Pharmacokinetics of gallic acid and protocatechuic acid in humans after dosing with Relinqing (RLQ) and the potential for RLQ-perpetrated drug-drug interactions on organic anion transporter (OAT) 1/3.

CONTEXT: Relinqing granules (RLQ) are being used alone or in combination with antibacterial drugs to treat urological disorders. OBJECTIVE: This study investigates the pharmacokinetics of RLQ in humans and the potential for RLQ-perpetrated interactions on transporters. MATERIALS AND METHODS: Twelve healthy subjects (six women and six men) participated to compare single- and multiple-dose pharmacokinetics of RLQ. In the single-dose study, all 12 subjects received 8 g of RLQ orally. After a 7-d washout period, the subjects received 8 g of RLQ for seven consecutive days (t.i.d.) and then a single dose. Gallic acid (GA) and protocatechuic acid (PCA) in plasma and urine samples were analysed using LC-MS/MS. The transfected cells were used to study the inhibitory effect of GA (50-5000 µg/L) and PCA (10-1000 µg/L) on transporters OAT1, OAT3, OCT2, OATP1B1, P-gp and BCRP. RESULTS: GA and PCA were absorbed into the blood within 1 h after administration and rapidly eliminated with a half-life of less than 2 h. The mean peak concentrations of GA (102 and 176 µg/L) and PCA (4.54 and 7.58 µg/L) were lower in males than females, respectively. The 24 h urine recovery rates of GA and PCA were about 10% and 5%, respectively. The steady-state was reached in 7 d without accumulation. GA was a potent inhibitor of OAT1 (IC50 = 3.73 µM) and OAT3 (IC50 = 29.41 µM), but not OCT2, OATP1B1, P-gp or BCRP. DISCUSSION AND CONCLUSIONS: GA and PCA are recommended as PK-markers in RLQ-related pharmacokinetic and drug interaction studies. We should pay more attention to the potential for RLQ-perpetrated interactions on transporters.

Guttiferone K Exerts the Anti-inflammatory Effect on Mycobacterium Tuberculosis- (H37Ra-) Infected Macrophages by Targeting the TLR/IRAK-1 Mediated Akt and NF-κB Pathway.

Mycobacterium tuberculosis (Mtb) remains a great threat to global health, killing more people than any other single infectious agent and causing uncontrollable inflammation in the host. Poorly controlled inflammatory processes can be deleterious and result in immune exhaustion. The current tuberculosis (TB) control is facing the challenge of drugs deficiency, especially in the context of increasingly multidrug resistant (MDR) TB. Under this circumstance, alternative host-directed therapy (HDT) emerges timely which can be exploited to improve the efficacy of TB treatment and disease prognosis by targeting the host. Here, we established the in vitro infection model of Mtb macrophages with H37Ra strain to seek effective anti-TB active agent. The present study showed that Guttiferone K, isolated from Garcinia yunnanensis, could significantly inhibit Mtb-induced inflammation in RAW264.7 and primary peritoneal macrophages. It was evidenced by the decreased production of inflammatory mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Further studies with immunoblotting and immunofluorescence revealed that Guttiferone K obviously inhibits the nuclear factor-kappa B (NF-κB) both in RAW264.7 and primary peritoneal macrophages relying on the TLR/IRAK-1 pathway. Guttiferone K could also suppress the NLRP3 inflammasome activity and induce autophagy by inhibiting the protein kinase B (p-Akt) and mammalian target of rapamycin (mTOR) phosphorylation at Ser473 and Ser2448 in both cell lines. Thus, Guttiferone K possesses significant anti-inflammatory effect, alleviating Mtb-induced inflammation with an underlying mechanism that targeting on the TLR/IRAK-1 pathway and inhibiting the downstream NF-κB and Akt/mTOR signaling pathways. Together, Guttiferone K can be an anti-inflammatory agent candidate for the design of new adjunct HDT drugs fighting against tuberculosis.

The possible regulatory effect of the PD-1/PD-L1 signaling pathway on Tregs in ovarian cancer.

Aim of this study was to investigate the possible regulatory effect of the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway on Tregs in ovarian cancer. Immunohistochemistry was used to detect the expression of PD-L1 and PD-1 and the presence of FOXP3+ Tregs in ovarian cancer. Then, ovarian cancer HO8910 cells were subjected to transfection with PD-L1 siRNA in vitro. CCK-8, Transwell and wound healing assays were performed to detect the biological behaviors of ovarian cancer cells. Human T-cells isolated from human peripheral blood were cocultured with HO8910 cells, which were divided into the Control, TGF-ß, and TGF-ß+ anti-PD-L1 groups. The proportion of differentiated Tregs was detected by flow cytometry. Mouse models of ovarian cancer were established, and PD-L1 antibody therapy was administered. Tumor growth and Treg recruitment were observed. PD-L1, PD-1 and FOXP3+ Tregs were found in ovarian cancer tissue. Patients with tumors with an advanced stage and low differentiation and lymph node metastasis had significantly higher levels of PD-1, PD-L1 and FOXP3+ Tregs. After transfection with PD-L1 siRNA, HO8910 cells showed a significant reduction in PD-L1 expression, proliferation, migration and invasion. After T-cells were cocultured with ovarian cancer cells, the TGF-ß+ anti-PD-L1 group showed a substantial decline in the differentiation of T-cells into Tregs compared with the TGF-ß group. Moreover, mice in the anti-PD-L1 group had significantly reduced tumor growth rates, Treg proportions in the tumor microenvironment, and FOXP3 expression.

Metallothionein-1 suppresses rheumatoid arthritis pathogenesis by shifting the Th17/Treg balance.

It is now well accepted that an imbalance between the Th17 and regulatory T-cell responses is closely associated with the development of rheumatoid arthritis (RA). However, the precise regulatory mechanism for the differentiation of Th17 and Treg in RA is not well characterized. The present study showed that metallothionein-1 (MT-1), which is a low molecular weight protein that is involved in the detoxification of heavy metals and scavenging of free radicals, was upregulated in RA. Furthermore, the synovial inflammation and pathologic symptoms in collagen-induced arthritis and collagen antibody-induced arthritis mice were significantly suppressed when MT-1 was expressed intraarticularly. Further investigation revealed that MT-1 inhibited the differentiation of Th17 cells but enhanced that of Treg cells. Furthermore, it markedly decreased both STAT3 and RAR-related orphan receptor gamma t (RORγt) expression in vitro and in vivo. Collectively, our studies demonstrated that MT-1 might manifest as a protein involved in immunosuppression of RA pathogenesis by shifting Th17/Treg balance and may prove to be a potential therapeutic target for RA autoimmune diseases.

MCL Plays an Anti-Inflammatory Role in Mycobacterium tuberculosis-Induced Immune Response by Inhibiting NF-κB and NLRP3 Inflammasome Activation.

Mycobacterium tuberculosis (Mtb) remains a significant menace to global health as it induces granulomatous lung lesions and systemic inflammatory responses during active tuberculosis (TB). Micheliolide (MCL), a sesquiterpene lactone, was recently reported to have a function of relieving LPS-induced inflammatory response, but the regulative role of MCL on the immunopathology of TB still remains unknown. In this experiment, we examined the inhibitory effect of MCL on Mtb-induced inflammatory response in mouse macrophage-like cell line Raw264.7 by downregulating the activation of nuclear factor kappa B (NF-κB) and NLRP3 inflammasome. Evidences showed that MCL decreased the secretion of Mtb-induced inflammatory cytokines (IL-1ß and TNF-α) in a dose-dependent manner. Meanwhile, MCL dramatically suppressed Mtb-induced activation of iNOS and COX2 as well as subsequent production of NO. Furthermore, MCL inhibited Mtb-induced phosphorylation of Akt (Ser 473) in Raw264.7. According to our results, MCL plays an important role in modulating Mtb-induced inflammatory response through PI3K/Akt/NF-κB pathway and subsequently downregulating the activation of NLRP3 inflammasome. Therefore, MCL may represent as a potential drug candidate in the adjuvant treatment of TB by regulating host immune response.

Interleukin-37 is increased in ankylosing spondylitis patients and associated with disease activity.

BACKGROUND: Interleukin-37 (IL-37) has been known to play an immunosuppressive role in various inflammatory disorders, but whether it participates in the regulation of pathogenesis of ankylosing spondylitis (AS) has not been investigated. Here, we examined the serum levels of IL-37 and its clinical association in AS, and explored the anti-inflammatory effects of IL-37 on peripheral blood mononuclear cells (PBMCs) from AS patients. METHODS: The mRNA levels of IL-37, TNF-α, IL-6, IL-17, and IL-23 in PBMCs and their serum concentrations from 46 AS patients were examined by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunoassay (ELISA), respectively. The correlations between serum IL-37 levels with disease activity, laboratory values and pro-inflammatory cytokines in AS were analyzed by Spearman correlation test. PBMCs from 46 AS patients were stimulated with recombinant IL-37 protein, expressions of TNF-α, IL-6, IL-17 and IL-23 were determined by RT-PCR and ELISA. RESULTS: Compared to healthy controls (HC), AS patients and active AS patients showed higher levels of IL-37 in PBMCs and serum respectively. Strikingly, serum IL-37 levels were higher in AS patients with osteoporosis than those without. Serum levels of IL-37 were correlated with laboratory values as well as TNF-α, IL-6 and IL-17, but not IL-23 in patients with AS. The productions of pro-inflammatory cytokines such as TNF-α, IL-6, IL-17, IL-23 in PBMCs from AS patients were obviously attenuated after recombinant IL-37 stimulation, but not in the HC. CONCLUSION: The higher levels of IL-37 were found in AS patients, which were correlated with disease activity and AS related pro-inflammatory cytokines. More importantly, IL-37 inhibits the expressions of the pro-inflammatory cytokines from PBMCs in AS patients, indicating the potential anti-inflammatory role of IL-37 in AS.

Identification of plant-derived natural products as potential inhibitors of the Mycobacterium tuberculosis proteasome.

BACKGROUND: The Mycobacterium tuberculosis (Mtb) proteasome has been established as a viable target for the development of anti-tuberculosis agents. In this study, the inhibitory activities of 100 plant-derived natural products on the Mtb proteasome were analyzed to identify novel potential inhibitors. METHODS: The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC can be hydrolyzed by the proteasome to release free AMC, the fluorescence of which is proportional to the proteasome activity. The inhibitory activities of 100 natural products (each at a final concentration of 200 µM) were detected by this method using MG132 as a positive control. RESULTS: Twelve of these natural products (10 of which were flavonoids) inhibited the activity of the Mtb proteasome by more than 65%. Comparison of the structural differences between the flavonoids with good inhibitory activity and those without inhibitory activity revealed that the hydroxyl at the flavonoid C ring C-3 or the hydroxyl/methoxyl at the flavonoid A ring C-6 were critical for the inhibition of proteasomal activity. CONCLUSIONS: These data indicate that flavonoids represent a basis for rational structural design in the process of novel anti-tuberculosis drug discovery.

[Cloning, expression and analysis of Der f Mag 29 allergen of Dermatophagoides farinae].

The full-length Mag 29 gene of Dermatophagoides farinae was amplified by RT-PCR with a pair of specific primers. The PCR product was cloned into pCold TF DNA vector. The constructed plasmid pCold TF-Mag 29 was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. The full-length Mag 29 gene was 429 bp. A specific band (Mr 63,000) were detected in the whole cells, the supernatant, and the precipitate. Bioinformatics analysis revealed that Mag 29 protein was composed with 142 amino acid residues with a calculated molecular weight of Mr 15,100, and its secondary structure was composed of alpha helix (55.63%), extended strand (3.52%), and random coil (40.85%). The Mag 29 allergen was a hydrophilic and cytoplasmic protein, and shared a high degree homology with the heat shock protein 70 family.

Astragaloside IV attenuates neuroinflammation and ameliorates cognitive impairment in Alzheimer's disease via inhibiting NF-κB signaling pathway.

The inflammatory process plays a significant role in the pathophysiology of Alzheimer's disease (AD). Anti-neuroinflammatory cascade is now considered an important measure for AD treatment. Astragaloside IV (AS-IV), a saponin of Astragali radix, has shown significant anti-inflammatory properties and protective effects against neurodegenerative diseases. However, the mechanisms of AS-IV in treating Alzheimer's disease (AD) have not been fully determined. The experiment research was carried out to comprehensively confirm the beneficial effects and underlying molecular mechanisms of AS-IV to AD. In this research, BV-2 cells were cultured in vitro and treated by AS-IV under the stimulation of LPS, qRT-PCR was adopted to analyze the mRNA expression level of inflammatory factors. Western-blot was carried out to analyze the phosphorylation level of NF-κB signaling pathway. 5xFAD mice were administrated AS-IV mixed in the diet for 3 months. Behavioral experiments were adopted to analyze learning and memory abilities. Immunohistochemical staining was adopted to observe the proliferation of microglias and the accumulation of Aß plaques. AS-IV cut down the mRNA expression of IL-1ß, COX-2, iNOS and TNF-α in LPS-stimulated BV-2 cells by suppressing the phosphorylation of IκB and p65, and inhibited the phosphorylated p65 from entering the nucleus. AS-IV increased the frequency of recognizing new objects in the novel object recognition test, shortened the escape latency, raised the number of crossing platform in the Morris water maze, inhibited the hyperplasia of microglias, and reduced the production of senile plaques in 5xFAD mice. In brief, AS-IV ameliorates learning and memory impairment by relieving the intensity of neuroinflammatory response in AD. Therefore, AS-IV is very promising to be a herbal medicine for AD treatment.

Micheliolide attenuates neuroinflammation to improve cognitive impairment of Alzheimer's disease by inhibiting NF-κB and PI3K/Akt signaling pathways.

Inflammatory reaction in the brain activates glial cells, and over-activated glial cells secrete inflammatory mediators, which aggravates the inflammatory response in the brain and accelerates the development of Alzheimer's disease (AD) in turn. Numerous natural compounds from herbs can alleviate inflammation, and it is very promising to find anti-neuroinflammatory natural compounds. Micheliolide (MCL) is an asesquiterpene lactone. Studies have proved that MCL showed an obvious anti-inflammatory property. Nevertheless, whether MCL can treat AD has not been determined. In this research, AD model mice were fed with a diet supplemented MCL for 3 months, the cognitive ability and inflammatory state of mice were detected. We found that MCL raised the frequency of touching novel objects, cut down the escape latency, raised the number of crossing platform, inhibited the infiltration of inflammatory cells and the secretion of interleukin-1α (IL-1α), IL-12p40, IL-13, IL-17A, tumor necrosis factor-α (TNF-α), granulocyte colony stimulating factor (G-CSF), macrophage inflammatory protein-1α (MIP-1α) and monocyte chemotactic protein-1 (MCP-1) in peripheral blood samples, inhibited the hyperplasia of glial cells and the production of IL-1α, IL-4, G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), MIP-1α and MIP-1ß, and reduced the deposition of Aß peptides in the brain of AD mice. We also concluded that MCL dropped the expression of IL-1ß, TNF-α, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the phosphorylation of IκB, p65 and Akt in BV-2 cells. In conclusion, MCL alleviates the intensity of systemic inflammatory reaction via inhibiting nuclear transcription factor κ gene binding (NF-κB) and phosphoinositide-3-kinase/serine/threonine kinase (PI3K/Akt) pathways in glial cells, and improves the cognitive impairment of AD mice. Therefore, MCL could be a therapeutic candidate for AD.

Quercitrin improved cognitive impairment through inhibiting inflammation induced by microglia in Alzheimer's disease mice.

OBJECTIVE: Diets rich in quercitrin show a neuroprotective effect, but the mechanism is not very clear at present. The objective of this study is to explore the effect and mechanism of quercitrin in the treatment of alzheimer's disease (AD). METHODS: 5XFAD transgenic mice were fed with a diet supplemented with quercitrin for three consecutive months. Behavioral experiments were conducted to assess the cognitive ability, luminex liquid chip technology was used to assess the production of proinflammatory cytokines and immunohistochemistry was used to elucidate the activation of microglia. RESULTS: Quercitrin increased the frequency in exploring new objects, shortened the escape latency and increased the frequency crossing the platform in AD model mice. Quercitrin inhibited the activation and proliferation of microglia, inhibited the secretion of inflammatory cytokines and chemokines and reduced the accumulation of amyloid-ß plaques in AD model mice. CONCLUSION: Quercitrin improved mice cognitive impairment through alleviating the intensity of inflammatory response and is a promising medicinal plant extract in the treatment of AD.

The licorice flavonoid isoliquiritigenin attenuates Mycobacterium tuberculosis-induced inflammation through Notch1/NF-κB and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Glycyrrhiza is a small perennial herb that has been traditionally used to treat many diseases across the world. Licorice (Gancao in Chinese) is the dried root and rhizome of G. glabra, G. uralensis or G. inflata. Licorice plays an important role in traditional Chinese medicine (TCM), and is the most frequently used in Chinese herbal formulas. Isoliquiritigenin (ISL) is a flavonoid extracted from licorice, and has been evaluated for its various biological activities, including anti-inflammatory, anti-tumor and anti-oxidant activities. Excessive and persistent inflammation in the Mycobacterium tuberculosis (Mtb) infection is not conducive to the elimination of Mtb, but contributes to serious pulmonary dysfunction. AIM OF THE STUDY: This study aimed to examine the anti-inflammatory effects of ISL in the Mtb infection. METHODS: In vitro models of Mtb-infected macrophages were established. Murine macrophage Raw 264.7 cells and primary peritoneal macrophages were used in this study. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. The effects of ISL on the secretion levels of interleukin -1ß (IL-1ß), tumor necrosis factor -α (TNF-α), and interleukin -6 (IL-6) were detected by the enzyme-linked immunosorbent assay (ELISA). The expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were measured by the real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Western blot was used to assess the effects of ISL on the activation of NLRP3 inflammasome and Notch1/NF-κB and MAPK signaling pathways. Immunofluorescence assays was used to detected the translocation of phosphorylation of p65 subunit of NF-κB. RESULTS: It was revealed that ISL inhibited the secretion of IL-1ß and the activation of pore-forming protein (gasdermin D, GSDMD) by suppressing the activation of NLPR3 inflammasome induced by Mtb infection. ISL was also shown to have promising inhibitory effects on inflammatory factors, such as TNF-α, IL-6, iNOS and COX2. Regarding the anti-inflammatory mechanism of ISL, it was found that ISL exerted its anti-inflammatory effects by inhibiting the activation of Notch1/NF-κB and MAPK signaling pathways. CONCLUSION: ISL reduced Mtb-induced inflammation through the Notch1/NF-κB and MAPK signaling pathways. ISL might be used as a potential adjuvant drug to treat tuberculosis by adjusting host immune responses.

Serum IL-6 and TNF-α Levels Are Correlated with Disease Severity in Patients with Ankylosing Spondylitis.

OBJECTIVE: Previous studies have shown that a number of cytokines participate in the regulation of ankylosing spondylitis (AS). To investigate the potential role of interleukin (IL)-6 and tumor necrosis factor- α (TNF-α) in AS pathogenesis, this study examined the serum levels of IL-6 and TNF-α in patients with AS and its clinical association with disease activity. MATERIALS AND METHODS: The serum concentrations of IL-6 and TNF-α from 80 patients with AS and 46 healthy control patients (HCs) were examined by electrochemiluminescence immunoassay. The correlations between the serum IL-6 and TNF-α levels and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), computed tomography (CT) imaging-based classification, and laboratory indicators were analyzed using the Spearman correlation test. RESULTS: Compared to HCs, patients with AS showed higher levels of IL-6 and TNF-α. There was also a positive correlation between the serum IL-6 and TNF-α levels and the BASDAI, the progression of AS, and the CT imaging-based classification. The serum levels of IL-6 correlated closely with C-reactive protein and the erythrocyte sedimentation rate. More important, patients with AS with hip joint involvement exhibited a significant elevation of serum levels of TNF-α, and higher IL-6 was detected in patients with the involvement of joints other than the hip and sacroiliac joints. CONCLUSION: The serum levels of IL-6 and TNF-α can function as important indicators for auxiliary diagnosis and disease activity evaluation of AS.

Quercetin improves the adipose inflammatory response and insulin signaling to reduce "real-world" particulate matter-induced insulin resistance.

Numerous epidemiological data and experimental studies support a strong link between fine particulate matter (less than 2.5 mm in aerodynamic diameter, PM2.5) exposure and the development of insulin resistance/type 2 diabetes mellitus (T2DM). Quercetin (Que), a flavonoid compound with anti-inflammatory effects, has been confirmed to improve glucose metabolic disorders in rodents and humans. In this study, we investigated the underlying mechanisms of particulate matter (PM)-induced glucose metabolic disorder and subsequently examined the protective effect and mechanism of quercetin supplementation. Male C57BL/6 mice in the control group and PM group were exposed to ambient filtered air (FA) or PM (6 h/day, 7 days/week) for 18 weeks. Mice in the Que group were exposed to PM for 18 weeks and administered Que (50 or 100 mg/kg bw). Glucose tolerance, insulin sensitivity, and systemic and visceral white adipose tissue (vWAT) inflammatory responses were measured. The expression of proteins involved in insulin signal transduction in vWAT was assessed. Chronic PM exposure caused systemic and vWAT inflammation characterized by an increase in serum IL-6 and TNF-α levels and increased vWAT macrophage filtration, triggering NLRP3 inflammasome activation, impairing the classic glucose metabolism signal in vWAT, and inducing whole-body insulin resistance. Moreover, Que administration significantly alleviated systemic and vWAT inflammation, abolished NLRP3 inflammasome activation, and improved signaling abnormalities characteristic of insulin resistance in vWAT and adipocytes. Based on these findings, chronic PM exposure activated the NLRP3 inflammasome and subsequently caused systemic and WAT inflammation and impaired insulin signaling in vWAT and adipocytes. Most importantly, Que administration inhibited NLRP3 inflammasome-mediated inflammation and insulin signaling in vWAT to improve these adverse effects.

Tanshinone IIA alleviates NLRP3 inflammasome-mediated pyroptosis in Mycobacterium tuberculosis-(H37Ra-) infected macrophages by inhibiting endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanshinone IIA (Tan), extracted from Salvia miltiorrhiza Bunge, is a perennial herbal plant widely used as a folk remedy in Asian countries. Several studies have proved that Tanshinone IIA possesses many biological activities, such as anti-inflammatory, free-radical scavenging abilities, antioxidant properties, liver protection, and anti-cancer properties. AIM OF THE STUDY: The objective of the present study was to examine the anti-inflammatory effects of Tan. MATERIALS AND METHODS: The in vitro infection model of Mycobacterium tuberculosis-infected macrophages with the H37Ra strain was established. Murine macrophage Raw 264.7 and human monocyte THP-1 were used for the experiments. Cell viability was determined by the MTT assay. Western blot and lactate dehydrogenase (LDH) activity assays were used to detect the effects of Tan on cell pyroptosis and the level of NLRP3 inflammasome activation. Western blot, Co-immunoprecipitation and Immunofluorescence assays were used to observe the effect of Tan on the expression level of TXNIP. Immunofluorescence assays were applied to explore the effect of Tan on mtROS. Western blot and agarose gel electrophoresis were adopted to observe the effect of Tan on endoplasmic reticulum stress. The siRNA technique was applied to knockdown the expression levels of PERK/peIF2α, IRE1α and ATF6, and Western blot assay was employed to explore the NLRP3 inflammasome activation and possible molecular regulation mechanism of Tan. RESULTS: This study demonstrated that Tan decreased Mtb-induced cell pyroptosis by measuring GSDMD-N and LDH release provoked by NLRP3 inflammasome activation. Additionally, Tan inhibited endoplasmic reticulum stress (ERS), mitochondrial damage, and TXNIP protein expression, all of which acted as upstream signals of NLRP3 inflammasome activation in Mtb-infected macrophages. Significantly, NLRP3 inflammasome activation was suppressed by knocking down ERS pathway proteins, which further clarified that Tan partly targeted ERS to exert anti-inflammatory and immunoregulatory actions. CONCLUSION: This research confirms Tan's anti-inflammatory and immunoregulatory mechanisms in Mtb-infected macrophages by downregulating NLRP3 inflammasome activation-mediated pyroptosis provoked by ERS. Tan may function as an adjuvant drug to treat TB by adjusting host immune responses.

Andrographolide exerts anti-inflammatory effects in Mycobacterium tuberculosis-infected macrophages by regulating the Notch1/Akt/NF-κB axis.

Tuberculosis is a serious public health problem aggravated by the slow progress in the development of new anti-tuberculosis drugs. The hyper-reactive TB patients have suffered from chronic inflammation which could cause deleterious effects on their bodies. Therefore, it is imperative to develop an adjunctive therapy based on inflammatory modulation during Mycobacterium tuberculosis (Mtb) infection. The present study aims to investigate the immune regulatory effects of Andrographolide (Andro) on Mtb-infected macrophages and its underlying mechanisms. The results showed that Andro inhibits the production of IL-1ß and other inflammatory cytokines in a dose-dependent manner. The down-regulation of IL-1ß expression causes the declining expression of IL-8 and MCP-1 in lung epithelial cells which were co-cultured with Mtb-infected macrophages.  The inhibition of the activation of NF-κB pathway, but not the inhibition of MAPK signaling pathway, accounts for the anti-inflammatory role of Andro. Further studies elucidated that Andro could evoke the activation of autophagy to degrade NLRP3, which ultimately inhibited inflammasome activation and subsequent IL-1ß production. Finally, the relevant results demonstrated that Andro inhibited the Notch1 pathway to down-regulate the phosphorylation of Akt/mTOR and NF-κB p65 subunit. Taken together, Andro has been found to suppress the Notch1/Akt/NF-κB signaling pathway. Both Akt inhibition-induced autophagy and inhibition of the NF-κB pathway contributed to restraining the activation of NLRP3 inflammasome and subsequent IL-1ß production. Then, the decreased production of IL-1ß influenced chemokine expression in lung epithelial cells. Based on these results, anti-inflammatory effect of Andro in TB infection is merit further investigation.

PCR-product microarray based on polyacrylic acid-modified surface for SNP genotyping.

PCR-product microarray has great efficiency in SNP genotyping, mutation screening and epigenetic analyzing from a large number of samples. The current PCR-product microarray technology is mostly based on the 3-D gel microarray technologies due to its high loading capacity for PCR products, while there is little progress for PCR-product microarray on planar glass, which gives low background and convenient fabrication. In this study, we improved the PCR-product microarray on planar glass by employing a polyacrylic acid-covered slide. The raw amino-modified PCR products were simply precipitated with ethanol and directly spotted for DNA immobilization. Three detection methods of hybridization, solid-single base extension and solid-multiple bases elongation were carried out for single nucleotide variation identification on the PCR-product microarray. The experimental results showed that the high immobilization yield for raw PCR product was achieved, and the high specificity and high ratio of S/N for genotyping on the microarray were obtained. SNP genotyping of cytochrome P450 2D6 gene in 30 individuals was successfully demonstrated. This study has significantly increased the performances of PCR-product microarray, which could improve its applications in SNP genotyping and mutation screening in large number of individuals.

miR-509-5p Downregulation Is Associated With Male Infertility And Acts As A Suppressor In Testicular Germ Cell Tumor Cells Through Targeting MDM2.

BACKGROUND: The dysregulation of microRNAs (miRNAs) has been linked with male infertility. miR-509-5p is highly expressed in testis and exerts suppressive effects on multiple types of human cancers. OBJECTIVES: Yet, whether miR-509-5p is connected with male infertility and plays a role in testicular germ cell tumor (TGCT) have not been explored. MATERIALS AND METHODS: This study detected miR-509-5p expression in germ cells from MA patients, and further characterize its functional roles in the proliferation and apoptosis of TGCT cells in vitro. RESULTS: We report that miR-509-5p is downregulated in germ cells from infertile men with maturation arrest (MA), which implies an inverse association between miR-509-5p level and male infertility. In addition, miR-509-5p suppresses proliferation and induces apoptosis of TGCT cells in vitro, suggesting that it exhibits tumor-suppressive effects on TGCT. Mechanistically, miR-509-5p targets the mouse double minute 2 (MDM2), an oncogenic factor in TGCT, and moreover, restored expression of MDM2 rescues miR-509-5p suppressive effects on TGCT cells, demonstrating that miR-509-5p suppresses TGCT cells through targeting MDM2. CONCLUSION: Collectively, these results implicate that miR-509-5p may participate in the pathogenesis of male infertility and TGCT through regulating proliferation and apoptosis, two critical cellular activities for spermatogenesis and TGCT tumorigenesis.

CONSORT-Assistive technology-180° rotating eating spoon improves the ability of eating of self-care patients with upper extremity dyskinesia: Rotating eating spoon improves eating ability.

OBJECTIVE: This study aims to develop an assistive technology-180° rotating feeding spoon that could improve the ability of eating of self-care patients with upper extremity dyskinesia. METHODS: The Brunnstrom 6-stage rating of hemiplegia was adopted. During the different recovery stages of the upper limbs, the patients orally ate using a feeding spoon with a non-rotatory head and a 180° rotating feeding spoon. The ability of these patients to eat by themselves was observed, and the basic activity of daily living (BADL) was assessed using the Barthel index (BI). RESULTS: The Brunnstrom assessment scale was used to analyze the results of the patient's upper limb function examination, and the results revealed that the 180° rotating feeding spoon could assist patients with different degrees of upper limb dysfunction when eating independently. CONCLUSIONS: The 180° rotating feeding spoon can assist patients with upper limb dysfunction when eating independently. For patients with different degrees of upper limb dysfunction, the spoon can provide different degrees of aid.

The Effect of Compound Danshen Dripping Pills on the Dose and Concentration of Warfarin in Patients with Various Genetic Polymorphisms.

PURPOSE: The combination of warfarin and compound Danshen dripping pill (CDDP) is helpful for patients with both coronary heart disease (CHD) and atrial fibrillation (AF). The main adverse drug reaction of warfarin is bleeding because of its narrow therapeutic index. The safety of a combination therapy with warfarin and CDDP is always a concern. Our previous research showed that the combination of warfarin and CDDP improved the quality of life for patients with both CHD and AF. This study describes the changes in dose and concentration of warfarin necessary and evaluates bleeding risk when warfarin is given concomitantly with CDDP. METHODS: An ultra-performance liquid chromatography-MS/MS method with a chiral column was developed to assay the concentration of S-warfarin and R-warfarin in human plasma simultaneously. The method was applied to compare the concentration of warfarin in patients taking warfarin combined with CDDP and without CDDP. International normalized ratio (INR) values were monitored to evaluate bleeding risk. Paired t tests were then used to compare the dose and the concentration in 2 periods. Moreover, patients with VKORC1, CYP2C9*3, CYP4F2, EPHX1, and PROC gene polymorphisms were evaluated to determine interactions. FINDINGS: The results indicate that the dose of warfarin had no significant change with or without CDDP. Also, the peak concentrations of S-warfarin and total warfarin were significantly different in CYP4F2 C/C patients, but there was no significant difference identified in other genetic groups. No bleeding occurred in the study. IMPLICATIONS: The dose of warfarin would be sustainable when combined with CDDP, because CDDP did not affect concentration of warfarin significantly in most patients and the change of INR was not significant. CHINA CLINICAL TRIAL REGISTRY IDENTIFIER: ChiCTR-ONRC-13003523.