The impact of HIV self-testing on risk behaviors among men who have sex with men: a mixed-methods study.

Background: Men who have sex with men (MSM) have a high prevalence of HIV and a low rate of HIV testing in China. HIV self-testing (HIVST) presents a viable strategy for expanding HIV testing among MSM. However, the impact of HIVST on risk behaviors among MSM remains controversial. Our study sought to ascertain this impact. Methods: From April 2021 to January 2022, a mixed-methods study was conducted in Qingdao City, employing both quantitative and qualitative methodologies. The quantitative component entailed a cohort study among MSM who had used HIVST. Generalized estimating equations fitting Poisson regressions were used to analyze the changes in risk behaviors of MSM in short time after HIVST (ST-HIVST) and longer time after HIVST (LT-HIVST) compared to before HIVST. Subsequently, we conducted in-depth interviews with 18 MSM who completed the follow-up to delve deeper into the impact of HIVST on MSM. Results: A total of 410 MSM were recruited in the cohort, of whom 83 were lost to follow-up. Compared to before HIVST, there were no significant changes in risk behaviors in ST-HIVST (p > 0.05), while the proportion of recreational drugs abuse (20.7% vs. 33.3%), commercial sex (14.6% vs. 22.9%), and unprotected anal sex (95.9% vs. 98.5%) increased significantly in LT-HIVST (p < 0.05). Specific changes varied across demographic characteristics. According to qualitative interviews, MSM might have decreased risk perception and increased risk behaviors after HIVST. Conclusion: The use of HIVST may promote MSM to engage in risk behaviors. In the future, customized HIVST promotion programs need to be developed to expand HIV testing among MSM and simultaneously control their risk behaviors.

Awareness and use of HIV pre-exposure prophylaxis and factors associated with awareness among MSM in Beijing, China.

Human immunodeficiency virus (HIV) sexual transmission among men who have sex with men (MSM) has increased markedly in Beijing, China in the past decade. Pre-exposure prophylaxis (PrEP) is a highly efficacious biomedical prevention strategy that remarkably reduces HIV-transmission risk. This study examined PrEP awareness among MSM and the factors influencing it. From April to July 2021, respondent-driven sampling was used to conduct a cross-sectional survey among MSM in Beijing, China. Demographic, behavior, and awareness data regarding PrEP were collected. The factors influencing PrEP awareness were assessed using univariate and multivariable logistic regression. In total, 608 eligible responders were included in the study. Among the respondents, 27.9% had PrEP awareness, 3.3% had taken PrEP, and 57.9% expressed interest in receiving PrEP, if required. Greater odds of PrEP awareness were associated with higher education level (adjusted odds ratio [aOR] 3.525, 95% confidence interval [CI] 2.013-6.173, P < 0.0001), greater HIV-related knowledge (aOR 3.605, 95% CI 2.229-5.829, P < 0.0001), HIV testing (aOR 2.647, 95% CI 1.463-4.788, P = 0.0013), and sexually transmitted infections (aOR 2.064, 95% CI 1.189-3.584, P = 0.0101). Lower odds of PrEP awareness were associated with higher stigma score (aOR 0.729, 95% CI 0.591-0.897, P = 0.0029). The findings indicate sub-optimal awareness and low utilization of PrEP in Beijing and highlight PrEP inequities among MSM with stigma. Strengthening the training of peer educators in disseminating PrEP knowledge and reducing stigma are critical for improving PrEP awareness.

TGF-ß1 contributes to the hepatic inflammation in animal models with nonalcoholic steatohepatitis by Smad3/TLR2 signaling pathway.

Non-alcoholic fatty liver disease (NAFLD) is increasingly affecting human health and the economy worldwide due to various factors. Here, we found that the expression of TGF-ß1 and TLR2 was significantly up-regulated in liver samples from both rats and mice nonalcoholic steatohepatitis (NASH) models. By constructing corresponding cell model, we found that TGF-ß1 challenge can positively regulate the expression of TLR2 and p-Smad2/3, and the dual luciferase reporter gene system and EMSA assay confirmed the existence of Smad3 binding site (-916 ∼ -906) in the promoter region of TLR2. The overexpression and interference changes of Smad2/3 further verified the above experimental results. Taken together, these findings suggest that TGF-ß1 promotes TLR2 transcription and its target gene expression via Smad3, leading to malignant exacerbation of liver inflammation in NASH, which provides new insights into the treatment of NASH.

Induction of PDCD4 by albumin in proximal tubule epithelial cells potentiates proteinuria-induced dysfunctional autophagy by negatively targeting Atg5.

Autophagy dysfunction is a hallmark of type 1 diabetes. However, the precise molecular mechanism of proteinuria-induced dysfunctional autophagy remains unclear. Herein, we investigated the role of programmed cell death 4 (PDCD4) in the regulation of autophagy in the pathogenesis of diabetic kidney disease (DKD) in vivo and in vitro. RT-qPCR, immunohistochemistry (IHC), and western blotting demonstrated an upregulation of Pdcd4 mRNA and protein in streptozotocin (STZ)-induced DKD rats, as compared to the control. In addition, IHC and western blotting of a unilateral ureteral obstruction mouse model showed an upregulation of PDCD4 in the disease group, as compared to their respective controls. IHC analysis of kidney biopsy samples of human DKD patients showed an upregulation of PDCD4 compared to the control. Western blotting of the STZ-induced DKD rat tissues displayed a low microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, as compared to the control. It was found that albumin overload in cultured PTECs upregulated the expression of PDCD4 and p62 and decreased the expression of LC3-II and autophagy-related 5 (Atg5) proteins. The knockout of Pdcd4 in cultured PTECs could reduce albumin-induced dysfunctional autophagy, as evidenced by the recovery of Atg5 and LC3-II protein. The forced expression of PDCD4 could further suppress the expression of the crucial autophagy-related gene Atg5. Evidence suggests that endogenous PDCD4 promotes proteinuria-induced dysfunctional autophagy by negatively regulating Atg5. Therefore, PDCD4 may be a potential therapeutic target in DKD.

Inhibition of miR-188-5p alleviates hepatic fibrosis by significantly reducing the activation and proliferation of HSCs through PTEN/PI3K/AKT pathway.

Persistent hepatic damage and chronic inflammation in liver activate the quiescent hepatic stellate cells (HSCs) and cause hepatic fibrosis (HF). Several microRNAs regulate the activation and proliferation of HSCs, thereby playing a critical role in HF progression. Previous studies have reported that miR-188-5p is dysregulated during the process of HF. However, the role of miR-188-5p in HF remains unclear. This study investigated the potential role of miR-188-5p in HSCs and HF. Firstly, we validated the miR-188-5p expression in primary cells isolated from liver of carbon tetrachloride (CCl4 )-induced mice, TGF-ß1-induced LX-2 cells, livers from 6-month high-fat diet (HFD)-induced rat and 4-month HFD-induced mice NASH models, and human non-alcoholic fatty liver disease (NAFLD) patients. Furthermore, we used miR-188-5p inhibitors to investigate the therapeutic effects of miR-188-5p inhibition in the HFD + CCl4 induced in vivo model and the potential role of miR-188-5p in the activation and proliferation of HSCs. This present study reported that miR-188-5p expression is significantly increased in the human NAFLD, HSCs isolated from liver of CCl4 induced mice, and in vitro and in vivo models of HF. Mimicking the miR-188-5p resulted in the up-regulation of HSC activation and proliferation by directly targeting the phosphatase and tensin homolog (PTEN). Moreover, inhibition of miR-188-5p reduced the activation and proliferation markers of HSCs through PTEN/AKT pathway. Additionally, in vivo inhibition of miR-188-5p suppressed the HF parameters, pro-fibrotic and pro-inflammatory genes, and fibrosis. Collectively, our results uncover the pro-fibrotic role of miR-188-5p. Furthermore, we demonstrated that miR-188-5p inhibition decreases the severity of HF by reducing the activation and proliferation of HSCs through PTEN/AKT pathway.

Epigallocatechin Gallate Protects Mice against Methionine-Choline-Deficient-Diet-Induced Nonalcoholic Steatohepatitis by Improving Gut Microbiota To Attenuate Hepatic Injury and Regulate Metabolism.

Epigallocatechin gallate (EGCG) has been regarded as a protective bioactive polyphenol in green tea against nonalcoholic steatohepatitis (NASH), but the mechanism remains poorly deciphered. Herein, we assessed the role and mechanism of EGCG on gut microbiota and the metabolism in NASH development. Forty-eight male C57BL/6J mice were fed with either a methionine-choline-sufficient diet or a methionine-choline-deficient (MCD) diet with or without EGCG administration for 4 weeks. Liver injury, inflammation, lipid accumulation, and iron overload were examined. 16S ribosomal RNA sequencing was used to detect the fecal microbiome. In our research, we observed that EGCG notably improved MCD-diet-derived gut microbiota dysbiosis, as proved by a distinctively clustered separation from that of the MCD group and by the decrease of the Oxalobacter, Oscillibacter, Coprococcus_1, and Desulfovibrio genera and enrichment of norank_f__Bacteroidales_S24_7_group, Alloprevotella, and Bacteroides. Spearman-correlation heatmap analysis indicated that Bacteroides and Alloprevotella induced by EGCG were strongly negatively correlated with lipid accumulation. Functional enzymes of the gut microbiome were predicted by PICRUSt based on the operation classification unit. The results revealed that 1468 enzymes were involved in various metabolic pathways, and 371 enzymes showed distinct changes between untreated and EGCG-treated mice. Long-chain-fatty-acid-CoA ligase ACSBG played a distinct role in fatty acid metabolism and ferroptosis and was significantly negatively correlated with Bacteroides. Altogether, the salutary effect of EGCG on NASH might be via shifting gut flora and certain enzymes from genera. Our study thus takes a step toward NASH prevention and therapy.

Programmed cell death factor 4 (PDCD4), a novel therapy target for metabolic diseases besides cancer.

Programmed cell death factor 4 (PDCD4) is originally described as a tumor suppressor gene that exerts antineoplastic effects by promoting apoptosis and inhibiting tumor cell proliferation, invasion, and metastasis. Several investigations have probed the aberrant expression of PDCD4 with the progression of metabolic diseases, such as polycystic ovary syndrome (PCOS), obesity, diabetes, and atherosclerosis. It has been ascertained that PDCD4 causes glucose and lipid metabolism disorders, insulin resistance, oxidative stress, chronic inflammatory response, and gut flora disorders to regulate the progression of metabolic diseases. This review aims to summarize the latest researches to uncover the structure, expression regulation, and biological functions of PDCD4 and to elucidate the regulatory mechanism of the development of tumors and metabolic diseases. This review has emphasized the understanding of the PDCD4 role and to provide new ideas for the research, diagnosis, and treatment of tumors and metabolic diseases.

Effects and Mechanisms of Autophagy Induced by Solubilized-Cholesterol in Hepatocytes: A Comparative Study Among Solvents.

Cholesterol, the principal sterol in mammalian cells, has been reported to play a role in the pathogenesis of several diseases through autophagy. Due to its insoluble characteristic, all in vitro cholesterol experiments are performed using dimethyl sulphoxide, methyl-ß-cyclodextrin, and ethanol co-solvents. To investigate whether the types of solvents have different effects on cholesterol-induced cell behaviors, we analyzed the effects and mechanisms of autophagy induced by solubilized-cholesterol in hepatic cells. We found that both solubilized-cholesterol and involved solvents could induce autophagy. Solubilized-cholesterol could further enhance the LC3-II expression with or without the pre-treatment with lysosomal blockers compared with the single-solvent groups, indicating that cholesterol could sensitize cells to solvents-induced autophagy. Besides, solubilized-cholesterol and single-solvent treatment could repress the activation of AKT-mTOR pathway. Furthermore, cholesterol solubilized in methyl-ß-cyclodextrin could induce apoptosis while other solubilized-cholesterol or single solvent groups could not, suggesting that different dissolve methods may affect the cytotoxic of cholesterol. These results strongly suggest that the effect of solvent should be taken into consideration in further in vitro cholesterol studies.

The Human Novel Gene LNC-HC Inhibits Hepatocellular Carcinoma Cell Proliferation by Sequestering hsa-miR-183-5p.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed cancer and the leading cause of cancer mortality. Several lines of evidence have demonstrated the aberrant expression of long noncoding RNAs (lncRNAs) in carcinogenesis and their universal regulatory properties. A thorough understanding of lncRNA regulatory roles in HCC pathology would contribute to HCC prevention and treatment. In this study, we identified a novel human lncRNA, LNC-HC, with significantly reduced levels in hepatic tumors from patients with HCC. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide) assays as well as colony formation and wound healing experiments showed that LNC-HC significantly inhibited the proliferation of the HCC cell line Huh7. Xenograft transplantation of LNC-HC-overexpressing Huh7 cells in nude mice resulted in the production of smaller tumors. Mechanistically, LNC-HC inhibited the proliferation of HCC cells by directly interacting with hsa-miR-183-5p. LNC-HC rescued the expression of five tumor suppressors, including AKAP12, DYRK2, FOXN3, FOXO1, and LATS2, that were verified as target genes of hsa-miR-183-5p. Overall, human LNC-HC was identified as a novel tumor suppressor that could inhibit HCC cell proliferation in vitro and suppress tumor growth in vivo by competitively binding hsa-miR-183-5p as a competing endogenous RNA (ceRNA). These findings suggest that LNC-HC could be a biomarker of HCC and provide a novel therapeutic target for HCC treatment.

Multidrug resistance affects the prognosis of primary epithelial ovarian cancer.

Multidrug-resistant tumor cells can tolerate different structures, functions and antidrug action mechanisms, therefore, allowing these cells to respond to various structurally unrelated mechanisms of different chemotherapy drugs and to exhibit cross-resistance. The present study aimed to investigate the role of Multi-drug resistance gene (MDR1), Placental glutathione S-transferase-P1 (GSTP1), Lung resistance protein (LRP) and Ras association domain family member 1 (RASSF1A) in primary epithelial ovarian cancer (PEOC). The mRNA (protein) expression levels of MDR1, product P glycoprotein, LRP and GSTP1 were evaluated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in all tissue samples, ovarian cancer cell line A2780 and A2780/DDP. Methylation-specific PCR (MSP) was used to detect RASSF1A gene methylation in all tissue samples. The resistance genes/proteins were either poorly or not expressed in A2780, however were highly expressed in A2780/DDP cell line. The expression of resistance genes/proteins decreased following different concentrations of zebularine-stimulated A2780/DDP. Hypermethylation and low expression of RASSF1A gene were detected in PEOC and A2780/DDP. Subsequent to being exposed to different concentrations of zebularine-stimulated A2780/DDP, the RASSF1A methylation level was decreased, while the unmethylation level was increased. The expression of RASSF1A gene/protein was gradually restored, and the gene/protein expression was enhanced with the increase in drug concentration. Multivariate logistic regression indicated that the expression level of gene LRP and GSTP1 was a risk factor for PEOC prognosis. Furthermore, the expression of LRP and GSTP1 in the negative-group survival curves was higher compared with the positive group. High expression of resistance genes may serve an important role in cancer primary resistance. Low expression caused by hyper-methylation of RASSF1A gene may serve an important role in cancer-acquired resistance in PEOC. The present study suggested that resistant gene expression may be a potential prognostic biomarker.

Apigenin Combined With Gefitinib Blocks Autophagy Flux and Induces Apoptotic Cell Death Through Inhibition of HIF-1α, c-Myc, p-EGFR, and Glucose Metabolism in EGFR L858R+T790M-Mutated H1975 Cells.

Cancer cells are characterized by abnormally increased glucose uptake and active bio-energy and biosynthesis to support the proliferation, metastasis, and drug resistant survival. We examined the therapeutic value of the combination of apigenin (a natural small-molecule inhibitor of Glut1 belonging to the flavonoid family) and gefitinib on epidermal growth factor receptor (EGFR)-resistant mutant non-small cell lung cancer, to notably damage glucose utilization and thus suppress cell growth and malignant behavior. Here, we demonstrate that apigenin combined with gefitinib inhibits multiple oncogenic drivers such as c-Myc, HIF-1α, and EGFR, reduces Gluts and MCT1 protein expression, and inactivates the 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, which regulates glucose uptake and maintains energy metabolism, leading to impaired energy utilization in EGFR L858R-T790M-mutated H1975 lung cancer cells. H1975 cells exhibit dysregulated metabolism and apoptotic cell death following treatment with apigenin + gefitinib. Therefore, the combined apigenin + gefitinib treatment presents an attractive strategy as alternative treatment for the acquired resistance to EGFR-TKIs in NSCLC.

Rosmarinic acid inhibits nicotine-induced C-reactive protein generation by inhibiting NLRP3 inflammasome activation in smooth muscle cells.

Atherosclerosis is widely known to be a chronic inflammatory disease. C-reactive protein (CRP), an important inflammatory factor, plays an essential role in the pathogenesis of atherosclerosis. Nicotine, the main addictive component of cigarette, has been shown to induce the production of CRP. The aim of this study was to investigate the effect of rosmarinic acid (RA), a polyphenol with antiinflammatory activity, on nicotine-induced elevation of CRP in vascular smooth muscle cells (VSMCs). We found that pretreatment of VSMCs with RA attenuated nicotine-induced expression of CRP in a time- and dose-dependant manner. In addition, RA also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and reactive oxygen species (ROS) production resulting from nicotine treatment in VSMCs. To confirm these findings in vivo, we constructed a nicotine-induced atherosclerosis rat model. RA did not significantly reduce the serum nicotine level of the rats, whereas it significantly decreased the levels of serum lipids, including concentrations of cholesterol, triglycerides, and low-density lipoprotein cholesterol, and the serum level of CRP. RA also led to diminished nicotine-induced activation of NLRP3 inflammasome and elevation in the CRP level in the aortic tissue of the model rats. The results of this study suggested a protective role of RA in nicotine-induced atherosclerosis by inhibiting the ROS-NLRP3 inflammasome-CRP axial, and RA therefore represented a potential effective therapeutic approach to atherosclerosis, in particular for those who smoke.

Tumor necrosis factor-α and diabetic retinopathy: Review and meta-analysis.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is produced by multinuclear giant cells and acts as local intensification signals in pathological processes associated with chronic eye inflammation. This meta-analysis was performed to provide a better understanding of the relationship between TNF-α and diabetic retinopathy (DR). METHOD: Online electric databases were searched to retrieve all relevant articles published before October 2017. The standard mean difference (SMD) and their 95% confidence intervals (CI) were included and then pooled with a random effects model. RESULTS: A total of 16 articles with 1286 participants were included in this meta-analysis. No difference in the level of TNF-α was found between DR patients and healthy controls (SMD = 0.39, 95% CI = -0.09 to 0.68, P = 0.01). Subgroup analysis showed that with respect to the level of TNF-α, the association was significant for studies conducted in Europe (SMD: 0.57, 95% CI: 0.11-1.02, P = 0.01), patients with type 1 DM (SMD: 1.06, 95% CI: 0.09-2.04, P = 0.03), studies based on serum samples (SMD: 0.57, 95% CI: 0.12-1.02, P = 0.01) and studies with a sample size >50 (SMD: 0.39, 95% CI: 0.03-0.75, P = 0.04). CONCLUSION: The results this meta-analysis indicated that the level of TNF-α in DR patients was significantly different from that in the healthy controls, so TNF-α represents a candidate biomarker for DR.

Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.

Application of embolization microspheres in interventional therapy of malignant non-hypervascular tumor of liver.

OBJECTIVE: To investigate the efficacy and safety of transarterial embolization (TAE) using embolization microspheres in the treatment of non-hypervascular malignant liver tumors. METHODS: Patients with malignant non-hypervascular liver tumors, who were treated with TAE using embolization microspheres, were selected and analyzed retrospectively. The technical success rate, tumor response, and complications were assessed. RESULTS: Six patients were included in the study: 1 patient each with hepatocellular-cholangiocarcinoma, intrahepatic cholangiocarcinoma, hepatic metastasis after resection of common bile duct carcinoma, liver metastasis from colon cancer, liver metastasis from esophageal cancer, and liver metastasis from pancreatic cancer. The technical success rate was 100%. At 1 and 3 months after TAE, tumor local reactions were seen in 6/6 and 2/6 patients, respectively, and the tumor necrosis rates were 48%-73% and 22%-68%, respectively. The main complications were those related to the embolization syndrome, including 1 case of liver abscess and 1 case of severe pain on the first day after embolization. CONCLUSION: TAE with embolization microspheres is safe and effective in non-hypervascular liver tumors. It is a feasible option for palliative therapy of these tumors.

Elevated expression of placental growth factor is associated with airway-wall vascular remodelling and thickening in smokers with asthma.

The increased expression of placental growth factor (PlGF) in chronic obstructive pulmonary disease and allergy-related asthma suggests its role in the pathogenesis of these diseases. In asthmatic smokers, airway remodelling is accompanied by an accelerated decline in lung function. However, whether PlGF contributes to the persistent airflow obstruction and vascular remodelling typically seen in asthmatic smokers is unknown. In this study we measured lung function, airway-wall thickening, and PlGF levels in serum and induced sputum in 74 asthmatic and 42 healthy smokers and never-smokers. Using human lung microvascular endothelial cells (HLMECs), we evaluated the in vitro effects of PlGF on each step of vascular remodelling, including proliferation, migration, stress-fibre expression, and tubule formation. Our data showed significantly higher serum and sputum PlGF levels in asthma patients, especially asthmatic smokers, than in healthy controls. Serum and sputum PlGF levels correlated negatively with post-bronchodilator forced expiratory volume in 1 s (FEV1) and the FEV1/forced vital capacity, but positively with airway-wall thickening. Stimulation of HLMECs with rhPlGF promoted all of the steps of airway-microvascular remodelling. These findings provide insights into the influence of cigarette smoking on the structural changes in the airways of asthmatics and the important pathogenic role played by PlGF.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.

BACKGROUND: NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening. METHOD: We recombined the reporter gene vector with inserting the "neo" transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela. RESULT: In this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes. CONCLUSION: In conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

[Bidirectional regulation of Pam3CSK4?induced inflammatory response by ATP?binding cassette transporter A1 knockdown in mouse mononuclear macrophages in vitro].

OBJECTIVE: To investigate the regulatory effect of ATP?binding cassette transporter A1 (ABCA1) knockdown on inflammatory response induced by Pam3CSK4 in mouse mononuclear macrophage RAW264.7 cell line. METHODS: A mouse mononuclear macrophage RAW264.7 cell line with stable ABCA1 knockdown was constructed and stimulated with Toll?like receptor 2 (TLR2) ligand Pam3CSK4, and the changes in the transcriptional levels of the proinflammatory and anti-inflammatory cytokines were analyzed in this cell model. RESULTS: In RAW264.7 cells, ABCA1 knockdown significantly up-regulated Pam3CSK4 stimulation?induced expressions of IL?1ß, TNF?α and IL?6 and also enhanced the expression of transcription factor cAMP?dependent transcription factor 3 (ATF3) without obviously affecting the expressions of the transcription factors ATF1, ATF2, ATF4 or ATF5. CONCLUSION: ABCA1 knockdown in macrophages may have both proinflammatory and anti?inflammatory effects. ABCA1 knockdown up?regulates the transcription of ATF3 possibly through a mechanism that is different from that for the other members of the ATF protein family.

Pristane induces autophagy in macrophages, promoting a STAT1-IRF1-TLR3 pathway and arthritis.

Autophagy is involved in both innate and adaptive immune regulation. We propose that autophagy regulates activation of TLR3 in macrophages and is thereby essential for development of pristane-induced arthritis. We found that pristane treatment induced autophagy in macrophages in vitro and in vivo, in spleen cells from pristane injected rats. The induced autophagy was associated with STAT1 phosphorylation and expression of IRF1 and TLR3. Blocking the pristane activated autophagy by Wortmannin and Bafilomycin A1 or by RNAi of Becn1 led to a downregulation of the associated STAT1-IRF1-TLR3 pathway. Most importantly, the development of arthritis was alleviated by suppressing either autophagy or TLR3. We conclude that pristane enhanced autophagy, leading to a STAT1-IRF1 controlled upregulation of TLR3 expression in macrophages, is a pathogenic mechanism in the development of arthritis.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na+-K+-ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na+-K+-ATPase activity was detected by SensoLyteR FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na+-K+-ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na+-K+-ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit is prepared.