damage, oxidative stress and immune balance; and in order to investigate the mechanism focused on the TLR4/MAPKs/NF-kB signaling pathway. The second objective evaluated the anti-arthritic activity of HMLE, in a clinical study which involved the first ever human feeding trial in rheumatoid arthritis patients.ResultsTolerable concentrations of HMLE for RAW264.7 cells were determined ranging from 0 to 125μg/mL. Within this concentration range, effective nitric oxide (NO) production inhibitory activity and relieving excessive inflammation activity were observed. Moreover, HMLE exhibited significant down-regulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) mRNA expression and inhibition of NF-kB activation, which suggests the potential of intestinal protective effects of HMLE.Supplementation of HMLE at a dose of 300mg/kg body weight for C57BL/6 mice for an 6 week period significantly increased the proportions of EPA and DHA in erythrocyte membrane fatty acids (p<0.05 for all). After the LPS challenge, mice administered control olive oil supplemented with HMLE exhibited a significantly lower level of intestinal damage than mice in control group, which was evidenced by higher villus height/crypt depth ratio, lower levels of neutrophil infiltration, decreased leakage of diamine oxidase (DAO) from mucosal to plasma and up-regulated tight junction protein (Occludin and Claudin-1) mRNA expression (p<0.05 for all); HMLE supplementation also significantly improved the immune imbalance induced by the LPS challenge on both protein level and mRNA level; HMLE supplementation exhibited high activity on alleviating the increased mRNA expression of TLR4 and MyD88 induced by LPS challenge (p<0.05), but no effects were observed on the downstream IL-1R-associated kinase 4 (IRAK4) and TNF receptor-associated kinase 6 (TRAF6) (p>0.05). Nevertheless, the inhibitory effects on the activation of mitogen-activated protein kinases (MAPKs, including p38, JNK and ERK) and nuclear factor kappa-B (NF-kB), which are targets of TLR4 signaling pathway, were still observed in mice receiving HMLE.In the study investigating the effects of HMLE on inflammatory bowel disease, experimental ulcerative colitis was successfully induced by administrating 3% dextran sulphate sodium (DSS). Mice received a gavage of control oil (olive oil) or supplemented with HMLE or reference drug (sulfasalazine pyridine, SASP) during the whole experimental period. Mice in HMLE group had a significantly attenuated inflammatory response, evidenced by significant alleviation of weight loss and DAI increasing (p<0.05 for both); the improvement in body weight was significantly better than for those mice in the SASP group. For the protection of the colon damage, HMLE exhibited significant benefits in a dose-dependent manner, which were evidenced by the alleviation of colon shortening, villus abruption, inflammatory infiltration, hyperemia, edema, ulcerations and necrosis and improved overall histopathological score (p<0.05 compared with control group). The beneficial effects of HMLE on oxidative stress was reflected by higher superoxidase dismutase (SOD) activity (p<0.05), normalized glutathione (GSH) concentration (p<0.05), reduced inducible nitric oxide synthase (iNOS) expression and NO production (p<0.05) in colon tissue. HMLE also protected against colon inflammation as assessed by lower pro-inflammatory factors secretion and mRNA expression (p<0.05) including TNF-a, IL-1(3 and IL-6, higher IL-10 secretion and mRNA level, and lower colon inflammatory mediators such as PGE2 production and COX-2 mRNA expression (p<0.05), compared with control group. Moreover, the increased mRNA expression of TLR4, MyD88, IRAK4 and TRAF6 in DSS treated colon was inhibited (p<0.05 for all) by HMLE as well as the reference drug (SASP), and therefore blocking the activation of two major targets, MAPKs and NF-κB, which suggests a potential mechanism underlying the beneficial effects of HMLE.The clinical human trial investigated whether HMLE could improve clinical conditions of rheumatoid arthritis patients. Fifty rheumatoid arthritis patients were randomly assigned to receive HMLE capsules (200mg HMLE+200mg corn oil per capsule) or receive placebo capsules (400mg corn oil per capsule) for 6 months. Forty-two subjects and fifty subjects were included in per-protocol and intention-to-treat analyses, respectively. Significant differences in changes on disease activity score (DAS28) and clinical disease activity index (CDAI) after the 6-month intervention (p<0.01) were observed in both analyses, with more evident efficacy shown in per-protocol populations (ΔDAS28= 0.47; ΔCDAI= 4.17;p<0.05 for both), which favored the benefits of the HMLE group. TNF-α, IL-1β and PGE2 but not IL-6, were significantly decreased in both groups, and the decrements were much larger in the HMLE group for TNF-α and PGE2 after 6 months from baseline (p<0.05 for both). IL-10 was significantly increased in both groups and the change was much more evident in the HMLE group (p<0.05).ConclusionsThe present studies showed HMLE supplementation is beneficial to LPS-induced intestinal damage and DSS-induced ulcerative colitis, via inhibition of oxidative stress, inflammatory response and TLR/MAPKs/NF-κB activation, which are promising for further experimental studies on treating inflammatory bowel disease. Additionally, HMLE supplementation also exhibited benefits for the clinical conditions of rheumatoid patients in relation to improvement in the balance between pro- and anti-inflammatory factors, which indicated its potential to serve as adjunctive treatment for rheumatoid arthritis. |
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