Objective To explore the aptamer specific binding blood group antigen-binding adhesin (BabA) of Helicobacter pylori (H.pylori) for blocking of H.pylori adhering host cell. Methods H.pylori strain was cultured and its genome was extracted as templates to amplify the BabA gene by PCR with designed primers. The BabA gene obtained was cloned and constructed into prokaryotic expression plasmid, which was induced by isopropyl beta-D-galactoside (IPTG) and purified as target. The single stranded DNA (ssDNA) aptamers that specifically bind to BabA were screened by SELEX. Enzyme-linked oligonucleotide assay (ELONA) was used to detect and evaluate the characteristics of candidate aptamers. The blocking effect of ssDNA aptamers on H.pylori adhesion was subsequently verified by flow cytometry and colony counting at the cell level in vitro and in mouse model of infection, respectively. Meanwhile, the levels of cytokines, interleukin 6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), IL-10 and IL-4 in the homogenate of mouse gastric mucosa cells were detected by ELISA. Results The genome of H.pylori ATCC 43504 strains was extracted and the recombinant plasmid pET32a-BabA was constructed. After induction and purification, the relative molecular mass (M_r) of the recombinant BabA protein was about 39 000. The amino acid sequence of recombinent protein was consistent with BabA protein by peptide mass fingerprint (PMF). Five candidate aptamers were selected to bind to the above recombinent BabA protein by SELEX. The aptamers A10, A30 and A42 identified the same site, while A3, A16 and the above three aptamers identified different sites respectively. The aptamer significantly blocked the adhesion of H.pylori in vitro. Animal model experiments showed that the aptamers can block the colonization of H.pylori in gastric mucosa by intragastric injection and reduce the inflammatory response. The levels of IL-4, IL-6, IL-8 and TNF-α in gastric mucosal homogenates in the model group with aptamer treatment were lower than that of model group without treatment. Conclusion Aptamers can reduce the colonization of H.pylori in gastric mucosa via binding BabA to block the adhesion between H.pylori and gastric mucosal epithelial cells.
Animals ; Mice ; Helicobacter pylori/genetics* ; Interleukin-4 ; Interleukin-6 ; Interleukin-8 ; Tumor Necrosis Factor-alpha ; Stomach ; Oligonucleotides ; Adhesins, Bacterial/genetics* ; Blood Group Antigens

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.
Animals ; Cattle ; Male ; Rats ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Body Weight ; Codonopsis/chemistry* ; Interleukin-6/blood* ; NF-kappa B/genetics* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Plant Extracts/therapeutic use* ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/pharmacology*

OBJECTIVE: To investigative the detection value of EB virus DNA (EBV-DNA), interleukin-2 (IL-2), and interleukin-6 (IL-6) level in peripheral blood of children with infectious mononucleosis (IM). METHODS: A total of 59 children clinically confirmed with IM in Anhui Provincial Children's Hospital from January 2018 to September 2020 were enrolled as IM group, while other 30 healthy children undergoing physical examination during the same period were enrolled as healthy group. The level of EBV-DNA load, IL-2, and IL-6 were compared between the two groups, and their diagnostic values for IM children were explored. According to the median level of EBV-DNA load, positive children were divided into high viral load group and low viral load group. The hepatomegaly and splenomegaly, and levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-2, and IL-6 were compared between the two groups. The relationship between EBV-DNA load and IL-2, IL-6 levels were explored. RESULTS: The positive rate of EBV-DNA was 67.80% in IM group, which was significantly higher than 10.00% in healthy group (P<0.001), and the levels of serum IL-2 and IL-6 were also significantly higher than healthy group (P<0.001). The results of ROC curve analysis showed that AUC of IL-2 combined with IL-6 and EBV-DNA load was 0.948, which was significantly greater than that of IL-2, IL-6, and EBV-DNA load alone (0.847, 0.728, 0.789) (P<0.001). The cut-off value of IL-2 and IL-6 was 15.545 pg/ml and 56.560 pg/ml, respectively. Both the proportions of cases with moderate to severe hepatomegaly and splenomegaly in high viral load group were significantly higher than those in low viral load group (P<0.01, P<0.05). The levels of ALT, AST, and IL-2 in high viral load group were significantly higher than those in low viral load group (P<0.001), as well as IL-6 (P<0.01). In high and low viral load groups, EBV-DNA load was positively correlated with IL-2 and IL-6 (in high viral load group, r_IL-2=0.598, r_IL-6=0.416; in low viral load group, r_IL-2=0.621, r_IL-6=0.527, P<0.001). CONCLUSION The detection of EBV-DNA load combined with IL-2 and IL-6 can improve the diagnostic accuracy of IM, and EBV-DNA load, IL-2 and IL-6 levels are related to the disease progression.
Child ; DNA, Viral ; Epstein-Barr Virus Infections ; Hepatomegaly ; Herpesvirus 4, Human ; Humans ; Infectious Mononucleosis/diagnosis* ; Interleukin-2/blood* ; Interleukin-6/blood* ; Splenomegaly

The aim of this paper was to observe the effect of Di'ao Xinxuekang(DXXK) on TLR4/MyD88/NF-κB signaling pathway in atherosclerotic rats, and to explore its anti-atherosclerotic mechanism. Sixty SD rats were randomly divided into normal group, model group, atorvastatin group(4.0 mg·kg~(-1)), and DXXK groups(100, 30, 10 mg·kg~(-1)), with 10 rats in each group. The atherosclerosis model was induced by high fat diet plus vitamin D_2. Experimental drugs were administered intragastrically once daily for 8 weeks starting from the 9 th week. Biochemical analyzers were used to detect levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) in blood lipid. The levels of serum tumor necrosis factor(TNF)-α, interleukin(IL)-6 and IL-1β were detected by ELISA. Pathological changes of aortic tissues were observed by using Sudan Ⅳ and HE staining. The mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 in aortic tissues were detected by RT-PCR and Western blot, respectively. As compared with the model group, TC, TG, and LDL-C levels in serum were significantly decreased, HDL-C content was significantly increased, and levels of TNF-α, IL-6, and IL-1β in serum were significantly decreased in atorvastatin group and DXXK high and middle dose groups. Aortic lesions in atorvastatin group and DXXK group were significantly improved, and the mRNA and protein expressions of TLR4, MyD88, NF-κB p65 in the aorta were decreased. DXXK has a preventive and therapeutic effect on atherosclerosis in rats, and its mechanism may be related to inhibiting inflammatory reaction by regulating TLR4/MyD88/NF-κB signal transduction, thereby inhibiting the progression of atherosclerosis.
Animals ; Aorta/pathology* ; Atherosclerosis/drug therapy* ; Atorvastatin ; Drugs, Chinese Herbal/pharmacology* ; Interleukin-6/blood* ; Interleukin-8/blood* ; Lipids/blood* ; Myeloid Differentiation Factor 88/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 4/metabolism* ; Transcription Factor RelA/metabolism* ; Tumor Necrosis Factor-alpha/blood*

This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Chalcone ; analogs & derivatives ; pharmacology ; Creatine Kinase ; blood ; Interleukin-6 ; metabolism ; L-Lactate Dehydrogenase ; blood ; Male ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Wistar ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.
Animals ; Cathepsin B ; antagonists & inhibitors ; physiology ; Dipeptides ; pharmacology ; Gene Knockout Techniques ; Hepatocytes ; Inflammation ; metabolism ; Interleukin-18 ; blood ; Interleukin-1alpha ; blood ; Interleukin-1beta ; blood ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; pathology ; Mice ; Sepsis ; etiology ; metabolism ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; blood

Indoor animal husbandry environments are inevitably contaminated with endotoxins. Endotoxin exposure is associated with various inflammatory illnesses in animals. This cross-sectional study evaluated the relationship between the degree of endotoxin exposure and the cellular and humoral immune profiles of fattening pigs. Blood samples were taken from the jugular vein of 47 pigs from ten pig farms in Korea. Whole blood cell counts and plasma immunoglobulin (Ig) classes were determined. Peripheral-blood mononuclear cells were stimulated in vitro with concanavalin A for 48 h, and cytokines released into culture supernatants were measured. The barns in which the pigs lived were assessed for endotoxin levels in the total and respirable dust by using the limulus amebocyte lysate kinetic QCL method. Low and high endotoxin exposures were defined as ≤ 30 and > 30 EU/m³, respectively. Compared to pigs with low endotoxin exposure (n = 19), highly exposed pigs (n = 28) had higher circulating neutrophil and lymphocyte (particularly B cells) counts, IgG and IgE levels, interferon-gamma (IFNγ) and interleukin (IL)-4 productions, and lower IgA levels and tumor necrosis factor-alpha (TNFα) production. The IL-4, IFNγ, and TNFα levels significantly correlated with endotoxin level and/or pig age. Constant exposure of pigs to high levels of airborne endotoxins can lead to aberrant immune profiles.
Agriculture ; Animal Husbandry ; Animals ; Blood Cell Count ; Concanavalin A ; Cross-Sectional Studies ; Cytokines ; Dust ; Endotoxins ; Horseshoe Crabs ; Housing ; Immunity, Cellular ; Immunoglobulin A ; Immunoglobulin E ; Immunoglobulin G ; Immunoglobulins ; In Vitro Techniques ; Interferon-gamma ; Interleukin-4 ; Interleukins ; Jugular Veins ; Korea ; Lymphocytes ; Methods ; Neutrophils ; Plasma ; Swine ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the effect of leukodepleted blood transfusions on peripheral blood Th1/Th2 cell balance in patients with acute lymphoblastic leukemia (ALL).

METHODSFifty-seven ALL patients in our hospital from March 2016 to August 2017 were selected, 31 of them received routine blood transfusion were enrolled in group A, and 26 patients received depleted-blood leukotransfusion were enrolled in group B, 36 cases in normal physical examination at the same period were enrolled in control group. And the basic clinical characteristics of patients were recorded; the ratio of Th1/Th2 cells in peripheral blood of patients was analyzed by flow cytometry;the serum levels of IL-2, IFN-γ, IL-4, IL-10 were detected by ELISA method; the mRNA levels of T-bet and GATA-3 in lymphocytes were detected by RT-PCR;the protein levels of T-bet and GATA-3 in lymphocyte were detected by Western blot.

RESULTSThe Th1/Th2 ratio in peripheral blood of ALL patients significantly related with patient age and risk grade (P<0.05).After treatment,the change of Th1/Th2 ratio in group A showed no statistical difference from Th1/Th2 ratio before treatment (P>0.05), while the Th1/Th2 ratio in group B increased (P<0.05);the levels of IL-2, IFN-γ, IL-4 and IL-10 secreted from Th1 and Th2 cells of ALL patients in A group were not changed significantly(P>0.05), while the levels of IL-2 and IFN-γ secreted from Th1 cells of ALL patients in group B increased, the levels of IL-4 and IL-10 secreted from Th2 cells in group B decreased with statistical difference (P<0.05); the RT-PCR and Western blot showed that the expression levels of T-bet mRNA and T-bet protein in group A were lower than those in control group, while the expression levels of T-bet mRNA and T-bet protein in group B were higher than those in group A (P<0.05); the expression levels of mRNA and GATA-3 protein in group A were higher than those in control group, the expression levels of mRNA and GATA-3 protein in group B were lower than those in group A (P<0.05).

CONCLUSIONThe leukoreduced blood transfusion helps to improve the balance of Th1/Th2 cells in peripheral blood and improve the immune function of patients, which may closely relate with the expression levels of T-bet and GATA-3.

Blood Transfusion ; GATA3 Transcription Factor ; Humans ; Interferon-gamma ; Interleukin-4 ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Th1 Cells ; Th2 Cells

The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN) in vivo of lipopolysaccharide (LPS)-treated rat. Tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral DA neurons at 3 and 7 day post-LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 day and remained elevated at 7 day post-LPS. IL-4 immunoreactivity was detected exclusively in microglia. IL-4 neutralizing antibody (NA) significantly increased survival of DA neurons in LPS-treated SN in vivo by inhibiting microglial activation and production of proinflammatory mediator such as IL-1β as assessed by immunihistochemical, RT-PCR and ELISA analysis, respectively. Accompanying neuroprotection are IL-4NA effects on decreased disruption of blood-brain barrier and astrocytes. The present data suggest that endogenously expressed IL-4 from reactive microglia may be involved in the neuropathological processes of degeneration of DA neurons occurring in Parkinson's disease.
Animals ; Antibodies, Neutralizing ; Astrocytes ; Blood-Brain Barrier ; Dopamine* ; Dopaminergic Neurons* ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Interleukin-4* ; Interleukins ; Lipopolysaccharides ; Microglia ; Neurons ; Neuroprotection ; Parkinson Disease ; Rats ; Substantia Nigra* ; Tyrosine 3-Monooxygenase

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology