| China is rich in squid resources, and due to the high nutritional value of squid, it is popular for chinese consumers. However, squid ink sac, which is the waste product during processing, causes pollution and becomes extra burden to squid processing enterprises. Hence, searching for bioactive components in squid ink and utilizing them, is the first but the most important step to settle this problem down. The study of functional components in squid ink and their bioactivities will be of important scientific significance and provide a theoretical basis for high value utilization of squid. Intestinal mucosal immunity, as a main part of the immune system, is challenged by the external environment any minute, such as food, air, water, in which there are countless components and bacteria etc. Under the stress conditions, such as chemotherapy, burn, malnutrition, high-pressure work, it could cause intestinal mucosal immunity damaged, even accompanied with some severe results, enteritisand infection. In this study, raw polysaccharide product OBP was extracted from Ommastrephes bartrami and based on that we purified the most abundant polysaccharide component SIP. Employing the mouse model of chemotherapeutic cyclophosphamide induced intestinal injury, the protective effects of OBP and SIP on mucosal immunity were investigated.First, the effect of OBP on intestinal microbiota in chemotherapy treated mice was studied. Intestinal commensal bacteria and host form a micro ecological structure, which is ainterdependent and interactive system. Once the homeostasisstate system is destroyed, intestine colonization resistance is damaged, which would cause pathogen colonization and invasion. Based on the 16S rDNA clone library, PCR-RFLP, real-time PCR methodologies, we tested the intestinal flora change. It shows that Cy caused the intestinal flora dysfunction, the increase of microbial quantity. Nonetheless, OBP intake could ameliorate intestinal flora dysfunction, including down-regulating the abnormal bacterial proliferation, protecting the probiotic Bifidobacterium and suppression of conditional pathogenic Bacteroides. It implies that dietary OBP could protect intestinal microbiota from chemotherapy injury.Chemotherapy drugs could also do harm to tight junctions and adheren junctions, which induces epithelium damage, potential bacteria relocation and infection. Thus, we studied the effect of OBP on intestine epithelial barrier, including on tight junction proteins and adheren junction proteins. The results show that dietary OBP enhanced ZO-1 and E-cadherin expression. This was also testified by histological observation. Meanwhile,200mg/kg OBP enhanced tight junction protein ZO-2, ZO-3, Cingulin expression, comparing to that in the Cy control mice. Even though Claudin-6, Claudin-7 were not affected by OBP, OBP increased Claudin-2 expression. It implies that dietary OBP could protect intestinal epithelial barrier from chemotherapy injury.Intestinal SIgA is a non-inflammatory immunoglobulin isotype, which is composed of IgA, J chain, pIgR. It fights against bacteria, virus, pathogens invasion and protects mucosal adaptive defense. In current contribution, we studied the protective effect of OBP on SIgA secretion and it shows that OBP could promote SIgA secretion against Cy damage. And the higher production of SIgA in OBP treated mice relied on the greater expression of IgA, J chain both of which was secreted by plasma cell and pIgR which was expressed in intestinal epithelial cell. Furthermore, TNF-a mRNA level of small intestine was relatively higher in OBP high dose group mice, which was responsible for the higher expression of pIgR. Moreover, IL-6 and IL-10 mRNA expression were increased by OBP treatment, which could lead to the terminally differentiation stages of B cells to IgA+ plasma cells. The enhanced immunoglobulin secretory pathway, which can be confirmed by enhanced unfolded protein response effectors XBP-1s and Bip, also contributed to the greater existence of IgA, J chain secreted by plasma cells in OBP group mice. And this highly developed secretion machinery in plasma cells depended on IRE-1 mediated XBP-1s pathway. Overall, OBP specifically alleviated Cy induced serum IgA level dropping down, which are mainly existed in mucosa system, but did not ameliorated serum IgG concentration, which are presented principally in serum for host defense.The most abundant polysaccharide component SIP from OBP was isolated. It is verified that the SIP was the functional component which promoted SIgA secretion in intestine. After exploring the related mechanisms, we found that the higher expression of IL-6, IL-10 in OBP mice was because of the effect of SIP on immune cells. It was testified in vitro on macrophage cell line RAW264.7, spleen lymphocytes.To study whether the enhancement effect of SIP on SIgA secretion is also because that SIP stimulates the pIgR mediated dIgA/pIgA transcytosis speed, we mimicked the transcytosis process in in viro study. We successfully constructed MDCK cell line with high expression of human pIgR, and the three-dimensional cell model. Firstly, dIgA and pIgA, which were substrate of pIgR transcytosis, were isolated and quantified by AKTA system from MM patient’s serum. By three-dimensional cell culture, we studied the localization of pIgR which was localized at the basolateral side of MDCK cell correctly by immunofluorescence labeling and confocal laser tomography techniques. The results showed that the SIP had no apparent impact on the transcytosis speed of SIgA formation by epithelial cells. However, it stimulated the epithelial cell line Caco-2 proliferation in vitro and pIgR expression in vivo. Taken together, SIP promoted the immunoglobulin secretory pathway by stimulating the IgA related host mucosal immune response, but not relied on the transcytosis speed of intestinal epithelium.The molecularmechanisms of squid ink polysaccharide in protecting intestinal mucosal immunity and barrierswere clarified in current study. It will provide a theoretical basis for developing healthy food products based on squid ink polysaccharide.This will not only bring good news to the chemotherapy treated patients, but also promote the high-value utilization of squid ink resources. Our results may have important implications for enhancement effect of immunopotentiating squid ink polysaccharide on intestinal mucosa immunity to against intestinal disorders involving infection and inflammation. |
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