This study aims to investigate the therapeutic effects of the Coptidis Rhizoma-Scutellariae Radix on cytokine storm secondary lung injury(CSSLI) induced by CpG1826 in mice, and to elucidate the potential molecular mechanisms by which its major active components, i.e., coptisine and wogonin, alleviate CSSLI by inhibiting the mitochondrial permeability transition pore(mPTP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) inflammasome pyroptosis pathway. In vivo, a mouse model of CSSLI was established by CpG1826 induction. Pulmonary edema was assessed by lung wet-to-dry weight ratio(W/D), lung injury was evaluated by hematoxylin-eosin(HE) staining, and ultrastructural changes in lung tissue were observed by transmission electron microscopy(TEM). The levels of interleukin(IL)-1β, high mobility group box 1 protein(HMGB1), IL-18, and IL-1α in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that the decoction of the Coptidis Rhizoma-Scutellariae Radix significantly reduced pulmonary edema, alleviated lung injury, and decreased the concentrations of related cytokines in BALF more effectively than either single herb alone, thereby improving CSSLI. In vitro, a CpG1826-induced CSSLI model was established in mouse alveolar macrophage MH-S cells. Calcein-AM quenching was used to screen for the most effective monomer components from the herb pair in inhibiting mPTP opening. Coptisine(5, 10, 20 μmol·L~(-1)) and wogonin(10, 20, 40 μmol·L~(-1)) markedly inhibited mPTP opening, with optimal effects and a clear dose-dependent pattern. These components suppressed mPTP opening, thereby reducing the release of mitochondrial DNA(mtDNA) and the accumulation of reactive oxygen species(ROS), effectively reversing the CpG1826-induced decrease in mitochondrial membrane potential(MMP). Further studies revealed that both coptisine and wogonin inhibited pyroptosis and downregulated the expression of key proteins in the NLRP3/Caspase-1/gasdermin D(GSDMD) pathway. In conclusion, the Coptidis Rhizoma-Scutellariae Radix improves CpG1826-induced CSSLI in mice, and this effect is associated with the inhibition of the mPTP/NLRP3 pyroptosis pathway, providing scientific evidence for its clinical application and further development.
Animals ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Pyroptosis/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Male ; Lung Injury/immunology* ; Cytokines/immunology* ; Scutellaria baicalensis/chemistry* ; Oligodeoxyribonucleotides/adverse effects* ; Mice, Inbred C57BL ; Coptis chinensis

OBJECTIVE: The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain (RBD) recombinant protein formulated with CpG 1826 plus alum dual adjuvant. METHODS: The BA.2 variant RBD (residues 308-548) fusing TT-P ₂ epitope was obtained from prokaryotic expression system, purification technology and dialysis renaturation, which was designated as Sot protein. The soluble Sot protein formulated with CpG 1826 plus alum dual adjuvant was designated as Sot/CA subunit vaccine and then the BALB/c mice were intramuscularly administrated with two doses of the Sot/CA subunit vaccine at 14-day interval (day 0 and 14). On day 28, the number of effector T lymphocytes secreting IFN-γ and IL-4 in mice spleen were determined by enzyme-linked immunospot (ELISpot) assay. The serum IgG, IgG1 and IgG2a antibodies were examined by enzyme-linked immunosorbent assay (ELISA). In addition, the level of neutralizing antibodies (NAbs) induced by Sot/CA subunit vaccine was also evaluated by the microneutralization assay. RESULTS: The high-purity soluble Sot protein with antigenicity was successfully obtained by the prokaryotic expression, protein purification and dialysis renaturation. The Sot/CA subunit vaccine induced a high level of IgG antibodies and NAbs, which were of cross-neutralizing activity against SARS-CoV-2 BA.2 and XBB.1.5 variants. Meanwhile, Sot/CA subunit vaccine also induced a high level of effector T lymphocytes secreting IFN-γ (635.00 ± 17.62) and IL-4 (279.20 ± 13.10), respectively. Combined with a decreased IgG1/IgG2a ratio in the serum, which indicating Sot/CA subunit vaccine induced a Th1-type predominant immune response. CONCLUSION The Sot protein formulated with CpG 1826 plus alum dual adjuvant showed that the excellent cellular and humoral immunogenicity, which provided a scientific basis for the development of BA.2 variant subunit vaccines and references for the adjuvant application of subunit vaccines.
Animals ; COVID-19 Vaccines/immunology* ; Alum Compounds/pharmacology* ; Mice, Inbred BALB C ; Vaccines, Subunit/immunology* ; Mice ; SARS-CoV-2/immunology* ; Oligodeoxyribonucleotides/administration & dosage* ; Female ; Adjuvants, Immunologic ; COVID-19/immunology* ; Antibodies, Viral/blood* ; Immunogenicity, Vaccine ; Spike Glycoprotein, Coronavirus/immunology* ; Antibodies, Neutralizing/blood* ; Adjuvants, Vaccine ; Immunoglobulin G/blood*

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.
Animals ; Astrocytes/metabolism* ; DNA Demethylation ; Epigenesis, Genetic ; GATA1 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Oligodeoxyribonucleotides/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7/metabolism* ; Visceral Pain/metabolism*

To prepare AS1411 targeted nano-ultrasonic contrast agent with liquid core, and to evaluate its ability for ultrasonic contrast enhancement and targeting MCF-7 cell in vitro.
 Methods: The modified solvent evaporation, self-synthesized membrane material and perfluorobrominane (PFOB) was used to form nano-ultrasonic contrast agent with PFOB core (nanoparticles, NP); then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) catalysis was used to connect AS1411 to the surface of NP to prepare NP-AS1411. The transmission electron microscopy was chosen to check the morphology of NP-AS1411. The size, surface charge, encapsulation efficiency, biocompatibility, the contrast grey value and the stability of NP-AS1411 and NP were compared. Whether AS411 was attached to the surface of NP was checked by gel electrophoresis. Fluorescence microscopy and flow cytometry were performed to examine the targeting ability of AS1411.
 Results: NP-AS1411 was a shell-nuclear structure under the electron microscope. Its size was at (245.4±16.5) nm, which was larger than that of NP (P=0.05). There was no significant difference in surface charge and encapsulation efficiency between NP-AS1411 and NP (P>0.05). In the MTT experiment, the cell viability decreased significantly at high concentration of NP-AS411 (25 mg/mL) after incubation for 24 h compared with the control group (0 mg/mL ) (P<0.05). The contrast gray value of NP-AS1411 was at 86.1+ 6.7, which was significantly higher than that of deionized water (P<0.05), and equivalent to that of NP (P>0.05). The contrast grey value of AS1411-NP was 80.1±9.2 after keeping at room temperature for 24 h, which showed no obviously change comparing with that before the treatment (P>0.05). The size of NP-AS1411didn't change too (P>0.05). The results of gel electrophoresis demonstrated that the AS1411 connecting to the surface of NP was the most when the molar ratio of NP:AS1411 was at 40:1. Flow cytometry analysis confirmed that NP and NP-AS1411 were combined with MCF-7 cells separately but the fluorescence produced by the combination of NP-AS1411 and MCF-7 was more intense.
 Conclusion: The modified solvent evaporation and EDC/NHS catalysis could successfully prepare ultrasound contrast agents with aptamer-conjugated nanoparticles with liquid core. The targeted ultrasonic contrast agents with liquid core possess good ultrasonic contrast enhancement ability in vitro, stability and specificity as well.
Cell Survival ; Contrast Media ; chemical synthesis ; Fluorocarbons ; Humans ; MCF-7 Cells ; Microscopy, Electron, Scanning Transmission ; Nanoparticles ; chemistry ; ultrastructure ; Oligodeoxyribonucleotides ; chemical synthesis

OBJECTIVETo investigate the effects of adipose-derived stem cells (ADSC) and non-methylated CpG-oligodeoxynucleotides (CpG-ODN) on the expression of peripheral blood CD4CD25regulatory T (Treg) cells in young mice with food allergy, as well as their immune intervention effects.

METHODSA total of 40 female BALB/c mice were randomly divided into control group, allergic group, ADSC treatment group, and CpG-ODN treatment group, with 10 mice in each group. A mouse model of food allergy was established by intraperitoneal injection and intragastric administration of ovalbumin (OVA) for sensitization and challenge. The mice in the control group were treated with normal saline at the same dose; the mice in the ADSC treatment group were given intraperitoneal injection of ADSC (1×10cells for each mouse) before and after OVA challenge, and those in the CpG-ODN treatment group were given intraperitoneal injection of non-methylated CpG-ODN solution (40 μg for each mouse) at 1 hour before challenge by gavage. The allergic symptom scores were determined for each group after model establishment. ELISA was used to measure the serum level of OVA-IgE. Flow cytometry was used to measure the percentage of peripheral blood CD4CD25Treg cells. Hematoxylin and eosin staining was used for the pathological analysis of the jejunum.

RESULTSThe allergic group had significantly higher allergic symptom scores and serum level of OVA-IgE than the control group (P<0.05). There were no significant differences in the allergic symptom score and the serum level of OVA-IgE between the ADSC treatment group and the CpG-ODN treatment group (P>0.05), but these two groups had significantly lower allergic symptom scores and serum level of OVA-IgE than the allergic group and significantly higher allergic symptom scores and serum level of OVA-IgE than the control group (P<0.01). The allergic group had a significantly lower percentage of peripheral blood CD4CD25Treg cells than the control group (P<0.05). The ADSC treatment group and the CpG-ODN treatment group had a significantly higher percentage of peripheral blood CD4CD25Treg cells than the allergic group (P<0.05); there were no significant differences between these two groups or between them and the control group (P>0.05). Pathological results showed structural damage and edema in the jejunal villi, a large number of eosinophils, and lymphocyte infiltration in the allergic group, while the ADSC treatment group and the CpG-ODN treatment group had less structural damage and edema in the jejunal villi, a lower number of eosinophils, and less lymphocyte infiltration.

CONCLUSIONSADSC and non-methylated CpG-ODN have a certain effect in the treatment of food allergy and can increase the percentage of peripheral blood CD4CD25Treg cells and reduce the level of OVA-IgE. They may be associated with the induction of immune tolerance and these two treatment have comparable effects. Detailed mechanisms of action still need further investigation.

Adipose Tissue ; cytology ; Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; Food Hypersensitivity ; immunology ; therapy ; Immunoglobulin E ; blood ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides ; pharmacology ; Ovalbumin ; immunology ; Stem Cell Transplantation ; T-Lymphocytes, Regulatory ; drug effects ; immunology

PURPOSE: It remains unknown whether local inhibition of Nuclear factor-kappa B (NF-κB) could have therapeutic value in the treatment of allergic rhinitis (AR). This study aimed to evaluate the effect of selective NF-κB inhibition using NF-κB decoy oligodeoxynucleotides (ODNs) for the local treatment of AR in ovalbumin (OVA)-sensitized wild-type mice. METHODS: BALB/c mice were sensitized with OVA and alum, and then challenged intranasally with OVA. NF-κB decoy ODNs were given intranasally to the treatment group, and NF-κB scrambled ODNs were given to the sham treatment group. Allergic symptom scores, eosinophil infiltration, cytokine levels in the nasal mucosa, nasal lavage fluid, and spleen cell culture, serum total and OVA-specific immunoglobulins, as well as intercellular adhesion molecure-1 (ICAM-1) in the nasal mucosa, were analyzed. RESULTS: NF-κB decoy ODNs significantly reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. They also suppressed serum levels of total IgE, OVA-specific IgE, and IgG1. IL-5 and TNF-α levels and the expression of ICAM-1 were decreased in the nasal mucosa of the treatment group compared to the positive control and sham treatment groups. In addition, IL-6 levels were significantly decreased in the nasal lavage fluid of the treatment group. Furthermore, NF-κB decoy ODNs significantly reduced expression of the systemic Th2 cytokines, IL-4 and IL-5 in spleen cell culture. CONCLUSIONS: This study demonstrates for the first time that local NF-κB inhibition using NF-κB decoy ODNs suppressed the allergic response in a murine AR model. This shows the therapeutic potential of local NF-κB inhibition in the control of AR.
Animals ; Anti-Allergic Agents ; Cell Culture Techniques ; Cytokines ; Eosinophils ; Immunoglobulin E ; Immunoglobulin G ; Immunoglobulins ; Intercellular Adhesion Molecule-1 ; Interleukin-4 ; Interleukin-5 ; Interleukin-6 ; Mice ; Nasal Lavage Fluid ; Nasal Mucosa ; NF-kappa B ; Oligodeoxyribonucleotides* ; Ovalbumin ; Ovum ; Placebos ; Rhinitis, Allergic* ; Spleen

OBJECTIVEThis study aims to synthesize MTO1 (a kind of oligodeoxynucleotides) and N-isopropylacrylamide-modified polyethylenimines (PEN) complexes (MT01/PEN) as well as to investigate the effect of the complexes on the expression of osteoprotegerin (OPG) and the receptor activator of nuclear factor κB ligand (RANKL) in the human osteoblast-like cell line MG63.

METHODSMG63 cells were transfected by MT01/PEN complexes formed with three different mass ratios (1:2, 1:4, 1:6) of MT01 to PEN. MT01 and MT01-s were used as positive control. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were performed to estimate the amount of OPG and RANKL released into the culture media and in MG63 at 24, 48, 72 h.

RESULTSMG63 responded to the MT01/PEN complexes by significantly upregulating the OPG on the protein and mRNA levels (P < 0.05). The protein and mRNA levels of RANKL were lower in most of the groups with complexes, and the OPG/RANKL ratio were higher (P < 0.05). MG63 were affected by the MT01/PEN complexes with different mass ratios, particularly when the ratio was 1:6.

CONCLUSIONMT01 can enhance the promotion of ossification by establishing the delivery system with PEN.

Acrylamides ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oligodeoxyribonucleotides ; Osteoblasts ; Osteoprotegerin ; Polyethyleneimine ; RANK Ligand ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

OBJECTIVEThe present study was to evaluate the effects of nuclear factor of kappa B decoy oligodeoxynucleotides on murine multiple myeloma models.

METHODSThe severe combined immunodeficient mice were injected subcutaneously with RPMI-8226 myeloma cells. When tumors became measurable, the mice were divided into 2 treatment groups who respectively received 5 µg/g or 10 µg/g liposome-NF-κB decoy ODN compounds, and one control group was selected; the control group received 10 µg/g liposome-NF-κB mutant decoy ODN compounds, twice per week for 4 weeks. The mice were killed when they died or the tumor diameter became >2 cm.

RESULTSThe liposome-NF-κB decoy ODN could efficiently suppress NF-κB DNA binding activity and inhibited the expression of IL-6. As compared with the control group, the two liposome-NF-κB decoy ODN-treated groups showed more remarkably survival time and smaller tumor volume.

CONCLUSIONIn vivo transfection of NF-κB decoy ODN may provide a new therapeutic strategy for multiple myeloma.

Animals ; DNA ; Disease Models, Animal ; Genetic Therapy ; Interleukin-6 ; Liposomes ; Mice ; Multiple Myeloma ; Oligodeoxyribonucleotides ; Transfection

OBJECTIVETo explore the effect of non-methylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) on serum transforming growth factor (TGF)-β and immune regulation in ovalbumin (OVA)-sensitized young mice.

METHODSThirty female BALB/c mice (2-3 weeks old) were randomly divided into control, model, and CpG-ODN intervention groups. A young mouse model of food allergy was established by OVA sensitization. Normal saline of the same volume was used for replacement in the control group. The mice in the intervention group were intraperitoneally injected with CpG-ODN solution 1 hour before every OVA sensitization. Allergic symptoms were observed and scored for each group. The jejunal tissue was histopathologically examined with hematoxylin-eosin staining. Serum OVA-IgE level was measured using ELISA. Serum concentrations of interleukin (IL)-4, interferon (IFN)-γ, and TGF-β were determined by CBA.

RESULTSAllergic symptoms were observed in the model group and the jejunal tissue showed the pathological characteristics of type I allergic reaction. The allergic symptom scores in the model and CpG-ODN intervention groups were significantly higher than in the control group (P<0.01). The serum levels of OVA-IgE, IL-4, and TGF-β were significantly higher in the model group than in the control and CpG-ODN intervention groups (P<0.05). The CpG-ODN intervention group had significantly higher serum levels of OVA-IgE, IL-4, and TGF-β than the control group (P<0.05). Compared with the control and CpG-ODN intervention groups, the model group had a significantly reduced IFN-γ level (P<0.05).

CONCLUSIONSThe serum TGF-β level is increased in the young mouse model of OVA-sensitized food allergy and is involved in the allergy mechanism. Non-methylated CpG-ODN can reduce the serum TGF-β level in sensitized young mice and play an immunoregulatory role in food allergy.

Aging ; Animals ; DNA Methylation ; Female ; Food Hypersensitivity ; drug therapy ; immunology ; Immunoglobulin E ; blood ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides ; pharmacology ; Ovalbumin ; immunology ; Transforming Growth Factor beta ; blood

OBJECTIVEThis aimed to investigate the effect of specific sequence oligodeoxynucleotide MT01 on the biological properties of osteoblasts invaded by Porphyromonas gingivalis (P. gingivalis ) by evaluating proliferation, cell cycle, and apoptosis.

METHODSMG63 osteoblasts were recovered and incubated with MT01, CpG ODN, metronidazole (MNZ), and gentamicin (GEN) for 3 h. P. gingivalis (the multiplicity of infection was 100:1) was added subsequently and cocultured for another 24 and 48 h. Cells with PBS comprised the blank group, whereas cells with P. gingivalis comprised the negative controls. Six experimental groups were established: PBS group, P. gingivalis group, MT01+P. gingivalis group, CpG ODN+ P. gingivalis group, MNZ+P. gingivalis group, and GEN+P. gingivalis group. The proliferative ability was measured by methyl thiazolyl tetrazolium assay, and the percentages of apoptosis and cell cycle were examined by flow cytometry.

RESULTSCompared with the blank group, proliferation increased significantly in the MT01+P. gingivalis group (P < 0.05). The ratio of cells was lower at the G₁ phase and higher at the S phase in the MT01+P. gingivalis group compared with the results in the P. gingivalis group (P < 0.05). Early cell apoptosis in the MT01+P. gingivalis group was significantly lower than that in the P. gingivalis group (P < 0.05).

CONCLUSIONMT01 can promote the proliferation, reduce the ratio of the G₁phase, increase the ratio of the S phase, and inhibit the early apoptosis of osteoblasts invaded by P. gingivalis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gentamicins ; pharmacology ; Humans ; Metronidazole ; pharmacology ; Oligodeoxyribonucleotides ; pharmacology ; Osteoblasts ; cytology ; drug effects ; Porphyromonas gingivalis ; pathogenicity