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Low molecular weight (6.5 kDa) glycyrrhiza polysaccharide (GP) shows a good immunomodulatory activity, however, its regulatory mechanism is still unclear. Using cyclophosphamide (CTX)-induced immunosuppressed mice as model, GP was verified to be able to reverse the CTX-induced intestinal structure damage. GP could increase the number of goblet cells, CD4+, CD8+ T lymphocytes and mucins, especially maintain the balance of Helper T lymphocyte 1/ Helper T lymphocyte 2 (Th1/Th2). Moreover, GP could alleviate immunosuppression through down-regulating extracellular regulated protein kinases/p38/nuclear factor kappa-Bp50 (ERK/p38/NF-κBp50) pathways, increasing short-chain fatty acids (SCFAs) level and secretion of cytokines such as interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-22 (IL-22), transforming growth factor-β3 (TGF-β3) and immunoglobulin M (IgM), immunoglobulin M (IgG) and Secretory immunoglobulin A (SIgA). The treatment of GP increased the total species and diversity of intestinal microbiota. Notably, immunoregulation of GP is closely related to the composition and diversity of intestinal microbiota. Microbiota analysis indicated that GP promoted the proliferation of beneficial bacteria such as Muribaculaceae_unclassified, Alistipes, Lachnospiraceae_NK4A136_group, Bacteroides, Ligilactobacillus and Clostridia_vadinBB60_group, conversely reduced the abundance of Proteobacteria and CTX-derived bacteria (Clostridiales_unclassified, Candidatus_Arthromitus, Firmicutes_unclassified and Clostridium). These results strongly displayed that GP enhance the immunity through these routes: increasing immune cell and cytokines, stimulating immune-related pathways, modulating intestinal microbiota and metabolites. Thus, GP shows a great potential to be an immune enhancer or a natural medicine for the treatment of intestinal mucosal injury.

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Methods and Materials Forty male specific pathogen-free (SPF) BALB/c mice, aged 6 weeks old (weight 20 ± 2.0 g). After one week of adaption under temperature (23 ± 2 °C), 55 ± 5% humidity and ordinary diet, the mice were randomly divided into five groups (n=8), control group (CON), model group (MOD), low dose GP group (100 mg/kg bw, GPL), high dose GP group (200 mg/kg bw, GPH) and lentinan (Solarbio, China) treated positive group (20 mg/kg bw, LNT). The immunosuppression model was established through intraperitoneally injected with CTX (Macklin, China) (100 mg/kg bw) once a day on the 1st to 3rd days. For the next ten days, CON and MOD groups were intragastrically given normal saline once a day, and GPL, GPH and LNT groups were given different doses of GP or lentinan. On the 14th day, feces and orbital blood were collected. Serum was collected at 4◦C by centrifuging at 15000 rpm for 15 min. Colon tissue was isolated. Part of intestinal tissue was taken and placed in 10% formalin for pathological analysis. The remaining samples were stored in a −80◦C refrigerator. The collected intestine tissues were fixed with 10% formalin and embedded in paraffin, then sliced and stained using the standard method of hematoxylin and eosin (H&E) to observe the histopathological changes. After incubation of tissue sections with primary antibody (anti-CD4+ and CD8+) and secondary antibody, the changes of CD4+ and CD8+ T lymphocytes were observed by optical microscopy. The contents of immune cytokines (IL-2, IL-10, IL-4, IL-22, IFN-γ and TGF-β3), chemokines (MCP-1 and MIP-1α) and immunoglobulins (IgG and IgM) in serum and sIgA in colon tissue were measured using ELISA mothed as described in Jiangsu Jingmei Biotechnology Co., LTD. RT-qPCR was used to detect the mRNA levels of p38, ERK, p50, T-bet and GATA3, with β-actin as the reference gene. Total RNA was extracted from intestinal tissues using Trizol reagent, and then was reverse transcribed to cDNA using M5 Super plus qPCR RT kit (Juomei, China). Total bacterial DNA was extracted from fresh mice feces by CTAB method and evaluated by 2% agarose gel electrophoresis. PCR products were purified by AMPure XT beads (Beckman, USA) and then assessed using Agilent 2100 Biochromateter (Agilent, USA) and Illumina (Kapa Biosciences, Woburn, MA, USA) library quantitative kits, then to sequencing by NovaSeq 6000 sequencer. The concentration of SCFAs was determined by GC. The filtrates were analyzed by using Shimadzu GC-2014 system (Shimadzu Co. Ltd, Kyoto, Japan) equipped with an SH-Rtx-Wax capillary column (30 m × 0.25 mm × 0.25 μm, Shimadzu Co. Ltd, Kyoto, Japan) and a hydrogen flame ion detector (FID). The injection volume was 1 μl. The temperature of FID detector and injection port was 240℃. The initial column temperature was 100 °C for 4 min, elevated at a speed of 10 ℃/min to 170℃ and maintained for 1 min, then elevated at a speed of 50 ℃/min to 220℃ and kept for 5 min.

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