关键词: 铁皮石斛多糖, 自然衰老小鼠, 肠道屏障, 肠菌代谢物

Abstract:

Objective To investigate the protective effect of Dendrobium officinale polysaccharides （DOP） on intestinal mucosal barrier damage， and to elucidate the possible mechanism. Methods Ten female C57BL/6 mice， aged 5 months， were selected as young group； twenty femal C57BL/6 mice， aged 15 months， were randomly divided into aged group and DOP treatment group （200 mg·kg^-1， DOP group）， with 10 mice in each group. The mice in DOP group were administrated with DOP by gavage. The body mass， food intakes and hanging time of the mice in various groups were detected. HE staining was used to observe the pathomorphology of intestinal and spleen tissues of the mice in various groups. Immunohistochemical staining was used to detect the expressions of intestinal atresin 1 （ZO-1） and Mucin 2（MUC2） in intestinal tissue of the mice in various groups. The intestinal baterial metabolite medium（IBMM） were prepared to intervene the Caenorhabditis elegans （C.elegans）， and the C.elegans were randomly divided into Young-IBMM group， Aged-IBMM group， and DOP-IBMM group.Immuno-fluorescence method was used to analyze the intestinal lipofuscin accumulation levels on the 1st day and the 12th day of the C. elegans in various groups. Brilliant blue staining was used to assess the intestinal leakage on the 1st day and the 12th day of C. elegans in various groups.The Caco-2 cells were randomly divided into Young-IBMM， Aged-IBMM and DOP-IBMM groups， and Western blotting method was used to detect the expression levels of ZO-1，Occludin，tumor necrosis factor-α（TNF-α），interleukin-6 （IL-6），phosphorylated myosin light chain（p-MLC），myosin light chain kinase（MLCK）proteins in the Caco-2 cells in various groups. Results Compared with young group， the body mass of the mice in aged group was increased （P<0.05）， the amount of food intake was decreased （P<0.05）， and the hanging time was decreased （P<0.05）； compared with aged group， the body mass of the mice in DOP group was significantly decreased （P<0.01）， the amount of food intake was increased （P<0.05）， and the hanging time was significantly extended （P<0.01）. The HE staining results showed that compared with young group， the thickness of intestinal mucosa of the mice in aged group became thinner， the goblet cells were reduced， the intestinal villi were disordered with different lengths， a large amount of hemosiderin was found on the surface of the spleen， the cell components in the red medullary were reduced， and the lymphatic sheath and lymphatic nodes around the intra-white pulp artery remained or almost disappeared； compared with aged group， the thickness of the intestinal mucosa of the mice in DOP group was increased， the goblet cells were increased， the length of the intestinal villi was consistent and neatly arranged， the overall function of the red pulp of the spleen was improved， and the components of the white pulp were increased. The immunohistochemical staining results showed that compared with young group， the expression levels of ZO-1 and MUC2 proteins in intestinal tissue of the mice in aged group were significantly decreased （P<0.05 or P<0.001）； compared with aged group， the expression levels of ZO-1 and MUC2 proteins in the intestinal tissue of the mice in DOP group were significantly increased （P<0.05 or P<0.001）. The immuno-fluorescence analysis showed that compared with Young-IBMM group， the intestinal lipofuscin accumulation level of C.elegans in Aged-IBMM group was significantly increased （P<0.001）；compared with Aged-IBMM group，the intestinal lipofuscin accumulation level of C.elegans in DOP-IBMM group was significantly reduced （P<0.001）. The brilliant blue staining showed that compared with Young-IBMM group， the bright blue dye leaked into the whole body of C.elegans from intestinal tissue in Aged-IBMM group， and the intestinal structure became blurred and was difficulted to be observed；compared with Aged-IBMM group， the leakage of bright blue dye of C.elegans in DOP-IBMM was reduced. The Western blotting results showed that compared with Young-IBMM group， the expression levels of TNF-α， IL-6， p-MLC， and MLCK proteins in the Caco-2 cells in Aged-IBMM group were significantly increased （P<0.01 or P<0.001）， and the expression levels of ZO-1 and Occludin proteins were significantly decreased （P<0.05 or P<0.01）； compared with Aged-IBMM， the expression levels of TNF-α， IL-6， p-MLC and MLCK proteins in the Caco-2 cells in DOP-IBMM group were significantly decreased （P<0.01）， and the expression levels of ZO-1 and Occludin proteins were significantly increased （P<0.05 or P<0.01）. Conclusion DOP has an ameliorating effect on intestinal mucosal barrier damage in the aged mice，and its mechanism may be related to the improvement of intestinal barrier damage by regulating intestinal bacterial metabolites， inhibiting the p-MLC/MLCK signal pathway， restoring the expression of tight junction complexes， and reducing the level of intestinal inflammation.

Key words: Dendrobium officinale polysaccharides, Natural aging mice, Intestinal barrier, Intestinal bacterial metabolites