Structure elucidation,immunomodulatory activity,antitumor activity and its molecular mechanism of a novel polysaccharide from Boletus reticulatus Schaeff

A new water-soluble heteropolysaccharide with a molecular weight of 15 k Da was isolated from the fruiting bodies of Boletus reticulatus Schaeff.Structural characterization results revealed that B.reticulatus Schaeff polysaccharide(BRS-X)had a backbone of 1,6-linkedα-D-galactose and 1,2,6-linkedα-D-galactose which branches were mainly composed of a terminal 4-linkedβ-D-glucose and the ratio of D-galactose and D-glucose was 5:1.Bioactivity assays indicated that BRS-X displayed a strong proliferative activity in T cells and B cells and promoted the secretion of immunoglobulin G(Ig G),Ig E,Ig D and Ig M.In addition,BRS-X could facilitate the proliferation and phagocytosis of RAW264.7 cells and could significantly inhibit the growth of tumors in S180-bearing mice.The results of transcriptome sequencing analysis illustrated that total 46 genes enriched in MAPK and total 34 genes enriched in PI3 K/Akt signaling pathways in BRS-X group.The protein VEGF and VEGFR expression were significantly reduced under the treatment with BRS-X.These findings provide a scientific basis for the edible and medicinal value of BRS-X.

Isolation, chemical characterization, and immunomodulatory activity of naturally acetylated hemicelluloses from bamboo shavings

Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing. In this study we investigated the isolation, chemical characterization, and immunostimulatory activity in vitro of the hemicelluloses from bamboo shavings. Shavings were first pretreated by steam explosion. The optimal pretreatment was found to be steam explosion at 2.2 MPa for 1 min. Following this pretreatment, the yield of hemicelluloses reached（2.05±0.22）%（based on the dry dewaxed raw materials）, which was 5.7-fold higher than that of untreated samples. Bamboo-shavings hemicellulose（BSH） was then prepared by hot water extraction and ethanol precipitation from the steam-exploded shavings. Purification of BSH by anion-exchange chromatography of diethylaminoethanol（DEAE）-sepharose Fast Flow resulted in a neutral fraction（BSH-1, purity of 95.3%, yield of 1.06%） and an acidic fraction（BSH-2, purity of 92.5%, yield of 0.79%）. The weight-average molecular weights（Mw） of BSH-1 and BSH-2 were 12 800 and 11 300 g/mol, respectively. Chemical and structural analyses by Fourier transform infrared spectroscopy（FT-IR）, 1D（^1H and ^13C） and 2D（heteronuclear single quantum correlation（HSQC）） nuclear magnetic resonance（NMR） spectra revealed that BSH-1 was O-acetylated-arabinoxylan and BSH-2 was O-acetylated-（4-Omethylglucurono）-arabinoxylan. BSH-1 had a higher content of acetyl groups than BSH-2. For the immunomodulatory activity in vitro, BSH and BSH-2 significantly stimulated mouse splenocyte proliferation while BSH-1 had no effect; BSH, BSH-1, and BSH-2 markedly enhanced the phagocytosis activity and nitric oxide production of the murine macrophage RAW264.7 in a dose-dependent manner. Our results suggest that the water-extractable hemicelluloses from steam-exploded bamboo shavings are naturally acetylated and have immunostimulatory activity.

Immunomodulatory activity of polysaccharides from Brassica rapa by activating Akt/NF-κB signaling

Objective:To investigate the immunomodulatory activity of polysaccharides from the roots of Brassica rapa.Methods:The crude polysaccharide from roots of B.rapa(BRP)was extracted and purified to further investigate the active fraction of BRT for inducing macrophage phagocytosis.Results:Effects on RAW264.7 cells demonstrated that BRP behaved better phagocytic capacity and had potent immunomodulatory activity,including increasing production of nitric oxide(NO),tumor necrosis factor a(TNFa)and upregulating mRNA levels of inducible NO synthase(iNOS)and TNFa.Furthermore,modulation of macrophage by BRP was indicated to be mediated via the activation of Akt and nuclear factor-kappa B(NF-κB).Conclusion:The beneficial effects of BRP could be used as an immunotherapeutic adjuvant in treatment of inflammatory diseases.

Correlation of conformational changes and immunomodulatory activity of lentinan under different subcritical water temperature

In order to reveal the intrinsic nature of the reduction or disappearance of biological activity in the processing and preparation of polysaccharides,and realize the effective control of highly active polysaccharides,the correlation between conformation and immunomodulatory activity of lentinan under different subcritical water temperatures was investigated.The results showed that,compared with the lentinan extracted by hot water(HW-L),with the increase of temperature(100–160°C),the molecular weight of lentinans decreased(5.041×105–1.754×106 g/mol)and the rigidity of the chain weakened.The chains gradually loosened and a rigid-flexible transition occurred.At 160°C,the conformation of lentinan completely changed from triple helix to random coil.In-vitro cell experiments showed that,lentinan at 120°C could more efficiently promote the proliferation and phagocytosis of RAW264.7 cells,and significantly promote the secretion of NO,TNF-α,IL-6 and IL-1β,exhibiting stronger immunomodulatory activity.But the activity of lentinan decreased sharply at 150–160°C.These results suggest a significant correlation between polysaccharide chain conformation and immunomodulatory activity under different subcritical water temperature conditions.By adjusting the temperature of subcritical water,the chain conformation of polysaccharides can be controlled,resulting in more efficient extraction of highly active polysaccharides.

The immunomodulatory activity of levan in RAW264.7 macrophage varies with its molecular weights

Levan is a fructose homopolymer obtained from both plants and microorganisms.Microbial levan is typically produced in a variety of molecular weights(MW).Previous studies have shown that different MW of levan exhibit different biological activities.Though levan was reported for its immunomodulatory effect by increasing nitric oxide(NO)production in RAW264.7 macrophage cells,whether its MW can affect this activity remains unknown.In this study,the effect of different levan MW from Bacillus subtilis on its immunoregulatory properties was investigated.B.subtilis was cultivated using various cultivation parameters to obtain levan of different MW.Three weight average MW of levan,>8×10^(5) Da(M1),1.5×10^(4) Da(M2)and 3.7×10^(3) Da(M3),were selected to test for their effect on modulating NO levels in RAW264.7 cells.Our results showed that M2 increased NO production in a dose-dependent manner,and more effectively than that of M1 and M3.Consistent with these findings,M2 significantly stimulated inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX2)protein expression,while less pronounced effects were observed in the cells treated with M1 and M3.Therefore,our results demonstrate that various MW of levan have varying effects on its immunoregulatory activities in RAW264.7 cells.Furthermore,our findings suggest that the size of levan should be taken into account when employing levan as one of the active ingredients in functional foods and immunomodulatory products.

Single-cell transcriptome analysis reveals the regulatory effects of artesunate on splenic immune cells in polymicrobial sepsis

Sepsis is characterized by a severe and life-threatening host immune response to polymicrobial infection accompanied by organ dysfunction.Studies on the therapeutic effect and mechanism of immunomodulatory drugs on the sepsis-induced hyperinflammatory or immunosuppression states of various immune cells remain limited.This study aimed to investigate the protective effects and underlying mechanism of artesunate(ART)on the splenic microenvironment of cecal ligation and puncture-induced sepsis model mice using single-cell RNA sequencing(scRNA-seq)and experimental validations.The scRNA-seq analysis revealed that ART inhibited the activation of pro-inflammatory macrophages recruited during sepsis.ART could restore neutrophils’chemotaxis and immune function in the septic spleen.It inhibited the activation of T regulatory cells but promoted the cytotoxic function of natural killer cells during sepsis.ART also promoted the differentiation and activity of splenic B cells in mice with sepsis.These results indicated that ART could alleviate the inflammatory and/or immunosuppressive states of various immune cells involved in sepsis to balance the immune homeostasis within the host.Overall,this study provided a comprehensive investigation of the regulatory effect of ART on the splenic microenvironment in sepsis,thus contributing to the application of ART as adjunctive therapy for the clinical treatment of sepsis.

In vitro cytotoxic and toxicological activities of ethanolic extract of Kaempferia galanga Linn. and its active component,ethyl-p-methoxycinnamate, against cholangiocarcinoma

Objective:To evaluate the cytotoxic,apoptotic,mutagenic and immunomodulatory activities of Kaempferia galanga Linn.(KG)extract and ethyl-p-methoxycinnamate(EPMC)in vitro.Methods:The present study investigated the cytotoxic[using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe nyl-2-H-tetrazolium bromide test],apoptotic(using a mitochondrial membrane potential assay),mutagenic(using a micronucleus test)and immunomodulatory(using flow cytometry)activities of the ethanolic extract of KG and its bioactive component,EPMC,against two cholangiocarcinoma(CCA)cell lines,CL-6 and HuCCT1,and one normal human cell line,OUMS-36 T-1 F.Results:Both KG extract and EPMC exhibited moderate cytotoxic activity against both CCA cells.The cytotoxic activity was supported by their concentration-dependent induction of apoptosis.CL-6 was most sensitive(3–4 fold)and selective to 5-fluorouracil(5-FU),compared with KG extract and EPMC[median half inhibiting concentration(IC50)and selectivity index(SI)were 23.01 lg/mL and 17.32;78.41 lg/mL and 4.44;100.76 lg/mL and 2.20,respectively for 5-FU vs.KG extract vs.EPMC].HuCCT1 was relatively more sensitive and selective to 5-FU and EPMC than KG extract[median IC50 and SI were 66.03 lg/mL and6.04;60.90 lg/mL and 3.65;156.60 lg/mL and 2.23,respectively for 5-FU vs.EPMC vs.KG extract].EPMC produced relatively potent cytotoxic activity against polymorphonuclear cells(IC50=92.20 lg/mL).KG extract and EPMC exhibited concentration-dependent mutagenic activity,as well as inhibition of tumor necrosis factor-a and interleukin-6.Conclusion:Considering cytotoxic,apoptotic,immunomodulatory and mutagenic activities,further development of KG as a drug candidate is likely to focus on the oral pharmaceutical formulation of a standardized KG extract rather than isolated compounds.

Dihydroartemisinin beneficially regulates splenic immune cell heterogeneity through the SOD3-JNK-AP-1 axis

The immunomodulatory potential of dihydroartemisinin(DHA) has recently been highlighted;however, the potential mechanism remains to be clarified. Single-cell RNA sequencing was explored in combination with cellular and biochemical approaches to elucidate the immunomodulatory mechanisms of DHA. In this study, we found that DHA induced both spleen enlargement and rearrangement of splenic immune cell subsets in mice. It was revealed that DHA promoted the reversible expansion of effective regulatory T cells and interferon-γ^(+)cytotoxic CD8^(+)T cells in the spleen via induction of superoxide dismutase 3(SOD3) expression and increased phosphorylation of c-Jun N-terminal kinases(JNK) and its downstream activator protein 1(AP-1) transcription factors. Further, SOD3 knockout mice were resistant to the regulatory effect of DHA. Thus, DHA,through the activation of the SOD3-JNK-AP-1 axis, beneficially regulated immune cell heterogeneity and splenic immune cell homeostasis to treat autoimmune diseases.

Effect of aqueous extract of Polygonum minus leaf on the immunity and survival of African catfish(Clarias gariepinus)

Objective:To establish immunomodulatory potential of aqueous extract of Polygonum minus(P.minus)leaves,the haematological effects and lysozyme activity of aqueous extract of P.minus leaf on Clarias gariepinus was studied.Methods:The fish were grouped and administered with the P.minus aqueous leaf extract intraperitoneally.Blood and serum samples were collected from each group and examined for various blood parameters.The turbidimetric assay for lysozyme activity using M.luteus and the survival rate of fish against A.hydrophila was carried out.Results:There was no significant impact(P>0.05)on white blood cell count,red blood cell count,mean corpuscular volume,mean corpuscular hemoglobin,mean corpuscular hemoglobin concentration and lysozyme activity was significantly(P<0.05)higher than control.The serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase levels were significantly lower than control(P<0.05).The highest dose group showed longest survival.Conclusions:From this study,it is considered that the aqueous extract of P.minus can be used as an immunostimulant in African catfish,but continuous administration may require maintaining the protection.