铁皮石斛多糖通过调节肠菌代谢物对老年小鼠肠道黏膜屏障损伤的改善作用

摘要/Abstract

摘要：

目的探讨铁皮石斛多糖（DOP）对老年小鼠肠道黏膜屏障损伤的保护作用，并阐明其可能的作用机制。方法选择10只雌性5月龄C57BL/6小鼠为年轻组，将20只C57BL/6小鼠（15月龄）随机分为老年组和老年鼠DOP（200 mg·kg^-1·d^-1）处理组（DOP组），每组10只。DOP组小鼠采用DOP灌胃，检测各组小鼠体质量、摄食量和悬挂时间；苏木精-伊红（HE）染色观察各组小鼠肠道和脾脏组织病理形态表现；免疫组织化学染色检测各组小鼠肠道组织中闭锁小带蛋白-1（ZO-1）和黏蛋白2（MUC2）的表达情况。制备肠菌代谢物培养基（IBMM）干预秀丽线虫（C.elegans），将C.elegans随机分为年轻IBMM组（Young-IBMM）、老年IBMM组（Aged-IBMM）和DOP-IBMM组，采用免疫荧光法分析各组C.elegans第1和12天的肠道脂褐素累积水平；亮蓝染色评估各组C.elegans第1和12天的肠道渗漏情况。制备IBMM干预Caco-2细胞，将Caco-2细胞分为Young-IBMM组、Aged-IBMM组和DOP-IBMM组，Western blotting法检测各组Caco-2细胞中ZO-1、闭合蛋白（Occludin）、肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、磷酸化肌球蛋白轻链（p-MLC）和肌球蛋白轻链激酶（MLCK）蛋白表达水平。结果与年轻组比较，老年组小鼠体质量增加（P<0.05），摄食量降低（P<0.05），悬挂时间变短（P<0.05）；与老年组比较，DOP组小鼠体质量明显减轻（P<0.01），摄食量增加（P<0.05），悬挂时间明显延长（P<0.01）。HE染色，与年轻组比较，老年组小鼠肠黏膜厚度变薄，杯状细胞减少，肠绒毛长短不一且排列无序，脾脏表面可见大量铁血黄素存在，红髓内细胞成分减少，白髓内动脉周围淋巴鞘和淋巴小结残存或几乎消失；与老年组比较，DOP组小鼠肠黏膜厚度增加，杯状细胞增多，肠绒毛长度一致且排列整齐，脾脏红髓整体功能改善，白髓成分增加。免疫组织化学染色，与年轻组比较，老年组小鼠肠道组织中ZO-1和MUC2蛋白表达水平明显降低（P<0.05或P<0.001）；与老年组比较，DOP组小鼠肠道组织中ZO-1和MUC2蛋白表达水平明显升高（P<0.05或P<0.001）。免疫荧光法，与Young-IBMM组比较，Aged-IBMM组C.elegans肠道内脂褐素累积水平明显升高（P<0.001）；与Aged-IBMM组比较，DOP-IBMM组C.elegans肠道内脂褐素累积水平明显降低（P<0.001）。亮蓝染色法，与Young-IBMM组比较，Aged-IBMM组C. elegans亮蓝染料渗漏至线虫全身，肠道结构变得模糊不清，不易观察；与Aged-IBMM组比较，DOP-IBMM组C.elegans亮蓝染料渗漏减少。Western blotting法，与Young-IBMM组比较，Aged-IBMM组Caco-2细胞中TNF-α、IL-6、p-MLC和MLCK蛋白表达水平明显升高（P<0.01或P<0.001），ZO-1和Occludin蛋白表达水平明显降低（P<0.05或P<0.01）；与Aged-IBMM组比较，DOP-IBMM组Caco-2细胞中TNF-α、IL-6、p-MLC和MLCK蛋白表达水平明显降低（P<0.01），ZO-1和Occludin蛋白表达水平明显升高（P<0.05或P<0.01）。结论 DOP对老年小鼠肠道黏膜屏障损伤有改善作用，其作用机制可能与通过调节肠菌代谢物、抑制p-MLC/MLCK通路、恢复紧密连接复合物表达、降低肠道炎症水平、进而改善肠道屏障损伤有关。

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 mechanisms. 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 mucus secretion protein （MUC2） in the intestinal tissue of the mice in various groups. The intestinal baterial metabolite medium（IBMM） were prepared to intervene the 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 intestinal tissue of the mice 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 of the mice in aged group； 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 the 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 was reduced in DOP-IBMM. 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 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

中图分类号:

R965

李宏, 王慧, 王隶书, 王超楠, 徐晓浩, 孙立伟. 铁皮石斛多糖通过调节肠菌代谢物对老年小鼠肠道黏膜屏障损伤的改善作用[J]. 吉林大学学报(医学版), 2024, (): 1-9.

Hong LI, Hui WANG, Lishu WANG, Chaonan WANG, Xiaohao XU, Liwei SUN. Ameliorating effect of Dendrobium officinale polysaccharides on intestinal mucosal barrier damage in elderly mice by regulating intestinal microbial metabolites[J]. Journal of Jilin University(Medicine Edition), 2024, (): 1-9.

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Group	Body mass(m/g)	Food take(m/g)	Hanging time(t/s)
Young	22.00±0.21	23.96±0.16	67.88±1.60
Aged	40.44±0.46^*	17.78±0.19^*	21.05±0.90^*
DOP	36.95±0.87^△△	19.99±0.37^△	47.20±0.85^△△

Group	Expressiong level of ZO-1	Expressiong level of MUC2
Young	0.13±0.01	0.25±0.01
Aged	0.08±0.06^*	0.17±0.01^**
DOP	0.16±0.07^△	0.20±0.01^△△

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