ABSTRACT
In situ vaccine (ISV) can provoke systemic anti-tumor immunity through the induction of immunogenic cell death (ICD). The development of ISV technology has been restricted by the limited and suboptimal ICD driven tumor antigen production which are currently relying on chemo-drugs, photo-/radio-sensitizers, oncolytic-virus and immunostimulatory agents. Herein, a sulfate radical (SO4 ·-) based ISV is reported that accomplishes superior tumor immunotherapy dispense from conventional approaches. The ISV denoted as P-Mn-LDH is constructed by intercalating peroxydisulfate (PDS, a precursor of SO4 ·-) into manganese layered double hydroxide nanoparticles (Mn-LDH). This design allows the stabilization of PDS under ambient condition, but triggers a Mn2+ mediated PDS decomposition in acidic tumor microenvironment (TME) to generate in situ SO4 ·-. Importantly, it is found that the SO4 ·- radicals not only effectively kill cancer cells, but also induce a necroptotic cell death pathway, leading to robust ICD signaling for eliciting adaptive immunity. Further, the P-Mn-LDH can activate the stimulator of interferon genes (STING) pathway to further boost anti-tumor immunity. Collectively, the P-Mn-LDH based ISV exhibited potent activity in inhibiting tumor growth and lung metastasis. When combined with immune checkpoint inhibitor, significant inhibition of distant tumors is achieved. This study underpins the promise of SO4 ·- based vaccine technology for cancer immunotherapy.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Viral pneumonia is a highly pathogenic respiratory infectious disease associated with excessive activation of the complement system. Our previous studies found that the anticomplement polysaccharides from some medicinal plants could significantly alleviate H1N1-induced acute lung injury (H1N1-ALI). The leaves and twigs of Tamarix chinensis Lour. are traditionally used as a Chinese medicine Xiheliu for treating inflammatory disorders. Interestingly, its crude polysaccharides (MBAP90) showed potent anticomplement activity in vitro. AIM OF THE STUDY: To evaluate the therapeutic effects and possible mechanism of MBAP90 on viral pneumonia and further isolate and characterize the key active substance of MBAP90. MATERIALS AND METHODS: The protective effects of MBAP90 were evaluated by survival tests and pharmacodynamic experiments on H1N1-ALI mice. Histopathological changes, viral load, inflammatory markers, and complement deposition in lungs were analyzed by H&E staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC), respectively. An anticomplement homogenous polysaccharide (MBAP-3) was obtained from MBAP90 by bio-guided separation, and its structure was further characterized by methylation analysis and NMR spectroscopy. RESULTS: Oral administration of MBAP90 at a dose of 400 mg/kg significantly increased the survival rate of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with MBAP90 (200 and 400 mg/kg) could decrease the lung index, lung pathological injury, the levels of excessive proinflammatory cytokines (IL-6, TNF-α, MCP-1, IL-18, and IL-1ß), and complement levels (C3c and C5b-9). In addition, MBAP-3 was characterized as a novel homogenous polysaccharide with potent in vitro anticomplement activity (CH50: 0.126 ± 0.002 mg/mL), containing 10.51% uronic acids and 9.67% flavonoids, which were similar to the composition of MBAP90. The backbone of MBAP-3 consisted of â4)-α-D-Glcp-(1â, â3,4,6)-α-D-Glcp-(1â, and â3,4)-α-D-Glcp-(1â, with branches comprising α-L-Araf-(1â, α-D-GlcpA-(1â, â4,6)-α-D-Manp-(1â and â4)-ß-D-Galp-(1 â . Particularly, O-6 of â4)-ß-D-Galp-(1â was conjugated with a flavonoid, myricetin. CONCLUSIONS: MBAP90 could ameliorate H1N1-ALI by inhibiting inflammation and over-activation of the complement system. These polysaccharides (MBAP90 and MBAP-3) with relative high contents of uronic acid and flavonoid substituent might be vital components of T. chinensis for treating viral pneumonia.
Subject(s)
Acute Lung Injury , Influenza A Virus, H1N1 Subtype , Pneumonia, Viral , Tamaricaceae , Animals , Mice , Complement System Proteins , Polysaccharides/pharmacology , Polysaccharides/therapeutic use , Polysaccharides/chemistry , Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Uronic Acids/pharmacology , Uronic Acids/therapeutic use , Flavonoids/pharmacologyABSTRACT
Traditional Chinese medicine (TCM) is recognized as a complex matrix, and improved analytical methods are crucial to extract the key indicators and depict the interaction and alteration of the complex matrix. Shenqi Fuzheng Injection (SQ), a water extract of Radix Codonopsis and Radix Astragali, has demonstrated preventative effects on myotube atrophy induced by chemotherapeutic agents. To achieve the improved analytical capability of complex biological samples, we established a highly reproducible, sensitive, specific, and robust gas chromatography-tandem mass spectrometry (GC-MS) method to detect glycolysis and tricarboxylic acid (TCA) cycle intermediates with optimized factors in the extraction and derivatization process. Our method detected fifteen metabolites and covered most intermediate metabolites in glycolysis and TCA cycles, including glucose, glucose-6-phosphate, fructose-6-phosphate, dihydroxyacetone phosphate, 3-diphosphoglycerate, phosphoenolpyruvate, pyruvate, lactate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, and malate. Through methodological verification of the method, it was found that the linear correlation coefficients of each compound in the method were greater than 0.98, all of which had lower limits of quantification, the recovery rate was 84.94-104.45%, and the accuracy was 77.72-104.92%. The intraday precision was 3.72-15.37%, the interday precision was 5.00-18.02%, and the stability was 7.85-15.51%. Therefore, the method has good linearity, accuracy, precision, and stability. The method was further applied to study the attenuating effects of the SQ in a chemotherapeutic agents-induced C2C12 myotube atrophy model to evaluate the changes in the tricarboxylic acid cycle and glycolytic products under the action by the complex systems of TCM and disease model. Our study provided an improved method to explore TCM's pharmacodynamic constituents and action mechanisms.
Subject(s)
Citric Acid Cycle , Glycolysis , Humans , Gas Chromatography-Mass Spectrometry/methods , Citric Acid , AtrophyABSTRACT
BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of â4,6)-α-Glcp-(1â, â3,4)-α-Glcp-(1â, â4)-α-Glcp-(1â, â4,6)-α-Galp-(1â, â5)-α-Araf-(1â, α-Rhap-(1â, α-Araf-(1â, α-GlcpA-(1â, â4)-ß-Manp-(1â, ß-Manp-(1â and â3)-ß-Manp-(1â. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.
Subject(s)
Acute Lung Injury , Extracellular Traps , Hedyotis , Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Animals , Chemokines/metabolism , Complement C3a/metabolism , Complement C3c/metabolism , Complement Inactivator Proteins , Cytokines/metabolism , Extracellular Traps/metabolism , Histones , Interleukin-6/metabolism , Ligands , Lipopolysaccharides , Mice , Peroxidase/metabolism , Polysaccharides/pharmacology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
A homogeneous polysaccharide coded as CPP-1 was extracted and purified from the root of Codonopsis pilosula (Franch.) Nannf. by water extraction, ethanol precipitation, and column chromatography. Its structure was analyzed by HPGPC-ELSD, HPLC, GC-MS, FT-IR, and NMR techniques. The results indicated that CPP-1 was composed of mannose (Man), glucose (Glc), galactose (Gal), and arabinose (Ara) at a molar ratio of 5.86 : 51.69 : 34.34 : 8.08. The methylation analysis revealed that the main glycosidic linkage types of CPP-1 were (1â)-linked-Glc residue, (1â3)-linked-Glc residues, (1â4)-linked-Gal residue, (1â2,3,4)-linked-Glc residue, (1â)-linked-Man residue, (1â3,4)-linked-Glc residue, and (1â)-linked-Ara residue. In vivo efficacy trial illustrated that CPP-1 supplements could alleviate HFD-induced mice obesity significantly, as well as improve obesity-induced disorders of glucose metabolism, alleviate insulin resistance, and improve the effects of lipid metabolism. The findings indicate that this polysaccharide has the potential for the treatment of obesity.
Subject(s)
Codonopsis , Animals , Codonopsis/chemistry , Diet , Dietary Carbohydrates , Galactose , Humans , Mannose , Mice , Obesity/drug therapy , Polysaccharides/chemistry , Polysaccharides/pharmacology , Spectroscopy, Fourier Transform InfraredABSTRACT
A novel homogeneous polysaccharide named GEP-1 was isolated and purified from Gastrodia elata (G. elata) by hot-water extraction, ethanol precipitation, and membrane separator. GEP-1, which has a molecular weight of 20.1 kDa, contains a polysaccharide framework comprised of only glucose. Methylation and NMR analysis showed that GEP-1 contained 1,3,6-linked-α-Glcp, 1,4-linked-α-Glcp, 1,4-linked-ß-Glcp and 1,4,6-linked-α-Glcp. Interestingly, GEP-1 contained citric acid and repeating p-hydroxybenzyl alcohol as one branch. Furthermore, a bioactivity test showed that GEP-1 could significantly promote the growth of Akkermansia muciniphila (A. muciniphila) and Lacticaseibacillus paracasei (L.paracasei) strains. These results implied that GEP-1 might be useful for human by modulating gut microbiota.
Subject(s)
Gastrodia/chemistry , Gastrointestinal Microbiome/drug effects , Plant Extracts/chemistry , Polysaccharides/pharmacology , Akkermansia/drug effects , Carbohydrates , Dietary CarbohydratesABSTRACT
Establishing the identity of bioactive compounds to control the quality of Traditional Chinese Medicines is made more challenging by the complexity of the metabolite matrix, the existence of isomers, and the range of compound concentration and polarity observed between individual samples of the same plant in a multicomponent preparation. In addition, LC-MS analysis has limited capability for the separation and analysis of potentially important trace compounds and isomers, which hinders the comprehensive metabolite characterization of functional foods and Traditional Natural Medicine. To facilitate and improve the chemical composition characterization and enhance metabolite discernment, a comprehensive strategy was developed which integrates ion mobility mass spectrometry (IMS) with offline two-dimensional liquid chromatography based on hydrophilic interaction chromatography (HILIC) and conventional reversed phase (RP) C18 chromatography. Through application of the HILIC × RP offline 2D-LC approach, trace compounds were enriched and separated promoting a more efficient and detailed analysis of the matrix complexity. Comprehensive non-targeted multidimensional data (Rt1D, Rt2D, MS, CCS and MS/MS) and data-independent-acquisition (DIA) mass data of the metabolites in complex food and drug samples were obtained in the IMS-DIA-MS/MS mode on a Waters-SYNAPT G2-Si mass spectrometer with an ESI source. Through the application of high-efficiency neutral loss (NLs) and diagnostic product ions (DPIs) filter strategies, information from DIA mass data permitted the rapid detection and identification of compounds. The identification coverage of metabolites with low-quality MS/MS data was also improved. In the absence of analytical standards, Collision Cross Section (CCS) prediction and matching strategies based on theoretical chemical structures provided a method to distingish isomers. To demonstrate the efficacy of the technique this comprehensive strategy was applied to the compound characterization of Gastrodia Rhizoma (GR). Characterization of 272 compounds was achieved, including 146 unreported compounds. The results affirm that this comprehensive five-dimensional data collection strategy has the capacity to support the in-depth study of the high level of chemical diversity in Traditional Chinese Medicines.
Subject(s)
Drug Discovery , Gastrodia , Medicine, Chinese Traditional , Chromatography, High Pressure Liquid , Data Collection , Drug Discovery/methods , Gastrodia/chemistry , Rhizome/chemistry , Tandem Mass SpectrometryABSTRACT
Two homogeneous polysaccharides, GEP-3 and GEP-4, were purified from Gastrodia elata, a precious traditional Chinese medicine. Their structural characteristics were obtained using HPGPC, PMP-HPLC, LC/MS, FT-IR, NMR, and SEM methods. GEP-3 was 1,4-glucan with molecular weight of 20 kDa. Interestingly, GEP-4 comprised of a backbone of â[4)-α-Glcp-(1]10â[4)-α-Glcp-(1â]5[6)-ß-Glcp-(1]11â6)-α-Glcp-(3â and two branches of ß-Glcp and p-hydroxybenzyl alcohol citrate, with repeating p-hydroxybenzyl alcohol attached to the backbone chain at O-6 position of â4,6)-α-Glcp-(1â and O-1 position of â3,6)-α-Glcp-(1â. GEP-4 is a novel polysaccharide obtained and characterized for the first time. Bioactivity test indicated that both of them significantly promote the growth of Akkermansia muciniphila (Akk. muciniphila). Furthermore, GEP-3 and GEP-4 promoted the growth of Akk. muciniphila from high-fat diet (HFD) fecal microbiota. These results indicated that GEP-3 and GEP-4 were potential Akk. muciniphila growth promoters.
Subject(s)
Gastrodia , Plant Extracts/pharmacology , Polysaccharides/pharmacology , Akkermansia/drug effects , Akkermansia/growth & development , Akkermansia/isolation & purification , Animals , Diet, High-Fat , Disease Models, Animal , Feces/microbiology , Gastrodia/chemistry , Gastrointestinal Microbiome , Mice , Molecular Structure , Non-alcoholic Fatty Liver Disease/microbiology , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Polysaccharides/chemistry , Polysaccharides/isolation & purificationABSTRACT
An acidic homogeneous polysaccharide (HD-PS-1) was purified from Hedyotis diffusa (Willd.) Roxb. HD-PS-1 possessed a backbone chain of â[4)-ß-Glcp-3-OAc-(1]6â[6)-ß-Manp-(1]2â6)-α-Galp-(1â[4)-α-Galp-(1]2â, with three branches of ß-Manp-(1â3)-ß-GlcpA, α-Rhap-(1â3)-α-Rhap and α-Galp attached to the backbone chain at O-4 position of 1,4,6-linked ß-Manp, O-3 position of 1,3,6-linked α-Galp and O-3 position of 1,3,4-linked α-Galp, respectively. HD-PS-1 exhibited significant anticomplement activity (CH50: 0.084⯱â¯0.009â¯mg/mL, AP50: 0.176⯱â¯0.013â¯mg/mL). It was found that the presence of uronic acids is important to anticomplement activity of HD-PS-1, given that the reduced HD-PS-1 showed weaker activity (CH50: 0.456⯱â¯0.008â¯mg/mL, AP50: 0.572⯱â¯0.010â¯mg/mL). Preliminary mechanism study indicated that HD-PS-1 interacted with C3 and C4 in the complement activation cascade. In addition, a neutral homogeneous polysaccharide (HD-PS-2) was also purified and characterized. HD-PS-2 displayed antioxidant activity by scavenging DPPH· radicals without anticomplement activity.
Subject(s)
Complement System Proteins/metabolism , Hedyotis/chemistry , Polysaccharides/chemistry , Carbohydrate Sequence , Dose-Response Relationship, DrugABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. is a heat-clearing Chinese medicine and well-known for its antimalarial constituent, artemisinin. It has gained increasing attention for its anti-inflammatory and immunoregulatory activities. Interestingly, the crude polysaccahrides of A. annua exhibited potent anticomplement activity. This study was to isolate and characterize its anticomplement homogeneous polysaccharides from A. annua, and reveal the relationship between structures and anticomplement activities of the isolated polysaccharides. MATERIALS AND METHODS: Water-soluble crude polysaccharides from the aerial parts of A. annua were extracted and fractionated by DEAE-cellulose and Sephacryl S-300 gel permeation chromatography. Homogeneity, molecular weight, monosaccharide composition, methylation and NMR analysis were performed to characterize the structures of homogeneous polysaccharides. Their anticomplement activities and targeting components in the complement activation cascade were evaluated by hemolytic assays. RESULTS: Three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were obtained from A. annua. AAP01-1 was composed of seven monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. AAP01-2 and AAP01-3 had similar monosaccharides with AAP01-1, except the absence of glucuronic acid. They were all branched acidic heteropolysaccharides with different contents of galacturonic acid (8%, 28% and 15% for AAP01-1, AAP01-2 and AAP01-3, respectively). AAP01-2 showed potent anticomplement activity with CH50 value of 0.360⯱â¯0.020â¯mg/mL through the classical pathway and AP50 value of 0.547⯱â¯0.033â¯mg/mL through the alternative pathway. AAP01-3 exhibited slightly weaker activity (CH50: 1.120⯱â¯0.052â¯mg/mL, AP50: 1.283⯱â¯0.061â¯mg/mL), while AAP01-1 was inactive. Moreover, AAP01-2 acted on C1q, C3, C4, C5 and C9 components and AAP01-3 interacted with C3, C4 and C5 components in the activation cascade of complement system. CONCLUSION: These results indicated that the relatively high contents of galacturonic acid were important for anticomplement activities of the polysaccharides from A. annua. The anticomplement polysaccharides are another kind of bioactive constituents conferring heat-clearing effects of A. annua.
Subject(s)
Artemisia annua/chemistry , Complement Activation/drug effects , Complement Inactivating Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Polysaccharides/pharmacology , Animals , Biological Assay , Complement Inactivating Agents/chemistry , Complement Inactivating Agents/isolation & purification , Complement System Proteins , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Guinea Pigs , Hemolysis/drug effects , Hexuronic Acids/chemistry , Hexuronic Acids/isolation & purification , Hexuronic Acids/pharmacology , Models, Animal , Molecular Structure , Plant Components, Aerial/chemistry , Rabbits , Structure-Activity RelationshipABSTRACT
In previous studies, crude Houttuynia cordata polysaccharides showed beneficial effects on acute lung injury in vivo, a syndrome in which anti-complementary activities played an important role. Anti-complementary activity-guided fractionation of H. cordata polysaccharides led to the isolation of two highly branched homogeneous polysaccharides, HC-PS1 and HC-PS3, with a molecular weight of 274â530 and 216â384 Da, respectively. The polysaccharides were purified by chromatography on DEAE-cellulose and Superdex columns. Their structural characterization was performed by IR, GC-MS, methylation, NMR, and SEM analysis. Both HC-PS1 and HC-PS3 are composed of eight types of monosaccharides, including rhamnose, arabinose, mannose, glucose, glucuronic acid, galactose, galacturonic acid, and xylose. The main linkages of the sugar residues in HC-PS1 include terminal Rhap, terminal and 1,5-linked Araf; 1,3,6-linked and 1,4,6-linked Manp; terminal, 1,4-linked, 1,3-linked, 1,3,6-linked and 1,4,6-linked and 1,3,4,6-linked Glcp; and terminal, 1,4-linked and 1,6-linked Galp. The main monosaccharide linkages in HC-PS3 are similar to that of HC-PS1, except the additional 1,3,4-linked Manp and the absence of 1,3,6-linked Glcp. HC-PS1 and HC-PS3 were found to inhibit complement activation through both the classical and alternative pathways with 50% inhibition concentrations of 0.272â-â0.318 mg/mL without interfering with the coagulation system. Preliminary mechanism studies indicated that both HC-PS1 and HC-PS3 inhibited the activation of the complement system by interacting with C2, C4, and C5. The results suggest that HC-PS1 and HC-PS3 could be valuable for the treatment of diseases associated with the excessive activation of the complement system.