Exploring the immune-inflammatory mechanism of Maxing Shigan Decoction in treating influenza virus A-induced pneumonia based on an integrated strategy of single-cell transcriptomics and systems biology.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza virus. Maxing Shigan Decoction (MXSGD) is a commonly used traditional Chinese medicine prescription for the prevention and treatment of influenza. However, its mechanism remains unclear. METHOD: The mice model of influenza A virus pneumonia was established by nasal inoculation. After 3 days of intervention, the lung index was calculated, and the pathological changes of lung tissue were detected by HE staining. Firstly, transcriptomics technology was used to analyze the differential genes and important pathways in mouse lung tissue regulated by MXSGD. Then, real-time fluorescent quantitative PCR (RT-PCR) was used to verify the changes in mRNA expression in lung tissues. Finally, intestinal microbiome and intestinal metabolomics were performed to explore the effect of MXSGD on gut microbiota. RESULTS: The lung inflammatory cell infiltration in the MXSGD group was significantly reduced (p < 0.05). The results of bioinformatics analysis for transcriptomics results show that these genes are mainly involved in inflammatory factors and inflammation-related signal pathways mediated inflammation biological modules, etc. Intestinal microbiome showed that the intestinal flora Actinobacteriota level and Desulfobacterota level increased in MXSGD group, while Planctomycetota in MXSGD group decreased. Metabolites were mainly involved in primary bile acid biosynthesis, thiamine metabolism, etc. This suggests that MXSGD has a microbial-gut-lung axis regulation effect on mice with influenza A virus pneumonia. CONCLUSION: MXSGD may play an anti-inflammatory and immunoregulatory role by regulating intestinal microbiome and intestinal metabolic small molecules, and ultimately play a role in the treatment of influenza A virus pneumonia.

Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-induced ferroptosis.

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide. Influenza A virus (IAV) has been found to activate multiple programmed cell death pathways, including ferroptosis. Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation, leading to cell death. However, little is known about how influenza viruses induce ferroptosis in the host cells. In this study, based on network pharmacology, we predicted the mechanism of action of Maxing Shigan decoction (MXSGD) in IAV-induced ferroptosis, and found that this process was related to biological processes, cellular components, molecular function and multiple signaling pathways, where the hypoxia inducible factor-1(HIF-1) signaling pathway plays a significant role. Subsequently, we constructed the mouse lung epithelial (MLE-12) cell model by IAV-infected in vitro cell experiments, and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage, increased reactive oxygen species (ROS) release, increased total iron and iron ion contents, decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4 (GPX4), increased expression of acyl-CoA synthetase long chain family member 4 (ACSL4), and enhanced activation of hypoxia inducible factor-1α (HIF-1α), induced nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in the HIF-1 signaling pathway. Treatment with MXSGD effectively reduced intracellular viral load, while reducing ROS, total iron and ferrous ion contents, repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway. Finally, based on animal experiments, it was found that MXSGD effectively alleviated pulmonary congestion, edema and inflammation in IAV-infected mice, and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

Research on Pachymaran to Ameliorate CsA-Induced Immunosuppressive Lung Injury by Regulating Microflora Metabolism.

Pachymaran (PCP), the major medicinal constituent of Poria cocos, has a regulatory effect on immunosuppressive lung injury, but its mechanism of action with respect to gut microorganisms and their metabolites is not clear. The aim of this study was to investigate the protective effect of PCP against immunosuppressive lung injury caused by cyclosporine A (CsA), and to reveal its possible mechanism of action via the comprehensive analysis of 16S rRNA and LC-MS. We demonstrated that PCP was effective at alleviating CsA-induced immunosuppressive lung injury by restoring the organ indices and lung tissue morphology and structure. PCP significantly altered the composition of the gut and lung microbiota in mice with CsA-induced immunosuppressive lung injury by increasing the number of beneficial bacteria from the Eubacterium nodatum group, Eubacterium ventriosum group, Akkermansia, and Ruminococcus, and reducing the pathogenic Rikenellaceae RC9 gut group to fulfill its immunomodulatory role. In lung tissue microecology, PCP intervention significantly reduced the abundance of Chryseobacterium, Lawsonella, Paracoccus, and Sediminibacterium and increased the abundance of Alloprevotella. The LC-MS results showed that PCP alleviated the CsA-induced immunosuppression of lung tissue injury. The model serum metabolite Americine decreased the expression of PC(O-18:1(4Z)/0:0). Our results suggest that PCP may be involved in regulating the composition, function, and metabolism of the gut and lung microbiota to reverse CsA-induced immunosuppressive lung injury.

Non-coding RNAs in breast cancer: with a focus on glucose metabolism reprogramming.

Breast cancer is the tumor with the highest incidence in women worldwide. According to research, the poor prognosis of breast cancer is closely related to abnormal glucose metabolism in tumor cells. Changes in glucose metabolism in tumor cells are an important feature. When sufficient oxygen is available, cancer cells tend to undergo glycolysis rather than oxidative phosphorylation, which promotes rapid proliferation and invasion of tumor cells. As research deepens, targeting the glucose metabolism pathway of tumor cells is seen as a promising treatment. Non-coding RNAs (ncRNAs), a recent focus of research, are involved in the regulation of enzymes of glucose metabolism and related cancer signaling pathways in breast cancer cells. This article reviews the regulatory effect and mechanism of ncRNAs on glucose metabolism in breast cancer cells and provides new ideas for the treatment of breast cancer.

A graphene-oxide-based fluorometric assay for norA gene transcription in MRSA using Nb.BbvCI-assisted target recycling and T7 exonuclease-triggered cascade dual recycling signal amplification.

We describe a graphene oxide (GO)-based bioassay for the fluorometric determination of norA gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This approach is based on Nb.BbvCI-assisted target recycling (NATR) and T7 exonuclease (T7 Exo)-triggered cascade dual-recycling signal amplification (TTCDRSA). The system included GO, a capture probe (CP), an assistant probe (AP), two carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP2), endonuclease Nb.BbvcI, and exonuclease T7. In the presence of a target, AP, together with the target RNA, can hybridise with CP via partial complementarity to one another and open its hairpin structure to form a triple complex that is recognised by Nb.BbvCI. Once the CP is cleaved, the released AP and target RNA can walk on the carboxylated graphene oxide (CGO) surface to bind with another CP which induces the next round of cleavage, accumulating many trigger probes (TPs). The TPs then activate TTCDRSA with the assistance of T7 Exo, HP1, and HP2 to produce large amounts of free FAMs. These free FAMs are repelled by GO and exhibit enhanced fluorescence signals at excitation/emission wavelengths of 480/514 nm. The limit of detection (LOD) of the bioassay was calculated to be 0.37 fM, and the linear range of the method ranged from 1 fM to 1 nM. More importantly, the bioassay also exhibited high sensitivity and selectivity for target RNA detection in real samples, which may open a new promising avenue for monitoring drug efflux and studying the mechanisms of drug actions.

Immune infiltration patterns and identification of new diagnostic biomarkers GDF10, NCKAP5, and RTKN2 in non-small cell lung cancer.

This study aimed to identify potential biomarkers for non-small cell lung cancer (NSCLC) and analyze the role of immune cell infiltration in NSCLC. R software was used to screen differentially expressed genes (DEGs) from NSCLC datasets obtained from the Gene Expression Omnibus (GEO) database, and functional correlation analysis was performed. The machine learning algorithms were used to screen the potential biomarkers of NSCLC. The diagnostic values were assessed through receiver operating characteristic (ROC) curves. The protein and mRNA expression levels of potential biomarkers were verified based on the Human Protein Atlas (HPA) database and qRT-PCR. CIBERSORT was used to evaluate the infiltration of immune cells in NSCLC tissues, and the correlation between potential biomarkers and infiltrated immune cell was analyzed. Finally, specific siRNAs were utilized to reduce the GDF10, NCKAP5, and RTKN2 expression in A549 and H1975 cells. The proliferation ability of A549 and H1975 cells was detected by MTT assay. A total of 848 upregulated DEGs and 1308 downregulated DEGs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were mainly related to cell division. Disease ontology (DO) enrichment analysis showed that the diseases with these DEGs were mainly lung diseases, including NSCLC. In addition,three potential biomarkers were identified: GDF10, NCKAP5, and RTKN2. Immune cell infiltration analysis showed that resting NK cells, activated dendritic cells, and Tregs may be involved in the pathogenesis of NSCLC. Meanwhile, GDF10, NCKAP5, and RTKN2 were negatively correlated with Tregs and naïve B cells but were positively correlated with activated dendritic cells and resting NK cells. Immunohistochemical staining showed that the expression of GDF10, NCKAP5, and RTKN2 in the lung tissue of patients with NSCLC was lower than that of normal lung tissue. qRT-PCR also confirmed that the mRNA expression of three biomarkers in NSCLC cell lines A549 and H1975 were significantly lower than those in human normal lung epithelial cells BEAS-2B. An MTT assay showed that GDF10, NCKAP5, and RTKN2 knockdown significantly promoted the proliferation of A549 and H1975 cells. The in vitro experiments showed that GDF10, NCKAP5, and RTKN2 played the inhibitory effects on NSCLC cell lines proliferation. Hence, GDF10, NCKAP5, and RTKN2 can be used as diagnostic biomarkers for NSCLC.

A graphene-oxide-based aptasensor for fluorometric determination of chloramphenicol in milk and honey samples utilizing exonuclease III-assisted target recycling and Nb.BbvCI-powered DNA walker cascade amplification.

Herein, a graphene oxide (GO)-based fluorescence aptasensor was developed for the sensitive and selective detection of chloramphenicol (CAP), based on exonuclease III (Exo III)-assisted target recycling and Nb.BbvCI-driven DNA walker cascade amplification. Interactions between CAP, hairpin1(HP1), hairpin2 (HP2), and 3'-amino modified hairpin3 (HP3) labeled with carboxyfluorescein (FAM) and covalently coupled to GO enabled efficient CAP detection. CAP was quantitatively assayed by measuring fluorescence at excitation/emission wavelengths of 480/514 nm, resulting from the accumulation of released FAM. A good linear range of 1 fM to 1 nM and a limit of detection (LOD) of 0.875 fM (signal-to-noise (S/N)= 3) were achieved. This aptasensor can distinguish the CAP from interference antibiotics with good specificity and selectivity, even if the concentration of the interfering substance is ten-fold higher than the target concentration. Moreover, the developed fluorescence aptasensor was successfully applied for the detection of CAP in spiked milk and honey samples. Thus, this method is potentially applicable for assaying CAP in foods and provides a promising strategy for the development of fluorescence aptasensors for environmental sample analysis.

[Maxing Shigan Decoction improves lung and colon tissue damage caused by influenza virus infection through JAK1/2-STAT1 signaling pathway].

Based on Janus kinase 1/2-signal transducer and activator of transcription 1(JAK1/2-STAT1) signaling pathway, this study explored the immune mechanism of Maxing Shigan Decoction in alleviating the lung tissue and colon tissue damage in mice infected with influenza virus. The influenza virus infection was induced in mice by nasal drip of influenza virus. The normal group, model group, oseltamivir group, antiviral granule group, and Maxing Shigan Decoction group were designed. After intragastric administration of corresponding drugs or normal saline for 3 or 7 days, the body mass was measured, and lung index, spleen index, and thymus index were calculated. Based on hematoxylin-eosin(HE) staining, the pathological changes of lung tissue and colon tissue were observed. Enzyme-linked immunosorbent assay(ELISA) was used to detect serum levels of inflammatory factors interleukin-8(IL-8) and interferon-γ(IFN-γ), Western blot and real-time quantitative polymerase chain reaction(RT-qPCR) to determine the protein and mRNA levels of JAK1, JAK2, STAT1, interferon regulatory factor 9(IRF9), and IFN-γ in lung tissue and colon tissue. The results showed that after 3 and 7 days of administration, the body mass, spleen index, and thymus index were lower(P<0.05 or P<0.01), and the lung index was higher(P<0.01) in the model group than in the normal group. Moreover, the model group showed congestion, edema, and infiltration of a large number of lymphocytes and macrophages in the lung tissue, irregular structure of colon mucosa, ulceration and shedding of epithelial cells, and infiltration of a large number of inflammatory cells. The model group had higher levels of serum IFN-γ(P<0.01), higher protein and mRNA expression of JAK1, JAK2, STAT1, IRF9, IFN-γ in lung tissue(P<0.05 or P<0.01), higher level of JAK2 protein in colon tissue(P<0.01), and higher protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) than the normal group. Compared with the model group, Maxing Shigan Decoction group had high body mass, spleen index, and thymus index(P<0.05 or P<0.01), low lung index(P<0.05 or P<0.01), and significant alleviation of pathological injury in lung and colon. Moreover, lower serum level of IFN-γ(P<0.05 or P<0.01), protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue(P<0.05 or P<0.01), JAK2 protein level in colon tissue(P<0.01), and protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) were observed in the Maxing Shigan Decoction group than in the model group. After 3 days of administration, the level of serum IL-8 in the model group was significantly higher than that in the normal group(P<0.01), and the level in the Maxing Shigan Decoction group was significantly reduced(P<0.01). In conclusion, Maxing Shigan Decoction can significantly up-regulate body mass, spleen index, and thymus index, down-regulate lung index, reduce the levels of IL-8 and IFN-γ, and down-regulate protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue and protein and mRNA levels of JAK2, STAT1, and IRF9 in colon tissue, and alleviate pathological damage of lung tissue and colon tissue. The mechanism is the likelihood that it inhibits the activation of JAK1/2-STAT1 signaling pathway to alleviate the damage to lung and colon tissue damage.

Graphene-oxide-based bioassay for the fluorometric determination of agrC gene transcription in methicillin-resistant Staphylococcus aureus that uses nicking-enzyme-assisted target recycling and a hybridization chain reaction.

Herein, we report the development of a graphene-oxide-based (GO-based) fluorescent bioassay for determining agrC gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). The design is based on nicking-enzyme-assisted (Nb.BbvcI-assisted) target recycling amplification (NATR) and a hybridization chain reaction (HCR). The system consists of a helper probe (HP), a molecular beacon (MB) probe, four hairpins, and endonuclease Nb.BbvcI, which plays a role in target recycling and signal amplification. In the absence of the target, all of the carboxyfluorescein-labeled (FAM-labeled) hairpins are adsorbed through π-stacking interactions onto the surface of GO, resulting in FAM signal quenching. When the target is added, three nucleic acid chains hybridize together to form a triple complex that is recognized by Nb.BbvCI. The MB probe is then cleaved by Nb.BbvCI to generate an HP/target complex and two new DNA fragments; the former is hybridized to another MB probe and enters the next round of reaction. The two newly reproduced DNA fragments induce a HCR with the assistance of hairpins 1-4 to create double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and generate strong fluorescence at excitation/emission wavelengths of 480/514 nm. Importantly, synergy between FAM and the dsDNA-SYBR Green I duplex structure led to significantly amplified fluorescence and enhanced sensitivity. The bioassay showed a detection limit of 7.5 fM toward the target and a good linearity in the 10 fM to 100 pM range. The developed method was applied to monitor biofilm formation and study the mechanism of drug action, with satisfactory results obtained.

Targeted inhibition of methicillin-resistant Staphylococcus aureus biofilm formation by a graphene oxide-loaded aptamer/berberine bifunctional complex.

Biofilm formation is known to promote drug resistance in methicillin-resistant Staphylococcus aureus (MRSA), which is closely related to persistent infections in hospital settings. In this study, a DNA aptamer specific to penicillin-binding protein 2a (PBP2a) with a dissociation constant (Kd) of 82.97 ± 8.86 nM was obtained after 14 cycles of systematic evolution of ligands by exponential enrichment (SELEX). Next, a bifunctional complex containing the aptamer intercalated by berberine into the double-strand region was prepared and adsorbed onto the surface of graphene oxide (GO) by π-stacking interactions. The GO-loaded aptamer/berberine bifunctional complex showed significantly higher inhibition of MRSA biofilm formation than the control. Furthermore, this study shows that the complex possesses anti-biofilm activity, which can be attributed to the ability of the aptamer to reduce cell-surface attachment by blocking the function of PBP2a and berberine to attenuate the level of the accessory gene regulator (agr) system, which plays an important role in mediating MRSA biofilm formation. Therefore, the simultaneous delivery of berberine and PBP2a-targted aptamer using GO may have potential for the treatment of chronic infections caused by MRSA biofilms. It also provides a new avenue for multitarget treatment of bacterial biofilms.

Extracellular vesicle-derived miR-1249-5p regulates influenza A virus-induced acute lung injury in RAW246.7 cells through targeting SLC4A1.

Acute lung injury (ALI) is characterized by tissue damage that leads to pulmonary epithelial membrane dysfunction and macrophage activation. Currently however, the exact mechanism by which the initial mediators of mouse lung epithelial (MLE-12) cells induce inflammation remines unclear. We constructed co-culture systems of MLE-12 cells with mouse macrophage cells RAW246.7 which were realized by the supernatant and Transwell chamber. In previous study, we successfully constructed an influenza A virus-induced MLE-12 cells model. Extracellular Vesicles (EVs) from cells supernatant were isolated by differential ultracentrifugation and confirmed by transmission electron microscopy. High-throughput sequencing results showed that MLE-12 cells stimulated by influenza A virus had higher level of miR-1249-5p. The results were validated by RT-qPCR analysis. The research aimed to investigate the roles and mechanisms of miR-1249-5p in ALI. RAW246.7 cells were transfected with miR-1249-5p mimic/inhibitor. The concentrations of TNF-α, IL-6 were determined by ELISA and the uptake of EVs was monitored by confocal laser scanning microscope. Western blotting detected changes in the SLC4A1 and NF-κB signaling pathway. The results indicated that miR-1249-5p played an important role in ALI, and further investigation of its target gene SLC4A1 and NF-κB signaling pathway provides ideas for new therapeutic targets and strategies for ALI.

Immunomodulatory effect of pachymaran on cyclosporine A (CsA)-induced lung injury in mice / 数字中医药（英文）

@#Objective To investigate the immunomodulatory effect of pachymaran on cyclosporine A (CsA)-induced lung injury in mice. Methods (i) Fifty male BALB/c mice were randomly divided into five groups (10 mice in each group): normal control (NC) group, 30, 45, and 60 mg/kg CsA groups, and lipopolysaccharide (LPS) group. Except for the NC group, other groups underwent CsA modeling. The NC group was treated with phosphate-buffered saline (PBS), the LPS group with 10 mg/kg LPS eight hours before mice euthanized, and the 30, 45, and 60 mg/kg CsA groups with corresponding doses of CsA for seven consecutive days. After treatment, the body and organ mass of each group were weighed, and the lung, thymus, and spleen indexes were calculated. Hematoxylin-Eosin (HE) staining was performed to observe histopathological changes in the lungs of the mice. The protein expression levels of interleukin (IL)-2 and IL-1β in the blood were detected using enzyme-linked immunosorbent assay (ELISA), and those of surfactant protein D (SP-D), IL-2, and IL-6 in lung tissues were detected by immunohistochemistry (IHC). The mRNA expression levels of SP-D, IL-1β, IL-6, and myeloperoxidase (MPO) in the lung tissues were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). (ii) Another 60 BALB/c mice were divided into six groups (10 mice in each group) : NC group, model control (MC) group, 50, 100, and 200 mg/kg pachymaran groups, and polyinosinic-polycytidylic acid [poly(I:C)] group. Except for the NC group, other groups underwent 45 mg/kg CsA modeling. The NC and MC groups were treated with distilled water, the pachymaran groups with corresponding doses pachymaran, and the poly(I:C) group with 0.1 mg/kg poly(I:C) for seven days.The mice were euthanized to obtain tissues and serum for detection. Detection methods were identical to those described in (i) above. Results (i) CsA (30 mg/kg) increased the lung index of mice (P < 0.001), and decreased the spleen index (P < 0.01), thymus index (P < 0.05), and the serum level of IL-2 (P < 0.05). CsA (45 mg/kg) decreased the spleen, thymus indexes, and the serum level of IL-2 (P < 0.01) in mice, and increased the serum level of IL-1β (P < 0.05) and the protein level of lung SP-D (P <0.001). CsA (60 mg/kg) increased the lung index of mice (P < 0.01), the serum level of IL-1β (P < 0.05), the protein level of lung SP-D (P < 0.01), and the mRNA levels of lung MPO and SP-D ( P < 0.05), and decreased the thymus index of mice (P < 0.01). HE staining showed that 30, 45, and 60 mg/kg CsA, and LPS caused pathological changes in the lung tissue of mice. (ii) After pachymaran intervention in MC mice, the spleen and thymus indexes (P < 0.05) were increased in the 100 and 200 mg/kg pachymaran groups, and the lung index was decreased (P < 0.05). Moreover, 50 mg/kg pachymaran increased the thymus index (P < 0.05) and decreased the lung index (P < 0.01) in MC group. Pachymaran (50, 100, and 200 mg/kg) improved lung tissue injury, reduced the serum level of IL-1β (P < 0.001), and the mRNA levels of MPO and SP-D in lung tissues (P < 0.05) of mice. Pachymaran (100 mg/kg) increased the protein level of lung IL-2 (P < 0.01), decreased the protein level of lung SP-D (P < 0.01), and the mRNA level of IL-1β (P < 0.001) in the lung tissues of mice. Pachymaran (200 mg/kg) increased the serum level of IL-2 (P < 0.01) and lung IL-6 of mice (P < 0.05). Pachymaran (50 and 200 mg/kg) increased the mRNA level of IL-6 in the lung tissues of mice (P < 0.05). Conclusion While the immune function of mice was suppressed by CsA, the lung tissue was also damaged. Pachymaran can improve the immunosuppression induced by CsA and improve the lung tissue injury in immunosuppressed mice.

Graphene-based fluorometric determination of agrD gene transcription in methicillin-resistant Staphylococcus aureus using exonuclease III-aided target recycling and DNA walker cascade amplification.

A graphene-based bioassay is described for the fluorometric determination of agrD gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method includes exonuclease III (Exo III)-assisted target recycling and DNA walker cascade amplification. Hairpin1 (HP1) consists of a capture probe (CP) and DNA walker sequence. In the absence of the target, 5'-amino modified hairpin2 (HP2) labeled with carboxyfluorescein (FAM) at its 3' terminus is covalently linked to graphene via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, resulting in the quenching of the FAM signal. The stem-loop structure of HP1 opens when the target is added to form partially complementary DNA/RNA hybrids. Exo III then initiates the target recycling process by cleaving the CP and DNA walker cascade reaction by automatic walking. This iterative reaction causes the FAM to dissociate from the graphene, and the fluorescence can be measured at excitation/emission wavelengths of 480/514 nm. Therefore, the target can be assayed by fluorescence. This method has a linear relationship with the concentration of target within the range 1 fM to 100 pM with a detection limit of 1 fM. The developed bioassay was used to monitor biofilm formation and investigate the mechanism of drug action with satisfactory results. Schematic representation of the graphene-based fluorescent bioassay for agrD gene transcription in methicillin-resistant Staphylococcus aureus by using exonuclease III-aided target recycling and DNA walker cascade amplification.

Zhuifeng Tougu capsules improve rheumatoid arthritis symptoms in rats by regulating the toll-like receptor 2/4-nuclear factor kappa-B signaling pathway.

OBJECTIVE: To investigate the efficacy of Zhuifeng tougu capsules (, ZFTG) in the treatment of rheumatoid arthritis (RA) in rats and study its mechanism, focusing on the toll-like receptor 2/4-nuclear factor kappa-B (TLR2/4-NF-κB) signaling pathway. METHODS: Type Ⅱ collagen and an artificial climate box were used to construct the rat model of collagen-induced arthritis with wind-cold-dampness arthralgia syndrome. The rats were divided randomly into a control group, wind-cold-dampness syndrome model group, and high-, medium-, and low-dose ZFTG groups. The methotrexate (MTX) control group was treated with the corresponding drug intervention for 28 d. The joint temperature, pain threshold, joint swelling degree, and arthritis index (AI) score were measured. The production of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factors (RFs) in the blood was detected by enzyme-linked immunosorbent assay. The protein expression of TLR2, TLR4, and NF-κB in synovial tissues was detected by Western blotting, and the mRNA expression of TLR2, TLR4, and NF-κB was detected by real-time polymerase chain reaction. RESULTS: Compared with the model group, the joint temperature in each treatment group, the MTX control group, and MTX group recovered, the degree of foot swelling, pain threshold, AI score decreased, serum CRP, ESR, RF level and the levels of TLR2, TLR4, and NF-κB in synovial tissue were decreased (P < 0.05). Among them, the curative effect in the medium-dose and MTX groups was more evident (P < 0.01). CONCLUSION: ZFTG has a significant effect on RA in rats, and its mechanism may involve regulating CRP levels, the ESR, and RFs via the TLR2/4-NF-κB signaling pathway.

Exploring the Mechanism of Zhibai Dihuang Decoction in the Treatment of Ureaplasma Urealyticum-Induced Orchitis Based on Integrated Pharmacology.

Background: Ureaplasma urealyticum (UU) infection is the most common cause of male infertility. Zhibai Dihuang Decoction (ZBDHD) can improve the rate of forwarding motility sperm, sperm deformity rate, seminal plasma zinc and refined berry sugar levels. Methods: The potential targets of ZBDHD are obtained from The Encyclopedia of Traditional Chinese Medicine (ETCM). Orchitis-related targets were collected from the Genecards and OMIM databases. The Cytoscape and the Database for Annotation, Visualization and Integrated Discovery (DAVID) were utilized to construct and analyzed the networks. Finally, a rat model of orchitis caused by UU infection was used to detect related indicators of mitochondrial energy metabolism using TUNEL apoptosis detection technology, loss cytometry, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and Western Blot. Results: A total of 795 ZBDHD targets and 242 orchitis-related targets were obtained. The "ZBDHD- orchitis PPI network" was constructed and analyzed. ZBDHD can regulate signaling pathways and biological processes related to mitochondrial energy metabolism. The results of experimental studies have shown that ZBDHD maintains the integrity of sperm mitochondrial respiratory chain function by enhancing mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities, promotes the synthesis of mitochondrial ATP, and improves sperm energy supply, thereby improving the motility, vitality and survival rate of sperm, and effectively improving the quality of semen in UU-infected rats (p < 0.05). Conclusion:This study discovered the multi-pathway mechanism of ZBDHD intervention in UU-induced orchitis through integrated pharmacological strategies, which provides a reference for further research on the mechanism of ZBDHD intervention in orchitis in the direction of mitochondrial energy metabolism.

Aptamers used for biosensors and targeted therapy.

Aptamers are single-stranded nucleic acid sequences that can bind to target molecules with high selectivity and affinity. Most aptamers are screened in vitro by a combinatorial biology technique called systematic evolution of ligands by exponential enrichment (SELEX). Since aptamers were discovered in the 1990s, they have attracted considerable attention and have been widely used in many fields owing to their unique advantages. In this review, we present an overview of the advancements made in aptamers used for biosensors and targeted therapy. For the former, we will discuss multiple aptamer-based biosensors with different principles detected by various signaling methods. For the latter, we will focus on aptamer-based targeted therapy using aptamers as both biotechnological tools for targeted drug delivery and as targeted therapeutic agents. Finally, challenges and new perspectives associated with these two regions were further discussed. We hope that this review will help researchers interested in aptamer-related biosensing and targeted therapy research.

Fluorometric determination of agrA gene transcription in methicillin-resistant Staphylococcus aureus with a graphene oxide-based assay using strand-displacement polymerization recycling and hybridization chain reaction.

A graphene oxide (GO)-based fluorescent bioassay was developed to quantify agrA gene transcription (its mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method is based on the use of Klenow fragment (KF)-assisted target recycling amplification and hybridization chain reaction (HCR). A triple complex was designed that contained a capture probe (CP), a trigger probe (TP), and a help probe (HP), which were partially complementary to one another. In the absence of the target, all the oligonucleotides labeled with carboxyfluorescein (FAM) are adsorbed onto the surface of GO by π-stacking interactions. This adsorption quenches the FAM signal. On the contrary, the target RNA causes the triple complex to disintegrate and initiates strand-displacement polymerization reaction (SDPR) and HCR in the presence of the appropriate raw materials, including the primer, KF, dNTPs, hairpin 1 (H1), and hairpin 2 (H2), generating double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and produce strong fluorescence, measured at excitation/emission wavelengths of 480/514 nm. The fluorescent signal is greatly amplified by SYBR Green I (SGI) due to the synergistic effect of dsDNA-SGI. The target was assayed with this method at concentrations in the range 10 fM to 100 pM, and the detection limit (LOD) was 10 fM. This method also displayed good applicability in the analysis of real samples. It provides a new way of monitoring biofilm formation and studying the mechanisms of drug actions. Graphical abstract Schematic representation of the graphene oxide-based fluorescent bioassay for agrA gene transcription in methicillin-resistant Staphylococcus aureus by using strand-displacement polymerization recycling and hybridization chain reaction.

Curcumin Nicotinate Selectively Induces Cancer Cell Apoptosis and Cycle Arrest through a P53-Mediated Mechanism.

Curcumin is an anticancer agent, but adverse effects and low bioavailability are its main drawbacks, which drives efforts in chemical modifications of curcumin. This study evaluated antiproliferative activity and cancer cell selectivity of a curcumin derivative, curcumin nicotinate (CN), in which two niacin molecules were introduced. Our data showed that CN effectively inhibited proliferation and clonogenic growth of colon (HCT116), breast (MCF-7) and nasopharyngeal (CNE2, 5-8F and 6-10B) cancer cells with IC50 at 27.7 µM, 73.4 µM, 64.7 µM, 46.3 µM, and 31.2 µM, respectively. In cancer cells, CN induced apoptosis and cell cycle arrest at G2/M phase through a p53-mediated mechanism, where p53 was activated, p21 and pro-apoptotic proteins Bid and Bak were upregulated, and PARP was cleaved. In non-transformed human mammary epithelial cells MCF10A, CN at 50 µM had no cytotoxicity and p53 was not activated, but curcumin at 12.5 µM activated p53 and p21 and inhibited MCF10A cell growth. These data suggest that CN inhibits cell growth and proliferation through p53-mediated apoptosis and cell cycle arrest with cancer cell selectivity.

Pathological Relationship between Intestinal Flora and Osteoarthritis and Intervention Mechanism of Chinese Medicine.

Chinese medicine (CM) has a good clinical effect on osteoarthritis (OA), but the mechanism is not very clear. Evidence-based medicine researches have shown that intestinal flora plays a role in the pathogenesis and succession of OA. Intestinal flora affects the efficacy of CM, and CM can affect the balance of intestinal flora. This paper focuses on the relationship between intestinal flora, intestinal microenvironment, brain-gut axis, metabolic immunity and OA, and preliminarily expound the significance of intestinal flora in the pathogenesis of OA and the mechanism of CM intervention. The above discussion will be of great significance in the prevention and treatment of OA by CM from the perspective of intestinal flora.

Fluorometric determination of mercury(II) via a graphene oxide-based assay using exonuclease III-assisted signal amplification and thymidine-Hg(II)-thymidine interaction.

A highly sensitive and selective fluorometric method is described for determination of mercury(II). It is based on (a) the use of graphene oxide (GO) acting as a quencher of the fluoresence of the carboxy-fluorescein (FAM), and (b) of Hg(II)-triggered cleavage of the newly formed nucleic acid sequences harbored blunt 3'-hydroxyl termini by exonuclease III (Exo III) that leads to signal amplification. Two DNA probes are used, viz. a capture probe (CP) and a help probe; HP) that is partially complementary. In the absence of Hg(II), the FAM-labeled hairpin (signal probe, SP) is adsorbed onto the surface of GO via π-stacking interactions. CP blocks the release of the HP for binding to SP. This results in quenching of the green fluorescence of the label. Upon addition of Hg(II), the linear structure of CP is converted to a hairpin structure due to the formation of thymidine-Hg(II)-thymidine duplexes. HP is released from the CP/HP hybrids, and this causes SP to be released from from GO and fluorescence to be recovered. The signal is strongly amplified by using Exo III-assisted targeting and recycling of HP. Hence, Hg(II) can be detected via the strong increase in fluorescence. The method has a linear response in the 0.1 to 30 nM Hg(II) concentration range and a 10 pM detection limit. It was applied to the determination of Hg(II) in three (spiked) Chinese medicines. Graphical abstract Schematic representation of fluorescence sensing strategy for Hg2+ by using graphene oxide as a quencher and exonuclease III-assisted signal amplification.